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Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2013:6 93–102
Diabetes, Metabolic Syndrome and Obesity: Targets and erapy
Synergistic effects of metformin, resveratrol,
and hydroxymethylbutyrate on insulin sensitivity
Antje Bruckbauer
1
Michael B Zemel
1,2
1
NuSirt Sciences Inc,
2
Department
of Nutrition, University of Tennessee,
Knoxville, TN, USA
Correspondence: Michael B Zemel
11020 Solway School Rd, Knoxville,
TN 37931, USA
Tel +1 865 206 6154
Email mzemel@nusirt.com
Background: The purpose of this study was to determine whether a mixture of the polyphenol,
resveratrol, and the leucine metabolite, hydroxymethylbutyrate (HMB), acts synergistically
with low doses of metformin to impact insulin sensitivity and AMP-activated protein kinase-
dependent outcomes in cell culture and in diabetic mice.
Methods: C2C12 skeletal myotubes and 3T3-L1 adipocytes were treated with resveratrol
0.2 µM, HMB 5 µM, and metformin 0.1 mM alone or in combination. db/db mice were treated
for 2 weeks with high (1.5 g/kg diet), low (0.75 g/kg diet), or very low (0.25 g/kg diet) doses
of metformin alone or in combination with a diet containing resveratrol 12.5 mg and CaHMB
2 g/kg.
Results: The combination of metformin-resveratrol-HMB significantly increased fat oxidation,
AMP-activated protein kinase, and Sirt1 activity in muscle cells compared with metformin
or resveratrol-HMB alone. A similar trend was found in 3T3L1 adipocytes. In mice, the two
lower doses of metformin exerted no independent effect but, when combined with resveratrol-
HMB, both low-dose and very low-dose metformin improved insulin sensitivity (HOMA
IR
),
plasma insulin levels, and insulin tolerance test response to a level comparable with that found
for high-dose metformin. In addition, the metformin-resveratrol-HMB combination decreased
visceral fat and liver weight in mice.
Conclusion: Resveratrol-HMB combined with metformin may act synergistically on AMP-
activated protein kinase-dependent pathways, leading to increased insulin sensitivity, which
may reduce the therapeutic doses of metformin necessary in the treatment of diabetes.
Keywords: diabetes, AMP-activated protein kinase, Sirt1, fat oxidation
Introduction
The rapid growth in prevalence of type 2 diabetes mellitus is mainly attributable to
changes in lifestyle and diet, and has been concomitant with an increase in obesity.
1
It is also recognized as one of the major causes of other chronic morbidities, such as
cardiovascular and kidney disease, and of early mortality, thus posing a serious pub-
lic health problem.
2
Therefore, identification of safer approaches to improve insulin
sensitivity is important as a means to control diabetes and to improve the efficacy of
lifestyle approaches for the reduction of obesity and risk of diabetes.
Metformin, a biguanide, either alone or as part of combination therapy, is the drug
of choice for oral treatment of diabetes, particularly in overweight and obese people.
3
Its main action is to lower blood glucose levels by inhibiting hepatic gluconeogenesis as
well as increasing insulin sensitivity.
3,7
Metformin acts, in part, via activation of AMP-
activated protein kinase (AMPK), thereby stimulating oxidation of fat in muscle.
4–6
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A daily oral dose of 2000–2500 mg/day is typically required
for optimal effect
8
and oral bioavailability is dose-dependent,
with decreased bioavailability at higher doses, suggesting
an active, saturable absorption process.
9
Although severe
adverse effects are rare, up to 30% of patients report gas-
trointestinal symptoms, including diarrhea, cramps, nausea,
and vomiting, which can cause severe discomfort and lead to
discontinuation of the drug.
10
Therefore, finding strategies to
reduce the dose of metformin required without compromising
its efficacy is a useful approach for managing and reducing
adverse events.
AMPK is a key regulator of cell and whole body energy
metabolism. Increases in the AMP/ATP ratio activate AMPK,
resulting in increased glucose uptake and a shift from ana-
bolic to catabolic ATP-producing pathways,
11
including fat
oxidation. AMP binding results in a conformational change
which promotes Thr 172 phosphorylation by upstream
kinases, including tumor suppressor liver kinase B1 and the
Ca
2+
/calmodulin-dependent kinase (CamKKβ) and prevents
its dephosphorylation by protein phosphatases.
