α-Lipoic acid regulates lipid metabolism through induction of sirtuin 1 (SIRT1) and activation of AMP-activated protein kinase
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Sirtuin 1 (SIRT1) is a longevity-associated protein, which regulates energy metabolism and lifespan in response to nutrient deprivation. It has been proposed to be a therapeutic target for obesity and metabolic syndrome. We investigated whether α-lipoic acid (ALA) exerts a lipid-lowering effect through regulation of SIRT1 activation and production in C2C12 myotubes.
ALA-stimulated AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), adipose triacylglycerol lipase (ATGL) and fatty acid synthase (FAS) production, as well as intracellular triacylglycerol accumulation and fatty acid β-oxidation were analysed in the absence or presence of a SIRT1 inhibitor (nicotinamide), SIRT1 small interfering (si) RNA and an AMPK inhibitor (compound C) in C2C12 myotubes. Mice with streptozotocin/nicotinamide-induced diabetes and db/db mice fed on a high-fat diet were used to study the ALA-mediated lipid-lowering effects in vivo.
ALA increased the NAD+/NADH ratio to enhance SIRT1 activity and production in C2C12 myotubes. ALA subsequently increased AMPK and ACC phosphorylation, leading to increased palmitate β-oxidation and decreased intracellular triacylglycerol accumulation in C2C12 myotubes. In cells treated with nicotinamide or transfected with SIRT1 siRNA, ALA-mediated AMPK/ACC phosphorylation, intracellular triacylglycerol accumulation and palmitate β-oxidation were reduced, suggesting that SIRT1 is an upstream regulator of AMPK. ALA increased ATGL and suppressed FAS protein production in C2C12 myotubes. Oral administration of ALA in diabetic mice fed on a high-fat diet and db/db mice dramatically reduced the body weight and visceral fat content.
ALA activates both SIRT1 and AMPK, which leads to lipid-lowering effects in vitro and in vivo. These findings suggest that ALA may have beneficial effects in the treatment of dyslipidaemia and obesity.
KeywordsAMPK ATGL α-Lipoic acid SIRT1
AMP-activated protein kinase
Adipose triacylglycerol lipase
Fatty acid synthase
Forkhead box O
Liver kinase B1
Small interfering RNA
α-Lipoic acid (ALA) is an endogenous cofactor in many multi-enzyme complexes that catalyse the oxidative decarboxylation of α-keto acids such as pyruvate and α-ketoglutarate. ALA is a powerful antioxidant derived from plants and animal tissues . It has been shown to reduce body weight by suppressing hypothalamic AMP-activated protein kinase (AMPK) . It increases insulin sensitivity and skeletal muscle fatty acid oxidation by activating AMPK in diabetes-prone Otsuka Long Tokushima fatty rats [3, 4]. It also increases glucose uptake in 3T3-L1 adipocytes , skeletal muscle cells  and skeletal muscles of ob/ob mice . Treatment with ALA ameliorates severe hypertriacylglycerolaemia by inhibiting triacylglycerol synthesis and VLDL-triacylglycerol secretion in Zucker diabetic fatty rats . Thus, ALA has potent beneficial effects on obesity, type 2 diabetes mellitus and dyslipidaemia.
Sirtuin 1 (SIRT1) is a class III NAD+-dependent histone/protein deacetylase that regulates lipid metabolism and lifespan by deacetylating lysine residues on various transcription factors . It deacetylates the transcription factor of forkhead box O protein (FOXO) , p53  and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) to control metabolic functions  and biological processes  similar to those observed with energy restriction. Decreased SIRT1 production may have a critical role in the pathogenesis of type 2 diabetes  and insulin resistance, which is associated with SIRT1 downregulation of type 2 diabetes . SIRT1 is regulated by NAD+, the concentration of nicotinamide (NA), and the activity of nicotinamide phosphoribosyltransferase (NAMPT). When ALA is taken up by cells, it is converted into dihydrolipoate by mitochondrial lipoamide dehydrogenase or the thioredoxin/thioredoxin reductase system, which couples the conversion of NADH into NAD+ and increases the NAD+/NADH ratio. NA, an inhibitor of SIRT1, can be converted into NAD+ by phosphoribosylation catalysed by NAMPT, and NAMPT is activated by nutrient deprivation, exercise and AMPK activation . Therefore, nutrient deprivation may increase SIRT1 activity by increasing the NAD+/NADH ratio and by decreasing the abundance of NA. However, whether ALA regulates SIRT1 activity or production has not been determined.
