Supplementary Methods
Dose-response effects of SN 28127 and SN 29220 on C3H/HeN mouse body weight and
food and water intake
To determine whether the weight reduction effects of SN 28127 and SN 29220 were
dose-dependent, single injections of either SN 28127 or SN 29220 (0.47 mol/kg, 1.4
mol/kg, or 4.2 mol/kg) or 42% DMSO in water (vehicle control) were administered to
male C3H/HeN mice (~50 day old mice) that had been housed in groups of four per cage
(2 cages = 8 mice per group) since they were ~40 days old. The mice had unrestricted
access to normal chow and tap water. Mouse body weight and food and water intake
were measured 2-3 times per week for up to 60 days post injection. Food and water
intake were averaged per mouse for each cage. At ~60 days post injection, mice were
euthanized, weighed, and body length (nose-anus) and tail length measured. Blood and
organs were collected for further analyses. Plasma was prepared and frozen. Organs
were either fixed for histology or snap frozen for RNA preparation.
Characterization of SN 29220 effects on C3H/HeN mouse metabolic phenotype and gene
expression at 2, 10, 30, and 160 days post drug injection
To characterize the effects of SN 29220 (1.4 mol/kg) over a maximum of 160 days post
injection, male C3H/HeN mice (~40 days old) were housed in groups of four per cage (2
cages = 8 mice per group) and had unrestricted access to normal chow and tap water.
Mouse body weight and food and water intake were monitored 2-3 times per week for up
to 160 days. Food and water intake were averaged per mouse for each cage. At each time
point (2, 10, 30 and 160 days post injection), mouse body and tail length were measured
and blood and organs collected from eight mice per group for further analyses. Plasma
was prepared and frozen. Organs were either fixed for histology or snap frozen for RNA
preparation.
Effect of SN 29220 on high-fat diet induced obesity and high-fat diet induced type 2
diabetes in male C57B/6J mice
1
The overall aim of this experiment was to determine whether SN 29220 could reverse
high-fat diet-induced obesity and type 2 diabetes in male C57BL/6J mice. C57BL/6J
mice, but not C3H/HeN mice, are sensitive to high-fat diet induced obesity 1 and
therefore the C57BL/6J mouse strain was used for these experiments. Mice were
randomly selected to start either a normal chow or high-fat diet at weaning. Male
C57BL/6J mice were housed in groups of four per cage (2 cages = 8 mice per group) and
had unrestricted access to either a low-fat (10 kcal% fat, D12450Bi, Research Diets, Inc.,
New Brunswick, NJ) or high-fat (60 kcal% fat, D12492i, Research Diets, Inc.) diet from
day 21 (weaning) and tap water. At either 80 days (mild obesity model) or 120 days
(morbid obesity model) half of the mice on each diet (24 mice) were injected i.p. with
either vehicle or SN 29220 (0.42 mol/kg body weight or 1.4 mol/kg body weight).
Following a single injection, mouse body weight and food and water intake were
monitored three times per week over approximately 60 days. Food and water intake were
averaged per mouse for each cage.
A small number of male C57BL/6J mice used for assessing SN 29220 effects on high-fatdiet induced obesity were also tested for glucose and insulin tolerance, post 42% DMSO
or SN 29220 injection. On day 42 post injection, two mice on low-fat diet and treated
with DMSO, three mice on high-fat diet and treated with DMSO, and three mice on highfat diet and treated with 1.4 mol/kg SN 29220, were fasted overnight. The following
morning glucose tolerance tests (GTTs) were performed on these mice. A week later
insulin tolerance tests (ITTs) were performed on the same mice. After a further oneweek these mice underwent MRI to measure body composition.
At ~60 days post injection all mice on either the low-fat or high-fat diets were
euthanized, weighed, and body length (nose-anus) and tail length measured. Blood and
organs were collected for further analyses. Plasma was prepared and frozen. Organs
were either fixed for histology or snap frozen for RNA preparation.
Distribution of radiolabelled SN 29220 in male C3H/HeN mouse blood, urine, feces and
tissues
2
3
H-SN 29220 2 was injected i.p. into ~ 50 day old male C3H/HeN mice to investigate the
retention of drug in tissues. A pilot study was conducted in which four mice were
injected with 3H-SN 29220 (1.4 mol/kg) and one of these mice was culled at each of
four different time points (4h, 1 day, 3 days and 9 days) post-injection. For the 4h, 1 day
and 3 day time points, mice were housed individually in metabolic cages and urine and
feces collected. The urine volume and weight of feces were collected. The fourth mouse
was housed alone in a standard mouse cage.
To measure 3H levels in urine, 100 L urine samples were added to 10 mL EmulsifierSafe (Perkin Elmer Life and Analytical Sciences, Boston, MA), shaken vigorously and
counted in a Packard Tri-Carb 1500 liquid scintillation counter (PerkinElmer, Vic,
Australia). Feces samples were dried at 50ºC for 48h, weighed and crushed before
rehydrating in water (100 L/20 mg dry faeces) for 30 min at room temperature.
Rehydrated faeces were solubilized with 1mL Soluene 350 (NZ Scientific Ltd, Auckland,
NZ) for 12 h at 37 ºC followed by the addition of 0.5 mL isopropyl alcohol and left for a
further 12 h at 37 ºC. Before counting 3H in the scintillation counter, 200 L of 30%
hydrogen peroxide was added to each sample and the samples were left for 10 min at
room temperature followed by 15 min at 37 ºC. Blood was collected in 600 L heparin
microtainer tubes, spun at 4000 rpm at 4 ºC for 10 min, and plasma collected. Plasma
was prepared from blood samples and then 50 L plasma was added to 6 mL Optiphase
Supermix scintillation fluid (Perkin Elmer Life and Analytical Sciences), shaken and
counted in the Packard Tri-Carb 1500 liquid scintillation counter. Tissues collected from
each mouse for monitoring 3H levels were: brain, pituitary gland, thymus, heart, lungs,
brown fat, stomach, liver, gall bladder, duodenum, ileum, caecum, colon, retroperitoneal
fat, adrenal glands, kidneys, subcutaneous fat (flanking the belly), skeletal muscle,
gonadal fat, testes, and visceral fat. Tissues were weighed and then 100–150 mg of each
tissue was solubilized. To 100-150 mg samples of each tissue, 1.5 mL of Soluene 350
was added and the samples were left at 37 ºC overnight. Following this, the samples
were cooled to room temperature for 1 h. Samples that appeared as a pale yellow colour
had 10 mL Hionic-Fluor (Perkin Elmer Life and Analytical Sciences) added and then
they were then vortexed vigorously before being counted in the Packard Tri-Carb 1500
3
liquid scintillation counter. Samples that appeared as a red/orange colour were bleached
for 10 min at room temperature with 0.2–0.4 mL 30% hydrogen peroxide. They were
then left at 37 ºC for 15 min followed by 1 h at room temperature before 10 mL HionicFluor was added and samples counted in the scintillation counter.
