Physiol Rev 95: 513–548, 2015
doi:10.1152/physrev.00013.2014
PHYSIOLOGY OF PROGLUCAGON PEPTIDES: ROLE OF
GLUCAGON AND GLP-1 IN HEALTH AND DISEASE
Darleen A. Sandoval and David A. D’Alessio
Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Cincinnati, Ohio
Sandoval DA, D’Alessio DA. Physiology of Proglucagon Peptides: Role of Glucagon
and GLP-1 in Health and Disease. Physiol Rev 95: 513–548, 2015;
doi:10.1152/physrev.00013.2014.—The preproglucagon gene (Gcg) is expressed
by specific enteroendocrine cells (L-cells) of the intestinal mucosa, pancreatic islet
␣-cells, and a discrete set of neurons within the nucleus of the solitary tract. Gcg
encodes multiple peptides including glucagon, glucagon-like peptide-1, glucagon-like peptide-2,
oxyntomodulin, and glicentin. Of these, glucagon and GLP-1 have received the most attention
because of important roles in glucose metabolism, involvement in diabetes and other disorders,
and application to therapeutics. The generally accepted model is that GLP-1 improves glucose
homeostasis indirectly via stimulation of nutrient-induced insulin release and by reducing glucagon
secretion. Yet the body of literature surrounding GLP-1 physiology reveals an incompletely understood and complex system that includes peripheral and central GLP-1 actions to regulate energy
and glucose homeostasis. On the other hand, glucagon is established principally as a counterregulatory hormone, increasing in response to physiological challenges that threaten adequate blood
glucose levels and driving glucose production to restore euglycemia. However, there also exists a
potential role for glucagon in regulating energy expenditure that has recently been suggested in
pharmacological studies. It is also becoming apparent that there is cross-talk between the proglucagon derived-peptides, e.g., GLP-1 inhibits glucagon secretion, and some additive or synergistic
pharmacological interaction between GLP-1 and glucagon, e.g., dual glucagon/GLP-1 agonists
cause more weight loss than single agonists. In this review, we discuss the physiological functions
of both glucagon and GLP-1 by comparing and contrasting how these peptides function, variably in
concert and opposition, to regulate glucose and energy homeostasis.
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INTRODUCTION
PREPROGLUCAGON GENE...
PHYSIOLOGICAL IMPORTANCE...
RECEPTOR DISTRIBUTION AND...
SECRETION OF PROGLUCAGON...
EFFECTS OF ␣-CELL PRODUCTS ON...
REGULATION OF GLUCOSE...
REGULATION OF ENERGY...
ROLE OF PROGLUCAGON PEPTIDES IN...
ROLE OF PROGLUCAGON PEPTIDES...
CONCLUSION
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I. INTRODUCTION
Obesity and type 2 diabetes mellitus (T2DM) are major
public health burdens of modern society, providing a growing medical and economic challenge. There is a pressing
need for new therapies that improve glycemia and/or body
weight and that have driven recent research in academic and
industrial laboratories. Over the last 10 years multiple
drugs have been developed for the treatment of T2DM that
utilize the signaling systems of products of the preproglucagon gene (Gcg). Of these, most widely recognized are those
that utilize the glucagon-like peptide 1 (GLP-1) signaling
system. There is also long-standing interest in glucagon antagonists to treat T2DM, but recent developments indicate
a potential role for glucagon agonists as components of
multireceptor agents for obesity and diabetes. While there
are many excellent reviews on GLP-1 physiology and pharmacology (53, 95, 174, 211) or glucagon physiology and
pharmacology (61, 101, 142, 148, 195), few have addressed the physiology and pharmacology of both of these
peptides in one review. These two closely related compounds, with distinct roles in metabolism, are now both
compelling drug targets, and understanding their physiology is timely. Moreover, the considerable effort expended
on study of the Proglucagon (ProG) peptides over the past
several decades has revealed a more complex set of interactions and overlapping regulation for glucagon and GLP-1
that may also be important in diabetes pharmacology.
II. PREPROGLUCAGON GENE EXPRESSION
AND ORGAN-SPECIFIC PROCESSING
Gcg is genetically expressed in a specific population of enteroendocrine cells (L-cells) of the intestinal mucosa, pancreatic islet ␣-cells, and a discrete set of neurons within the
nucleus of the solitary tract (NTS; Refs. 150, 197). Studies
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DARLEEN A. SANDOVAL AND DAVID A. D’ALESSIO
on the regulation of Gcg transcription stretch back over
three decades, although the process is still not completely
understood. The Gcg promoter has four enhancer elements
and a cAMP response element. Gcg expression is increased
by elevations of cAMP or exposure to amino acids through
these elements (133, 449), which are also targets for a number of homeodomain protein transcription factors (198).
There is mounting evidence for cell-specific regulation of
Gcg expression in the pancreas and intestine. In islet ␣-cells,
Pax6 and MafB promote, whereas insulin inhibits, Gcg
transcription through specific intermediates at distinct
DNA binding regions (139, 198). In contrast, insulin increases Gcg expression in intestinal endocrine cells (60,
446). The latter effect is mediated by effectors of the canonical Wnt signaling pathway, ␤-catenin and transcription
factor 7 like 2 (TCF7L2), a pathway that seems to be specific for control of intestinal Gcg transcription (60). Beyond
nutrient and hormonal regulation, it is well established that
bowel resection or injury causes a large increase in Gcg
expression, although the mediators of this response are not
clear. The existence of separate mechanisms to regulate
transcriptional control of Gcg parallels the distinct patterns
of prohormone processing in the major cell types producing
ProG peptides.
cells and neurons of the NTS predominance of PC1/3 expression yields GLP-1, oxyntomodulin, and GLP-2 as the
physiologically relevant products (237, 401, 416). PC2 is
also found within the brain but is not colocalized with Gcg,
and only trace amounts of glucagon have been detected in
the CNS (237), supporting the idea that PC1/3 processing of
Gcg predominates there. There is PC1/3 expression in
␣-cells, albeit at lower levels than PC2, and increasing evidence for islet production of GLP-1. Within the intestine,
the density of Gcg expression and ProG synthesis increases
from the proximal to distal gut and expression is highest in
the colon.
III. PHYSIOLOGICAL IMPORTANCE OF
GLUCAGON AND GLP-1
In addition to glucagon and GLP-1, ProG contains the bioactive proteins glucagon like peptide-2 (GLP-2) and oxyntomodulin as well as fragments such as glicentin, glicentinrelated pancreatic polypeptide (GRPP), and major proglucagon fragment (MPGF) that are of unclear functional
significance (FIGURE 1). The relative amounts and forms of
these ProG peptides in any one cell type depend on tissuespecific posttranslational modification by prohormone convertases (PC). In the ␣-cell, high PC2 expression produces
predominantly glucagon (173), while in the intestinal L-
It is now generally accepted that GLP-1 has a broad role in
glucose homeostasis, in great part through stimulation of
nutrient-induced insulin release (227, 275) and by reducing
glucagon secretion (435). In contrast, the best-recognized
function of glucagon as a counterregulatory hormone that
is released when glucose levels decrease below basal levels,
and stimulate hepatic glucose production (88). A role for
both peptides has been proposed in the pathogenesis of
T2DM. On the one hand, the reduced GLP-1 levels sometimes observed in T2DM patients (414) have led to a
suggestion that inappropriately low levels of GLP-1 reduce the insulin response to a given glucose load. On the
other hand, postprandial GLP-1 levels are increased by
⬃10-fold after roux-en-Y gastric bypass and vertical
sleeve gastrectomy, two highly efficacious bariatric surgeries associated with improved if not resolved diabetes
and normalized body weight in obese patients (26, 298).
In contrast, elevated glucagon levels are implicated in the
Preproglucagon
PS
Proglucagon
GRPP
Glucagon
IP1
GLP-1
IP2
GLP-2
GRPP
Glucagon
IP1
GLP-1
IP2
GLP-2
Psck2
dominant
α-cell
Glicentin-related
pancreatic peptide
Major proglucagon
fragment
Glucagon
GLP-1
GRPP
GLP-2
IP1
Intervening peptide 1
GLP-1
IP2
Psck1/3
dominant
GLP-2
Glucagon
Oxyntomodulin
IP2
GRPP
514
IP1
Glucagon
IP1
FIGURE 1. Posttranslational processing of
preproglucagon. The preproglucagon gene
encodes proglucagon, a peptide that is differentially processed based on the relative activities of the prohormone convertases 1/3 and
2. In the ␣-cells of the pancreatic islet, prohormone convertase 2 (Psck2) predominates
and glucagon, glicentin-related pancreatic
polypeptide (GRPP), intervening peptide 1
(IP1), and a proglucagon fragment are the
more prevalent products. In the intestinal Lcell and specific CNS neurons, prohormone
convertase 1 (Psck1) action is relatively
greater and proglucagon is cleaved to GLP-1,
GLP-2, oxyntomodulin, glicentin, and IP2.
Most recent evidence indicates that ␣-cells
have some PC 1/3 activity, and it is likely that
neurons and L-cells also have PC 2.
Glicentin
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GLUCAGON AND GLP-1
high basal glucose production seen with T2DM (72). As
a result, numerous effective pharmaceutical compounds
to treat T2DM are either on the market or in development that increase GLP-1 action, and/or reduce the activity of glucagon (148). However, the actions of glucagon have been less tractable, and no glucagon receptor
antagonists have yet reached the clinic. Surprisingly, dual
glucagon and GLP-1 agonists are in development that
have balanced actions of the two peptides such that
weight loss and improved glucose control have been reported in preclinical models (79). In the following sections of this review, we will discuss the physiological
functions of both glucagon and GLP-1 with a goal of
describing how these related signaling systems act in
health and disease, and their potential as therapeutic
agents.
IV. RECEPTOR DISTRIBUTION AND
FUNCTION
A. GLP-1 Receptor Distribution and
Signaling
The GLP-1 receptor (GLP-1r) is found within the pancreas,
lung, adipose tissue, kidney, heart, vascular smooth muscle,
and in a number of specific nuclei in the CNS (54, 135).
Most of what is known about GLP-1r signaling comes from
studies of ␤-cell function. The stimulation of insulin secretion by activation of the GLP-1r is primarily due to activation of a stimulatory G protein, generation of cAMP, and
promotion of the activities of protein kinase A (PKA) and
the exchange protein activated by cAMP 2 (EPAC2) (175).
These mediators control transcription of key genes including proinsulin, glucose transporter-2 (GLUT2), subunits of
the ATP-sensitive potassium channel (KATPase) and the secretory vesicle related protein ␣-SNAP, likely through
cAMP responsive element binding protein (CREB) (449).
However, the GLP-1r also activates other pathways to influence gene transcription including those mediated by extracellular signal-regulated kinase 1/2 (Erk 1/2), and phosphoinositol 3-kinase (PI3K). Increased PKA and EPAC2
activity potentiate the closure of the KATPase initiated by
␤-cell glucose metabolism, regulate membrane electrical
activity, and promote Ca2⫹ influx, to amplify glucosestimulated insulin exocytosis (175, 252). A key feature of
GLP-1r signaling is the dependence on increases in glucose flux into the ␤-cell to be fully manifest, i.e., GLP-1 is
a weak insulin secretagogue at basal concentrations of
ambient glucose. The mechanism for this important phenomenon has not yet been explained, but there is evidence that PKA has variable actions on the KATPase that is
dependent on the energy state of the cell (240). In the
fasting state, when intracellular ADP levels are elevated,
PKA hyperpolarizes the ␤-cell membrane by increasing
KATPase conductance, but when ADP levels are reduced
by increased glucose metabolism, PKA contributes to
KATPase closure and depolarization. The minimal effectiveness of GLP-1 to stimulate insulin secretion under
conditions of basal or low glucose is a characteristic of
other GI hormones as well (68). Given the benefit of
insulin secretagogues that are muted when their action
could cause hypoglycemia, it is notable that the mechanism for this has not been more fully developed.
Recent findings implicate the transcription factor TCF7L2 in
GLP-1 ␤-cell signaling, an important connection in that
genome-wide association studies have noted TCF7L2
polymorphisms to be linked with the risk for T2DM.
While the control of GLP-1r expression is not fully understood, there is now evidence to support a role for
TCF7L2 and the Wnt signaling pathway in ␤-cell synthesis of the receptor. Moreover, a component of GLP-1rmediated gene transcription is dependent on ␤-catenin/
TCF7L2 (242). Finally, humans with specific single nucleotide polymorphisms of the TCF7L2 gene have
diminished insulin responses to oral glucose and intravenous GLP-1 (310, 355, 411). The latter findings, in particular, raise the possibility that the GLP-1 system has a
role in the pathogenesis of diabetes.
The role of GLP-1 in the ␤-cell goes beyond its role as an
insulin secretagogue, and considerable attention has been
given to actions related to ␤-cell growth and differentiation.
However, to some degree, the signaling mechanisms overlap. Specifically, GLP-1r activation of the PKA pathway in
the ␤-cell also contributes to ␤-cell replication and reduces
apoptosis. Indeed, GLP-1r activation benefits ␤-cell survival in the presence of multiple apoptotic conditions including hyperglycemia, hyperlipidemia, inflammatory cytokines, and oxidative stress (97). However, the PKA pathway does not seem to be the principle means by which the
GLP-1r controls ␤-cell growth and apoptosis. For example,
in ␤-MIN6 cells, GLP-1 administration reduces H2O2-induced apoptosis both through cAMP and ERK1/2/PI3K signaling (179). Upstream of ERK1/2 activation are both PKA
and an adaptor protein, ␤-arrestin, that binds to the GLP-1r
and is necessary for full GLP-1r signaling (380). In ␤-MIN6
cells, GLP-1 induces a cAMP-dependent activation of
ERK1/2 within minutes, followed by a later ␤-arrestin-dependent increase of ERK1/2 signaling that occurs over an
hour (324). Moreover, in MIN6 cells, ␤-arrestin is necessary for the antiapoptotic effects of GLP-1. Activation of
both ␤-arrestin and Akt, a prosurvival kinase, contributes
to initiation of ␤-catenin-dependent Wnt signaling, a pathway established in cancer biology to be critical for cell proliferation and survival. Activation of ␤-arrestin and Akt
leads to accumulation of cytosolic ␤-catenin and subsequent translocation to the nucleus where it forms a complex
with TCF7L2 (242), a transcription factor that activates
expression of Wnt target genes (433). Wnt signaling via
␤-catenin and TCF7L2 is necessary for the full effects of the
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DARLEEN A. SANDOVAL AND DAVID A. D’ALESSIO
long-acting GLP-1 agonist Ex4, as both siRNA silencing of
␤-catenin and a dominant negative insertion of TCF7L2 in
INS-1 cells blunted the ability of Ex4 to stimulate ␤-cell
proliferation (242). In addition, pancreatic-specific deletion
of TCF7L2 impairs GLP-1-induced insulin secretion from
isolated mouse islets (73a). These data illustrate the wideranging signaling pathways induced by GLP-1r activation
to regulate islet function. The findings from in vitro studies
demonstrate that GLP-1r activation is connected to the key
pathways that are essential for regulation of ␤-cell proliferation (242). One of the reasons that this area of GLP-1
physiology has received intense study is the potential clinical application to T2DM, a progressive disease that is
thought to involve increased rates of ␤-cell death. However,
although much has been learned about ␤-cell growth from
this work, a connection to human disease has not been
established.