AMPK, a fuel-sensing enzyme, exerts effects similar to SIRT1 on cellular energy metabolism and mitochondrial biogenesis [17, 18]. Once activated, AMPK phosphorylates acetyl-CoA carboxylase (ACC), thereby blocking its activity. This in turn decreases malonyl-CoA concentration, leading to enhanced mitochondrial fatty acid β-oxidation. The AMPK signalling pathway is thought to be a natural response to reduce dyslipidaemia and to ameliorate insulin resistance . Therefore, activation of SIRT1/AMPK signalling may offer significant pharmacological benefits in treating dyslipidaemia, obesity and metabolic syndrome. Because of the aforementioned metabolic actions of SIRT and AMPK, we investigated whether ALA regulated lipid metabolism through the SIRT1 and AMPK signalling pathway. We demonstrated that ALA activated and induced SIRT1 production and function as an upstream regulator of AMPK. ALA also enhanced ATGL and suppressed fatty acid synthase (FAS) production, which in turn reduced lipid accumulation in C2C12 cells exposed to high glucose. These data suggest that ALA may exert beneficial effects in treating dyslipidaemia and obesity of the metabolic syndrome.
DMEM, FCS, glutamine, gentamicin, penicillin and streptomycin were purchased from Life Technologies (Gaithersburg, MD, USA). Rabbit polyclonal antibodies to FAS, acetyl-lysine and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Gentex (Irvine, CA, USA). Antibodies to phospho-AMPK, AMPK, ATGL, phospho-ACC and ACC were obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit polyclonal anti-SIRT1, mouse polyclonal anti- liver kinase B1 (LKB-1) and horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 5-Aminoimidazole-4-carboxamide riboside (AICAR), compound C and NA were purchased from Calbiochem-Novabiochem (San Diego, CA, USA). HRP-conjugated anti-rabbit IgG was purchased from Bio-Rad (Hercules, CA, USA). SIRT1 assay kits were from BIOMOL (Plymouth Meeting, PA, USA).
Culture of C2C12 myotubes and preparation of cell lysates
C2C12 skeletal muscle cells were cultured in DMEM supplemented with 10% heat-inactivated FCS and penicillin (100 U/ml)/streptomycin (100 mg/ml). After reaching confluence, C2C12 cells were differentiated to myotubes by the addition of 2% horse serum for 96 h as described by Lagouge et al , and these myotubes were treated with various concentrations of the indicated agents and incubated for the indicated periods in a 5% CO2 humidified incubator at 37 °C. Cells were scraped off using a rubber policeman to obtain cell lysates for the experiments.
Immunoprecipitation and western blotting
Proteins from cell lysates were separated by SDS-PAGE and transferred to poly(vinylidene difluoride) membranes for immunoblotting. Membranes were blocked with blocking solution containing 3% BSA and 0.1% Tween 20 in PBS for 1 h at room temperature followed by incubation with the primary and secondary antibodies. For immunoprecipitation, the agarose beads were conjugated with antibody to LKB-1. Protein (500 μg) from cultured cells was incubated with cross-linked LKB-1 beads overnight, and the immunoprecipitates were boiled with sample loading buffer containing 0.5 mol/l TRIS/HCl (pH 6.8), 4.4% (wt/vol.) SDS, 20% (vol./vol.) glycerol, 2% (vol./vol.) 2-mercaptoethanol and bromophenol blue in distilled/deionised water for 5 min before SDS-PAGE. Immunodetection was performed using a LumiGLO chemiluminescence kit (Amersham International, Amersham, UK). Levels of phosphorylation and abundance were quantified by scanning densitometry using a model GS-700 imaging densitometer (Bio-Rad), normalised to levels of total protein.