Based on data obtained from the pilot study, a more extensive study was undertaken
using 4 male ~50 day old C3H/HeN mice for each time point. The mice were housed in
standard cages and had unrestricted access to routine chow and tap water. Mice were
injected with 3H-SN 29220 (1.4 mol/kg). At 2, 9, 20, 30 and 40 days post injection,
groups of 4 mice were euthanized and blood and tissues collected. Radioactivity in
plasma and tissues was measured as described for the pilot study.
Measurements for body size, body weight and food and water intake
Mice were identified by ear tagging under anaesthetic (3% halothane/O2) prior to
treatment. Each mouse was weighed carefully by removing it from its cage, placing it in
a cardboard box on top of a balance and then returning it to its cage. Food pellets and
water were pre-weighed for each mouse cage. Food and water intake data were derived
by subtracting weights of food pellets and water remaining for each cage at set time
points. Prior to weighing the food, partly digested food pellets were retrieved from inside
the cage and these were included in the food weight. Baseline body weight and food and
water intake were measured for 7-10 days prior to drug-treatment to determine that the
animals were eating and gaining weight normally. Body length (nose-anus) and tail
length (anus-tail tip) were measured using callipers to the nearest mm when mice were
euthanized.
Organ weights and blood chemistry
Mice were faster overnight (~16 h) and then euthanized with halothane anaesthesia.
Blood was collected from the retro-orbital sinus into EDTA-coated vacutainer blood
collection tubes (Becton Dickson Vacutainer Systems, New Jersey, USA). The blood
was kept on ice until the tubes were centrifuged at 4000 rpm for 10 min at 4 °C. An
aliquot of each plasma sample was sent to A+ Labs, Auckland City Hospital, for analysis
4
of glucose, sodium, potassium, chloride, urea, albumin, total protein, globulin, alkaline
phosphatase (ALP), aspartate aminotransferase (AST), alanine transaminase (ALT),
amylase, cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein
(LDL) cholesterol, and triacylglycerols. Following cervical dislocation, organs (brain,
pituitary gland, heart, liver, lungs, adrenal glands, spleen, kidneys, stomach,
retroperitoneal fat, visceral [omental] fat, gonadal fat, subcutaneous [flanking the
abdomen] fat, brown fat, ovaries, testes, and small intestine) were dissected and
individually weighed. Tissues were either fixed for immunohistochemistry or in situ
hybridization, or snap frozen for protein and gene expression studies.
Measurement of hormone levels
Plasma insulin levels were measured using a Mercodia Ultrasensitive mouse insulin
ELISA kit (Mercodia AR, Sweden). Plasma leptin levels were measured using a
Quantikine® mouse leptin ELISA kit (R & D Systems, Pharmaco Ltd, NZ).
Measurement of urinary glucose and fecal triacylglycerol and cholesterol levels
To determine whether SN 29220 treated mice lose energy through excreting it as either
glucose in the urine and/or as lipids in the feces, male C57BL/6J mice aged 50 days were
injected with either vehicle or SN 29220 (1.4 mol/kg). Body weights were measured
weekly and between 59 and 67 days post injections, the mice were housed individually in
metabolic cages with water ad lib but without food for 5 hours between 9am and 2pm.
Urine and feces were collected and weighed. Glucose was measured in undiluted urine
samples using a Roche Modular Platform P module and a Roche glucose enzymatic
colorimetric assay kit at the A+ Lab, Auckland City Hospital. The method used for
measurement of fecal lipids was adapted from Lee et al 3. Feces were diluted 1:15 (w/v)
in stool diluent (10mL/L Triton X-100, 6 mL/L Brij® 30 and 0.1 mL/L HCl in isotonic
saline, 150 mmol/L) and thoroughly mixed by pipetting and vortexing and then leaving
for 30 minutes at room temperature before centrifugation at 1050g for 15 minutes. The
supernatants were transferred to auto analyser cups and then samples were assayed using
a Roche Modular Platform P module and Roche triacylglyceride and cholesterol
enzymatic kits the A+ Lab, Auckland City Hospital. The method was validated by
5
spiking aliquots of mouse feces with 0.18 - 0.27 mol triacylglycerol and 0.46 - 0.69
mol cholesterol in the form of a Roche Diagnostics CFA calibrator (Roche Diagnostics,
Auckland, NZ). We demonstrated > 96% cholesterol recovery and 100% triacylglycerol
recovery.
Immunohistochemistry
Pancreas was fixed in Bouin’s fluid while all other tissues were fixed in 10% neutral
buffered formalin. Following tissue fixation and processing the tissues were wax
embedded. Tissue sections (5 m) were cut on a Leica CM1900 cryostat (Biostrategy,
Auckland, NZ), and attached to polysine-coated microscope slides (ThermoFisher,
Auckland, New Zealand). Sections were stained with Hematoxylin and Eosin (H & E).
In situ hybridization of neuropeptide gene expression in mouse brain
Brains were fixed in 4% paraformaldehyde at 4 ºC for 7 days. Sucrose (10% wt/vol) was
added 16 h before freezing the brains in OCT (Sakura, Torrance, CA) embedding
medium. Brains were stored at -80 ºC until they were cut on a cryostat. Five crosssectional series of sections (20 m) from each brain were cut on a Leica CM1900
cryostat and mounted onto polysine-coated slides. Sections were hybridized with 33Plabelled cRNA antisense mouse proopiomelanocortin (POMC), 33P-labelled cRNA
antisense mouse neuropeptide Y (NPY) or 33P-labelled cRNA antisense mouse agouti
gene related peptide (AGRP). The POMC and AGRP DNA templates for making
riboprobes were a generous gift from Professor J. K. Elmquist 4. The POMC construct
was originally developed in the laboratory of Professor R. A. Steiner 5. The mouse NPY
DNA template was constructed following reverse transcriptase (RT-PCR) on C57BL/6J
mouse hypothalamic total RNA using forward primer, 5’-gatgaattctctcacagaggcaccca-3’
and reverse primer, 5’-gaaacgtcgacaagtcgggagaacaa-3’. The forward and reverse primers
encode for ECoR1 and Sal1 restriction sites, respectively. The DNA fragment was
subcloned into pBKS and sequenced to confirm that it was NPY DNA. Adjacent brain
series from each mouse were hybridized with one of these three cRNA probes. Sections
were hybridized in 65% formamide in 0.26 M NaCl, 1.3 X Denhardt’s, 13 mM Tris HCl
(pH 8), 1.3 mM EDTA, 13% dextran sulfate at 60-65 ºC for 18 h. Sections were washed
6
and exposed to Hyperfilm MP (GE Healthcare Life Sciences, Auckland, NZ) alongside
14
C radioactive scale bars (GE Healthcare Life Sciences) for 2 days. Following the
development of the autoradiograds, the slides were coated in Hypercoat LM1
photographic emulsion (GE Healthcare Life Sciences) and kept in the dark at 4 °C for 7
days after which time the slides were developed and the sections were NissL stained.
