Mitochondria, Oxidative Stress,
and the Pathophysiology of Type 2
Diabetes
The Ascent of Man
( and Type 2 Diabetes )
Total Prevalence of Diabetes
in the United States(All Ages,
2005):
Total: 20.8 million people
(7 percent of the population)
Diagnosed: 14.6 million people
Undiagnosed: 6.2 million people
Incidence of Diabetes, United
States:
In 2005: 1.5 million new cases of diabetes
were diagnosed in people aged 20 years or
older
Cost of diabetes in the United
States:
Total (direct and indirect : $132 billion
Direct medical costs: $92 billion
Indirect
costs:
$40 billion
(from the National
Diabetes
Information
Clearing House)
(disability, work loss, premature mortality).
Sloth or Gluttony ?
Human Migration According to Mitochondrial DNA Diaspora
(Pure Motherly Lineages)
Mitochondrial Eve ?
Western Europe
Africa
South America
North America
(upside down)
You are here

India
China
Australia
Russia
Carb-free
Food Quest
Fuel gathering and
energy expenditure
MAP of human race migrations, according to the Mitochondrial DNA. The Numbers
represent thousands of years before present time (time units used in Radiocarbon
dating). The Blue line represents area covered in ice or tundra during the last great ice
age. The Letters represent mitochondrial DNA haplotypes (pure maternal lineages);
Haplogroups can be used to define genetic populations and are often geographically
oriented.
What ARE Mitochondria ?
EndoSymbiotic Bacteria
“The Little Friends Within”




Maternally Inherited Co-Incident DNA
Aerobic Cellular Respiration (90%)
Chloroplast’s Gas Exchanger (O2)(CO2)
Signaling System
The “ PowerHouses of the Cell ”
 Oxidative - Phosphorylative Coupling
 Endothermic Heat Source
 Generation of ATP
Mitochondrial Chemiosmosis
The BioEnergetics of ATP Generation
H+
H+
H+
ADP
H+
Cytosol
ATP
H+
+iP
H+
H+
Cytosol
Oxidative - Phosphorylation
H+
H+
H+
H+
H+
H+
H+
H+

 ATP
COUPLING: ATP synthesis (phosphorylation) must
be coupled to the generation of a H+ reservoir/pH
gradient (oxidation) between the mitochondrial
membranes.
Elemental
ATP
ADP +
iP
(phosphate)
Remember this
H+

ATP-Synthase
ATP-Synthase:
Hydrogen-Powered
H+
H+
H+ H+
What Causes Insulin
Resistance?
What Causes Pancreatic ßeta Cell
Impairment?
Insulin Resistance:
Skeletal Muscle
 Triglyceride Content of skeletal
muscle is directly proportional to
Insulin Resistance
 Increased Circulating Free Fatty
Acids (FFAs) result in suppression
 of Insulin-mediated Glucose Uptake
 Triglyceride accumulates either by
 1) Fat overload or
 2) Diminished ß-oxidation
Intramyocellular Lipid (IMCL) more closely correlates with insulin
resistance than BMI, waist-to-hip ratios, or total body fat(mcGarry)
Which Came First?
Insulin Resistance or Fatty Acid accumulation?
With hyperinsulinemic-euglycemic clamp,
Artificial elevation of plasma FreeFatty Acid
concentration in otherwise healthy humans leads to:
1)
22-61% increase in Intramyocellular Lipid content
(Brechtel et al. 2001)
2)
40-50% loss of either oxidative glucose disposal or
Muscle glycogen synthesis (Boden 1995)
With hyperinsulinemic-euglycemic clamp,
Glucose infusions in healthy, young, lean yet insulin resistant
Offspring of patients with type-2 diabetes:
1)
60% decrease in glucose uptake within skeletal muscle insulinresistant offspring of T2DM parents
2)
80% increase in the intramyocellular lipid content (IMCL)
3)
Co-incident 30% reduction in mitochondrial phosphorylation
(Petersen et.al. NEJM 2004)
Mitochondrial Dysfunction
Loss of Oxidative-Phosphorylation with
subsequent declines in ATP generation
Petersen et.al. PLoS 2005
MRS (magnetic resonance spectroscopy) assessment of ATPSynthase flux and intramyocellular inorganic phospate in
healthy,normoglycemic (i.e. not “diabetic”) lean Insulin-resistant
offspring of T2DM patients versus non-insulin resistant controls
Results:
1)
2)
3)
4)
5)
Insulin stimulated Glucose uptake declined 50% in IR group
2-Fold increase in IntraMyocellularLipid Content in IR Group
90% increrase in ATP synthesis rate in Control Group vs.