Similar to GLP-1r actions in the ␤-cell, CNS GLP-1r signaling is dependent on nutrient availability. Williams et al.
(434) were the first to demonstrate that the ability of peripherally administered Ex4 to reduce food intake was
blunted in fasted compared with fed animals. This has been
confirmed in other studies where fasting, glucose deprivation with 2-deoxyglucose, or diets high in fat or fructose
and low in carbohydrate, also blunt the anorectic action of
GLP-1r agonists (44, 163, 277, 352). Part of this response is
dependent on leptin, since administration of this peptide to
increase low, fasting levels to those typical of the fed state,
restores the anorectic action of Ex4 (434). In addition, a
recent body of work suggests that CNS fuel-sensing pathways also play a critical role in GLP-1 actions in the brain.
For example, AMPK, a highly conserved fuel-sensing enzyme, is activated when cellular fuels are depleted and thus
is activated by fasting, stimulates nutrient entry into the cell,
and upregulates several metabolic pathways that regenerate
ATP levels. Pharmacological activation of AMPK specifically within the CNS blunts the anorectic action of GLP-1r
agonists in hypothalamic and/or hindbrain nuclei (44, 163,
352). Suppression of food intake by Ex4 in the hindbrain
involves multiple intracellular signaling pathways including
rapid suppression of AMPK and stimulation of the MAPK
(163) and PI3K/Akt pathways (343). Finally, it is important
to note that in rodents Ex4 has an effect to lower food
intake that is 100-fold more potent than GLP-1 (21). It is
not clear whether this is due to differences in rates of metabolism of GLP-1 and Ex4 in the brain, or other factors
such as distinct interactions of the peptides with the GLP-1r
(255). Regardless, understanding how neurons are activated by GLP-1r agonists is of physiological and clinical
importance.
The distribution of the CNS GLP-1r includes neurons in the
hypothalamus, the hindbrain, and amygdala, and there is
evidence from studies in rodents that GLP-1 mediates distinct functions in specific populations of neurons. For ex-
516
ample, GLP-1r located within either the paraventricular
nucleus of the hypothalamus or the hindbrain suppress food
intake without causing visceral illness, the term given to
specific behaviors rats and mice exhibit when exposed to
toxins that is analogous to nausea and malaise (219). In
contrast, GLP-1 administered to the arcuate nucleus does
not affect feeding but reduces hepatic glucose production
(353). Lastly, GLP-1 injected specifically in the amygdala
causes visceral illness but not anorexia (219). Based on
these findings, suppression of food intake by GLP-1 may
be mediated by neural populations distinct from those
that cause visceral illness in rats, or nausea in humans.
This finding has relevance to clinical medicine where beneficial effects of pharmacological GLP-1r agonists on satiety and weight loss can be limited by gastrointestinal
side effects.
It is unknown how or whether the peripheral and central
GLP-1r systems interact. The half-life of GLP-1 is ⬍2 min in
humans and rodents, limiting the time over which plasma
GLP-1 can directly activate CNS neurons (168). An alternative route for circulating GLP-1 to stimulate the brain is
through visceral afferent neurons, traveling primarily in the
vagus nerve. GLP-1r mRNA is expressed in the neural cell
bodies of the nodose ganglion (281, 406) that contribute
the afferent tracts supplying the thoracic and abdominal
viscera, and are contained in the vagus. GLP-1 increases
vagal afferent neuronal activity (41, 203), and hepatic portal venous infusion of a GLP-1r antagonist, exendin-[9 –39]
(Ex9), impairs glucose tolerance (406). While some studies
report that infusions of GLP-1 or Ex9 directly into the portal vein do not affect food intake in rats (216, 344), there
has been one report that portal venous administration of
GLP-1 reduces feeding (25). Furthermore, both bilateral
subdiaphragmatic truncal vagotomy in rats, and afferent
denervation of mice with capsaicin, significantly blunts the
anorectic effects of peripherally administered GLP-1 and
Ex4, respectively (1, 344, 390). Since Ex4 has a plasma
half-life of 30 min, it has a greater chance to reach the CNS
via the circulation. This may explain why vagotomy blunts
the effect of Ex4 on food intake acutely, but has little effect
on suppression of food intake over several hours (229).
Interestingly, the patterns of cFos activation in the brain of
Ex4-treated animals with and without vagotomy differ,
supporting a model whereby vagal afferents are necessary
for the full effect of Ex4 on the CNS (204).
GLP-1r are also located within the intestine, making paracrine activation of adjacent gastrointestinal nerve terminals
a plausible signaling pathway. Intraperitoneal injections of
Ex4 activate both submucosal and myenteric neurons of the
rat duodenum (421), but it is not known whether this is due
to direct or indirect activation. More recent data demonstrate that subdiaphragmatic vagotomy has a potent inhibitory effect on glucose tolerance and blunts GLP-1-induced
suppression of glucose intake while a specific ablation of the
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hepatic branch of the vagus has no effect (161). These data
suggest that the major source of vagal activation may be
from intestinal rather than hepatic innervation. Thus, while
more research is needed to understand the complexity of
GLP-1r signaling in the gastrointestinal tract, a functional
neuronal link between the peripheral and CNS GLP-1 systems is suggested as a means of regulating both satiety and
glucose homeostasis. While understanding these pathways
is in the early stages, they may become important pharmacological targets as enterally available GLP-1r agonists are
under development.
B. Tissue Distribution of the Glucagon
Receptor
Both the sequences of glucagon and the glucagon receptor (GCGR) are highly conserved across mammalian species (14). Like GLP-1, binding of glucagon to the GCGR
activates adenylyl cyclase through G proteins of the Gs
subtype with subsequent generation of cAMP and activation of PKA and other downstream mediators as the
major mode of intracellular signaling (311). Evidence for
a GCGR was first demonstrated using hepatic membrane
preparations (314, 315, 337), and the richest source of
glucagon binding is in the liver and kidney (14). Lesser
binding occurs in heart, adipose tissue, the CNS, adrenal
gland, and spleen. GCGR gene expression is greatest in
liver, with more modest mRNA levels in the kidney,
heart, spleen, ovary, and the pancreatic islets (54, 103).
Consistent with the relative receptor expression, the liver
and kidney play the major role in glucagon clearance,
accounting for ⬃70% of the removal from the circulation
(14, 89, 373). Much of glucagon removal is through receptor-mediated endocytosis, but there is evidence for
endovascular proteolysis by dipeptididyl peptidase IV
(318) and enzymatic degradation at the level of the
plasma membrane (31, 117). The half-life of glucagon in
circulating plasma is relatively short: 2, 5, and 7 min in
rats, dogs, and humans, respectively (14, 316).
The expression of the glucagon receptor gene (Gcgr) in
islet cells and hepatocytes increases in response to a rise
in ambient glucose (297). Conversely, exposure to chronically elevated glucagon levels decreases expression. This
latter response is likely mediated through signaling pathways initiated by cAMP in that it is mimicked by forskolin and reversed by somatostatin (2). Studies of Gcgr
expression are generally consistent with binding studies
and support a model whereby glucagon signaling is partially regulated by the available number of receptors. But
while changes in GCGR expression could be important
for modulating glucagon action, to date there are no
definitive studies connecting the expression level of the
GCGR with relative glucagon action. In contrast to the
GCGR, pancreatic GLP-1r expression does not change
with glucose or cAMP (2).
V. SECRETION OF PROGLUCAGON
PEPTIDES BY ␣- AND L-CELLS
A. GLP-1 Secretion
Intestinal GLP-1 is secreted postprandially by enteroendocrine L-cells which increase in density along the intestine,
from relatively few in the duodenum to progressively
greater numbers in the jejunum, and the highest amount in
the ileum and colon (16). L-cells are located within the
intestinal epithelium and have apical processes that extend
into the gut lumen giving these cells direct access to ingested
nutrients (107), and indeed, carbohydrates (217), proteins
(217), and lipids (447) all individually stimulate GLP-1 secretion. While studies with isolated enteroendocrine cells,
or intestinal cell lines, suggest direct effects of nutrients to
stimulate GLP-1 release, the degree to which this occurs in
vivo is not clear. Neural, endocrine, and paracrine mechanisms have all been proposed to explain the response of
L-cells to nutrients (286).
The molecular basis for GLP-1 secretion from L-cells is not
as clearly defined as it is for some other hormones such as
insulin release from islets. Nevertheless, there have been
some parallels reported. For example, as in ␤-cells, KATP
channels and the sulfonylurea receptor are expressed in Lcells (296, 331), and closure of KATP channels, regulated by
increases in ATP as a consequence of glucose metabolism,
leads to GLP-1 secretion (301, 331). However, the role of
the KATP channel may differ between L-cell populations as
the KATP blocker glibenclamide stimulated glucose-induced
GLP-1 secretion in ileum, but not colon explants (130). It is
important to note that in clinical trials sulfonylureas do not
significantly affect GLP-1 secretion from T2DM subjects.
Current evidence suggests that a variety of G protein-coupled receptors (GPCR) play a key role in GLP-1 secretion.
Gs-coupled receptors that increase cAMP and activate PKA,
and its associated exchange proteins, are associated with
GLP-1 release both in vitro and in vivo (38, 39, 332). One of
these, the G protein-coupled bile acid receptor 1, or TGR5,
is a target of bile acids that stimulates release of GLP-1
(187, 381). Although bile acids also act on a nuclear transcription factor, farnesoid X receptor, TGR5 is the primary
receptor population responsible for bile acid-induced
GLP-1 secretion (206). Interestingly, increasing bile acids
within the intestinal lumen are well-known to increase
ProG peptides. For example, infusion of bile directly into
the ileum of dogs increases gut glucagon-like immunoreactivity (282) and infusion of a specific bile salt, deoxycholate,
increases plasma enteroglucagon levels when infused into
the colon in humans (3). Recent in vitro data using both
primary enteroendocrine cells and an intestinal cell line in-
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DARLEEN A. SANDOVAL AND DAVID A. D’ALESSIO
dicated that although bile acids do activate TGR5 and promote GLP-1 release, pharmacological TGR5 agonists are
more potent secretagogues and also enhance L-cell responses to calcium and glucose-induced GLP-1 secretion
(302) compared with naturally occurring bile acids.
Other GPCRs link lipid absorption to L-cell secretions as
well. Specifically, GPR119, GPR120, and GPR40 are activated by long-chain fatty acids and their derivatives (178),
and this increases GLP-1 secretion. GPR119 is a Gs-coupled
receptor that is expressed in ␤-cells, the CNS, and is also
highly colocalized to L-cells within the gastrointestinal
tract. GPR119 agonists increase GLP-1 secretion (63) while
GPR119 knockout mice have blunted GLP-1 responses to
an oral glucose load (233). In contrast to GPR119, a Gscoupled receptor, GPR120 and GPR40 are Gq-coupled receptors, and act primarily through PKC. Activation of both
GPR40 and GPR120 is associated with GLP-1 secretion
(106, 166, 183, 248); however, their role may be more
pharmacological than physiological. Members of these
fatty acid binding GPCR are under active investigation as
drug targets for the treatment of T2DM.
Still another set of nutrient-sensing receptors linked to
GLP-1 secretion are “sweet taste receptors” that are expressed both in the tongue, where they play a role in sweet
taste perceptions, and also in the GI tract (191), where the
function is less clearly understood. However, when intestinal sweet taste receptors are activated with a nonnutritive
agonist, GLP-1 is secreted, and mice null for one such sweet
taste receptor, ␣-gustducin, have reduced GLP-1 responses
to an oral glucose load (191). ␣-Gustducin is also a GPCR,
but its second messenger system is not understood. While
these data demonstrate the potential of chemo-sensors in
linking carbohydrate availability to L-cell secretion of
GLP-1, it should be noted that this research is in its early
stages, and the findings from different studies have not been
consistent.
The higher density of L-cells in the lower intestine suggests
that GLP-1 secretion after meals comes mostly from the
ileum and colon. Indeed, while nutrient infusions into ligated sections of the duodenum and ileum, respectively,
both cause significant increases in portal vein concentrations of GLP-1, ileal infusion causes significantly greater
excursions of GLP-1 (165). However, the upper gut may
have important indirect effects on GLP-1 secretion from the
lower gut. For example, both concentrations of plasma
GLP-1 levels rise rapidly after a meal (71) prior to nutrient
transit to the distal gut (18, 115). This disparity between the
rates of nutrient passage along the gut and postprandial
GLP-1 secretion suggests feed-forward neural or paracrine
signals from the duodenum to the ileum that result in increased GLP-1 secretion (37, 152). The case for a neural
mechanism to connect the presence of nutrients in the upper
intestine to L-cells lower in the gut has been suggested by a
518
number of experimental models. Nutrient-induced GLP-1
secretion with duodenal infusions is delayed when the gut is
transected/reanastomosed below the duodenum compared
with when it is simply ligated; the latter manipulation presumably maintains neural innervation (336). Bilateral subdiaphragmatic vagotomy in conjunction with transection
completely abolishes nutrient-induced GLP-1 secretion,
suggesting a gut-brain-gut axis in regulating GLP-1 secretion. In rodents, human enteroendocrine cells, and the isolated perfused porcine ileum model, cholinergic agonists,
which bind to receptors in both the enteric and parasympathetic nervous system, act via types 1, 2, and 3 muscarinic
receptors to stimulate GLP-1 secretion (12, 37, 153, 332).
Conversely, norepinephrine and ␣-adrenergic agonists inhibit (153) and ␤-adrenergic agonists stimulate (99, 312)
GLP-1 secretion, suggesting that enteric, parasympathetic,
and sympathetic neural innervation are all important in the
regulation of GLP-1 secretion. In humans, the rise in plasma
GLP-1 following ingestion of oral glucose is blunted by
atropine (18, 115). While effects of atropine on GI motility
cannot be discounted in this effect, there was a significant
reduction of plasma GLP-1 corrected for plasma glucose.
Thus, while there are many details yet to understand, the
bulk of published evidence supports a neural contribution
to GLP-1 secretion (312).
Recent evidence also suggests pharmacological stimulation
of GLP-1 secretion. The commonly used anti-diabetes drug
metformin has been shown to increase plasma GLP-1 levels
in T2DM subjects (284), an effect that is thought to be
mediated by direct luminal effects of the drug. In addition,
GLP-1 seems to affect L-cell secretion. In studies with human subjects receiving the GLP-1r antagonist exendin-(9 –
39) (Ex9), plasma concentrations of GLP-1 are consistently
higher when GLP-1 action is blocked (346, 348, 439), suggesting a physiological role for GLP-1 to inhibit its own
secretion. While it is possible that this is due to a reduction
of receptor-mediated clearance by Ex9, there are compatible findings from clinical trials with dipeptidyl-peptidase 4
(DPP-4) inhibitors that are not likely to affect this process.