The sections were then viewed under both light-field and dark-field on a Zeiss Axioskop
2 Plus microscope (Carl Zeiss NZ Ltd, Auckland, NZ). The films were scanned using a
Canon Scan 5000F (Canon NZ Ltd, Auckland, NZ) and ImageJ
(http://imagej.nih/ij/features.html) was used to calculate volumes of the scanned signals.
A calibration curve was plotted of volume versus kBq/g for the 14C radioactive scale bar
on each film.
Real time PCR
Aurum™ total RNA mini kit (BioRad, Hercules, CA, USA) was used to extract RNA
from liver and Aurum™ total RNA fatty and fibrous tissue mini kit (BioRad) was used to
extract RNA from retroperitoneal fat and skeletal muscle. All extracted RNA was
quantitated using an Eppendorf BioPhotometer (Global Science Ltd, Auckland, NZ),
visually checked for intact RNA on a 1.2% agarose gel and finally checked for genomic
DNA contamination following PCR of the intron-less melanocortin 4 receptor. A twostep Real-Time PCR was performed using TaqMan® gene expression assays (Applied
Biosystems, Foster City, CA). An ABI PRISM® 7900HT fast real-time PCR system
(Applied Biosystems) was used to detect and measure the accumulation of fluorescent
emissions. Primers and probe sets for target genes (Peroxisome proliferative activated
receptor coactivator  [PPAR, Fatty acid synthase [FASn], Stearoyl coenzyme A
desaturase 1 [SCD1], Uncoupling protein 1 [UCP1], Uncoupling protein 2 [UCP2],
Uncoupling protein 3 [UCP3], Forkhead box protein 01 [FOXO1], Carnitine
palmitoyltransferase 1 [CPT1], Carnitine palmitoyl transferase 2 [CPT2],
Diacylglycerol-acyltransferase 1 [DGAT1], Peroxisome proliferative activated receptor
coactivator  [PPAR, Acyl-coenzyme A dehydrogenase 1 [ACAD1], Acetyl-coenzyme
A acyltransferase 2 [ACAA2], Lipoprotein lipase [LPl]) and house keeping genes
(HydroxyMethylBilane Synthase [HMBS], Hypoxanthine Guanine Phosphoribosyl
7
Transferase [HPRT], Actin- [ActB], TATA box Binding Protein [TBP], and
Glyceraldehyde 3-Phosphate Dehydrogenase [GAPDH]) were purchased as single tube
Assays-on-Demand™ containing forward and reverse unlabelled PCR primers and a
fluorescent reporter FAM-labelled TaqMan® MGB probe. The Applied Biosystems
amplification efficiencies reported for these assays were 100 ± 10%. The fractional cycle
number at which the fluorescence passes the threshold (CT-value) was used to calculate
the input amount of target gene and reference gene. The stability of five housekeeping
genes for each tissue was investigated via geNorm v3.5 software
(http://allserv.ugent.be/~jvdesomp/genorm/index.html) and only the stable housekeeping
genes were used for normalization of expression for the genes of interest.
Glucose and insulin tolerance tests
Mice were fasted for 16 h prior to undergoing GTTs or ITTs. Blood glucose was
monitored using a glucometer (ACCU-CHEK Advantage, Roche Diagnostics NZ Ltd,
Auckland, NZ) on a drop of blood collected from the tail tip of each mouse. For the
GTTs, each mouse received a single i.p. injection of 20% D-glucose (Health Support Ltd,
Auckland, NZ) (2 g/kg body weight) immediately after the basal glucose measurement.
At 30 min intervals over 180 min following the glucose injection, blood glucose was
monitored on a drop of blood collected from the tail tip of each mouse. For the ITTs,
each mouse received a single i.p. injection of human insulin 0.25 U/kg body weight)
(Roche Diagnostics NZ Ltd) immediately after the basal glucose measurement. At 30
min intervals over 180 min following the glucose injection, blood glucose was monitored
on a drop of blood collected from the tail tip of each mouse.
Magnetic resonance imaging of mouse body composition
Magnetic resonance imaging (MRI) was used to compare body fat composition between
C57BL/J male mice on a high-fat diet (60%kcal) for 100 days from weaning and then
treated with a single injection of either 42% DMSO (control mice) or SN29220 (1.4
mol/kg). The MRI was performed ~55 days post-treatment on anaesthetized (ketamine
and 2% xylazine) mice using a Varian 4.7T system interfaced with a Unity Inova
spectrometer (Varian Inc., Palo Alto, USA). Images were acquired using a birdcage-
8
design radio-frequency coil with inner diameter of 72 mm (m2m Imaging, New Jersey,
USA). A gradient-echo pulse sequence (TR = 610 ms, TE = 3.8 ms, flip angle = 70 º,
matrix = 256 x 128) was used to acquire a saturation pulse (flip angle = 90º, duration =
15 ms) followed by a gradient-echo host sequence identical to that used for the PDweighted images. Both PD and fat saturated images were obtained for a series of
contiguous 2 mm thick coronal slices for each mouse. The number of slices varied with
each animal size and was chosen so that the entire volume of the animal was covered.
Body fat composition was quantified by processing of the MR data using ImageJ.
Supplementary Results
SN 28127 significantly reduced heart and stomach mass for C3H/HeN, a/a, AVY/a and
NZG/Kgm mice and reduced liver, kidney, spleen, pancreas, brain, lung and small
intestine mass in only some of these strains
At 160 days post-injection, the weights of heart and stomach were significantly reduced
in SN 28127-treated C3H/HeN, a/a, AVY/a and NZG/Kgm mice compared with the
DMSO-treated mice (Figure S4). Compared to DMSO-treated mice, SN 28127
treatment significantly reduced liver weights in C3H/HeN, AVY/a and NZG/Kgm mice,
significantly reduced kidney weights in C3H/HeN, a/a and AVY/a mice, significantly
reduced spleen weights in C3H/HeN and AVY/a mice and significantly reduced pancreas
weights in AVY/a and NZG/Kgm mice (Figure S4). SN 28127 treatment significantly
reduced brain weights for C3H/HeN and a/a mice, lung weight for NZG/Kgm mice and
small intestine weight for AVY/a mice, compared to DMSO-treated control mice (Figure
S5). SN 28127 treatment had no significant effects on testes or adrenal gland weights for
all four mice strains (Figure S5).