5% increase in mitochondrial ATP synthesis rate in IR Group
Insulin-induced phosphate increases correlated with insulinstimulated increases in ATP synthesis in both groups
Mechanism of InAction
Loss of Mitochondrial phosphorylation results in the accumulation of
triglycerides and other Fatty Acid metabolites, particularly DAGs, Fatty
AcylCoA, and ceramides that embed in the cellular membrane leading to a
co-incident loss of insulin sensitivity. Deep pathologic mechanism involves
the activation of Protein Kinaase C-Theta (PKC-) and the serine/threonine
phosphorylation of the Insulin-Receptor Substrate (IRS-1) inhibitting GLUT4
translocation resulting in decreased glucose removal from bloodstream
Morino et.al. 2006
Pancreatic ßeta Cell:
Glucose sensing, Mitochondria, and Insulin release
Beta-cell mitochondria serve as
fuel sensors that link
glucose exposure to
insulin release:
1)
GLUT2 transporters
introduce glucose to
mitochondria causing
increased ATP:ADP
ratio
2)
K+ gate closure-->
depolarization
3)
Voltage sensitive Ca++
channels open
4)
Exocytosis of insulin
storage granules to
bloodstream
[1]
GLUT2
[2]
K+
ATP:
ADP
Ca++
[4]
Insulin
[3]
ß-Cell Impairment:
Loss of glucose-stimulated insulin release
Superoxide radicals induce
UCP-2 upregulation
contributing to proton leak,
ATP:ADP, and loss of
glucose stimulated insulin
release: 1) Hyperglycemia/Fatty acid
excess causes chemiosmotic overload and
superoxide (O2-) generation
2)
3)
4)
ATP : ADP
(SuperOxide Radical)
O2- as oxidative SIGNAL,
upregulates UCP-2 with
decreased ATP production
Resulting in loss of insulin
secretory response
UCP-2 upregulation
decreases SO- generartion =
tight negative feedback
WAKE UP !!!
Uncoupling Protein 1 (UCP1)
• Produced in brown adipose tissue of newborn humans and hibernating
mammals. (we eventually lose our “baby fat” ) Unique to mammals.
• Diverts energy from ATP generation to Adaptive Thermogenesis (Heat
Release) Proton partioning
• Thermogenic uncoupling occurs in response to overeating and cold
• Decreases mitochondrial production of Oxidative damage
• Adaptive response to cold temperature mediated by norepinephrine
with secondary effect of regulating energy balance.
• Increased expression due to Fatty acid-induced Superoxide
generation from mitochondria. Inhibited by purine nucletide.
• NOT found in skeletal muscle.
• UCP1 expression in the smooth muscle cells can increase hypertension
and dietary athersclerosis w/o effecting cholesterol levels (was this the
problem with the GLITzares ask Dina)PPAR-gamma agonists
• UCP1 expression also causes INCREASED Superoxide expression
in the vasculature = oxidative stress-induced vascular damage.
Uncoupling Protein 2 (UCP2)
•
•
•
•
•
•
•
•
•
•
•
UCP1 homologue, but expressed in white adipose tissues of humans
during feeding, rather than brown adipose tissue of hibernating animals.
Widely expressed among mammals.