Transfection of SIRT1-specific small interfering (si) RNA
C2C12 cells were seeded at 5 × 105 cells per 6 cm plate and allowed to adhere overnight. SIRT1-specific siRNA was transfected into cells using lipofectamine (Invitrogen, Grand Island, NY, USA) after 48 h of differentiation. After 48 h, transfected cells were treated with ALA (300 μmol/l) for 24 h. Cell lysates were collected in parallel for the western blot analysis.
Measurement of SIRT1 activity
SIRT1 activity was assayed by a Fluor-de-Lys fluorescence assay kit (BIOMOL) according to the manufacturer’s instructions. Briefly, the assay was performed by incubating recombinant human SIRT1 protein and substrates, including a fluorogenic acetylated Lys382 p53 peptide (50 μmol/l) and NAD+ (100 μmol/l) at 37 °C for 30 min according to the manufacturer’s instructions. The fluorescence intensity was measured using a Fluoroskan Ascent microplate fluorimeter (Thermo Electron, Milford, MA, USA). Negative controls included ‘no enzyme’ and ‘time zero’ controls, in which Developer II solution plus 2 mmol/l NA was added before mixing of the substrates with or without the SIRT1 enzyme. SIRT1 activity was calculated with the corrected arbitrary fluorescence units of the tested compounds to the ‘no enzyme’ control and expressed as fluorescent units relative to the control. To rule out ALA possession of autofluorescence or interference of NA itself with the fluorescent signal, the Developer II solution was incubated with the Fluor-de-Lys deacetylated standard or the tested compounds in the absence of SIRT1 enzyme or substrates. The deacetylated standard dose-dependently increased the fluorescent rate, whereas the tested compounds did not alter the fluorescent intensity, indicating that the change in p53 deacetylation caused by these compounds depends on the specific SIRT1 activity.
After differentiation, cells were resuspended in medium supplemented with [9,10-3H]palmitate in a mixture of the palmitate and 10% BSA at a 1: 2 volume ratio. In total, 3.3 μl [9,10-3H]palmitate and 6.7 μl BSA were used per ml of cell culture medium. Each sample used 0.5 × 106 cells in 1 ml medium supplemented with the [9,10-3H]palmitate/BSA mixture and cultured for 24 h in 24-well plates. After 24 h, the supernatant fraction was applied to an ion-exchange column (Dowex 1X8-200; Sigma, St Louis, MO, USA), and 3H-labelled water was recovered by elution with 2.5 ml water. A 0.75 ml aliquot was then used for scintillation counting.
Measurement of intracellular triacylglycerol content
Intracellular triacylglycerol was assayed using a colorimetric assay (GPO-Trinder Reagent A; Sigma) . Briefly, 30 μl triacylglycerol standard or supernatant fraction was added to a 96-well flat-bottom polystyrene plate, and 300 μl triacylglycerol reagent was then added to the microplate. The plate was incubated for 5 min, and the absorbance was determined at 520 nm with a Gen5 data collector (BioTek Instruments, Highland Park, Winooski, VT, USA). Intracellular triacylglycerol levels were normalised to total protein and expressed as μg lipid/mg protein.
NAD+/NADH ratio measurement
The NAD+/NADH ratio was measured from whole-cell extracts of C2C12 myotubes or liver using the Biovision NAD+/NADH quantification kit (Biovision, San Francisco, CA, USA). To detect total NAD+ (NADt [NAD+ + NADH]), 50 μl extracted sample (cell or tissue) was transferred to labelled 96-well plates. Into each well of the NADH standard, 100 μl of the NAD+ Cycling Mix was added at room temperature for 5 min to convert NAD+ into NADH. Then 10 μl NADH developer was added to each well for 1–4 h. The absorbance was determined at 450 nm with a Biotex Microplate Spectrophotometer. To detect NADH, NAD+ needs to be decomposed before the reaction by heating at 60 °C for 30 min. The sample readings were applied to the NADH standard curve, and the amounts of NADt and NADH were determined using the equation. At the same time, the NAD+/NADH ratio was calculated as: (NADt − NADH)/NADH. These readings were normalised to the cell number (cell sample) and protein concentration (tissue sample).