12
When puri-
fied recombinant AMPK activity from bacterial cell lysates
was measured, AMP stimulated the activity of AMPK by
up to 10-fold and upstream kinases by up to 100-fold, and
the combination of AMP with the upstream kinases by up to
1000-fold.
13
In addition, there is a bidirectional interaction
between the sirtuin, Sirt1, and AMPK, leading to mutual
activation.
14,15
The sirtuins, Sirt1 and Sirt3, are also well
known regulators of glucose and lipid metabolism. Their
activation converges on the same pathways activated by
AMPK
16
and results in increased mitochondrial biogenesis
and fatty acid oxidation. Because lipid and glucose metabo-
lism is dysregulated in type 2 diabetes mellitus, AMPK and
sirtuin modulators have been suggested to be promising
therapies.
We have recently demonstrated that a nutritional mixture
of resveratrol, a naturally occurring polyphenol found in the
skin of red grapes, and either leucine or HMB, a naturally
occurring metabolite of leucine, produces profound improve-
ment in insulin sensitivity, muscle glucose, and palmitate
uptake, as well as a reduction in inflammatory and oxidative
stress in mice with diet-induced obesity; notably, these effects
occurred at concentrations that were without effect when the
compounds were provided independently of each other.
17
These effects are mediated by activation of AMPK, Sirt1, and
Sirt3.
17
Thus, they converge upon the same metabolic path-
ways activated by metformin. Consequently, this study was
designed to determine whether a blend of resveratrol-HMB
would act synergistically with metformin to control diabetes
in diabetic mice. The intention was to develop a formulation
that uses very low levels of metformin in combination with
the resveratrol-HMB blend in order to lower the effective
dose of metformin required for management of diabetes.
Materials and methods
Cell culture
3T3-L1 preadipocytes were incubated at a density of
8000 cells/cm
2
(10 cm
2
dish) and grown in the absence of
insulin in Dulbecco’s modified Eagle’s medium (25 mM
glucose) containing 10% fetal bovine serum and 1% pen-
icillin-streptomycin (adipocyte medium) at 37°C in a 5%
CO
2
atmosphere. Confluent preadipocytes were induced to
differentiate using a standard differentiation medium (DM2-
L1, Zen-Bio Inc, Research Triangle Park, NC, USA). The
preadipocytes were maintained in this differentiation medium
for 3 days and subsequently cultured in adipocyte medium
for a further 8–10 days to allow at least 90% of cells to
reach full differentiation before treatment. The medium was
changed every 2–3 days, and differentiation was determined
microscopically as inclusion of fat droplets.
C2C12 muscle cells were incubated at a density of
8000 cells/cm
2
(10 cm
2
dish) and grown in Dulbecco’s
modified Eagle’s medium containing 10% fetal bovine serum
and antibiotics at 37°C in a 5% CO
2
atmosphere. Cells were
grown to 100% confluence, transferred into differentia-
tion medium (Dulbecco’s modified Eagle’s medium with
2% horse serum and 1% penicillin-streptomycin), and fed
with fresh differentiation medium every day until myotubes
were fully formed (6 days).
Treatment concentrations for all cell experiments were
200 nM resveratrol (Sigma-Aldrich, St Louis, MO, USA),
5 µM HMB (PureBulk Inc, Roseburg, OR, USA), and 0.1 nM
or 1 mM metformin (Sigma-Aldrich) as indicated. The incu-
bation time was 4–48 hours, depending on the experiment.
Fatty acid oxidation
Cellular oxygen consumption was measured using an XF24
analyzer (Seahorse Bioscience, Billerica, MA, USA) in
24-well plates at 37°C, as described by Feige et al,
18
with
slight modifications. Cells were seeded at 40,000 cells
per well, differentiated as described above, treated for
24 hours with the indicated treatments, washed twice with
nonbuffered carbonate-free pH 7.4 low glucose (2.5 mM)
Dulbecco’s modified Eagle’s medium containing carnitine
0.5 mM, equilibrated with 550 µL of the same medium
in a non-CO
2
incubator for 30 minutes, and then inserted
into the instrument for 15 minutes of further equilibration.
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Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2013:6
O
2
consumption was measured in three successive baseline
measures at five-minute intervals prior to injection of palmi-
tate (200 µM final concentration). Post-palmitate injection
measurements were taken over a 4-hour period with cycles
consisting of a 10-minute break and four successive measure-
ments of O
2
consumption.