DPP4 inhibitors reduce the metabolic inactivation of
GLP-1 and increase circulating concentrations of the intact peptide (32, 409). However, these drugs have been
noted to consistently reduce the levels of total GLP-1, a
measure reflective of GLP-1 secretion. It is not known
whether GLP-1 interacts directly with L-cells or whether
the influence is indirect.
A. Glucagon Secretion
Glucagon is the chief secretory product of ␣-cells and has
been the principle measure of ␣-cell function. Endogenous
glucagon levels are highest in the venous drainage of the
pancreas and the hepatic portal vein, consistent with the
liver as principle target. Hepatic clearance of glucagon has
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GLUCAGON AND GLP-1
been reported as being 20 – 40% of the portal content by
most (89, 192, 373), but not all (82) groups, with the kidney
contributing the major remaining portion of peripheral glucagon removal (89). There is no evidence that the other cell
types that express Gcg, either enteroendocrine L-cells or
neurons in the hindbrain, contribute to plasma glucagon
levels. Like the L-cell, a good case can be made for nutrient,
paracrine, and neural inputs in the control of glucagon secretion. The major physiological stimuli for glucagon release is mixed nutrient or protein ingestion, activation of the
autonomic nervous system (ANS), and hypoglycemia (101,
142). As suggested by the diversity of these regulatory factors, control of secretion at the level of the islet is complex
and multilayered. Similar to secretion of insulin by ␤-cells,
␣-cell secretion of glucagon occurs with release of a stored
pool of peptide initiated by specific stimuli. Moreover, the
␣-cell shares much of the secretory machinery associated
with insulin secretion from ␤-cells, including membrane
surface receptors, the KATP, specific ion channels, and exocytotic proteins. While there is convincing evidence to support a range of factors in the control of glucagon output,
important questions remain as to how these factors interact,
and under which circumstances.
Although controversial for many years, there is now good
evidence that changes in plasma glucose have direct effects
on glucagon secretion, mediated through changes in ␣-cell
electrical activity, that involve KATP channels (59, 143,
251). Importantly, increased availability of human islets for
β-Cell
research has been consistent with the results from rodent
islet studies, supporting intrinsic sensing of glucose as a
mechanism involved in ␣-cell secretion (251, 329). Initial
observations that ␣- and ␤-cells had similar glucose transport, metabolism, and inhibition of the KATP channels yet
opposite secretory responses was at first difficult to explain.
However, recent evidence suggests that differences in resting electrical characteristics and ion channel function
downstream of KATP channel closure can explain much of
the reciprocal pattern of glucagon and insulin secretion at
relative hypo- and hyperglycemia (251, 329, 341). This
model is depicted in FIGURE 2. In addition to glucose, it
has long been appreciated that amino acids are important
regulators of the ␣-cell. Protein meals or infusions of
amino acids stimulate glucagon release, and arginine is
commonly used to stimulate glucagon secretion in research studies.
There are substantial differences in glucagon release from
isolated ␣-cells compared with whole islets (118, 142,
237a). One major implication of this observation is that
other islet cells have important roles in ␣-cell regulation.
Endocrine cells in islets, which are exposed to high concentrations of local products either in the interstitium or microcirculation, have been implicated in the control of glucagon secretion. Insulin suppresses glucagon release (142),
acting either through the vasculature of the islet as a hormone, or through local cell-to-cell contact. Addition of insulin to isolated islets or pancreata reduces, while removal
α-Cell
Glucose
Endoplasmic
reticulum
Glucose
Glycolysis
Glycolysis
AcCoA
AcCoA
TCA
cycle
Ca2+
TCA
cycle
Ca2+
Mitochondria
ATP/ADP
ATP/ADP
Insulin
K+
Glucagon
K+
Ca2+
Ca2+
K+
Na+
K+
ep
D
ep
D
Exocytosis
a ti o n
Ca2+
a
ol
a
ol
riz
r iz
Exocytosis
a ti o n
Ca2+
Na+
FIGURE 2. Glucoregulation of glucagon versus insulin exocytosis. An increase in plasma glucose is utilized by
both ␣- and ␤-cells to generate ATP. The increase in ATP opens potassium ATP channels and subsequently
causes membrane depolarization. In ␤-cells, this activates calcium channels increasing intracellular calcium
and stimulating the release of insulin via exocytosis. While KATP channel activation also activates calcium
channels in ␣-cells, ␣-cells also express N-type sodium channels which are also activated by membrane
depolarization. This process blocks, rather than excites, exocytosis.
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DARLEEN A. SANDOVAL AND DAVID A. D’ALESSIO
of insulin with antibodies increases, glucagon release. Consistent with this, acute reduction of ␤-cell mass by 70%
with streptozotocin causes hyperglucagonemia and consequently hyperglycemia in mice (177). Genetic deletion of
the insulin receptor specifically from ␣-cells causes hyperglucagonemia and mild hyperglycemia, with increased glucagon release in response to arginine and insulin-induced
hypoglycemia (208). In healthy humans, increased insulin
secretion during a hyperglycemic glucose clamp suppressed the glucagon response (19), demonstrating the
potency of insulin inhibition of the ␣-cell. However, any
role of endogenous insulin on the ␣-cell response to low
glucose concentrations is likely to be tonic, since ␤-cell
secretion is minimal at glucose concentrations where
␣-cells only start to increase secretion (389). In keeping
with this, there is a body of work suggesting that a rapid
decline of islet insulin is necessary for appropriate glucagon secretion in response to hypoglycemia (176). The
issue of ␣-cell regulation by insulin as glucose levels fall
has not been fully resolved, but it seems that insulin does
contribute measurably to the suppression of glucagon
after meals (268). In an elegant study in dogs, Greenbaum et al. (140) demonstrated that insulin is necessary
for the suppression of glucagon, and stimulation of somatostatin, during progressive hyperglycemia. Other
compounds released from ␤-cells have been shown to
inhibit glucagon release, including zinc (118), ␥-aminobutyric acid (17), and glutamate (142), but the ultimate importance of these compounds is not clear.
The third major endocrine cell type in the islet, ␦-cells, produces somatostatin which suppresses ␣-cells primarily
through the somatostatin-2 receptor (384). ␦-Cells have
been proposed to have a paracrine role based on their anatomical features, as they radiate processes that extend
through the islet and abut other cell types. In addition, there
is evidence that the major product of ␦-cells, somatostatin14, acts locally in the islet. Exogenous somatostatin is a
potent inhibitor of glucagon secretion, and administration
of a somatostatin-2 receptor agonist reduces fasting glucagon and glucose concentrations (384). While deletion
of the prosomatostatin gene in transgenic mice does not
affect basal glucagon or insulin levels in vivo or in isolated islets, these animals have significantly greater glucagon secretion in response to arginine than controls, an
effect retained by isolated islets (158). Together these
data suggest that effects of somatostatin are minimal
under basal conditions but become important when nutrient stimuli are involved.
Several recent papers suggest that other ␣-cell products may
in fact regulate glucagon secretion. ␣-Cells from islets of
both primates and mice secrete glutamate and express ionotropic glutamate receptors (iGlutR; 47). In addition, glutamate stimulates release of glucagon, and the effect of glutamate seems to be important for a normal glucagon response
520
to low glucose concentrations since inhibition of iGlutR
impaired the hypoglycemic counterregulation in mice (47).
␣-Cells also express glucagon receptors, and when exposed
to increasing concentrations of glucagon increase intracellular cAMP and membrane capacitance, a surrogate for
exocytosis (250). While these experiments demonstrate glucagon receptor signaling in ␣-cells, it is not possible to discern whether this process stimulates or inhibits glucagon
secretion. Regardless, the addition of apparent autocrine
control to glucagon secretion lends another level of detail to
a complex system.
The autonomic nervous system (ANS) plays a central role in
the regulation of glucagon secretion, particularly as it relates to hypoglycemic counterregulation. Activation of both
limbs of the ANS, parasympathetic and sympathetic, increases the release of glucagon, as does epinephrine released
from the adrenal gland (389). Signaling through adrenergic
receptors increases cAMP in ␣-cells (80b), and catecholaminergic signaling seems to be synergistic with hypoglycemia in stimulating glucagon release (341). Several of
the peptides that act as postganglionic parasympathetic
neurotransmitters, such as acetylcholine, vasoactive intestinal polypeptide, gastrin releasing peptide, and pituitary adenylate cyclase activating polypeptide, can stimulate ␣-cell
secretion (8), and genetic disruption of autonomic neurons
in the islet predispose mice to hypoglycemia (342). Recent
work emphasizes the role of glucose sensing neurons centered in the ventromedial hypothalamus (VMH) as mediating the CNS counterregulatory response (102, 239). Activation of KATP channels in the VMH augments (263), and
closure of these channels (112) blunts, the glucagon and
epinephrine responses to hypoglycemia. Efferent output
from the VMH in response to hypoglycemia depends on the
relative GABA/glutamate tone in these nuclei (398, 452). It
seems likely that the brain response to hypoglycemia extends to other sites in the hypothalamus and hindbrain, but
at present the specific areas governing islet function are not
known.
Similar to insulin, glucagon release is also affected by the
actions of enteric peptides (100, 142). Glucose-dependent
insulinotropic polypeptide (GIP) stimulates glucagon release (80b, 100), likely through direct actions on the GIP
receptor expressed on ␣-cells (80b). Since GIP secreted after
meals is a potent stimulus for insulin secretion, which
would tend to decrease ␣-cell secretion, there may be opposing effects of direct and indirect actions in the postprandial state. Many other peptides expressed in the gastrointestional tract, including cholecystokinin, vasoactive intestinal polypeptide, and gastric releasing peptide (100), also
stimulate glucagon release. However, it is not clear whether
the mechanism is endocrine or neural since these peptides
also function as neurotransmitters and are components of
islet innervation (8).
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GLUCAGON AND GLP-1
Of great importance, the other major proG peptide, GLP-1,
is a key regulator of glucagon secretion. Activation of the
GLP-1 receptor inhibits glucagon release from isolated islets or ex vivo perfused pancreas (100). However, there is
some controversy over the mechanism whereby this occurs.
GLP-1 increases the secretion of hormones from both ␤and ␦-cells, and so could act indirectly to reduce glucagon
release (100, 142); this is considered to be a major part of
the inhibitory role of GLP-1 on the ␣-cell. In addition, there
is evidence that GLP-1 affects electrical activity and secretion of ␣-cells even in the absence of changes in somatostatin or insulin (80b). The major question debated in this area
is whether ␣-cells express the GLP-1 receptor. While some
groups have demonstrated the presence of very low level
expression of the GLP-1 receptor (80b, 333), others have
been unable to document any expression at all (300, 399).
Moreover, GLP-1 receptor activation generates cAMP
which tends to be associated with increased glucagon release. However, a recent study supports a model in which
low numbers of GLP-1 receptors on ␣-cells, with capacity to
generate proportionately small amounts of cAMP, mediate
suppressive effects through discrete inhibition of N-type
calcium channels (80b). This elegant formulation provides
one possible explanation as to how low and high intra-␣cellular cAMP concentrations can have opposite effects on
glucagon release. This model requires confirmation but has
the potential to rectify an area of controversy in glucagon
regulation and provide a template for examining the role of
other regulatory factors acting through GPCR.
dense integration of control by nutrient substrates, neural,
endocrine, paracrine, and autocrine inputs to secretion.
Work in recent years has shifted the debate from what is the
most important signal controlling ␣-cell secretion, to how the
myriad factors involved interact and work together. This
seems a likely direction of future research for regulation of
GLP-1 secretion as well. A key difference between the regulation of ␣- and L-cells is that it seems to be more complex in
the former. Because glucagon has a key role during fasting,
after meals, and hypoglycemia, whereas GLP-1 is released
primarily during nutrient absorption, ␣-cells are subject to a
greater range of controlling factors. While there appears to
be some overlap in regulatory forces such as ambient glucose, somatostatin, and ␤-cell constituents, it seems likely
that these also have specific roles as well. With evidence
growing that the ␣-cell secretes other important compounds
with key physiological effects, such as GLP-1, acetycholine,
and glutamate, the possibility for differential release of
these compounds will need to be considered in future work.
VI. EFFECTS OF ␣-CELL PRODUCTS ON
ISLET FUNCTION
In addition to regulation by systemic factors, the pancreatic
islet is subject to local control through paracrine and autocrine mechanisms (FIGURE 3). The ␣-cell participates in this
system, primarily by secreting products that regulate ␤-cell
function. Understanding research in this area requires consideration of the differences in islet architecture that exist
between rodents and humans (383), and the heterogeneity
of islet size and organization within species (33, 113). It is
well-established that in mice and rats the organization of
the islet includes a mantle of ␣- and ␦-cells on the periphery
of the islet, surrounding a core of ␤-cells. Estimates of relative cell composition in rodent islets are ⬃75– 80% ␤- and
20 –15% ␣-cells, and most ␤-cells have contact with other
␤-cells (46, 49, 383). In humans, the major endocrine cell
Overall, considerable progress has been made in the past
decade in the understanding of ␣-cell regulation. One interpretation of the totality of these data is as a complex, mulitlayered model controlling secretion of an important regulatory hormone. The current model of ␣-cell secretion
seems to be more similar to than different from the physiology of ␤-cells, and also shares some mechanisms with
L-cell secretion. A consistent theme across these cell types is
SNS
PNS
PC2
PC1/3
α-Cell
SNS
GlucaR
Glucagon
Mini-glucagon
Gcg
ProGIP
PNS
?
Insulin
GLP-1
GIP
GLP-1r
GIPr
FIGURE 3. Neuronal and paracrine regulation of ␣- and ␤-cell interactions. Activation of the sympathetic nervous system inhibits and parasympathetic activity stimulates ␤-cell release of insulin, while both
limbs of the autonomic nervous system increase glucagon secretion from the ␣-cell.
Paracrine action from the ␣-cell via GIP,
glucagon, and GLP-1 stimulates ␤-cell release of insulin. In contrast, mini-glucagon,
a product of glucagon cleavage, inhibits
␤-cell release by acting on an, as of yet,
unknown receptor.
β-Cell
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521
DARLEEN A. SANDOVAL AND DAVID A. D’ALESSIO
types are spread more heterogeneously throughout the islet,
most ␤-cells have contact with either ␣- or ␦-cells, and the
percentage of ␤-cells per islet is 40 – 60% (33, 46, 113).
Based on recent analyses of large numbers of human pancreata, the frequency of contacts between ␣- and ␤-cells
appears to be much greater than previously estimated, as is
the location of islet endocrine cells in close proximity to the
microvasculature (33). It is notable that in humans, smaller
islets have cellular architecture that resembles rodents, i.e.,
a mantle of ␣-cells surrounding a ␤-cell core with a higher
percentage of ␤-cells than larger islets; these smaller islets
also have greater relative insulin secretion (33, 113). These
recent studies on the organization of islet anatomy, particularly in humans, have shifted the focus from interactions
mediated through the microcirculation to control through
direct cell-to-cell contacts.