SN 28127 significantly increased blood ALP levels of CH/3HeN, a/a, AVY/a and
NZG/Kgm mice, significantly reduced blood cholesterol and HDL-cholesterol for AVY/a
mice only, and significantly reduced blood triacylglycerol levels for C3H/HeN and AVY/a
mice
At 160 days post SN 28127 injection, blood ALP levels were significantly increased in
C3H/HeN, a/a, AVY/a and NZG/Kgm mice compared with the DMSO-treated control
9
mice (Figure S4). SN 28127 treatment significantly reduced cholesterol and HDLcholesterol levels for AVY/a mice compared to DMSO-treated controls but had no effect
on cholesterol or HDL-cholesterol levels for the other mice strains (Figure S4 and
Figure S6). Blood triacylglycerols were significantly reduced following SN 28127
treatment compared to DMSO treatment, for C3H/HeN and AVY/a mice only, although
there was a trend for triacylglycerols to be reduced in a/a and NZG/Kgm mice (Figure
S4).
SN 28127 did not affect blood sodium, potassium, total protein, ALT or AST levels in any
of the four mouse strains. SN 28127 significantly increased blood albumin levels of
C3H/HeN mice, significantly increased blood urea levels of a/a mice, and significantly
reduced blood globulin levels of C3H/HeN and a/a mice
At 160 days post injection, blood sodium, potassium, total protein, ALT or AST levels
were no different between SN 28127 and DMSO-treated mice (Figure S6). Compared
with DMSO treatment, SN 28127 significantly increased blood albumin in C3H/HeN
mice only and significantly increased blood urea in a/a mice only (Figure S6). Blood
globulin levels were significantly reduced only in SN 28127 treated C3H/HeN and a/a
mice, compared with DMSO-treated control mice (Figure S6).
Effect of SN 28127 treatment on liver gene expression for C3H/HeN, a/a, AVY/a and
NZG/Kgm mice
At 160 days post injection, significant increases in CPT1, DGAT1 and
PPAR
for SN28127 treatment compared to DMSO-treated controls,
cted in livers from C3H/HeN mice but not in livers from a/a, AVY/a or
NZG/Kgm mice (Figure S7). In contrast to the SN 28127-induced increase in PPAR
mRNA in C3H/HeN mice, SN 28127 treatment significantly reduced PPAR mRNA in
the livers of AVY/a mice. SN 28127 treatment had no significant effects on PGC1,
DIO2, FASn, PDK4, SCD1 or FOXO1 liver gene expression in C3H/HeN mice (Figure
S7).
10
Effect of SN 28127 treatment on retroperitoneal fat gene expression for C3H/HeN, a/a,
AVY/a and NZG/Kgm mice
At 160 days post SN 28127 injection compared with DMSO injection, mRNA for the fat
cell differentiation gene, PPAR, and fatty acid synthesis genes, FASn and SCD1, were
significantly increased in retroperitoneal fat removed from C3H/HeN and AVY/a mice
(Figure S8). There was also a trend for PPAR, FASn and SCD1 to be increased at 160
days following the SN 28127 injection of a/a and NZG/Kgm mice but only SCD1 mRNA
was significantly increased by SN 28127 compared with DMSO controls in NZG/Kgm
mice. The mRNA for a gene involved in heat production, DGAT1, was significantly
increased in retroperitoneal fat of SN 28127-treated C3H/HeN and AVY/a mice compared
to DMSO-treated control mice (Figure S8). There was a similar trend, although not
significant, for an increase in DGAT1 mRNA expression in SN 28127-treated a/a and
NZG/Kgm mice compared with their DMSO-treated controls. SN 28127 treatment
appeared to cause an increase in UCP3 mRNA expression in retroperitoneal fat from all
four mice strains but the increase only reached significance for AVY/a mice. In contrast
to UCP3, UCP2 mRNA showed reduced expression in all four mice strains treated with
SN 28127 compared to mice treated with DMSO. However, UCP2 mRNA was only
significantly reduced in retroperitoneal fat of SN 28127-treated NZG/Kgm mice.
FOXO1 mRNA was significantly increased in SN 28127-treated C3H/HeN mice
compared to DMSO-treated control mice. There were no significant changes in UCP1,
PPAR, PGC1 or DIO2 gene expression in retroperitoneal fat of SN 28127 compared
with DMSO-treated C3H/HeN mice. There was a trend for CPT1 mRNA to decrease
with SN 28127 treatment in a/a, AVY/a and NZG/Kgm mice compared with DMSOtreated control mice but this trend did not reach significance (Figure S8).
No significant effect of SN 28127 treatment on skeletal muscle gene expression for
C3H/HeN mice
At 160 days post SN 28127 injection, there were no significant differences in C3H/HeN
mice skeletal muscle gene expression for UCP1, UCP2, UCP3, PDK4, CPT1 and PGC1
compared to expression of these genes in the DMSO control mice (Figure S9).
11
Effect of SN 28127 treatment on hypothalamic neuropeptide gene expression for
C3H/HeN mice at 160 days post drug injection
In situ hybridization experiments on mouse brain coronal sections showed that AGRP
mRNA expression was significantly up-regulated (p = 0.0117), POMC mRNA was
significantly down-regulated (p = 0.0159), and NPY mRNA expression was unchanged
(p = 0.1425) in the hypothalamic arcuate nucleus of C3H/HeN mice at 160 days post
SN28127 injection compared with control mice injected with DMSO (Figure S10).
Characterization of the effects of SN 29220 treatment on C3H/HeN mouse metabolic
phenotype at 2, 10, 30, 50 and 160 days post drug injection
The earliest changes observed were decreased plasma urea and increased plasma HDLcholesterol at 2 days post SN 29220 (1.4 mol/kg) injection, compared to DMSO-treated
control male C3H/HeN mice (Table S1). The decreased plasma urea was not sustained.
At 10 and 22 days post SN 29220 injection, plasma urea levels were not significantly
different from those exhibited by DMSO-treated control mice (Figures S11 and S12,
Table S1). By 30 days post SN 29220 injection, plasma urea levels were again
significantly decreased in SN 29220-treated mice compared to DMSO-treated controls
and the urea levels remained decreased for up to 160 days. The increased plasma HDLcholesterol was also not sustained. HDL-cholesterol levels were no different between SN
29220 and DMSO-treated mice 10 days post injection, while at 22 and 30 days post
injection, SN 29220-treated mice had significantly decreased HDL-cholesterol levels
compared with DMSO-treated control mice (Figure S13, Table S1). However, at 50 and
160 days post injection, SN 29220 treatment once again increased HDL-cholesterol levels
compared to DMSO-treated control mice although there was only a significant difference
observed at 160 days (Figures S13and S14, Table S1).