Sirt1 activity
Sirt1 activity was measured using the Sirt1 fluorimetric drug
discovery kit (BML-AK555, ENZO Life Sciences Interna-
tional Inc, Plymouth Meeting, PA, USA). The sensitivity and
specificity of this assay kit has been tested by Nin et al.
19
Using this assay, Sirt1 activity is assessed by the degree
of deacetylation of a standardized substrate containing an
acetylated lysine side chain. The substrate utilized is a peptide
containing amino acids 379–382 of human p53 (Arg-His-
Lys-Lys[Ac]), an established target of Sirt1 activity; Sirt1
activity is directly proportional to the degree of deacetylation
of Lys-382. Samples were incubated with peptide substrate
(25 µM) and NAD
+
(500 µM) in phosphate-buffered solution
at 37°C on a horizontal shaker for 45 minutes. The reac-
tion was stopped by addition of 2 mM nicotinamide and a
developing solution that binds to the deacetylated lysine to
form a fluorophore. Following 10 minutes of incubation at
37°C, fluorescence was read in a plate-reading fluorometer
with excitation and emission wavelengths of 360 nm and
450 nm, respectively. Resveratrol 100 mM served as a Sirt1
activator (positive control) and suramin sodium 25 mM as a
Sirt1 inhibitor (negative control). Endogenous Sirt1 activity
in muscle cells and mouse white adipose tissue was measured
using a modified assay with 5 µL of cell lysate. The lysates
were prepared by homogenizing cells in ice-cold RIPA
buffer with protease inhibitor mix (MP Biomedicals LLC,
Solon, OH, USA). After 10 minutes of incubation on ice, the
homogenate was centrifuged at 14,000 g and the supernatant
was used for further experiments. Data for endogenous Sirt1
activation were normalized to cellular protein concentration
measured using a bicinchoninic acid assay.
AMPK activity
AMPK activity in cells was measured using an AMPK assay
kit (CycLex Co, Ltd, Nagano, Japan) according to the manu-
facturer’s instructions. This assay provides a nonisotopic,
sensitive, and specific method in the form of an enzyme-
linked immunosorbent assay and uses anti-phospho-mouse
IRS-1 S789 monoclonal antibody and peroxidase-coupled
anti-mouse IgG antibody as a reporter molecule. The amount
of phosphorylated substrate is determined by measuring
absorbance at 450 nm. Differentiated cells were incubated
with the treatments indicated for 24 hours. The cells were
washed three times with ice-cold phosphate-buffered solu-
tion, lysed in cell lysis buffer for 90 minutes on ice, and
centrifuged at 3500 rpm for 15 minutes at 4°C. Next, 10 µL of
clear supernatant was used for each assay. Purified recombi-
nant AMPK active enzyme was included as a positive control
for phosphorylation, and 0.5 mM AICAR, a potent AMPK
activator, was included in some experiments as an additional
positive control. To calculate the relative AMPK activity of
the samples, an inhibitor control with compound C for each
sample was included once, and inhibitor control absorbance
values were subtracted from the test sample absorbance
values. Each experiment was assayed in triplicate from four
independent cell replicates.
Western blotting
The antibodies P-AMPKα (Thr-172) and AMPKα were
obtained from Cell Signaling Technology Inc (Danvers, MA,
USA) and EMD Millipore (Billerica, MA, USA), respectively.
3T3L1 adipocytes were treated as indicated, and the cells
were washed with ice-cold phosphate-buffered solution, then
lysed with ice-cold RIPA buffer and protease and phosphatase
inhibitor cocktails (Sigma-Aldrich), centrifuged at 14,000 g
at 4°C for 10 minutes, and the resulting supernatant was used
for experiments. Protein was measured using a bicinchoninic
acid kit (Thermo Scientific Inc, Waltham, MA, USA). For
Western blotting, equal amounts of protein per sample
(10 µg) were resolved on 10% gradient polyacrylamide gels
(Criterion precast gel, Bio-Rad Laboratories, Hercules, CA,
USA), transferred to polyvinylidene difluoride membranes,
incubated in blocking buffer (3% bovine serum albumin in
Tris-buffered saline), then incubated with primary antibody
(1:1000) overnight, washed, and incubated with secondary
horseradish peroxidase-conjugated antibody (1:10.000) for
one hour. Visualization and chemiluminescent detection was
done using BioRad ChemiDoc instrumentation and software
(Bio-Rad Laboratories), and band intensity was assessed
using Image Lab 4.0 (Bio-Rad Laboratories), with correction
for background and loading controls.