Neural regulation of islet function is well established, although the nature and magnitude of effects are still under
debate. In general, sympathetic nervous system activity is
thought to be important in settings where there is enhanced
demand for glucose, i.e., hypoglycemia and physical activity, and parasympathetic activity is important before and
during meal ingestion (8, 389, 396). Parasympathetic cholinergic signaling mediates cephalic insulin secretion, a response most clearly apparent in rats, but also in humans (5,
394). There is limited but suggestive evidence that glucagon
secretion can be mediated in this manner (105, 365, 377).
Recent data suggest a difference between the density of
autonomic innervation of rodent and human ␣- and ␤-cells,
with rats and mice having greater density of neural fibers in
islets (339). While this has led to the hypothesis that neural
regulation of islet secretion is more important in rodents,
and paracrine control in humans, this has not been formally
tested, and not all studies have demonstrated reduced innervation of human islet cells (141).
The GCGR is expressed by islet ␤-cells, and it has long been
appreciated that supraphysiological amounts of glucagon
stimulate insulin release in vitro and in vivo (207, 212,
274). Similar to other peptide secretagogues, glucagon
amplifies glucose-stimulated insulin secretion primarily
through mechanisms activated by increased cAMP, and
does so in both rodent and human islets (182, 274). In
isolated human islets studied in culture, antagonism of the
GCGR impairs insulin secretion in response to increased
glucose in the media, suggesting a tonic role for islet glucagon action in maintaining glucose competence (273); similar results have been reported in isolated rat ␤-cells and
islets (182, 207). Consistent with the actions of glucagon to
potentiate insulin secretion, dispersed ␤-cells had greater
secretory responses to elevated glucose when attached to an
␣-cell (440). Thus a body of evidence has been generated
that supports islet glucagon in the potentiation of insulin
secretion, most likely through paracrine and cell-to-cell interaction.
522
Mice with transgenic overexpression of the GCGR in
␤-cells have evidence of enhanced secretory function (129).
These mice have increased insulin release in response to
glucose and glucagon compared with wild-type controls, as
well as greater ␤-cell mass and insulin content. Consequently their blood glucose levels throughout the day are
modestly reduced, and they have markedly improved glucose tolerance. In contrast, mice with a global deletion of
the GCGR have a more complex islet phenotype. These
animals have enhanced insulin secretion to IP glucose, but
this effect appears to be due to increased production and
action of GLP-1, which is greatly increased in these animals
that develop ␣-cell hyperplasia and excess production of
ProG peptides (10). Mice with a global deletion of Gcg have
lower plasma insulin but similar blood glucose levels as
control animals (159). In response to an IP glucose load, the
Gcg knockouts have increased insulin secretion and improved glucose tolerance (120). However, a potential explanation for this observation is increased circulating GIP
concentrations and expression of GIP in the islets (120).
One interpretation of these findings is that a loss of the
actions of glucagon and GLP-1 is compensated for by another secretagogue. Although it would be useful to study the
question in mice with a ␤-cell specific knockout of the
GCGR, the results from genetic mouse models currently
available support glucagon as having a role in the regulation of insulin release.
While glucagon and GLP-1 are ␣-cell products that promote insulin secretion, a novel peptide fragment of glucagon, glucagon[19 –29] or miniglucagon, has been proposed
to have an inhibitory role (24, 74). Miniglucagon is a constituent of ␣-cell secretory granules that is released with
glucagon, but also produced from the metabolism of glucagon at target tissues like the liver. The fragment is produced
by the combined activity of proteases that cleave at the
Arg-Arg residues at glucagon(18,19) (117). Miniglucagon
does not act through the GCGR but rather has been proposed to work through an as yet unidentified receptor that
causes hyperpolarization of ␤-cells and attenuation of the
effect of secretagogues that require voltage-gated calcium
channels to stimulate insulin release (75). In the perfused rat
pancreas, subpicomolar concentrations of miniglucagon inhibit insulin release (74). Although work with miniglucagon has been limited and led primarily by one laboratory,
the potential for ␣-cells to make products that modulate
insulin secretion both up and down is interesting and fits
with the need for islet regulation across the fed and fasting
states.
ProG processing differs fundamentally in ␣-cells and enteroendocrine L-cells based on the predominant hormone
proconvertase present in these cell types, and the common
belief for many years was that this led to distinct, nonoverlapping production of ProG peptides. To summarize this
concept, PC2 in ␣-cells was believed to account for almost
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GLUCAGON AND GLP-1
total conversion of ProG to glucagon with little processing
of the COOH terminus of the molecule to the GLP peptides;
and PC1/3 in L-cells cut out GLP-1 and GLP-2 with little
NH2-terminal processing to give glucagon. In this diametric
formulation, the endocrine pancreas produced only glucagon and the gut only GLP-1/GLP-2. However, findings
from studies over the past decade have refined the view of
␣-cell ProG processing sufficiently that it is possible to consider an islet GLP-1 signaling system. Similarly, deletion of
PC 1/3 from enteroendocrine cells markedly impairs ProG
processing in the intestine, causing elevated tissue levels
of ProG and glucagon, suggesting a capacity for NH2-terminal processing by L-cells. This function has been raised as
a possible explanation for the parallel increases in postprandial GLP-1 and glucagon in subjects with gastric bypass
(201, 348).
While PC2 is essential for normal ProG processing in normal adult ␣-cells (122, 123), it is now clear that PC1/3
activity is also variably present as well (426). This has been
observed in embryonic and neonatal mice, with pregnancy,
and in a variety of prediabetic and diabetic states (214, 294,
397, 437). Thus GLP-1 seems to be generated from ProG in
the ␣-cell, albeit in lower amounts than glucagon. In cultured ␣-cell lines or isolated islets, high media glucose concentrations increase PC 1/3 expression and cellular GLP-1
content (265, 430). Moreover, intact GLP-1[7–36]amide is
secreted from isolated rat islets (262, 430), and from isolated human islets and ␣-cells (257), in culture. Interruption
of GLP-1r signaling in isolated rodent islets or pancreata,
using receptor antagonists or gene knockout, reduces basal
(262) and glucose-stimulated insulin secretion (116). These
findings have recently been corroborated in a mouse model
with ␤-cell-specific deletion of the GLP-1r (378). Based on
studies of ␤-cell lines or isolated islets with blockade or gene
deletion of the GLP-1r, it has been suggested that there is
constitutive activity of the GLP-1r in the absence of agonist
ligands (367). However, given the mounting evidence for
GLP-1 production in the islet, this proposal requires reconsideration. Finally, infusion of exendin-(9 –39) to fasting
humans, with unstimulated and low circulating GLP-1 levels, significantly decreases insulin secretion during glucose
clamps (346, 359). These findings suggest a role for GLP-1r
signaling that is independent of nutrient-induced L-cell secretion and can be taken as support for the action of local
islet GLP-1. Overall, there has been an accumulation of
evidence to support a role for local production of GLP-1 in
the islet in the regulation of insulin secretion as a paracrine
factor.
Beyond a role in normal islet function, ␣-cell GLP-1 seems
to be involved in response to stress and illness as well. In rats
treated with the ␤-cell toxin streptozotocin, there is an acute
increase in islet PC 1/3 and ProG expression, and increased
processing of the pro-peptide to GLP-1 (295). Moreover,
increased GLP-1 signaling in the islet was implicated in the
recovery from injury. In mice with a deletion of the glucagon receptor, ␣-cell hyperplasia leads to increased ProG
expression, with massive elevations of plasma GLP-1 (128).
These elevated levels of islet-derived circulating GLP-1 contribute to glucose lowering and delayed gastric emptying, as
well as glucose-stimulated insulin release (10). Islet GLP-1
production and action is also mediated by the cytokine
interleukin (IL)-6, which is released in response to exercise,
obesity, and diabetes (109, 110). Expression of the IL-6
receptor is relatively high on ␣-cells, and IL-6 signaling
increases ProG transcription and GLP-1 production in
mice. There are no available data to support this in humans,
but a recent study has demonstrated elevated levels of IL-6
and GLP-1, which were significantly correlated, in humans
with critical illness (202). IL-6 stimulated GLP-1 release
from ␣- and L-cells, increases insulin secretion through the
actions of GLP-1 (110), and is necessary for the maintenance of glucose tolerance in response to diet-induced obesity (109). These data suggest that IL-6, released from adipose tissue and skeletal muscle, regulates insulin secretion
in part through local production of GLP-1 in the islet. Finally, virally mediated transgenic overexpression of PC 1/3
in ␣-cells increased production of islet GLP-1, enhanced
insulin secretion, and protected islets from the detrimental
effects of high concentrations of cytokines (432). Islets producing more GLP-1 were also more effective in reducing
hyperglycemia when transplanted into diabetic mice. Overall, the findings from these and other studies suggest that a
paracrine system of islet GLP-1 signaling plays a role in
several adaptations to metabolic stress. Understanding the
control of the relative production of ␣-cell GLP-1 and glucagon would seem to hold promise for therapeutic development.
Not all factors released from ␣-cells and implicated in paracrine signaling are ProG-derived peptides. Recent studies
have demonstrated production of acetylcholine by ␣-cells
and regulation of insulin secretion through the ␤-cell muscarinic M3 receptor (340). In human islets, endocrine cells
positive for glucagon also contain the vesicular acetylcholine transporter and choline acetyltransferase. These proteins are contained in vesicles distinct from those containing
glucagon, suggesting separate secretory systems in the
␣-cell. Reduction of ambient glucose caused release of acetylcholine from a subset of islets, while increased glucose
did not. Experimental interventions to increase acetylcholine increased insulin release from islets, and this could be
blocked by muscarinic receptor antagonists. Likewise, reductions of acetylcholine release decreased insulin secretion
from cultured islets. These studies in isolated human islets
demonstrate that release of acetylcholine from ␣-cells, independent of autonomic nerves, provides paracrine regulation of ␤-cell secretion.
More recently, the incretin GIP has been demonstrated to be
produced and secreted from ␣-cells (119). This interesting
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523
DARLEEN A. SANDOVAL AND DAVID A. D’ALESSIO
finding builds on the coincident production of proGIP and
ProG in K/L enteroendocrine cells by demonstrating coexpression in the endocrine pancreas as well. In the ␣-cell,
proGIP is processed by proconvertase 2 into a truncated
GIP1–30 form that is distinct from the longer, GIP1– 42 produced in the gut. However, GIP1–30 is equipotent to fulllength GIP as an insulin secretagogue and like glucagon and
GLP-1 seems to contribute to glucose competence as interference with its action attenuates glucose-stimulated insulin
release. In keeping with this novel discovery in islets, mice
with a deletion of ProG gene have increased circulating GIP
levels, production and release of GIP from islets, and localization of GIP immunostaining to both ␣- and ␤-cells (120).
Expression of GIP in ␤-cells seems to be ectopic as it is not
seen in wild-type mice. These findings support the notion
that a component of the incretin effect is mediated locally in
the islet, by the classical incretins.
date are not conclusive as three studies did not demonstrate
an effect of Ex9 on gastric emptying rate (279, 293, 349),
while another reported a modest but significant effect (83).
The cause for this discrepancy is not clear, but differences in
caloric content of the meal, differing methodologies and
experimental conditions, and possibly variability among
healthy humans are possible explanations for the indefinite
results of the Ex9 studies. Interestingly, GLP-1 receptor
knockout mice do not have increased gastric emptying rate,
per se (232), suggesting that these receptors are not necessary for regulation of gastric emptying rate. While the physiological role of GLP-1 on gastric emptying rate remains to
be determined, the ability of pharmacological GLP-1 agonists to inhibit gastric emptying rate is incontrovertible, and
this offers a potential mechanism for controlling postprandial glycemic excursions when used in the treatment of
T2DM.
VII. REGULATION OF GLUCOSE
HOMEOSTASIS BY PROGLUCAGON
PEPTIDES
A number of studies have reported effects of GLP-1 on
glucose and lipid metabolism in the liver. However, this
area is much less clear than for glucagon because in contrast
to the GCGR which is expressed in abundance by hepatocytes, it has been very difficult to demonstrate the GLP-1r in
the liver (300, 322, 333). In addition, despite attempts by a
number of investigators, an alternative receptor to mediate
GLP-1 effects in the liver has not been convincingly demonstrated, nor do alternative forms of the known GLP-1r seem
to account for hepatic effects of the peptide. At present, it is
unclear whether differences in GLP-1r expression in the
liver are due to species differences, specifically between humans and mice, or whether the small amount of detection of
the GLP-1r is due to contamination of GLP-1r expressed in
the vasculature of the liver rather than the parenchyma. The
GLP-1r is expressed on hepatic portal vagal afferents leading to a more likely scenario that hepatic effects of GLP-1
are indirectly mediated by CNS action.
A. GLP-1
Over 50 years ago, it was discovered that insulin secretion
in response to a glucose load was greater with oral compared with intravenous glucose administration (266). It is
now understood that this response is the result of insulinotropic intestinal peptides, predominantly GIP and GLP-1,
which are secreted in response to ingested nutrients and
function to stimulate the pancreas to secrete insulin. This is
termed the incretin effect to describe the effect of GI factors,
incretins, to stimulate internal secretions, i.e., insulin. Although GLP-1 has several effects that contribute to glucose
homeostasis, its stimulation of insulin secretion is thought
to be the primary mode by which it lowers blood glucose.
However, it is important to note that physiologically and
pharmacologically GLP-1, and GLP-1r agonists, regulate
glycemia through both insulin-dependent and -independent
modes of action.
GLP-1 also reduces glucagon levels and inhibits gastric
emptying rate, two insulin-independent mechanisms by
which GLP-1 affects glucose homeostasis. By reducing glucose entry into the gut, GLP-1-induced inhibition of gastric
emptying rate reduces postprandial glucose excursions. Exogenous administration of GLP-1 certainly inhibits gastric
emptying rate in healthy (285, 290, 429) and T2DM subjects (435) and inhibits antral and stimulates pyloric motility (358), all contributing to a blunting of nutrient entry
into the intestine. These studies involve exogenous administration of GLP-1 which creates some difficulty in reconciling the physiological versus pharmacological effects. Several studies have infused the GLP-1 receptor antagonist exendin-(9 –39) (Ex9) during test meals to assess the effects of
endogenous GLP-1 on gastric emptying rate. The results to
524
Infusion of GLP-1 in humans during experimental conditions with clamped insulin and glucagon concentrations
reduces hepatic glucose production by 20 –30%, a common
finding by two different groups of investigators (321, 366).