UCP2 ubiquity in human tissue including macrophage-rich tissues
Critical increase (with UPC3) in metabolic adaptation to fasting regardless
of weight (lean or obese)
Fetal hepatic hematopoeisis in Kupffer cells until birth due to UCP2
Possibly critical for macrophage differentiation
Essentially Non-Thermogenic un-coupling
Still induced by superoxide and “anti-oxidant” by negative feedback
UPC homologue (with UPC3) of the skeletal muscle
Inducible neuroprotective protein by cellular redox of apoptotic
suppression
Extensive expression in Spleen, Lung and macrophages suggests immunity
or inflammatory dampening of ROS defenses
Negative regulation of insulin secretion in Pancreatic Beta -cell
contributing significantly to Type 2 Diabetes induced by superoxide
generation
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Uncoupling Protein 3 (UCP3)
Found ONLY in skeletal muscle in humans (with UCP2)
More homologous to UCP2 than to UCP1, and un-affected by cold
And yet: possibly thermogenic in skeletal muscle based on function in yeast
cells
Critical increase (with UPC2) in metabolic adaptation to fasting regardless of
weight (lean or obese). Player in energy balance
Increased expression due to fatty acid-induced Superoxide generation
from mitochondria. Inhibited by purine nucleotide.
Not primarily conserved in humans for uncoupling purposes (?) and not a
major determinant of metabolic rate
Increased by fatty acid entry into mitochondria beyond its capacity for
beta-oxidation. May be translocating fatty acids out of the matrix
Possiblle role in “proton partitioning” (reproductive vs. repair ?)
UCP3 overexpression may lead to hyperphagia without co-incident weight
gain
Muscle UPC3 increased by thyroid hormone and fasting
Increased ROS generation associated with diminished UPC3 uncoupling
effects. Yet still not required for bodyweight regulation, exercise tolerance,
fatty-acid oxidation, or cold-induced thermogenisis
Important in MDMA-induced hyperthermia
PGC-1
“peroxisome proliferator-activated receptor-Gamma”
Co-activator One
•
Nuclear protein whose expression is induced by COLD (through
adrenergic agonism), hi-caloric diets, and refeeding following fasting
•
Induced within Brown Adipose Tissue and Skeletal muscle
CAUSES:
1)
2)
3)
4)
Activates PPAR-Gamma to “proliferate peroxisomes”
UCP-1 upregulation or Adaptive Thermogenesis
Upregulatiiion for Genes of Oxidative-Phosphorylation
Stimulates DOUBLING of Mitochondrial content through NRF-1 and
mtTFA
PGC-1 links the external environment (cold, diet,
oxygen, environmental stressors) with
mitochondrial biogenesis thereby constituting
the Adaptive Interface between outside and
inside.
(Wu Z, Puigserver P, et al. Cell 1999)
Peroxisomes ??
Reactive Oxygen Species (ROS)
Reactive Oxygen Species:
Reactive Nitrogen Species:
Radical: SuperOxide (O2-)
Hydroxyl (OH-)
Peroxyl (RO2)
Hydroperoxyl (H O2-)
Radical: Nitric Oxide (NO)
Nitrogen Dioxide (NO2-)
NoN-Radical:
Peroxynitrite (ONOO-)
NoN-Radical:
Hydrogen Peroxide (H2O2)
Ozone (O3)
SuperOxide  Nitric Oxide  Peroxynitrite
Anti-Oxidants
“Any substance that, when present at low concentrations compared to those
of an oxidisable substrate, significantly delays or inhibits oxidation of this
substrate” Halliwell Biochem Pharmacol 1995
Endogenous Anti-Oxidants
•
•
•
•
•
•
Glutathione
Catalase
SuperOxide Dismutase
CoEnzyme Q-10
Cytochrome C Peroxidase
Glutathione peroxidase
Dietary Anti-oxidants
•
•
•
•
Vitamins A, C, E
Alpha-Lipoic Acid
N-acetyl cysteine
Polyphenols
– Green Tea (camellia sinensis)
– Olive Oil (Olea europaea)
•
•
•
Proanthocyanidins:
– Red wine (Vitis vinifera)
– Blueberries (Vaccinium spp.)