At 10 days post SN 29220 injection, retroperitoneal fat mass and spleen mass were
significantly decreased compared to measurements made on DMSO-treated control mice.
These differences were sustained through 22, 30, 50 and 160 days post treatment,
although the reduction in retroperitoneal fat mass at 50 days was not a significant
difference (Figures S13-S15, Table S1). Non-fasting plasma insulin levels were
significantly decreased at 10 days and 160 days post SN29220 treatment compared to
12
DMSO-treated mice, while no significant differences for non-fasting plasma insulin
levels were observed between SN 29220 and DMSO-treated mice at 22, 30 and 50 days
post injection (Figures S18 and S19).
SN 29220 treatment significantly decreased the following measurements compared to
DMSO-treated mice from day 22 through day 160 post injection: liver mass, kidney
mass, gonadal fat mass, plasma globulin, plasma amylase, and plasma triacylglycerols
(Figures S11-S15, Table S1). However, kidney mass and gonadal fat mass differences
were not significant at 50 days post drug injection (Figures S13 and S15, Table S1).
Visceral fat mass was significantly decreased by SN 29220 treatment at days 30, 50 and
160 post injection compared to DMSO-treated control mice (Figures S16 and S17,
Table S1). No measurements were obtained for visceral fat mass at 2, 10 and 22 days
post injection. Plasma potassium levels were significantly decreased by SN 29220 at
days 22 and 30 post injection compared to DMSO-treated control mice, but they were not
different to those levels measured for DMSO-treated mice at 50 and 160 days post
injection (Figures S11 and S12, Table S1). Plasma glucose and cholesterol levels were
significantly decreased by SN 29220 at days 22, 30 and 50 post injection compared to
DMSO-treated mice, but they were increased compared to plasma glucose and
cholesterol measurements for DMSO-treated mice at 160 days post injection (Figures
S13 and S14, Table S1).
SN 29220 treatment significantly increased plasma sodium and plasma chloride
measurements compared to DMSO-treated mice at day 22 through day 50 post injection
but not at 160 days post injection (Figures S11 and S12, Table S1). Plasma AST and
plasma ALT levels were both significantly increased by SN 29220 treatment compared to
DMSO-treated mice, at days 22 and 30 post injection only (Figures S11 and S12, Table
S1). SN 29220 treatment significantly increased plasma ALP compared to DMSOtreated mice at 30, 50 and 160 days post injection (Figures S11 and S16, Table S1).
Characterization of the effect of SN 29220 on C3H/HeN mouse retroperitoneal fat, liver
and skeletal muscle gene expression at 10 days post drug injection
13
Real time PCR was used to show that CPT1 mRNA expression was significantly reduced
in C3H/HeN mouse retroperitoneal fat 10 days after an i.p. injection of SN 29220 (1.4
mol/kg) compared to DMSO-treated mice (Figure S20). No significant differences
were found for the following twelve genes studies in retroperitoneal fat: PPAR, PPAR,
FASn, CPT2, DGAT1, SCD1, FOXO1, ACAD1, ACAA2, LPl, UCP2 and UCP3 (Figure
S20). Expression of a similar panel of genes was compared between SN 29220 and
DMSO-treated C3H/HeN mice 10 days post injection in liver and skeletal muscle and no
significant differences were found in either tissue (Figures S21 and S22).
Characterization of the effect of SN 29220 on C3H/HeN mouse hypothalamic
neuropeptide gene expression at 10 days post drug injection
NPY mRNA expression was significantly increased (p = 0.045) as detected in the
hypothalamus using in situ hybridization on C3H/HeN mouse brain coronal sections at 10
days post SN 29220 injection compared to DMSO-treated mice (Figure S23). No
significant differences were observed for AGRP and POMC mRNA expression in the
C3H/HeN mouse hypothalamus at 10 days post SN 29220 treatment compared to DMSOtreated mice (Figure S23).
SN 29220 treatment reversed high-fat diet induced obesity in male C57BL/6J mice
Despite marked differences in body weights for mice treated with DMSO and fed either a
low-fat or high-fat diet, their food intake was similar (Figure S24). SN 29220 (0.47
mol/kg or 1.4 mol/kg) treatment had little effect on food intake when given to mildly
obese mice, but food intake was reduced by 15 days post drug injection, when either dose
of SN 29220 was given to morbidly obese mice. Water intake for mice treated with
DMSO and fed a low-fat or high-fat diet was similar in the mice treated at ~80 days of
age (Figure S25A). However, C57BL/6J mice aged 120 days or drank less water on a
high-fat diet compared to age-matched mice on the low-fat diet (Figure S25B). SN
29220 treatment of either mildly or morbidly obese mice induced a dose-dependent
increase in water intake starting at ~15 days post drug injection and lasting for at least 45
days. The increase in water intake was transient for the mild obese mice and by the time
they were euthanized at 60 days post injection, their water intake was approaching that of
14
DMSO-treated mice. However, the morbidly obese mice were euthanized at 48 days post
injection and their water intake was still elevated at this time.
Magnetic resonance imaging of mouse body composition and fat cell histology: SN
29220 treatment reversed high-fat diet induced obesity in male C57BL/6J mice
SN 29220 (1.4 mol/kg) reduced fat mass of morbidly obese C57BL/6J mice by ~75% at
42 days post drug treatment as shown by MRI (Figure S26). Figure S26A shows a
representative control mouse fed a high-fat diet from weaning and injected with DMSO
when the mouse was aged 120 days. MRI was performed on the mouse 42 days after the
injection. The mouse weighed 50.6 g and analysis of the MRI images determined that this
mouse had a 54% fat ratio (fat mass/ lean mass x 100). Figure S26B shows a
representative SN 29220 (1.4 mol/kg)-treated mouse fed a high-fat diet from weaning
and injected with drug when the mouse was aged 120 days. MRI was performed on the
mouse 42 days after the injection. The mouse weighed 25.5g and analysis of the MRI
images determined that this mouse had a 12% fat ratio.
SN 29220 (1.4 mol/kg) treatment reduced fat cell size of retroperitoneal, visceral and
gonadal fat pad tissues, as demonstrated using H & E staining of fat pads removed from
C57BL/6J mice fed a high-fat diet from weaning, and then injected at 80 days of age with
either DMSO or SN 29220 (Figure S27). The histological staining was performed on
tissues removed ~ 60 days post drug or DMSO injection.