The mechanism explaining this observation is not clear and
is subject to the same questions about direct or indirect
actions of GLP-1 on the liver as have been raised for other
responses. The effect of GLP-1 on hepatic glucose production is not explained by differences in free fatty acid levels or
rates of lipolysis (366). There is accumulating evidence that
GLP-1 effects on hepatic glucose metabolism may be neurally mediated. Central nervous system administration of
GLP-1 (15) and specific administration directly into the
arcuate nucleus of the hypothalamus improves hepatic insulin sensitivity (353) and increases hepatic glycogen synthesis (223). Moreover, neural GLP-1 sensors have been
demonstrated in the vasculature of the hepatic portal venous system, where GLP-1 signaling has been observed to
affect fasting and post-challenge glycemia (15, 186, 187,
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GLUCAGON AND GLP-1
406). There is also some evidence that GLP-1 promotes
peripheral glucose uptake (15, 223), mostly related to neural mechanisms, although this finding has not been universal (366).
Interestingly, like peripherally administered GLP-1, activation of central GLP-1r regulates glucose homeostasis via
multiple mechanisms. Intracerebroventricular GLP-1 increases insulin secretion (223, 353) and inhibits gastric
emptying rate (184). The ability of CNS GLP-1r activation
to improve glucose homeostasis is maintained in mice fed a
high-fat diet via increasing insulin secretion and improving
hepatic insulin sensitivity (43). As GLP-1 is also made in the
hindbrain region of the CNS, it remains unknown whether
CNS action of GLP-1 is via locally or peripherally derived
GLP-1.
A number of studies have demonstrated that GLP-1 alters
other aspects of hepatic metabolism, namely, that it promotes fatty acid oxidation in the liver (27, 300, 388). These
effects are mediated in part through transcriptional effects
on key enzymes controlling ␤-oxidation (300). In obese
mice, administration of GLP-1 agonists reduces hepatic steatosis (86, 300), an effect reported in humans as well (145,
388); these effects appear to be independent of the effect of
chronic GLP-1 administration to reduce body weight.
While the effects of GLP-1 on hepatic fat metabolism have
not been directly compared with those of glucagon, there
are parallels with both peptides acting to shift the liver
towards lipid oxidation and away from lipogenesis.
B. Glucagon
The cardinal endocrine action of glucagon, to increase hepatic glucose production, has been known for nearly 100
years, dating from the time when pancreatic extracts were
first being tested as a treatment for diabetes (238). Subsequent work demonstrated effects of glucagon to counter
hypoglycemia and led to the general principle that it has a
role opposing that of insulin to maintain plasma glucose in
times of stress, fasting, or exercise (238). The development
of a radioimmunoassay sensitive enough to measure levels
in the blood provided insights into the major regulatory
influences on glucagon (404). The endocrine mechanism of
glucagon action is based on the effects of exogenous glucagon to increase hepatic glucose output in animals, humans,
and a number of in vitro systems, and removal of circulating
glucagon with a neutralizing antibody to reduce blood glucose (35, 195).
The actions of glucagon to increase hepatic glucose production are mediated primarily through the cAMP/PKA signaling pathway (195, 419). Glycogenolysis is promoted, and
glycogenesis inhibited, primarily through the activation of
phosphorylase kinase and its downstream target glycogen
phosphorylase (210, 443). The balance between glycogen
breakdown and synthesis is primarily a function of relative
insulin and glucagon effects on hepatocytes, i.e., the relative
cAMP signal, as well as the level of glycogen stores (443).
Genetic or pharmacological interventions that increase glycogen synthesis relative to glycogenolysis promote glucose
tolerance (210, 319), and the cellular physiology controlling the flux through these pathways is being explored for
drug targets. GCGR signaling also restrains glycolysis by
modifying glycolytic enzymes, particularly fructose-2,6 bisphosphatase, regulating the flux between glucose-6-phosphate and fructose biphosphate, and inhibiting pyruvate
kinase activity (195). Thus the immediate effects of increased GCGR signaling in the liver are reduction of glucose
oxidation and rapid mobilization of stored glucose for export to the circulation.
Central to the effect of glucagon on hepatic glucose metabolism is regulation of phosphoenolpyruvate carboxykinase
(Pepck) transcription. Pepck expression varies with the
metabolic state, with increases during fasting and suppression by insulin (195). Glucagon increases PEPCK levels
through CREB and Fox01, transcription factors downstream of PKA (436). PEPCK catalyzes the conversion of
the TCA cycle product oxaloacetate into phosphoenolpyruvate which is a key step in gluconeogenesis from lactate,
pyruvate, and alanine. Overexpression of Pepck can increase blood glucose (387), and deletion of Pepck decreases
gluconeogenesis (42, 138). While Pepck expression has
been used as a surrogate for glucagon action and/or rates of
gluconeogenesis, recent findings suggest a more complex
picture in which PEPCK mRNA or protein levels are only
one of several factors governing flux from the tricarboxylic
acid (TCA) cycle to blood glucose (42). In addition to
PEPCK, glucagon increases hepatic production of several
other key genes involved in glucose production including
those coding for peroxisome proliferator activated receptor-␥ coactivator 1 (PGC-1) and glucose-6-phosphatase
(G6P). Overall, glucagon has effects on several levels of
hepatic gluconeogenic gene expression that promote sustained glucose production even after the finite glycogen supply is depleted. Gluconeogenesis is an energetically demanding process, requiring 6 mol of high-energy phosphate
bonds for each mole of glucose produced, and is tightly
linked to TCA cycle activity (42). A recent paper supports
GCGR signaling as a means of connecting energy production with gluconeogenesis. In this report, glucagon mediated the enhanced rates of hepatic lipid oxidation characteristic of fasting, effects that were due to GCGR activation
of PPAR␣ and p38MAPK signaling to increase the expression of enzymes involved in ␤-oxidation (246). These findings are compatible with the long-standing view that glucagon contributes to hepatic fatty acid oxidation and ketogenesis at several metabolic steps that reduce cellular
malonyl CoA and disinhibit carnitine palmitoyl transferase
1 (121, 148). Importantly, elimination of the GCGR was
associated with increased liver triglyceride content during
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DARLEEN A. SANDOVAL AND DAVID A. D’ALESSIO
fasting, indicating that the effects of glucagon on ␤-oxidation could extend to hepatic lipid balance as well. These
findings are supported by the recent demonstration that
GCGR signaling is needed for the correction of hepatic
steatosis by exercise (29).
In another recent study, glucagon action, either from exogenous peptide or increases of endogenous peptide with fasting or exercise, shifted the balance of hepatic energetics as
reflected in adenine nucleotide levels, increasing AMP relative to ATP (28). This effect was dependent on signaling
through AMPK and required gluconeogenesis through
PEPCK for the full effect on AMP/ATP. The increase in
hepatocyte AMP/ATP due to GCGR signaling was proposed to be secondary to abrupt intracellular energy deficits, such as those occurring from suppression of glycolysis
or initiation of gluconeogenesis, two actions of glucagon.
Once these metabolic shifts occurred, activation of AMPK
mediated a robust program of effects to increase lipid oxidation in compensation. Among these were increased expression of PPAR␣ and fibroblast growth factor 21 (FGF21) (29, 246); FGF-21 has recently been demonstrated to
mediate some of the actions of glucagon on lipids and body
weight (149). Taken together, these findings support a
model whereby glucagon initiates an integrated response to
requirements for circulating glucose by mobilizing glycogen
for rapid use, and initiating gluconeogenesis for more extended provision. The energy costs of glucose anabolism are
paid by accelerating lipid oxidation, a process that also regulates hepatic triglyceride stores. This model has been described
more extensively in an excellent recent review (157).
Glucagon-stimulated fatty acid oxidation contributes to ketogenesis in settings where delivery of free fatty acids to the
liver are increased, such as extended fasting and uncontrolled type 1 diabetes (101). Recent work indicates that the
transcription factor Foxa2, which controls the expression
of genes involved in fatty acid oxidation and ketogenesis
(441, 450), is activated both by fasting and glucagon
(409a). Glucagon signaling leads to acetylation of Foxa2,
which facilitates its localization in the nucleus and transcriptional activity through pathways downstream of
cAMP (409a). Experimental activation of Foxa2 increased
fat oxidation and ketogenesis, and improved hyperglycemia, hyperinsulinemia, and hepatic steatosis in obese and
diabetic animals. Notably, insulin signaling leads to phosphorylation of Foxa2, its exit to the cytoplasm, and inactivation of transcription of its gene targets (442). This system
presents yet another example of opposing regulation by
insulin and glucagon to promote the metabolic shifting
from the fed to fasted state and back again.
The model emerging from preclinical studies in animal
models is generally consistent with physiological studies in
humans. Studies using somatostatin to inhibit insulin and
glucagon secretion, with selective replacement of one or
526
both hormones, have demonstrated that after an overnight
fast, glucagon is necessary to support normal fasting glucose levels (36, 65); basal insulin replacement without glucagon results in hypoglycemia. On the other hand, plasma
insulin and hepatic insulin action also play an important
role in postabsorptive glycemic regulation (327). Given that
plasma glucagon concentrations do not increase significantly until glucose concentrations drop below 4.5 mM, the
effect of glucagon on fasting hepatic glucose production is
not the result of stimulated secretion. Rather, the effects of
glucagon to promote glycogenolysis and initiate gluconeogenesis occur in the setting of decreased insulin action. In
other words, a threshold is crossed whereby the effect of
insulin to inhibit intrahepatocyte generation of cAMP is
surpassed by the action of glucagon to increase adenylylate
cyclase activity.
That glucagon-driven glycogenolysis and gluconeogenesis
follow different temporal patterns is well established. During fasting, hepatic glycogen stores are steadily depleted
such that glycogenolysis contributes ⬃50% of liver glucose
output in the postabsorptive state but less than 10% after
36 h of fasting (234, 379). In most acute experiments, using
pancreatic clamps as a means to control glucagon and insulin concentrations, it is the glycogenolytic effects of glucagon that are most apparent (328). Promotion of gluconeogenesis by glucagon requires the provision of glucose precursors, primarily lactate, alanine, and glycerol. Increased
delivery of these compounds to the liver is not directly regulated by glucagon, but requires the gradual disinhibition of
lipolysis and proteolysis due to reductions of circulating
insulin as the duration of fasting lengthens. With extended
periods of starvation, preservation of protein stores becomes paramount and gluconeogenesis is tightly controlled
by precursor supply (48).
While the importance of hepatic glucagon action is clear in
the fasting state, the role of glucagon action in prandial
metabolism is less clear. Suppression of plasma glucagon
levels contributes minimally to glycemia after glucose ingestion in humans (370). There is a paucity of information
available about postprandial glucagon effects with mixed
nutrient meals, which maintain or elevate plasma glucagon.
The question of glucagon regulation of blood glucose following meals awaits the more general availability of the
effective glucagon receptor antagonists that have been developed by industrial laboratories for targeted clinical research. Consistent with the model above that emphasizes a
niche in the regulation of fasting metabolism, glucagon does
not have important actions in the regulation of hepatic glucose uptake (419).
The hallmark setting for glucagon action is during hypoglycemic counterregulation, when levels increase and hepatic
glucose increases dramatically (69). Glycogenolysis provides the most rapid source of glucose for release, but glu-
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GLUCAGON AND GLP-1
coneogenesis is also activated, and combined with a supply
of glucose precursors liberated through the activity of catecholamines, also contributes meaningfully to the response
(146). Thus the synergy of catecholamines and hypoglycemia to stimulate glucagon release also combine in the endorgan response to return glucose levels to normal.
Glucagon is also released during exercise and contributes to
maintenance of blood glucose during this metabolic stressor
(28, 422). Similar to hypoglycemia, the effects of glucagon
interact with the varying amounts of catecholamines secreted
in response to exercise, and to the usually low circulating levels
of insulin in this setting, to ensure adequate glucose output.
With protracted exercise, the effects of glucagon to promote
lipid oxidation become increasingly important to preserve limited glucose and provide energy (423).
C. Genetic Models of the Proglucagon
System
Recent studies of mouse models that are null for genes encoding Gcg, the GCGR, prohormone convertases, and factors involved in ␣-cell development (Arx) and glucagon signaling (Gs) have provided new and important information
on the physiology of proglucagon peptides. Comparisons
among the phenotypes of these models have been one of the
important advances in the field in recent years.
Two different strains of Gcg knockout mice have been generated. One strategy involved insertion of a green fluorescent protein cDNA sequence and a diphtheria toxin cassette
which resulted in Gcg deficiency in the pancreas, intestine,
and presumably the CNS, although this was not specifically
examined (120, 159). These mice have improved IP and oral
glucose tolerance, increased glucose-stimulated insulin secretion (both in vivo and in vitro), and ␣-cell hyperplasia
(120). Interestingly, these mice have decreased levels of insulin when measured during their usual feeding (159), suggesting that the impact of Gcg expression on insulin secretion may be nutrient-dependent.
An inducible Gcg knockout mouse has recently been generated by inserting the human diphtheria toxin receptor
plus a green fluorescent protein into the Gcg gene (305).
This insertion does not affect function of the gene until the
animals are exposed to exogenous administration of diphtheria toxin which kills cells expressing its receptor. Interestingly, with this model Gcg was knocked down only in
enteroendocrine L-cells and ␣-cells, but not CNS neurons.
These mice had mild improvements of oral, but impaired IP,
glucose tolerance. The ability of diphtheria toxin to reduce
Gcg expression in this model was short-lived in the intestine
where cell turnover is more rapid compared with the pancreas. Thus studying these animals 1 wk after administration resulted in a pancreas-specific downregulation of Gcg,
and these animals had normal IP glucose tolerance (305).
Together with the data on the chronic deletion of Gcg, it
seems that L-cell expression of Gcg is essential for IP, while
glucagon is essential for oral, glucose tolerance. This is
striking given the fact that plasma levels of GLP-1 have
negligible increases during an IPGTT. However, the data do
leave open the question of the role of CNS Gcg in regulating
glucose homeostasis.
Another genetic model of reduced Gcg action is deletion of
the prohormone convertases that process Gcg-derived peptides to GLP-1 (and GLP-2, and oxyntomodulin; Pcsk1) or
glucagon (Pcsk2). Mice null for Pcsk1 do not produce
GLP-1 or GLP-2 and are smaller (reduced length and
weight) than wild-type controls, but only the heterozygote
mouse had any impairment of IP glucose tolerance (453).
Notably, patients with a Pcsk1 mutation are obese, hyperphagic, have reactive hypoglycemia, and have enteroendocrine dysfunction (114, 188, 215). In fact, Pcsk1 was
found to be the third most prevalent genetic contributor to
obesity (67). While the obesity is consistent with data supporting brain Gcg expression as crucial for regulation of
energy homeostasis (22), Pcsk1 also processes proopiomelanocortin, a key neurotransmitter in the regulation of energy homeostasis.
Pcsk2 null mice that do not produce glucagon from Gcg are
similar to whole body Gcg knockout animals with mild
hypoglycemia, improved glucose tolerance (122), and ␣-cell
proliferation (122, 123). This similarity is striking since
prohormone convertases are involved in the posttranslational processing of proinsulin and proopiomelanocortin,
among other peptides, yet it is the effects of the Gcg deletion
that are most pronounced.