Animals and diets
Diabetic 8–10-week-old male db/db mice (C57BLKS/J-
lepr
db
/lepr
db
) were purchased from Harlan Laboratories
(Indianapolis, IA). After acclimatization to their environment
for 5–7 days, they were randomized into six different dietary
groups with 10 animals per group and kept on their diet for
2 weeks. The metformin concentrations in the diets were
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Metformin, resveratrol, HMB, and insulin sensitivity
Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2013:6
based on literature values of high (300 mg/kg body weight),
low (150 mg/kg body weight), and very low (50 mg/kg body
weight) metformin studies in mice,
20–23
based on average food
consumption (8 g/day) and average body weight (40 g or
0.04 kg) for diet-induced mice; this was calculated to be
equivalent to a 1.5 g, 0.75 g, and 0.25 g metformin/kg diet,
respectively. The dietary groups were either the standard
diet (AIN 93G) only (control group), or the standard diet
combined with one of the three different concentrations of
metformin (high [300 mg/kg body weight], low [150 mg/kg
body weight], or very low metformin group [50 mg/kg body
weight]); the two lowest doses of metformin were provided
with or without a mixture of resveratrol (12.5 mg/kg diet)
and CaHMB 2 g/kg diet (low-dose metformin + resveratrol-
HMB and very low-dose metformin + resveratrol-HMB
groups, respectively).
The animals were housed in polypropylene cages at a
room temperature of 22°C ± 2°C and on a 12-hour light/dark
cycle. The animals had free access to water and food through-
out the experiment, and food intake was not measured. Body
weight was measured at days 0, 7, and 14 of the study period.
At the end of the 14-day treatment period, all animals were
fasted overnight and humanely euthanized with isoflurane
overdose and cervical dislocation. Blood and tissues were
collected for further experiments as described below. Liver
and visceral adipose tissue mass was weighed immediately
after removal.
The University of Tennessee is accredited by the
American Association for Accreditation of Laboratory
Animal Care. This study and all animal procedures were
performed under the auspices of a protocol approved by
the Institutional Animal Care and Use Committee and in
accordance with Public Health Service policy and recom-
mendations of the guide.
HOMA
IR
The homeostasis model assessment of insulin resistance
(HOMA
IR
) was used as a screening index for changes in
insulin sensitivity. HOMA
IR
is calculated via a standard
formula from fasting plasma insulin and glucose as follows:
HOMA
IR
= [Insulin (µU/mL) × glucose (mM)]/22.5. Plasma
glucose and insulin concentrations were measured using a
glucose assay kit from Cayman (Ann Arbor, MI, USA) and
an insulin kit from Millipore, respectively.
Insulin tolerance test
Insulin tolerance tests were performed at 2 pm on
day 7. The mice were injected intraperitoneally with insulin
(0.75 U/kg body weight) in approximately 0.1 mL of 0.9%
NaCl. A 5 µL drop of blood was taken from the cut tail
vein before injection of insulin and after 15, 30, 45, and
60 minutes for determination of blood glucose. Change in
blood glucose over the linear portion of the response curve
was then calculated.
Statistical analysis
All data are expressed as the mean ± standard error of the
mean. Data were analyzed by one-way analysis of variance,
and significantly different group means (P , 0.05) were sepa-
rated by the least significant difference test using GraphPad
Prism (GraphPad Software Inc, La Jolla, CA, USA).
Results
Palmitate oxidation, as measured by palmitate-stimulated
oxygen consumption rate, was significantly increased by
both metformin and the resveratrol-HMB combination in
muscle cells (P , 0.002, Figure 1), while the combination
of metformin and resveratrol-HMB showed a 30% greater
effect compared with either metformin or resveratrol-HMB
alone (P , 0.04, Figure 1A). A similar pattern was seen in
adipocytes (Figure 1B), where the combination of HMB with
either resveratrol or metformin increased the area under the
concentration-time curve by about 100%. The combination
of metformin and resveratrol-HMB enhanced this effect
by a further 16%. However, in contrast with muscle cells,
metformin alone did not show any effects in adipocytes.