No abnormalities were detected in spleen cell and tissue morphology
We did not investigate the eosinophil status of the mice in our study, but no clear
differences in spleen cell and tissue morphology were observed following preliminary
analysis of H&E-stained sections obtained from C3H/HeN mice 10 days after injections
of either DMSO or SN 29220 (1.4 mol/kg) (data not shown). Similarly no clear
differences were observed in Periodic acid-Schiff (PAS)-stained spleen sections obtained
from C3H/HeN mice at 2 and 20 days following injections of either DMSO or SN 29220.
However, we would not expect either of these stains to detect differences in eosinophil
status. Blood cell counts were performed on blood collected from just two DMSO and
15
two SN 29220 (1.4 mol/kg)-treated C3H/HeN mice at 10 days after treatment. White
cell, segmented neutrophil and lymphocyte counts appeared to be reduced by the drug,
while red cell count, hemoglobin and hematocrit were not affected (data not shown). It is
therefore unclear what is causing the decreased spleen mass, and what if any, role the
spleen plays in the drug-induced adipose tissue mass loss. There was nothing to indicate
a consistent increase in apoptosis in the white pulp of the drug-treated mice. However, it
is possible that a wave of apoptosis may have occurred at a very early time point
following administration of the drug.
Supplementary Figure Legends
Figure S1
Structures of SN 28127 and SN 29220.
Figure S2
SN 28127 reduced body weights of a/a, AVY/a and NZG/Kgm male mice, with
variable transient increases in food and water intake. Animals were housed
individually and at 50-70 days of age were injected i.p. with either 42% aqueous DMSO
(data shown in black) or SN 28127 (4.2 mol/kg) (data shown in red). Day 0 on the x
axis represents the day of injection. Body weight and food and water intake were
measured up to ~160 days post-injection and for body weight are shown as a percentage
of the body weight on the day of injection. Food and water intake are expressed as
weight per mouse per day. Data are shown as mean ± SEM; a/a (n = 6), AVY/a (n = 5-6)
and NZG/Kgm (n = 4-6).
Figure S3
SN 28127 effects on body weight, body size, plasma glucose, plasma insulin, plasma
leptin, and fat cell mass of a/a, AVY/a and NZG/Kgm male mice. Animals were
housed individually and at 50-70 days of age they were injected i.p. with either 42%
aqueous DMSO or SN 28127 (4.2 mol/kg). All data was obtained at ~160 days postinjection and are shown as mean ± SEM; a/a (n = 6), AVY/a (DMSO n = 6, SN 28127 n =
16
5) and NZG/Kgm (DMSO n = 6, SN 28127 n = 4). Mann Whitney U non-parametric
test; *, p < 0.05; **, p <0.01
Figure 4
SN 28127 effects on organ weights, plasma ALP, plasma cholesterol and plasma
triacylglycerols of C3H/HeN, a/a, AVY/a and NZG/Kgm male mice. SN 28127
significantly reduced heart and stomach mass of C3H/HeN, a/a, AVY/a and NZG/Kgm
male mice but only significantly reduced liver, kidney and pancreas mass of some of
these strains of mice. SN 28127 significantly increased plasma ALP of C3HeN, a/a,
AVY/a and NZG/Kgm male mice, significantly reduced plasma triacylglycerols of
C3H/HeN and AVY/a mice, but only significantly reduced cholesterol of AVY/a mice.
Animals were housed individually and at 50-70 days of age they were injected i.p. with
either 42% aqueous DMSO or SN 28127 (4.2 mol/kg). Plasma was prepared from
blood (mice fasted for 4 h prior to bleed) collected just prior to when mice were
euthanized at ~160 days post injection and tissues were dissected and weighed. Data are
shown as mean ± SEM; C3H/HeN (n = 5), a/a (n = 6), AVY/a (DMSO n = 6, SN 28127 n
= 5) and NZG/Kgm (DMSO n = 6, SN 28127 n = 4). Mann Whitney U non-parametric
test; *, p <0.05; **, p < 0.01
Figure S5
SN 28127 treatment did not affect testes or adrenal gland weights in any of the
mouse strains but significantly reduced brain weights for C3H/HeN and a/a strains,
reduced lung weight for NZG/Kgm strain and reduced small intestine weight for the
AVY/a strain. Animals were housed individually and at 50-70 days of age they were
injected ip with either 42% aqueous DMSO or SN 28127 (4.2 mol/kg). Tissues were
collected when mice were euthanized at ~160 days post injection. Data are shown as
mean ± SEM; C3HeN (n = 5), a/a (n = 6), AVY/a (DMSO n = 6, SN 28127 n = 5) and
NZG/Kgm (DMSO n = 6, SN 28127 n = 4). Mann Whitney U test; *, p < 0.05; **, p <
0.01
17
Figure S6
SN 28127 significantly increased plasma albumin and plasma urea of C3H/HeN and
a/a male mice respectively, and significantly reduced plasma globulin of C3H/HeN
and a/a mice and significantly reduced HDL-cholesterol of AVY/a mice. SN 28127
did not affect plasma sodium, potassium, total protein, ALT or AST in any mouse
strain. Animals were housed individually and at 50-70 days of age they were injected
i.p. with either 42% aqueous DMSO or SN 28127 (4.2 mol/kg). Plasma was prepared
from fasted blood (overnight fast) collected before mice were euthanized at ~160 days
post injection. Data are shown as mean ± SEM; C3H/HeN (n = 5), a/a (n = 6), AVY/a
(DMSO n = 6, SN 28127 n = 5) and NZG/Kgm (DMSO n = 6, SN 28127 n = 4). Mann
Whitney U test; *, p < 0.05; **, p < 0.01
Figure S7
SN 28127-induced alterations in liver gene expression detected using real time PCR
was mouse-strain dependent. Animals were housed individually and at 50-70 days of
age they were injected i.p. with either 42% aqueous DMSO or SN 28127 (4.2 mol/kg).