Comparing the phenotype of the Gcg null to the respective
knockout of Glp-1r and Gcgr provides important clues regarding the physiology of the ProG system. GCGR null
animals are phenotypically similar to Gcg null mice, with
lower fasting glucose levels, improved glucose tolerance,
and ␣-cell hyperplasia (10, 245). In contrast, GLP-1r
knockouts have impaired oral and IP glucose tolerance,
and no major changes in islet architecture (64, 128, 303,
363). GCGR null animals have enhanced whole body
insulin sensitivity (381), whereas the GLP-1r null animal
has specific impairments in hepatic but not peripheral
insulin action (15).
Interestingly, secretagogue-dependent increases in insulin
secretion and reduction in gastric emptying rate also play a
role in mediating improvements in overall glucose tolerance
in many of these models. In fact, a caveat to comparing the
phenotype of the Gcg null to the GCGR null mouse is the
increase in plasma and islet GLP-1 levels in the GCGR null
mouse (10). To eliminate this confound, dual genetic elimination of the GCGR and GLP-1r were generated. These
mice were found to have fasting glucose levels and IP glu-
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DARLEEN A. SANDOVAL AND DAVID A. D’ALESSIO
cose tolerance that was comparable to wild-type controls,
but maintained improved oral glucose tolerance, reduced
rates of gastric emptying, and impaired glucose-stimulated
insulin secretion seen in the GCGR null animals (10). Interestingly, the dual GLP-1/glucagon receptor null mice have
increased sensitivity to GIP-induced insulin secretion compared with the Gcg null mice that have increased plasma
levels of GIP (120).
In addition to the role of Gcg on function, there is also a role
for Gcg in differentiation of islets. For example, along with
the improvement in glucose homeostasis, another key feature common to Gcg null, Pcsk2, and GCGR null animals is
␣-cell hyperplasia (FIGURE 4). This seems to be a common
feature of the interruption of glucagon signaling, since it has
been described in mice treated with anti-GCGR antibody or
antisense oligonucleotides as well (144). In animals that
lack glucagon (Pcsk2 null mice), glucagon replacement has
been shown to reverse the ␣-cell proliferation, indicating
that loss of glucagon and/or glucagon signaling is required
for ␣-cell proliferation (425). This is supported by the fact
that mice with dual elimination of the GLP-1 and glucagon
receptors still have ␣-cell hyperplasia (10). Further research
has demonstrated that liver specific GCGR signaling (58,
245) is not only required but that there is an, as of yet
unknown, circulatory factor released when hepatic GCGR
signaling is absent that stimulates ␣-cell hyperplasia (245).
This was demonstrated by the fact that ␣-cell hyperplasia
occurred even in “normal” islets that were transplanted to
PS
Obesity
GRPP
Glucagon
Psck1/3 null
CNS
L-cells
IP1
GLP-1
IP2
GLP-2
the kidney capsule of whole body or liver-specific GCGR
null animals (245).
In summary, Gcg, GCGR, and the GLP-1r are all required
for various components of ␤-cell function, and hepatic
GCGR signaling is required for normal ␣-cell growth and
differentiation. Underscoring the importance of the Gcg
system, each of these models has developmental adaptations that result in upregulation of alternative enteroendocrine systems and contribute to the maintenance of glucose
homeostasis.
VIII. REGULATION OF ENERGY
HOMEOSTASIS BY PROGLUCAGON
PEPTIDES
A. GLP-1
The regulation of energy homeostasis involves the balance
between caloric expenditure to caloric ingestion. When
these two variables are matched calorie for calorie, body
weight is stable. This process is largely thought to be regulated by the CNS in response to peripheral levels of circulating and stored nutrients, hormones, and neuronal feedback from the periphery. While the roles of GLP-1 and
glucagon on glucoregulation largely oppose one another,
the two peptides have similar actions to cause negative energy balance, albeit through different sides of the energy
Preproglucagon null
Psck2 null
α-cells
GLP-1
Glucagon
α-cell hyperplasia
GLP-1r null
Glcar null
Islet cells
FIGURE 4. Summary of the genetics of
␣-cell hyperplasia. A number of mouse models have been generated to understand Gcg
biology. While models that target GLP-1 action tend to be obese (Psck1/3 null), or
have mild obesity and/or glucose intolerance (GLP-1r null), models that target glucagon signaling (Gcg null, GCGR null, GLP1r⫹Glar null) all have pronounced ␣-cell hyperplasia. While the GCGR null also has a
compensatory increase in GLP-1, the
GCGR/GLP-1r double KO still has ␣-cell hyperplasia indicating that increases in GLP-1
signaling are not responsible for this effect.
Liver-specific GCGR null animals also have
␣-cell hyperplasia indicating that hepatic receptors are necessary for the effect.
GLP-1r + Glcar null
Liver Glcar null
528
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GLUCAGON AND GLP-1
balance scale. However, the role of proglucagon peptides in
energy homeostasis has largely focused on GLP-1 rather
than glucagon. Part of this is based on the fact that the
posttranslational processing of neuronal Gcg is via PC1/3
and thus their primary product occurring naturally in the
CNS is GLP-1 (94, 150, 197).
Many of the hindbrain neurons expressing Gcg relay information from the viscera carried by primary afferent neurons
and have rich axonal projections to the hypothalamus,
hindbrain, and amygdala (150, 244), and also to vagal efferent catecholaminergic and serotonergic neurons (243).
While expression of Gcg is localized to cells in the hindbrain, GLP-1 has been detected throughout the hypothalamus by immunostaining of nerve fibers (197, 393, 416),
supporting its role as a ligand for GLP-1r in key area such as
the paraventricular and arcuate nuclei. In the paraventricular nucleus, GLP-1 immunoreactive terminals are found on
corticotropin release hormone neurons and in oxytocinergic neurons were stained positive for GLP-1 both within the
paraventricular nucleus and the supraoptic nucleus (393).
Thus, while the neuronal expression of Gcg seems to be
discrete, GLP-1r have widespread expression within the rodent CNS (54, 135). In contrast, it is unclear how much
glucagon is produced within the brain, and the relatively
less common CNS glucagon receptors have not been well
characterized (54).
Despite recent advances, much of the neurophysiology of
Gcg neurons remains unclear. The factors that control
hindbrain production and secretion of ProG products are in
an early stage of understanding. For example, ex vivo studies demonstrate that Gcg neuronal activity is stimulated by
leptin (167, 180), cholecystokinin (CCK), and epinephrine
(168), but not PYY or melanotan II, or most importantly,
GLP-1 (168). Consistent with this latter finding, GLP-1r are
not located on Gcg neurons, suggesting that peripheral
GLP-1 does not directly regulate activity of these neurons.
Using c-fos as a marker for in vivo neuronal activation
demonstrates that experimental gastric distention (417)
and noxious and nutrient stimuli (124, 335) increase Gcg
neuronal activation. Most studies surrounding Gcg neuronal function are pharmacological. For example, GLP-1 administration directly into the CNS has a potent, but shortlived, inhibitory effect on food intake that is blocked by
coadministration of Ex9 (392, 402). Peripherally administered GLP-1 has a limited effect on food intake in rats (292),
in great part due to its rapid inactivation in the circulation
(194), suggesting that the anorectic effect of GLP-1 is mediated primarily via CNS GLP-1 receptors.
CNS GLP-1 is also implicated in long-term energy balance
as production is responsive to changes in feeding status and
adiposity. Specifically, hindbrain Gcg gene expression and
hypothalamic GLP-1 protein and are reduced after acute
and chronic restriction of food intake (137, 180), while
high-fat diet and obesity increase hindbrain Gcg expression
(221). In addition, blocking CNS GLP-1 either by chronically blocking GLP-1r with CNS administration of Ex9 (22,
267, 402), or by using viral-mediated RNA interference to
reduce Gcg expression (22), increases adiposity; in contrast,
1 wk of CNS administration of GLP-1 in high-fat-fed mice
decreases adiposity independent of food intake (299).
GLP-1r KO mice also have increased adiposity, although
this effect is dependent on background strain (154, 363,
364). Notably, chronic intracerebroventricular Ex9 administration to wild-type C57BL/6J mice fed a high-fat, verylow-carbohydrate diet causes weight loss (221). These results contradict most other work indicating that GLP-1 signaling is associated with negative energy balance. One
explanation for this is that the effects of GLP-1r blockade
are counteracted by the shift in dietary macronutrients
through specific CNS nutrient-sensing (44, 162, 164, 277,
352).
The role of CNS GLP-1 in regulating long-term energy balance seems to be integrated with CNS leptin signaling. For
example, the reduction of hindbrain Gcg expression and
hypothalamic GLP-1 protein levels by fasting is prevented
by maintaining leptin concentrations with exogenous administration (137, 180). Leptin replacement also maintains
the anorexic ability of both GLP-1, and Ex4 that is typically
lost with fasting (434). Consistent with these in vivo responses, leptin depolarizes Gcg neurons (167), and interestingly, deletion of the leptin receptor from Gcg neurons results in mice that are hyperphagic with rapid weight gain
but increased metabolic rate and normal glucose tolerance
(362).
It is notable that the interaction between leptin and GLP-1
is bidirectional. For example, the GLP-1r antagonist Ex9
delivered directly into the fourth cerebral ventricle attenuates the anorexic effects of leptin (451). The interactions of
GLP-1 and leptin fit with current concepts of energy homeostasis, whereby visceral signals that have short-term actions
on satiety, and signals responsive to adiposity, are linked.
However, there may be differences between physiological
and pharmacological regulation. For example, neuronal activation in the hindbrain in response to administration of
Ex4, as reflected in c-Fos expression by NTS neurons, is
reduced rather than potentiated by leptin treatment (434).
Regardless, at present, it is reasonable to suggest that physiological fluctuations in leptin modulate the CNS GLP-1
system and the role of GLP-1 in long- and short-term energy
balance.
While there is a wealth of data demonstrating an important
role of GLP-1 to regulate food intake, the data on whether
it also regulates energy expenditure is less consistent. In
mice, 1 wk of centrally administered GLP-1 prevented
weight loss-induced reductions in energy expenditure
(299), suggesting a positive role for brain GLP-1 on energy
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DARLEEN A. SANDOVAL AND DAVID A. D’ALESSIO
homeostasis. However, longer term administration (1 mo)
of central GLP-1 decreases EE (222), and GLP-1R KO mice
have been found to have an increase in energy expenditure
(154). Because energy balance is a fine-tuned regulatory
system, the animal literature could conflict due to differences in background strain, type of diet, and energy status.
These latter points are supported by the fact that GLP-1 has
a blunted anorexic effect in fasted animals (44, 352, 434).
While in humans, exogenous GLP-1 infusion that raises
levels fourfold over baseline was associated with an increase
in energy expenditure, this only occurred when insulin levels were held constant, suggesting an indirect, rather than a
direct, effect of GLP-1 on energy expenditure (372). Finally,
long-acting GLP-1 agonists do not seem to regulate energy
expenditure in patients (34, 155). Taken together, we believe these data reflect that GLP-1’s effects on reducing food
intake are much more definitive than any effects on energy
expenditure.
B. Glucagon
It is well established that glucagon also leads to negative
energy balance, mostly based on studies with exogenous
administration of peptide, or pathological states where it is
secreted to high levels. Two key features about glucagon
modulation of energy balance that distinguishes it from
GLP-1 are 1) that glucagon both inhibits food intake and
stimulates energy expenditure and 2) glucagon does not
induce visceral illness at doses that cause anorexia (126). In
addition, while the effects of GLP-1 to regulate energy balance have not been attributed to actions in specific brain
regions, how glucagon influences the CNS is not well understood, and most studies were conducted over 20 years
ago before techniques for mapping specific brain functions
were developed. Regardless, it is now clear that pharmacological administration of glucagon increases resting energy
expenditure and decreases food intake across multiple species (51, 77, 78, 125, 172, 237b, 261, 280, 306, 351, 360,
386, 427).
Consistent with a physiological role to regulate food intake,
plasma glucagon levels increase postprandially (200, 235,
403), and administration of glucagon antibodies prior to a
meal, to immunoneutralize circulating peptide, increases
meal size (236, 237b). However, the mechanism for this
effect of glucagon has not been specifically delineated, and
most of what is known in this area comes from pharmacological studies. Like GLP-1, CNS administration of glucagon inhibits food intake (185, 228), although the precise
distribution of glucagon receptors in the CNS is lacking.
Early work focused on a peripheral site of action that was
tied to glucagon’s ability to increase hepatic glucose production and consequently blood glucose levels. However, a
study in rabbits demonstrated that glucagon suppressed
food intake without changing hepatic glycogen levels (408).
Interestingly, like GLP-1, activation of afferent neurons
530
that innervate the portal vein has also been implicated in
glucagon action. Infusion of glucagon into the portal vein
has a dose-dependent effect to reduce food intake (427).
Interestingly, complete vagotomy eliminates the ability of
glucagon to suppress food intake, while specifically sparing
the hepatic branch of the vagus preserves glucagon-induced
anorexia (127). Satiety signals converge on the hindbrain,
specifically the NTS and the area postrema, and lesions of
afferent vagal nerve terminals in the NTS also block the
ability of glucagon to reduce food intake (424). Together
with the data on GLP-1 action within the portal vein, these
data suggest that glucagon-induced satiety is via portal vein
action while GLP-1-induced satiety is via intestinal innervation.
Another alternative to a CNS site of glucagon action is
brown adipose tissue (BAT), a target organ that has come to
the forefront as a mediator of energy expenditure. BAT is a
highly metabolic tissue that, when stimulated, generates
heat that increases local and sometimes core body temperature. In some studies, glucagon increases both core body
temperature (90) and BAT mass and temperature (30, 90,
226, 444), indicative of an increase in energy expenditure.
BAT is highly activated during cold exposure as a way to
generate heat, and indeed, cold exposure increases both
plasma and BAT glucagon levels (147), suggesting a physiological role for glucagon in nonshivering thermogenesis.
Some suggest that the impact of glucagon on BAT is only
physiologically important during cold exposure as glucagon increases BAT temperature more potently in coldacclimatized rats compared with rats kept at room temperature (90).
Glucagon activates BAT in vitro (199), suggesting direct
regulation of this tissue. However, in vivo, propranolol, a
nonspecific ␤-adrenergic receptor antagonist (85), blocks
the effect of glucagon on BAT thermogenesis, suggesting an
important indirect role mediated through the adrenergic
nervous system as well. Coadministration of glucagon and
epinephrine additively stimulate energy expenditure, yet the
effect of glucagon is still present in adrenalectomized rats
(77), dissociating glucagon action from epinephrine levels
per se. One speculation is that glucagon interacts with the
SNS at other tissues (e.g., the CNS). Overall these data
suggest that glucagon and the sympathetic nervous system
have integrated effects on regulating energy expenditure via
BAT activity. Despite these interesting findings in animal
models, an important role of BAT in human energy balance
remains a debated issue.