Consistent with these observations, AMPK activity in
C2C12 was significantly increased by the combination of
metformin and resveratrol-HMB (P , 0.01, Figure 2B), but
not by low-dose metformin or HMB alone or by metformin-
HMB in the absence of resveratrol (Figure 2A). We also
assessed AMPK activation by measuring phosphorylation
at Thr-172. Consistent with our activity measurements, low-
dose (0.1 mM) metformin exerted no independent effect on
P-AMPK, but when low-dose metformin was combined with
either HMB or a resveratrol-HMB combination, it produced
an increase of about 40%, similar to that found with resver-
atrol-HMB (Figure 2D and E, P , 0.001). Expressing the
data as an P-AMPK/AMPK ratio produced similar results
(Figure 2F). In addition, Sirt1 activity was augmented to
a significant extent by the combination of metformin and
HMB compared with either treatment alone (Figure 2C,
P , 0.02).
Based on these in vitro data, we investigated the syner-
gistic effects of low-dose metformin and resveratrol-HMB on
insulin sensitivity in diabetic mice. Consistent with previous
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Bruckbauer and Zemel
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reports in the literature,
21,24,25
high-dose metformin reduced
plasma insulin by 27% (P , 0.02, Figure 3A) and HOMA
IR
by
35% (P , 0.025, Figure 3B), although no change was found
in plasma glucose levels in these highly insulin-resistant mice
(Table 1). In contrast, low-dose metformin and very low-dose
metformin exerted no significant independent effects, while
combining either the low-dose or very low-dose of metformin
with resveratrol and HMB resulted in significant decreases
in plasma insulin from 62 µU/mL to 43 µU/mL (P , 0.02,
Figure 3A), comparable with that seen with high-dose
metformin. Moreover, there was no significant difference
between the low-dose metformin-HMB combination versus
the very low-dose metformin-HMB combination. Consistent
with this observation, the HOMA
IR
index decreased from 29
units on the control diet to 19 on the low-dose metformin-
resveratrol-HMB blend and to 16 units on the very low-dose
metformin-resveratrol-HMB blend (P , 0.025, Figure 3B),
reflecting an improvement in insulin sensitivity comparable
with that found with high-dose metformin. Similar results
were found for the insulin tolerance test (Figure 4). Animals
on the control, low-dose, or very low-dose of metformin
showed minimal changes in blood glucose in response to
insulin challenge. In contrast, those on the standard metformin
dose and those on either low-dose or very low-dose metformin
combined with resveratrol-HMB showed decreases in blood
glucose of about 60 mg/dL over the 30-minute linear portion
of the response curve (P , 0.02). Moreover, the metformin-
resveratrol-HMB combination reduced visceral adiposity
(Table 1). Animals on the control diet had a mean visceral
fat mass of 4.5 g, and this was not affected by metformin at
any dose in the absence of resveratrol-HMB. Low-dose met-
formin combined with resveratrol-HMB and very low-dose
metformin combined with resveratrol-HMB reduced visceral
fat by about 20%, to 3.8 and 3.6 g, respectively (P , 0.03).
These treatments also reduced liver mass, from 2.78 g (con-
trol) to 2.35 g and 2.41 g, respectively (P , 0.05), while no
effect on body weight was seen (Table 1).
Discussion
Our results demonstrate potentiation of the effect of the
antidiabetic drug, metformin, using a mixture of resveratrol
and HMB, on fat oxidation and insulin sensitivity in vitro
188
AB
A and C
A and C
OCR
OCR
177
166
155
144
133
122
111
100
89
4000
CTRL
0.1MM MET
RESV/HMB
MET/R/HMB
CTRL
0.1MM MET
RESV/HMB
MET/HMB
MET/R/HMB
3500
3000
2500
2000
1500
1000
500
0
4000
a
a
b
b
c
a
b
b
b
3500
3000
2500
2000
1500
1000
500
0
78
164
155
147
138
129
120
112
103
94
86
77
1275277 103 128
Time (min) Time (min)
C2C12 3T3L1
OCR (%)AUC OCR (%)
OCR (%)AUC OCR (%)
153 179 204 229 255
CTRL
0.1 mm MET
RESV/HMB
MET/HMB
MET/R/HMB
2275175100 124148 172197 221 245
Figure 1 Synergistic effects of metformin and resveratrol-HMB on fatty acid oxidation in C2C12 muscle cells and 3T3L1 adipocytes. Differentiated C2C12 muscle cells (A)
and 3T3L1 adipocytes (B) were treated with the treatments indicated for 24 hours.