Tissues were collected and snap frozen after mice were euthanized at ~160 days post
injection. Real time PCR data was normalized to HMBS, Actin and TBP housekeeping
genes. Data are shown as mean ± SEM; C3HeN (n = 5), a/a (n = 6), AVY/a (DMSO n =
6, SN 28127 n = 5) and NZG/Kgm (DMSO n = 6, SN 28127 n = 4). *, p < 0.05; **, p <
0.01; ***, p < 0.001
Figure S8
SN 28127 induced alterations in retroperitoneal fat gene expression detected using
real time PCR was mouse-strain dependent. Animals were housed individually and at
50-70 days of age they were injected i.p. with either 42% aqueous DMSO or SN 28127
(4.2 mol/kg). Tissues were collected and snap frozen after mice were euthanized at
~160 days post injection. Real time PCR data was normalized to HMBS, Actin, TBP and
HPRT housekeeping genes. Data are shown as mean ± SEM; C3HeN (n = 5), a/a (n = 6),
AVY/a (DMSO n = 6, SN 28127 n = 5) and NZG/Kgm (DMSO n = 6, SN 28127 n = 4). *,
p<0.05; **, p < 0.01; ***, p < 0.001
18
Figure S9
SN 28127 did not induce any significant alterations in C3H/HeN skeletal muscle
gene expression detected using real time PCR. Animals were housed individually and
at 50-70 days of age they were injected i.p. either with 42% aqueous DMSO or with SN
28127 (4.2 mol/kg). Tissues were collected and snap after mice were euthanized at
~160 days post injection. Real time PCR data was normalized to HMBS, Actin and TBP
housekeeping genes. Data are shown as mean ± SEM; n = 5.
Figure S10
SN 28127 induced significant upregulation of AGRP mRNA and significantly
induced downregulation of POMC mRNA in C3H/HeN mouse hypothalamus, but
did not significantly alter NPY mRNA expression. Animals were housed individually
and at 50-70 days of age they were injected i.p. with either 42% aqueous DMSO or SN
28127 (4.2 mol/kg). Brains were collected after mice were euthanized at ~160 days
post injection and were fixed in 4% paraformaldehyde. In situ hybridization was
performed on 25 
thick coronal sections using mouse gene-specific anti-sense
riboprobes. Two representative autoradiograms from a total of five DMSO-treated and
five SN 28127-treated mice, that were exposed to film for 1 day (NPY) or 2 days (AGRP
and POMC), are shown for each of AGRP, NPY and POMC genes (all sections with
signal from all mice studied are shown in Figure S4). The highest signals for AGRP and
NPY were outside of the linear range and therefore the volume of signal could not be
accurately calculated for these two genes. Data are shown as mean ± SEM; Mann
Whitney U test; *, p = 0.0117 (AGRP) and *, p = 0.0159 (POMC)
Figure S11
Time course of SN 29220-induced changes in plasma sodium, potassium, chloride,
albumin, urea, globulin, ALT, AST, and ALP levels in male C3H/HeN mice. Animals
were housed in groups of 4 and at ~50 days of age they were injected i.p. with either 42%
aqueous DMSO or SN 29220 (1.4 mol/kg). Plasma was prepared from blood (mice
fasted overnight) collected just prior to when mice were euthanized at the times post
19
injection, indicated on the x axis. Data are shown as mean ± SEM; n = 8. Mann Whitney
U test; *, p < 0.05; **, p < 0.01; ***, p <0.001
Figure S12
Comparison of SN 29220-induced changes in plasma sodium, potassium, chloride,
albumin, urea, globulin, ALT and AST levels in male C3H/HeN mice at 30 days and
161 days post-drug injection. Animals were housed in groups of 4 and at ~50 days of
age they were injected i.p. with either 42% aqueous DMSO or SN 29220 (1.4 mol/kg).
Plasma was prepared from blood (mice fasted overnight) collected just prior to when
mice were euthanized at the times post injection, indicated on the x axis. Data are shown
as mean ± SEM; n = 8. Mann Whitney U test; *, p < 0.05; **, p < 0.01; ***, p <0.001
Figure S13
Time course of SN 29220-induced changes in kidney and spleen weights, and in
plasma glucose, amylase, cholesterol, HDL cholesterol, and triacylglycerol levels in
male C3H/HeN mice. Animals were housed in groups of 4 and at ~50 days of age they
were injected i.p. with either 42% aqueous DMSO or SN 29220 (1.4 mol/kg). Plasma
was prepared from blood (mice fasted overnight) collected just prior to when mice were
euthanized and tissues were collected at the times post injection, indicated on the x axis.
Data are shown as mean ± SEM; n = 8. Mann Whitney U test; *, p < 0.05; **, p < 0.01;
***, p < 0.001
Figure S14
Comparison of SN 29220-induced changes in kidney and spleen weights, and in
plasma glucose, amylase, cholesterol, HDL cholesterol, and triacylglycerol levels in
male C3H/HeN mice at 30 days and 161 days post-drug injection. Animals were
housed in groups of 4 and at ~50 days of age they were injected ip with either 42%
aqueous DMSO or SN 29220 (1.4 mol/kg). Plasma was prepared from blood (mice
fasted overnight) collected just prior to when mice were euthanized and tissues were
collected at the times post-injection indicated on the x axis. Data are shown as mean ±
SEM; n = 8. Mann Whitney U test; *, p < 0.05; **, p < 0.01; ***, p < 0.001
20
Figure S15
Comparison of SN 29220-induced changes in body weight, body length, tail length,
liver mass, gonadal fat mass and retroperitoneal fat mass in male C3H/HeN mice at
30 days and 161 days post drug injection. Animals were housed in groups of 4 and at
~50 days of age they were injected ip with either 42% aqueous DMSO or SN 29220 (1.4
mol/kg). Body weight, body length, and tail length were measured when mice were
euthanized and tissues were collected at the times post injection, indicated on the x axis.
Data are shown as mean ± SEM; n = 8. Mann Whitney U test; *, p<0.05; **, p<0.01;
***, p<0.001
Figure S16
Comparison of SN 29220-induced changes in plasma total protein, plasma ALP
levels, and visceral fat mass in male C3H/HeN mice at 30 days and 161 days post
drug injection. Animals were housed in groups of 4 and at ~50 days of age they were
injected i.p. with either 42% aqueous DMSO or SN 29220 (1.4 mol/kg). Plasma was
prepared from blood (mice fasted overnight) collected just prior to when mice were xX
axis. Data are shown as mean ± SEM; n = 8. Mann Whitney U test; *, p < 0.01; **, p <
0.01
Figure S17
Time course of SN 29220-induced changes in plasma total protein and visceral fat
mass in male C3H/HeN mice. Animals were housed in groups of 4 and at ~50 days of
age they were injected i.p. with either 42% aqueous DMSO or SN 29220 (1.4 mol/kg).