Given that glucagon increases energy expenditure and reduces food intake (FIGURE 5), it is not surprising that many
(30, 80a, 360) but not all (213) studies demonstrate weight
loss with chronic administration. While the glucagon receptor knockout mouse has a lean phenotype (10), they also
have developmental adaptations for the lack of glucagon,
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GLUCAGON AND GLP-1
GlcaR
Visceral
afferents
Energy
expenditure
Food
intake
Glucagon
BAT-GlcaR
Islet cell
FIGURE 5. Glucagon regulation of energy homeostasis. Glucagon
appears to activate vagal afferents that subsequently suppress food
intake via CNS mechanisms. However, glucagon also activates
brown adipose tissue (BAT) to increase energy expenditure. Thus
glucagon action can reduce body weight both by increasing energy
expenditure and reducing food intake.
one being an increase in plasma GLP-1 (10). Regardless, the
ability of chronic glucagon to suppress body weight makes
it an interesting target for treatment of obesity. The recent
investment of pharmaceutical companies towards combined therapeutics offers an opportunity to take advantage
of the weight-reducing effects of glucagon while offsetting
its hyperglycemic effects by combining with other hypoglycemic agents, such as GLP-1.
IX. ROLE OF PROGLUCAGON PEPTIDES IN
DIABETES AND OTHER PATHOLOGICAL
STATES
A. Pathophysiology of Glucagon in Diabetes
Similar to insulin secretion, glucagon release in persons
with diabetes is abnormal, and a number of defects have
been identified from studies of diabetic subjects. In states of
poorly controlled diabetes with severe insulin deficiency or
ketoacidosis, plasma glucagon concentrations are extremely high (101). However, fasting hyperglucagonemia
of a more modest degree is present in many T2DM patients
even without metabolic decompensation, and a good case
can be made that inappropriate fasting glucagon drives inappropriate HGP in diabetic patients (20, 326). Similar to
the diabetic ␤-cell, the ␣-cell in diabetic subjects has abnormal sensitivity to glucose with less suppression during hy-
perglycemia from enteral or parenteral sources, and plasma
glucagon levels after eating mixed nutrient meals are generally higher with T2DM (101, 405, 420). In contrast, there
is evidence for sluggish or reduced glucagon responses to
hypoglycemia (101, 131). It is not clear how this inappropriate response to low glucose is mediated, but the functional result suggests another form of ␣-cell glucose insensitivity. There is emerging evidence that ␣-cell dysfunction
in diabetes has a genetic basis as a common polymorphism
in the KIR6.2 gene, which predisposes to T2DM and is
related to blunted glucose-induced suppression of glucagon
(361, 400).
A number of studies in humans support a role for glucagon
in postabsorptive glucose regulation, with estimates that it
accounts for 40 –50% of fasting hepatic glucose production
(20, 84a, 241). These findings are compatible with those of
rodent models demonstrating important glucagon effects to
support fasting blood glucose levels (35, 128). In classic
studies performed soon after the discovery that somatostatin inhibits ␣-cell secretion, it was demonstrated that hyperglycemia and ketogenesis were substantially improved in
insulinopenic T1DM subjects when plasma glucagon was
reduced (132). Moreover, in T2DM subjects, suppression
of islet hormone secretion by somatostatin reduced basal
hepatic glucose production significantly, and this effect was
enhanced when insulin was given at basal levels (101). Together these findings exemplify the basis for concluding that
glucagon action contributes to pathogenic as well as physiological control of fasting glucose control.
In subjects with T2DM, plasma glucagon levels are not
normally suppressed after carbohydrate containing meals
or during oral glucose tolerance testing. Thus, in diabetic
subjects with persistent fasting hyperglucagonemia, postprandial hepatic glucose production remains elevated at
fasting levels, rather than making the abrupt postprandial
decline typical of nondiabetic subjects (15). In healthy subjects the suppression of glucagon secretion after meals is a
key factor in shifting hepatic metabolism from glucose production to a glucose clearance (42). Nondiabetic subjects
given somatostatin with infusions of glucagon to maintain
basal levels during a glucose bolus had significantly greater
glycemic excursions than when glucagon was allowed to
follow the normal postprandial decline (370). A similar
pattern was seen in subjects with T2DM (371). Overall a
substantial body of evidence indicates that failure to suppress fasting levels of glucagon after eating contributes to
hyperglycemia, an effect that is magnified in the setting of
reduced plasma insulin.
B. Pathophysiology of GLP-1 in Diabetes
In a now classic paper, Nauck et al. (288) reported that
subjects with T2DM had a blunted incretin effect, a find-
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531
DARLEEN A. SANDOVAL AND DAVID A. D’ALESSIO
ing generally interpreted to be a specific defect that contributes to hyperglycemia. This finding, which has been
widely cited and recently corroborated (278), raised
questions about the adequacy of incretin secretion, metabolism, and action in diabetic individuals. There have
been a large number of studies comparing GIP and GLP-1
secretion in diabetic and nondiabetic subjects (414). The
results have varied considerably, but there is now emerging consensus that incretin secretion in diabetic subjects
is comparable to nondiabetic subjects, and not likely to
be a primary cause of abnormal glucose regulation (50,
269, 286). Moreover, the metabolism of GLP-1 and GIP
in diabetic patients compared with controls is also similar (412), suggesting that differences in elimination do
not explain a reduced incretin effect. This leaves loss of
␤-cell sensitivity to GLP-1 or GIP as the likely explanation for a blunted incretin effect in T2DM. Consistent
with this hypothesis, the insulinotropic effect of GIP is
nearly absent in hyperglycemic subjects with T2DM
(270, 287, 415), and there is evidence that hyperglycemia
leads to decreased ␤-cell expression of the GIP receptor
(249, 431). In contrast to what is seen with GIP, GLP-1
retains potent activity to stimulate insulin secretion in
diabetes (287, 323, 325). While persons with T2DM
have decreased ␤-cell sensitivity to GLP-1 (220), supraphysiological doses enhance glucose-stimulated insulin
secretion, but GIP remains ineffective regardless of the
dose (170). The disparity in stimulating insulin release
from diabetic subjects is the clearest distinction between
GIP and GLP-1 as incretins, and is puzzling given the
great similarity in the ␤-cell signaling they induce. Recent
reports indicate that ␤-cell responses to supraphysiological amounts of GIP and GLP-1 can be enhanced in diabetic subjects by 4 wk of glycemic control (169, 171), and
this contributes to an improvement in the incretin effect
(11).
On the basis of current evidence, abnormalities in the
GLP-1 system do not play a primary role in the pathogenesis of T2DM. Early claims that diabetic individuals had
deficiencies of GLP-1 have not been substantiated, at least
for larger populations of subjects. And while the insulin
response to GLP-1 is impaired compared with nondiabetic
subjects, this is likely a function of general ␤-cell dysfunction, since studies with GLP-1r blockade show comparable
relative contributions of GLP-1 to prandial insulin secretion
in T2DM and nondiabetic humans (346, 439).
C. Tumors Secreting Proglucagon Peptides
and the Glucagonoma Syndrome
Islet ␣-cell tumors can arise sporadically or as a component
of the multiple endocrine neoplasia, type 1, syndrome.
The first definitive description of a glucagon-producing
tumor was in 1966 in a patient with a pancreatic mass
and dramatically elevated plasma glucagon concentra-
532
tions (264). ␣-Cell tumors are very rare with an incidence
of 1 per 20 –50 million (108, 218), and only a subset
manifest clinical symptoms related to the actions of secreted proglucagon peptides. A glucagonoma syndrome
has been described and several hundred cases reported in
the literature (108, 218, 382, 428). There are a few defining features including glucagon levels ⬎500 mg/ml and
diabetes; indeed, in the largest case series of glucagonoma syndrome, the presence of diabetes correlated
with plasma glucagon levels (428). The diabetes associated with glucagonoma syndrome is not insulinopenic
and can often be treated with diet and oral agents, and is
not associated with ketoacidosis. However, a review of
several larger case series suggests that the most common
presenting feature is in fact weight loss (108, 253, 428). It
is notable that patients with glucagonoma syndrome often have micronutrient deficiencies and hypoaminoacidemia, consistent with increased nutrient catabolism as
well as decreased nutrient intake. Thus these data are
consistent with the animal literature demonstrating that
glucagon regulates energy homeostasis.
D. Proglucagon Peptides in Bariatric
Surgery
The number of bariatric surgical procedures has surged in
the last two decades in response to the unrelenting increase
in the prevalence of obesity (40). Procedures such as gastric
bypass (GB) and vertical sleeve gastrectomy (VSG) cause
significant weight loss in most patients (66) and also reduce
obesity-related comorbidities (62, 134, 260, 376). Of great
interest has been the rapid correction of diabetic hyperglycemia by GB and VSG soon after surgery and before substantial weight loss (317, 356, 395). The magnitude and
reproducibility of these findings have spurred research into
the mechanism behind this effect, with GLP-1, and to a
lesser extent glucagon, receiving considerable attention as
potential mediators of metabolic changes after bariatric
surgery.
Persons with GB have enormous increases in postprandial
GLP-1 secretion compared with subjects without surgery
(190, 224, 230, 307, 348). Peak plasma levels of GLP-1 are
5- to 10-fold elevated, and this response persists for years
after surgery (76, 225, 231, 410). Of note, GLP-1 levels are
also substantially increased after VSG despite the significant
differences in surgical anatomy compared with GB (283,
307). The cause of increased L-cell secretion following surgery is unclear. The simplest explanation is that both GB
and VSG cause increased flux of ingested nutrients to the
small intestine (87, 338), and secretion of GLP-1 is known
to be dependent on rate of glucose entrance into the gut (56,
357). However, the details of this process are still unclear,
and this remains an area of active research.
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GLUCAGON AND GLP-1
The increased plasma GLP-1 in GB is associated with an
enhanced incretin effect. Nondiabetic subjects with GB had
a threefold greater contribution of GLP-1 to their postprandial insulin response than nonoperated controls (348). Similarly, diabetic subjects with GB also had a disproportionate
reduction of insulin secretion during infusion of a GLP-1R
antagonist (196, 201). While it has been proposed that
GLP-1 could contribute to the increased satiety following
GB, recent studies in mice with GLP-1R gene deletions have
demonstrated normal weight reduction following GB or
VSG (276, 438, 445).
With a greater number of patients having GB, the long-term
complications of this procedure have become more defined
(205). One of the more dramatic of these is a syndrome of
hyperinsulinemic hypoglycemia that occurs several years
after surgery (304, 368). Affected subjects have normal glucose regulation during fasting but recurrent hypoglycemia
1–2 h after eating. One hypothesis proposed for this syndrome is that it is due to enhanced GLP-1 sensitivity or
action in a subset of GB patients. Indeed, in one study
patients with recurrent postprandial hypoglycemia had
higher plasma GLP-1 levels compared with asymptomatic
GB subjects (136). This has not been the case in all such
comparisons (348, 350), a variability suggesting that
plasma GLP-1 is not the only factor involved in the hypoglycemic syndrome. A recent study demonstrates that
blocking the GLP-1r with Ex9 corrects postprandial hypoglycemia in affected subjects while having a much smaller
effect on blood glucose and insulin secretion in asymptomatic GB subjects (347). These results suggest that increased
GLP-1 action contributes to the post-GB hypoglycemic syndrome and raises the possibility that a combination of elevated plasma GLP-1 and enhanced sensitivity to its actions
mediate this effect.
Several groups have reported that postprandial glucagon
secretion is also increased following GB (52, 348, 350,
369). It is not currently understood why plasma glucagon is
two- to fourfold higher in GB patients and the observation
is generally inconsistent with the improved glucose tolerance in diabetic subjects after surgery. However, it is important to note that glucose dynamics are changed considerably following GB and VSG, with elevated gastric emptying (57), accelerated absorbance of enteral carbohydrate in
the early phase of meals, an early and elevated glucose peak,
and a rapid decline to or below baseline glycemia (52, 338,
350). One hypothesis for why plasma glucagon levels are
elevated in this setting is as a response to the need for a
shorter period of hepatic suppression of glucose production. This has been suggested in a recent study of GB subjects in whom the profile of endogenous glucose production
paralleled that of plasma glucagon (52). The mechanism by
which the distinct profile of plasma glucagon is regulated in
GB is not known, but is likely to provide new insights into
the regulation of ␣-cell secretion.
X. ROLE OF PROGLUCAGON PEPTIDES IN
THERAPEUTICS
A. Glucagon Antagonists and Agents That
Reduce Glucagon Signaling
Based on the evidence supporting glucagon as a significant
contributor to abnormal glucose regulation in T2DM, targeting glucagon action is reasonable therapeutic strategy
that has been pursued for the past 30 years. Despite compelling findings from clinical and preclinical work, there has
not yet been an approved drug that acts primarily by limiting glucagon secretion or signaling. This is due in great part
to problems that may be encountered when glucagon signaling is substantially reduced, problems that have been
reported in animal models and humans (9, 73). The mouse
models that have been developed to study the physiology of
glucagon action include animals with interruption of glucagon receptor function (128, 303) as well as ␣-cell secretion
(151, 160). These animal lines tend to have lower fasting
glucose and significantly improved glucose tolerance than
control mice and provide proof of principle that long-lasting blockade of glucagon signaling could be an effective
means of reducing diabetic hyperglycemia. However, despite preclinical data indicating that reduced glucagon action lowers, and resets, the level of glucose that animals
defend, there are concerns raised by the development of
␣-cell hyperplasia and profound elevations of circulating
proglucagon-related peptides in mice with GCGR or proglucagon deletion (9) and in humans with GCGR mutations
(448). In addition to the expansion of endocrine cells, animals deficient in glucagon signaling also have increased total pancreatic weight reflecting expansion of the exocrine
compartment. In addition to this apparent drive to cellular
growth that occurs when GCGR signaling is abolished, reductions in hepatic glucagon signaling also lead to reduced
lipid oxidation (151, 246). More recent work raises the
possibility that absent GCGR signaling reduces the tolerance and recovery of hepatocytes to toxic injury (375).
These findings provide a cautionary note to therapeutic
strategies that include long-term interference with glucagon
signaling, particularly in the liver.
Blockade of glucagon action in humans has been possible
with several small molecule GCGR antagonists developed
by pharmaceutical companies, but the information available from these clinical programs is limited. One of these
compounds, BAY27-9955, was demonstrated to block the
actions of exogenous glucagon in nondiabetic subjects
(308), but was not further developed and no information on
treatment of diabetic subjects with fasting hyperglycemia or
endogenous hyperglucagonemia was published. A small
molecule, MK0893, lowered fasting glucose significantly in
diabetic subjects in a dose-responsive manner, but these
results were only presented in preliminary form (111). Similar findings have been reported with LY2409021, with
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DARLEEN A. SANDOVAL AND DAVID A. D’ALESSIO
dose-dependent reductions in fasting and postprandial glucose (209). As the pharmacological approach most analogous to genetic models of reduced GCGR signaling, antagonists have obvious potential as drugs to lower glucose and
treat diabetes. However, the data from preclinical studies
raise concerns that adverse effects such as ␣-cell hyperplasia, hepatic steatosis, and susceptibility to hepatic toxicity
could occur in treated patients. Moreover, the effects of
these agents on glucose counterregulation would need to be
carefully assessed.
In reality, there are a number of available drugs to treat
diabetes that act in part by inhibiting glucagon secretion.