Notes: OCR was measured after palmitate injection (point A and C) and represented as a percentage of baseline. The AUC is calculated by subtracting the starting rate
(third baseline point) from each average rate point. Data are expressed as the mean ± standard deviation (n =4).Barswiththesamelettersuperscriptsarenotsignicantly
differentfromeachother;barswithnonmatchinglettersuperscriptsindicatesignicantdifferencesbetweengroups(P , 0.04).
Abbreviations: AUC, area under curve; CTRL, control; HMB, hydroxymethylbutyrate; MET, metformin; OCR, oxygen consumption rate; RESV, resveratrol.
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Metformin, resveratrol, HMB, and insulin sensitivity
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and in vivo. These effects were found with concentrations
of each compound that were too low to exert significant
independent effects, demonstrating a synergistic action. We
have recently shown that the amino acid leucine, as well as
its metabolite HMB, acts synergistically with the polyphe-
nol, resveratrol, on insulin sensitivity and fat metabolism.
17
These effects were mediated by activation of AMPK, Sirt1,
and Sirt3 in muscle cells and adipocytes. Therefore, we
hypothesized that the addition of another activator of this
pathway may act synergistically and may be able to enhance
this effect. Indeed, consistent with our previous data, the
combination of resveratrol with HMB enhanced fatty acid
oxidation by about 100% in adipocytes and by about 160%
in muscle when measured over a 2-hour period in this study.
However, addition of low-dose metformin, which had little or
no effect by itself, resulted in a further significant increase of
AMPK activity in C2C12
Absorbance/mg protein
Control
MET
HMB
Met-HMB
0
5
10
15
a
a
a
a
AMPK activity in C2C12
Absorbance/mg protein
Control
MET
MET/RESV/Leu
MET/RESV/HMB
0
2
4
6
aa
ab b
Sirt1 activity in 3T3L1
AFU/ ug protein
Control
MET
HMB
MET-HMB
0
50
100
150
200
c
b
b
a
P-AMPK
Band intensity
Control
MET (0.1 mM)
RESV/HMB
MET/HMB
MET/R/HMB
AICAR (1 mM)
0
50
100
150
200
ab
a
bc
c
c
c
P-AMPK/AMPK
Ratio
Control
MET (0.1 mM)
RESV/HMB
MET/HMB
MET/R/HMB
AICAR (1 mM)
0
1
2
3
a
ab
c
bc
bc
c
A
B
C
D
control Met R/HMB Met/HMB Met/R/HMB AICAR
P-AMPK
AMPK
E
F
Figure 2 Synergistic effects of metformin and resveratrol-HMB on AMPK and Sirt1 activity in C2C12 muscle cells and 3T3L1 adipocytes. Differentiated muscle cells and
adipocytes were treated with the treatments indicated. (A and B) AMPK activity in C2C12 cell lysates. (C) Sirt1 activity in 3T3L1 cell lysates. (D) Quantitative band analysis
of AMPK phosphorylation in 3T3L1 adipocytes. (E) Western blots of AMPK in 3T3L1 cell lysates using anti-phospho-AMPK-α (Thr172) and anti-AMPKα antibodies. AICAR
was used as a positive control. (F) P-AMPK/AMPK ratio in 3T3L1 adipocytes.
Notes: Data are expressed as the mean ± standard error of the mean (n =4).Barswiththesamelettersuperscriptsarenotsignicantlydifferentfromeachother;barswith
nonmatchinglettersuperscriptsindicatesignicantdifferencesbetweengroups(P , 0.05).
Abbreviations:AFU,arbitraryuorescentunits;AMPK,AMP-activatedproteinkinase;HMB,hydroxymethylbutyrate;MET,metformin;RESV,resveratrol.
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Bruckbauer and Zemel
Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2013:6
this effect; these effects were accompanied by corresponding
increases in AMPK and Sirt1 activity.
Consistent with our previous observations,
17
leucine
combined with resveratrol was able to exert effects similar
to those of the resveratrol-HMB combination; however, in
combination with metformin, HMB was superior (data not
shown). Although no significant difference between the
metformin-HMB and metformin-resveratrol-HMB combina-
tions was detected in adipocytes, there was an enhanced effect
of metformin-resveratrol-HMB in muscle cells.