Plasma was prepared from blood (mice fasted overnight) collected just prior to when
mice were euthanized and tissues were collected at the times post injection indicated on
the x axis. Data are shown as mean ± SEM; n = 8. Mann Whitney U test; *, p < 0.01;
***, p < 0.001
Figure S18
21
Time course of SN 29220-induced changes non-fasting plasma insulin levels in male
C3H/HeN mice. Animals were housed in groups of 4 and at ~50 days of age they were
injected i.p. with either 42% aqueous DMSO or SN 29220 (1.4 mol/kg). Plasma was
prepared from blood collected just prior to when mice were euthanized at the times post
injection indicated on the x axis. Data are shown as mean ± SEM; n = 8. Mann Whitney
U test; *, p < 0.05
Figure S19
Comparison of SN 29220-induced changes non-fasting plasma insulin levels in male
C3H/HeN mice at 30days and 161 days post injection. Animals were housed in groups
of 4 and at ~50 days of age they were injected i.p. with either 42% aqueous DMSO or SN
29220 (1.4 mol/kg). Plasma was prepared from blood collected just prior to when mice
were euthanized at the times post injection indicated on the x axis. Data are shown as
mean ± SEM; n = 8. Mann Whitney U test; **, p < 0.01
Figure S20
At 10 days post-injection, SN 29220 induced a significant decrease in CPT1 gene
expression detected using real time PCR, without significantly altering expression of
other genes in male C3H/HeN mouse retroperitoneal fat. Animals were housed in
groups of 4 and at ~50 days of age they were injected i.p. with either 42% aqueous
DMSO or SN 29220 (1.4 mol/kg). Tissues were collected and snap frozen when mice
were euthanized at 10 days post injection. Real time PCR data was normalized to
HMBS, Actin, TBP and HPRT housekeeping genes. Data are shown as mean ± SEM; n
= 8; Mann Whitney U test; **, p < 0.01
Figure S21
At 10 days post injection, SN 29220 had no significant effect on gene expression
detected using real time PCR in male C3H/HeN mouse liver. Animals were housed in
groups of 4 and at ~50 days of age they were injected i.p. with either 42% aqueous
DMSO or SN 29220 (1.4 mol/kg). Tissues were collected and snap frozen when mice
were euthanized at 10 days post injection. Real time PCR data was normalized to
22
HMBS, Actin, TBP and HPRT housekeeping genes. Data are shown as mean ± SEM; n
=8
Figure S22
At 10 days post injection, SN 29220 had no significant effect on gene expression
detected using real time PCR in male C3H/HeN mouse skeletal muscle. Animals
were housed in groups of 4 and at ~50 days of age they were injected i.p with either 42%
aqueous DMSO or SN 29220 (1.4 mol/kg). Tissues were collected and snap frozen
when mice were euthanized at 10 days post injection. Real time PCR data was
normalized to HMBS, Actin, TBP and HPRT housekeeping genes. Data are shown as
mean ± SEM; n = 8
Figure S23
At 10 days post injection, SN 29220 induced significant upregulation of NPY mRNA
but did not significantly alter either AGRP or POMC mRNA in C3H/HeN mouse
hypothalamus. Animals were housed in groups of 4 and at ~50 days of age they were
injected i.p. with either 42% aqueous DMSO or SN 29220 (1.4 mol/kg). Brains were
collected when mice were euthanized at 10 days post injection and were fixed in 4%
paraformaldehyde. In situ hybridization was performed on 25
thick coronal sections
using mouse gene-specific anti-sense riboprobes. Autoradiograms exposed to film for 1
day (NPY) or 2 days (AGRP and POMC) from all DMSO-treated and all SN 29220treated mice are shown in supplementary Figures S16 – S18. The highest signals for
NPY were outside of the linear range and therefore the volume of signal could not be
accurately calculated for NPY. Data are shown as mean ± SEM; Mann Whitney U test;
*, p = 0.0457
Figure S24
SN 29220 did not affect food intake when C57BL/6J mice were fed either a high-fat
or low-fat diet. Animals were housed in groups of 4 and at weaning (21 days) were fed
either low-fat (10 kcal %fat) or high-fat (60 kcal %fat) diets. A) Mild Obese Model:
Mice were injected i.p. with either 42% aqueous DMSO or SN 29220 at ~80 days of age.
23
B) Morbid Obese Model: Mice were injected i.p. with either 42% aqueous DMSO or SN
29220 at ~120 days of age. Day 0 on the x axis represents the day of injection. Food
intake was measured up to ~50 - 60 days post injection. Data are shown as average food
intake per mouse per day for groups of 8 mice.
Figure S25
SN 29220 transiently increased water intake when C57BL/6J mice were fed either a
high-fat or low-fat diet. Animals were housed in groups of 4 and at weaning (21 days)
they were fed either low-fat (10 kcal %fat) or high-fat (60 kcal %fat) diets. A) Mild
Obese Model: Mice were injected i.p. with either 42% aqueous DMSO or SN 29220 at
~80 days of age. B) Morbid Obese Model: Mice were injected i.p. with either 42%
aqueous DMSO or SN 29220 at ~120 days of age. Day 0 on the x axis represents the day
of injection. Water intake was measured up to ~50-60 days post injection. Data are
shown as average water intake per mouse per day for groups of 8 mice.
Figure S26
MRI images showed that SN 29220 reduced fat mass by ~75% of male C57BL/6J
mice fed a high-fat diet. Animals were housed in groups of 4 and at weaning (21 days)
they were fed a high-fat (60 kcal %fat) diet. Mice were injected i.p. with either 42%
aqueous DMSO or SN 29220 (1.4 mol/kg) at ~120 days of age and MRI was performed
at ~ 42 days post injection. A) Representative DMSO-treated mouse (weighed 50.6g and
had 54% fat by MRI). B) Representative SN 29220-treated mouse (weighed 25.5g and
had 12% fat by MRI). Image analysis was performed following scans for full anatomy
and scans for fat-suppressed anatomy. Images show RED as fat tissue and
GREEN/YELLOW as all tissues other than fat.
Figure S27
SN 29220 treatment of male C57BL/6J mice fed a high-fat diet reduced visceral,
retroperitoneal and gonadal fat cell size. Animals were housed in groups of 4 and at
weaning (21 days) they were fed a high-fat (60 kcal %fat) diet. Mice were injected i.p.
with either 42% aqueous DMSO or SN 29220 (1.4 mol/kg) at ~80 days of age and were
24
euthanized at ~ 62 days post injection. All images are representative of 3 mice. A, B)
Visceral Fat; C, D) retroperitoneal Fat; E, F) Gonadal Fat. Scale bar = 200M (20X
objective)
Figure S28
Excretion of 3H from 3H labeled SN 29220.
Three mice were housed individually in metabolic cages and at ~50 days of age they were
injected i.p with 3H-SN 29220 (1.4 mol/kg) (Day 0). One mouse was euthanized at
each time point; 4 h, 1 day and 3 days post injection.
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