Both sulfonylureas and exogenous insulin inhibit glucagon
secretion to a modest degree (309, 330), although the effects
are variable and the specific contribution of this action to
their therapeutic benefit has not been determined. In addition, two new classes of drugs, GLP-1 receptor agonists and
DPP-4 inhibitors, have both been demonstrated to lower
blood glucose at least partially through reductions of
plasma glucagon. Exogenous administration of GLP-1 reduces plasma glucagon levels, a response reported with the
early human use of this peptide (289). In a recent study islet
hormone secretion was blocked with an infusion of somatostatin, and insulin and glucagon were replaced to match
the levels seen after GLP-1 administration. The various
combinations of insulin and glucagon levels in the healthy
subjects studied supported glucagon lowering and insulin
stimulation as approximately equivalent in the GLP-1 control of fasting glucose (156). Administration of pharmacological amounts of GLP-1 to hyperglycemic type 1 diabetic
subjects with minimal ␤-cell function reduced blood glucose by ⬃4 mM, associated with a 40 –50% decrease in
plasma glucagon (291). Thus increasing plasma GLP-1 activity with either injectable analogs or by protecting endogenous peptide with DPP-4 inhibitors are approaches to reduce glucagon action.
Both exenatide and liraglutide lower blood glucose and hemoglobin A1c in T2DM subjects, improve insulin secretion,
and reduce plasma glucagon (93). Exenatide has effects similar to native GLP-1 to reduce glucagon concentrations in
subjects with type 1 diabetes (104), and this has been proposed to account for the lower fasting blood glucose in
these subjects. With chronic administration, exenatide and
liraglutide reduce plasma glucagon with concurrent increases of insulin. Although reductions of plasma glucagon
are likely to contribute to the therapeutic effect, it is not
possible at present to attribute the relative contribution of
this action. DPP4 inhibitors are also in use for the treatment
of diabetes, with a number of agents having similar efficacy
now available (81). DPP4 cleaves NH2-terminal dipeptides
from a number of naturally occurring peptides, but the
effects of DPP4 inhibitors to improve hyperglycemia in patients with T2DM seems to be mostly due to the protection
of plasma GLP-1. These drugs have similar actions as
534
GLP-1 receptor agonists on islet hormone secretion, with
their major effects being to stimulate insulin secretion, and
reduce plasma glucagon. With chronic use of DPP4 inhibitors and lower blood glucose levels, changes in plasma glucagon can appear to be greater than those of insulin (6).
Thus there has been a tendency to focus on the effects of
these agents on ␣-cells. However, the relative contribution
of glucagon suppression to diabetic control by these drugs
has also not been proven.
Recent work suggests that metformin, the most commonly
used oral medication to treat T2DM, may in fact lower
blood glucose as a hepatic glucagon antagonist. It has long
been appreciated that metformin reduces hepatic glucose
production in humans with diabetes (70, 84). This action is
independent of increased insulin secretion or insulin sensitivity and has been attributed to suppression of glycogenolysis (70) and/or gluconeogenesis (385). In cultured hepatocytes, metformin, or the related compound phenformin,
inhibits generation of cAMP or activation of PKA by
glucagon and reduce glucose production (272). These
effects were corroborated in vivo, with mice treated with
metformin resistant to the hyperglycemic effects of glucagon. These effects have not been validated in diabetic
humans but raise the possibility that antagonism of glucagon has been one of the most common means of treating diabetes heretofore. It is noteworthy that metformin
treatment is very well tolerated and has not been associated with hepatic toxicity in humans.
B. GLP-1 Agonists and DPP-4 Inhibitors
The effectiveness of GLP-1 to stimulate insulin secretion,
and activate other processes that lower blood glucose (reduce plasma glucagon, delay gastric emptying, suppress hepatic glucose production), is central to the development of
drugs that work through the GLP-1r system. Peptide agonists of the GLP-1r that are resistant to metabolism by
DPP4 are now widely used to treat diabetes (7, 93). The
development of incretin-based drugs has been one of the
major advances in diabetes medicine in recent years.
GLP-1r agonists recapitulate most of the physiological actions of native GLP-1, and also have pharmacological effects on satiety (247). Small molecule DPP4 inhibitors also
stimulate insulin and reduce glucagon, actions that can be
attributed to GLP-1r signaling (96), although the precise
mechanism of action of these agents is not yet fully understood.
Three GLP-1 receptor agonists are currently available for
the management of hyperglycemia in patients with type 2
diabetes (53, 93). Exenatide (Byetta), a synthetic replica of
exendin-4, has been available clinically since 2005 and is
administered twice daily. Liraglutide (Victoza), a GLP-1
analog modified to include a fatty acid side chain to facilitate albumin binding, has a longer half-life and is suitable
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GLUCAGON AND GLP-1
for once daily injection; this agent received approval in
2010. An extended-release form of exenatide, exenatide ER
(Bydureon), is administered weekly and has been available
since 2012. Because they are peptides, these agents are administered subcutaneously. The key pharmacological characteristics of the commercially available GLP-1 RAs are
summarized in TABLE 1. Exenatide BID and exenatide ER
are predominantly eliminated through glomerular filtration, while liraglutide is thought to be eliminated through a
receptor-based mechanism and not through renal clearance
(189, 254).
The development and study of GLP-1r agonists has provided new insights into the actions of native GLP-1. In
particular, effects of GLP-1r signaling on ␣-cell secretion,
gastric motility, and satiety have become even more apparent with pharmacological dosing in clinical studies. These
actions of GLP-1r agonists are believed to have a major role
in their therapeutic benefits, and may in some patients outstrip their insulinotropic actions. There remain major questions as to how the ␤-cell and non-␤-cell actions of GLP-1
are mediated, especially since the GLP-1r is expressed minimally, if at all, by putative target tissues like the ␣-cell,
gastric musculature, or the liver. Thus indirect mechanisms,
neural or paracrine (406, 418), have been proposed for
many of the responses to systemic GLP-1.
Another aspect of GLP-1 physiology that carries over to
pharmacology is the effect of GLP-1 agonists, but not inhibitors of DPP-4, to reduce food intake and cause weight
loss (413). In clinical trials with exenatide or liraglutide,
subjects predictably lose several kilograms of body weight
and frequently report a decrease in appetite (93). The neural
pathways leading to this response are not currently established, and both direct actions in the CNS and activation of
peripheral neural circuits have been proposed to mediate
this response (23). The effects of liraglutide on body weight
in diabetic patients have prompted investigation into its use
as a treatment for obesity in nondiabetic humans (13). It
remains an open question as to whether and how the satiety/anorectic actions of GLP-1r agonists are related to the
major side effects of these drugs, nausea and vomiting. It
has been noted that most patients have improvement in
nausea within the first few weeks of treatment with a
GLP-1r agonist, while weight loss can persist over months
and years. Better understanding of the neural mechanisms
of these processes would benefit the therapeutic utility of
these agents.
The trend in drug development for GLP-1r agonists is manipulation of pharmacokinetics to improve convenience
and compliance (259). Agents that have to be administered
once every week or 2 wk have the advantage of reducing the
number of injections patients need to take. Interestingly,
data are emerging to support pharmacodynamic differences
between constant GLP-1r agonism with long-acting agents
and activation/deactivation with short-acting agents. Thus
it appears that long-acting GLP-1 agonists have a greater
effect to suppress glucagon while short-acting agents have a
greater effect to delay gastric empting (92). Moreover, there
is evidence that a more constant exposure to GLP-1 action
causes less nausea than with rapid-acting compounds (45,
92, 181, 334). These findings have potential implications
for the use of different GLP-1r agonist formulations in particular patients, and it seems likely that there are important
underlying mechanisms to explain these effects.
The mechanism of action of DPP-4 inhibitors is still under
investigation. The simple view that doubling the amount of
active incretins in the circulation is sufficient to account for
the clinical effects has been questioned (72). Studies in mice
suggest that GLP-1 is active in the intestine where it acts
through afferent nerves to mediate actions to lower blood
glucose (418). DPP-4 activity has also been described in
pancreatic islets as well (91), so it is conceivable that treatment with DPP-4 inhibitors could act by protecting locally
produced GLP-1. Similar to the case for GLP-1r agonists,
the use of DPP-4 inhibitors to learn more about the physiology of glucose regulation is rich with possibility.
There has been some recent concern regarding possible adverse effects from GLP-1r agonists, and to a lesser extent
DPP inhibitors with diseases of the exocrine pancreas (4),
particularly pancreatitis. Preclinical studies have not con-
Table 1. Key pharmacological characteristics of commercially available GLP-1 receptor agonists
Characteristic
Exenatide BID (Byetta)
Liraglutide (Victoza)
Exenatide ER (Bydureon)
Exenatide contained in hydrolyzable
polymer microspheres
Description
Synthetic exendin-4
Administration
Subcutaneous injection twice daily
before meals
Human GLP-1 modified with
amino acid substitution
and acyl chain addition
Subcutaneous injection
once daily, any time
2.4 h
2.1 h
13 h
8–12 h
Half-life
Time to peak concentration
Subcutaneous injection, once
weekly, any time but immediately
after suspension
⬃2 wk
6–7 wk
GLP-1, glucagon-like peptide 1.
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DARLEEN A. SANDOVAL AND DAVID A. D’ALESSIO
sistently suggested a mechanism for this (97), and the epidemiology is not clear. The impending conclusion of several
large randomized controlled trials directed at the effects of
GLP-1r agonists on cardiovascular safety, but also monitoring for pancreatitis, is expected to provide more conclusive information on this potential adverse effect.
C. Dual Glucagon/GLP-1 Receptor
Coagonists
A novel and exciting recent approach to the treatment of
diabetes has been the development of hybrid peptides that
activate more than one receptor to generate an effect (345).
These agents are touted as mimicking the broad range of
hormonal changes associated with bariatric surgery, and
the need to utilize more than one ligand-receptor system to
achieve significant weight loss. Some of the first compounds
developed using this strategy were glucagon/GLP-1 coagonists, peptides engineered to activate the cognate receptors
of both peptides in different relative potencies (79, 80). The
rationale behind this line of drug development is that both
glucagon and GLP-1 bind specific and distinct receptor populations in the brain to cause satiety (23, 193), and activating these together might have synergistic results. In an initial
report, two hybrid peptides, one with balanced GCGR and
GLP-1R potency and a second with sevenfold greater activity at the GLP-1R, reduced body weight and fat, increased
energy expenditure, and dramatically improved glucose tolerance in obese mice and rats (80). The effects on weight
loss were significantly greater than that of a GLP-1R-only
agonist, and this additive response supports different activation of different neural pathways for glucagon and GLP-1
to cause weight loss. Very similar findings were reported
with a different GLP-1/glucagon coagonist including a careful demonstration of superiority of weight loss compared
with a GLP-1R agonist, and a convincing demonstration of
dependence on both receptors for full activity (313). In
humans, while GLP-1 infusion alone had no effect on energy expenditure, and glucagon infusion alone raised both
blood glucose levels and energy expenditure, coadministration of both peptides increased energy expenditure but did
not raise glucose levels (391).
It is clear from studies of GLP-1R⫺/⫺ mice and comparisons
of coagonists with different relative potencies at the two
receptors that GLP-1r agonism ameliorates the hyperglycemic effects of glucagon (71, 72, 289). Based on the limited
work in this area, peptides with balanced agonism for the
GLP-1/GCGRs seem to have the most therapeutic promise
(79, 80). While greater glucagon potency confers greater
energy expenditure and suppression of food intake, there
seems to be a threshold beyond which glucose control worsens despite weight loss. The potential for a GLP-1R/GCGR
coagonist to lower blood glucose has also been demonstrated with oxyntomodulin (98), a proglucagon product
that activates both receptors albeit less potently than the
536
cognate ligands. A single amino acid substitution at position 3 in oxyntomodulin abolishes agonism at the GCGR;
this analog does not activate fatty acid oxidation through
the GCGR, but has improved effects on glucose regulation
compared with the native compound. Thus the coupling of
GLP-1 activity, to mitigate any hyperglycemic effects, with
glucagon allows the catabolic actions of both peptides on
energy balance to give an augmented response compared
with either alone.
XI. CONCLUSION
This review challenges the simple model that Gcg encodes
peptides, particularly GLP-1 and glucagon, in a strictly tissue-specific fashion to regulate opposite ends of glucose
fluctuations, e.g., GLP-1 as an insulin secretagogue that
improves glucose homeostasis and glucagon as a counterregulatory hormone with a hepatic centric mode of action.
In fact, these peptides act at several levels to regulate glucose homeostasis but also have broader actions on energy
and nutrient metabolism that are sometimes in opposition
and sometimes complementary. Importantly, these two
proglucagon peptides have important pharmacological actions and are likely to be a focus of investigation and drug
development for the foreseeable future. Overall, a role for
both glucagon and GLP-1 in the pathogenesis and treatment of T2DM are possible, and this is underscored by the
fact that combined GLP-1/glucagon therapies are effective
hypoglycemic and weight loss agents (FIGURE 6).
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence: D.
D’Alessio, Duke University Medical Center, Dept. of Med-
GLP-1
sensitivity
Glucagon
Glucagon
antagonists
T2DM
Bariatric Surgery
Glucagon
GLP-1
GLP-1
agonists
Combined Therapy
Glucagon + GLP-1 agonist
FIGURE 6. The Gcg system has been implicated in both the cause
and cure of T2DM. Increased glucagon, and decreased GLP-1,
action have both been implicated in the pathology of T2DM, supporting the pursuit of glucagon antagonists and GLP-1 agonists to treat
T2DM. Interestingly, bariatric surgery, which causes drastic improvements in energy and glucose homeostasis, increases both
glucagon and GLP-1. Combined glucagon and GLP-1 agonists are
also in the pipeline for treatment of obesity and T2DM.
Physiol Rev • VOL 95 • APRIL 2015 • www.prv.org
GLUCAGON AND GLP-1
icine, Div. of Endocrinology/Metabolism, DUMC Box
3921, Durham, NC 27710 (e-mail: david.dalessio@duke.
edu).
17. Bailey SJ, Ravier MA, Rutter GA. Glucose-dependent regulation of gamma-aminobutyric acid (GABA A) receptor expression in mouse pancreatic islet alpha-cells. Diabetes 56: 320 –327, 2007.
18. Balks HJ, Holst JJ, Brabant G, Medizin ZI, Endokrinologie AK, Hannover MH. Rapid
oscillations in plasma glucagon-like peptide-1 (GLP-1) in humans?: cholinergic control
of GLP-1. J Clin Endocrinol Metab 82: 786 –790, 2014.
DISCLOSURES
D. A. Sandoval is on the scientific advisory board for Ethicon Endo Surgery and has research grants from Ethicon
Endo Surgery, Novo Nordisk, and Boehringer Ingelheim.
D. A. D’Alessio consults for Lilly, Merck, Novo Nordisk,
and Zealand and has research grants from MannKind Pharmaceuticals, Sanofi Aventis, and Ethicon Endo Surgery.
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