For this reason, we conducted our animal study using
only the resveratrol-HMB combination, and found that this
combination significantly potentiated the effects of low-
dose metformin on insulin sensitivity, resulting in efficacy
comparable with that of treatment with higher doses of
metformin. Although mean plasma glucose was highest in
the very low-dose metformin-resveratrol-HMB group, it
was not significantly different from that in the other groups,
while the insulin concentration and calculated HOMA
IR
were
significantly lower, indicating an improvement in insulin
sensitivity. Further, consistent with our previous results in
diet-induced obesity in mice,
17
combination with resveratrol-
HMB reduced visceral fat mass and liver weight, while no
effect on these outcomes were found using metformin alone.
Because the number of mice used in the animal study was
limited, we did not include a resveratrol-HMB group.
Therefore, we cannot rule out that these effects were caused
Table 1 Effects of indicated treatments on body weight, visceral fat mass, liver weight, plasma glucose, and food intake in mice
Control Standard
metformin
Low-dose
metformin
Very low-dose
metformin
Low-dose
metformin +
resveratrol-HMB
Very low-dose
metformin +
resveratrol-HMB
Body weight (g) (start)
45.0 ± 0.6
a
45.2 ± 0.8
a
45.1 ± 0.6
a
44.4 ± 0.9
a
45.6 ± 1.0
a
46.7 ± 0.6
a
Body weight (g) (end)
46.8 ± 0.7
a
48.6 ± 0.6
a
48.2 ± 0.4
a
46.3 ± 1.1
a
48.3 ± 1.0
a
47.9 ± 1.0
a
Plasma glucose (mg/dL)
481.3 ± 25.0
a
460.1 ± 24.7
a
482.3 ± 15.4
a
503.0 ± 17.0
a
433.7 ± 51.6
a
529.1 ± 18.6
a
Visceral fat (g)
4.5 ± 0.18
a
4.3 ± 0.29
a
4.6 ± 0.14
a
4.4 ± 0.13
a
3.8 ± 0.19
b
3.6 ± 0.23
b
Liver (g)
2.78 ± 0.16
a
2.76 ± 0.09
a
2.65 ± 0.09
a
2.68 ± 0.12
a
2.35 ± 0.13
b
2.41 ± 0.08
b
Notes: db/db mice were fed the indicated diets for 2 weeks. Data are expressed as the mean ± standard error of the mean (n = 10). Values with the same letter superscripts
ineachrowarenotsignicantlydifferentfromeachother;valueswithnonmatchinglettersuperscriptsineachrowindicatesignicantdifferencesbetweengroups(P , 0.03).
Abbreviation: HMB, hydroxymethylbutyrate.
60
30
a
b
a
a
b
b
15
0
a
Insulin (µU/mL)
A
B
HOMA
b
b
b
a
a
45
30
15
0
Control
Std MET
Low MET
Very low MET
Low MET+RESV/HMB
Very low MET+RESV/HMB
Figure 3 Synergistic effects of metformin and resveratrol-HMB on insulin sensitivity
in mice. Effects of standard dose, low-dose, and very low-dose of metformin
compared with low-dose metformin-resveratrol-CaHMB and with very low-dose
metformin-resveratrol-CaHMB on (A) plasma insulin and (B) HOMA
IR
in db/db
mice.
Notes: Data are expressed as the mean ± standard error of the mean (n = 10). Bars
with the same lettersuperscripts arenot signicantlydifferent fromeach other;
barswithnonmatchinglettersuperscripts indicate signicant differencesbetween
groups (P , 0.025).
Abbreviations: HOMA, homeostatic assessment of insulin resistance; HMB,
hydroxymethylbutyrate; MET, metformin; RESV, resveratrol.
30
a
b
b
b
a
a
Control
Std met
Low MET
Very low MET
Low MET+RESV/HMB
Very low MET+RESV/HMB
30-minute glucose change (mg/dL)
15
0
−15
−30
−45
−60
−75
Figure 4 Synergistic effects of metformin and resveratrol-HMB on insulin tolerance
test in mice.
Notes: Effects of standard dose, low-dose, and very low-dose metformin compared
with low-dose metformin-resveratrol-CaHMB and with very low-dose metformin-
resveratrol-CaHMB on 30-minute plasma glucose response to insulin (0.75 U/kg
body weight) in db/db mice. Data are expressed as the mean ± standard error of the
mean (n =10).Barswiththesamelettersuperscriptsarenotsignicantlydifferent
from each other; bars with nonmatching letter superscripts indicate signicant
differences between groups (P , 0.02).