– Chocolate (Theobroma Cacao)
Resveratrol (Japanese Knotweed )
Ginsengs (Panax quinguefolius,
Panax ginseng)
Mediterranean Miracle ?…
Olive Oil
( Olea europaea )
 Mono-Unsaturated Fat (oleic acid), +
antioxidant polyphenols
 THE Principle source of fat in Spain,
Greece, and Italy
 Olive oil can reduce the oxidative
effects of fried fats on phopholipid
membrane rancidity (C-C breakage)
 A Natural JUICE (while all other oils
from seed must be refined) Positive
effects on:
 Anti-oxidant Profile with Phenols,
Carotenes, Vit E
 Decreased Triacylglycerols
 Increased HDL
 Anti-Thrombotic, w/ decreased
platelet -aggregations
[International Consensus Report 2005]
4 Groups of Rats were fed a 40% fat diet (either beef
tallow, palm oil, olive oil, sunflower oil) to investigate
the effects of “non-shivering thermogenesis”
according to UPC mRNA expression:
RESULTS:
UCPs 1/2/3 were all upregulated in BAT (brown adipose
tissue) in the olive oil group
UCP3 in the Skeletal muscle was significantly more
upregulated in the Olive oil group
Saturated Fat was associated with increased food intake
No significant differences in Insulin, glucose, T3,
corticosterone, or Fatty acids were seen among the
groups. [ Rodriguez 2002 ]
PROANTHOCYANIDINS
In Vino Veritas
RED, RED WINE
•
In diabetics, Polyphenolics from Dealcoholized muscadine grape wine still
demonstrated:
–
 blood glucose
–
 insulin
–
 glycated hemoglobin
–  Vitamin C and E levels
–  monounsaturated fatty acid cell content
–  hepatic functioning (Banini,Nutrition 2006)
•
Red Wine Consumption (360 ml/day x 2
wks) resulted in 43% improvement in
glucose disposal compared to controls
(Napoli 2005)
•
Contains Resveratrol………..
Resveratrol
 Now considered to be a MINOR, not “major” ingredient of Red Wine
 Has recently been shown to Mimic the effects of Caloric Restriction:
 Energy shunt towards tissue repair (anti-oxidation) vs. reproduction
 30% fewer calories approximates a 30% increase in LifeSpan*
 2000-2100 calories per day but untested in Humans
 May be more beneficial than exercise
 SIRT1 genes co-ordinate the Repair Response: turning on Sirtuins
 SIRT genes 3, 4, 5 are in the mitochondria; 6 & 7 are in the nucleus
 SIRT 1 genes increase the # of mitochondria produced by neurons
 Sirtuins (their proteins) activate PGC-1
 PGC-1 may also generate extra chemicals to detoxify free radicals(st.pierre)
 Fasting induces deacetylation of PGC-1 BY SIRT1 Fatty Acid oxidation
 “It is a proven artifact that Resveratrol activates sirtuins” --Dr. DiStefano, Elixir
 “Resveratrol…. :
 Improves health and survival of mice on a high-calorie diet” (Baur etal, Nature 2006)
 Improves mitochondrial function and protects against metabolic disease by
activating SIRT1 and PGC-1” (Lagouge, Auwerx, et.al., Cell 2006)
 [as “Sirtuin Activators] mimic caloric restriction and delay aging in metazoans”
(Wood JG, Rogina B, Sinclair D, et.al. Nature 2004)
Japanese Knotweed
( Polygonum cuspidatum )
Knotweed as Invasive Weed (US)
(polygonum cuspidatum)
USDA Plant Database
“Food of the Gods”….
Chocolate !!
( Theobroma cacao )
DIABETES
 Significant reduction in Blood Glucose and
fructosamine levels following consumption (1%
dietary) of Cacoa Liquor Proanthcyanidin (CLPr)
equivalent to 2.5 Kg
[Makoto 2007]
 Flavonols in dark, not WHITE!, chocolate of
daily single bar 100 g (500 mg polyphenols) for
15 days increased insulin sensitivity (with coincident decrease in blood pressure) measured
by HOMA-IR
[Grassi 2005 Am J Clin Nutr.]