Abbreviations: HMB, hydroxymethylbutyrate; Met, metformin; RESV, resveratrol.
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Metformin, resveratrol, HMB, and insulin sensitivity
Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy 2013:6
simply by resveratrol-HMB treatment alone. However, the
demonstration of synergy in the in vitro studies of myotubes
suggests that low-dose metformin acts synergistically with
resveratrol-HMB in vivo as well.
The primary action of metformin is to lower blood glucose
concentration by inhibiting hepatic glucose production and
stimulating glucose disposal in skeletal muscle.
3
Metformin
also activates AMPK, although it is not clear whether this
is by direct or indirect stimulation.
26
It has been postulated
that inhibition of mitochondrial respiration at complex 1
resulting in decreased ATP production is responsible for
AMPK activation,
5,27
but this model would be contrary to
the finding of increased fatty acid oxidation in muscle with
metformin. Metformin also acts via inhibition of AMP deami-
nase, thereby inhibiting AMP catabolism and increasing the
cellular AMP/ATP ratio, resulting in phosphorylation and
activation of AMPK.
28
In addition, metformin may interact
directly with the γ-subunit of the AMPK complex, produc-
ing a structural change that promotes phosphorylation and
activation by upstream kinases.
6
Resveratrol has also been shown to activate AMPK by
two mechanisms, ie, high doses of resveratrol inhibit cAMP-
phosphodiesterase, thus increasing cellular cAMP concen-
trations and activating the upstream kinase CaMKKβ,
29
and moderate doses of resveratrol lead to Sirt1-dependent
activation of AMPK.
30
We have demonstrated direct effects
of HMB on Sirt1 activation in a cell-free system
31
as well
as the synergistic effects of resveratrol and HMB on Sirt1
both in vitro and in vivo.
17
Considering the two different
mechanisms of AMPK activation of these compounds, it is
possible that metformin in the low concentrations used in
our study acts as an allosteric effector to promote AMPK
phosphorylation by resveratrol and HMB.
Although the pathogenesis of insulin resistance in
type 2 diabetes mellitus is still not fully understood, there
is evidence that accumulation of intracellular lipid metabo-
lites in skeletal muscle and hepatocytes, as observed in
obesity or lipodystrophy, leads to impairment in insulin
signaling pathways.
33
Moreover, decreased mitochondrial
oxidative capacity may result in impaired lipid oxidation
in skeletal muscle in obese and diabetic individuals,
34,35
potentially exacerbating lipid overflow. Therefore, finding
strategies to improve mitochondrial function and to stimulate
catabolic oxidative pathways may contribute to both the
prevention and treatment of diabetes.
AMPK, as a key regulator of energy metabolism, mediates
the switch from anabolic processes to catabolic pathways.
AMPK activity is decreased in obese insulin-resistant patients
compared with obese insulin-sensitive patients.
36
Further,
lack of skeletal muscle AMPKα2 activity in transgenic
mice exacerbates the development of diet-induced glucose
intolerance and insulin resistance.
37
In contrast, activation of
AMPK improves symptoms of impaired glucose homeostasis
and insulin resistance.
38–40
Given that lifestyle interventions
such as caloric restriction or exercise, which are physiological
activators of AMPK, are difficult to incorporate or to maintain
in the daily routine of most people, finding agents mimicking
these effects are of great interest. Unfortunately, many of
these compounds require concentrations which are associated
with adverse effects. However, obtaining synergy from three
unrelated compounds permits the use of very low concentra-
tions of each individual component, reducing the likelihood
of adverse effects while preserving the effects of higher-dose
metformin on fat oxidation and insulin sensitivity.
Conclusion
In this study we have demonstrated the synergistic effects
of a mixture of resveratrol and HMB with a low dose of
metformin on activation of AMPK and Sirt1, with an associ-
ated increase in fat oxidation in muscle and adipose tissue
in cell culture. Consistent with our in vitro data, 2 weeks of
treatment with a combination of these compounds increased
insulin sensitivity and reduced adiposity in mice, and these
effects were comparable with those found with high-dose
metformin treatment. Therefore, this combination may be a
useful approach to lowering the therapeutic metformin con-
centration required and may be beneficial for the management
of type 2 diabetes.
Disclosure
Financial support for this study was provided by NuSirt
Sciences Inc, Knoxville TN. AB and MBZ are employees
and stockholders of NuSirt Sciences Inc.
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