Cacoa or dark-chocolate has been
shown to reduce the
cardiovascular risks of
– Atherosclerosis
– Hypertension
– Platelet adhesion
– LDL Oxidation
 Dark Chocolate flavonols decreased Insulin
resistance, LDL cholesterol ; improved flowmediated dilation ; and ameliorated insulin
sensitivity in patients with essential hypertension
[Grassi 2005 Hypertension ]
Apple Pie Ingredient…
Cinnamon
Cinnamomum zeylanicum
 Cinnamon ( C. cassia ) at
daily intake of 1, 3, or 6 grams
for 40 day duration led to
decrease serum glucose
levels of 18-29%
 Not dose Dependent (all
doses were equipotent)
 Also, Decreased serum
triglyceride levels of 23-30%
 Phenolic acids in C.
zeylanicum provide even
higher TEAC* concentrations
[Khan et.al. 2003]
* Total Equivalent Anti-oxidant Concentrations
“A compound in cinnamon called
methylhydroxy chalcone polymer (MHCP)
makes fat cells more responsive to insulin
by activating an enzyme that causes insulin
to bind to cells and inhibiting the enzyme
that blocks this process”. -- Dr. Richard A
Anderson USDA [Jarvill-Taylor KJ, et.al. 2001]
Panax Quinquefolius
American Ginseng*
( Panax quinquefolius )
“KING ROOT”
( avg $ / Pound = $400 )
 Anti-oxidant Ginsenosides identical
to research conducted on the
Korean Cousin (Panax ginseng)
 Over 2000 published articles
 Ancient Chinese indication for
“quenching thirst”
 Rank order of ginsenoside content:
leaf > berry > root (sustainable)
 Numerous studies demonstrating
anti-hyperglycemic effects and
improved insulin resistance
 Suspected pancreatic ß-cell
sparing effect ( Xie, 2005)
* Endangered - Habitat destruction, , Housing development,
Mountain Top Removal
Symbiosis
Mitochondrion
Chloroplast
CO2
Mitochondria in the Modern Mythos
Midi-chlorians
(
Mitochondria & Chloroplasts )
The boy nodded his understanding.
"Can I ask you something?”
The Jedi Master nodded.
"What are midi-chlorians?"
Wind whipped at Qui-Gon's long hair, blowing strands of
it across his strong face.
"Midi-chlorians are microscopic life-forms that reside
within the cells of all living things and communicate
with the Force.”
“They live inside of me?" the boy asked.
"In your cells.…. We are symbionts with the midichlorians."
"Symbi-what?"
"Symbionts. Life-forms living together for mutual
advantage. Without the midi-chlorians, life could not
exist, and we would have no knowledge of the Force.
Our midi-chlorians continually speak to us, Annie,
telling us the will of the Force."
"They do?"
Qui-Gon cocked one eyebrow. "When you learn to quiet
your mind, you will hear them speaking to you."
Anakin thought about it for a moment, then frowned. "I
don't understand."
Qui-Gon smiled, and his eyes were warm and secretive.
"With time and training, Annie, you will."
---from “Star Wars Episode I: The Phantom Menace”
novelization by Terry Brooks
May the Force Be With
You
THE END
Remember the waterwheel
Blueberries
( Vaccinium myrtillus )
Adrenalin stress types, under sympathetic nervous system stress, will trigger
peripeheral coversions of Thyroxine to metabolically active T3 resulting not only in
an upregulation of a basal metabolic rate but the increased expression of of all three
uncoupling proteins (Ricquier Biochem Journal on UCPs) ( Masaki FEBS letter
1997)( Gong DW T3 B3 adrenergic agonists J Biol Chem 1997) SAVE THIS!
PPAR -gamma can be upregulated by oleic acid perhaps:this could likely lead to
upregulation of UCPs in the BAT (as thiazollidinediones upregulate UCP3 in adipose
tissue yet unfortunately down regulate UCP2 in skeletal muscle)