The db/db mice on a C57BL/6 background (male, 14 weeks old, 50 g) were gifts from the Development Center for Biotechnology of Taiwan. The animals were given free access to water and were fed on a standard diet. ALA (200 mg/kg) or vehicle was administered orally in the afternoon (14:00–14:30 hours). The serum biochemical profiles, including triacylglycerol, cholesterol, insulin, HDL-cholesterol, LDL-cholesterol, aspartate aminotransferase and alanine aminotransferase, were determined with a Biochem-Immuno autoanalyser (Brea, CA, USA). Whole blood levels of biochemical variables, including HbA1c and glucose, were also measured with a Biochem-Immuno autoanalyser. The quality controls, calibrations and determining procedures were carried out according to the suppliers’ instructions. The glucose tolerance test and histopathological examinations were performed on day 90.
Streptozotocin/NA-induced diabetes mouse model
Male C57BL/6J (6 weeks old) mice were obtained from the Taiwan National Laboratory Animal Center. Streptozotocin (STZ, 100 mg/kg) was administered to C57BL/6J mice with NA (240 mg/kg), twice with an interval of 2 days, and then these mice were fed on a high-fat diet for 4 weeks to induce type 2 diabetes . Mice were randomly separated into three groups for treatment with 0, 50 or 200 mg/kg by oral administration. Plasma glucose concentration and body weight were measured on days 0, 7, 14 and 28. Biochemical measurements, glucose tolerance and pathological examinations were performed on day 30.
Liver and muscle were fixed and embedded in tissue-freezing medium (Leica Microsystems, Wetzlar, Germany) and stored at −80 °C. The frozen tissue was cut into 7 μm-thick sections and placed on glass slides. The tissue sections were stained with haematoxylin and eosin, Oil Red or Sudan III. Oil Red staining and Sudan III staining were counterstained with haematoxylin to visualise lipid droplets.
All data are expressed as the mean±SEM, and statistical significance (p < 0.05) between experimental groups was determined by a single-factor ANOVA for multiple groups or unpaired t test for two groups.
ALA increases SIRT1 activity and production by modulating the NAD+/NADH ratio
ALA stimulates AMPK phosphorylation and LKB-1 deacetylation
ALA-increased [9,10-3H]palmitate β-oxidation is mediated through SIRT1/AMPK signalling
ALA regulates lipid metabolism and lowers high-glucose-induced cellular triacylglycerol accumulation
ALA ameliorates the metabolic function of mice with STZ/NA-induced diabetes
Metabolic variables of STZ-treated mice fed on a high-fat diet (HFD) with or without ALA
HFD + ALA50
HFD + ALA200
Body weight (g)
26.71 ± 0.47
34.36 ± 1.47*
31.27 ± 1.25†
28.06 ± 0.58†
Epididymis fat weight (g)
0.53 ± 0.06
2.23 ± 0.20*
1.92 ± 0.27
1.06 ± 0.06†
Liver weight (g)
0.98 ± 0.08
1.03 ± 0.12
1.19 ± 0.08
1.14 ± 0.09
22.00 ± 2.55
54.60 ± 7.23*
19.71 ± 3.36†
18.00 ± 2.16†
Plasma glucose (mmol/l)
7.27 ± 0.41
11.44 ± 0.79*
11.10 ± 0.31
9.50 ± 0.33†
0.21 ± 0.10
0.72 ± 0.10*
0.63 ± 0.15
0.41 ± 0.06†
ALA regulates lipid-related proteins, the NAD+/NADH ratio, and lipid accumulation in STZ/NA-induced diabetic mice
To further confirm the changes in FAS and ATGL in vivo, we examined the levels of ATGL, FAS and phospho-AMPK in the liver of STZ/NA-induced diabetic mice. ALA treatment decreased FAS production and increased ATGL and phospho-AMPK levels (Fig. 5d). The NAD+ level was increased from 278 to 575 pmol/μg protein, and the NAD+/NADH ratio was increased by more than 50% (Fig. 5e–g).
Effects of ALA treatment in db/db mice
Effect of ALA on metabolic variables of db/db mice
Control (n = 4)
ALA (n = 4)
219.00 ± 30.87
162.00 ± 46.41
138.00 ± 17.32
127.00 ± 15.67
3.26 ± 0.30
2.29 ± 0.10*
3.15 ± 0.16
1.35 ± 0.12*
84.75 ± 8.17
82.00 ± 10.95
18.75 ± 1.71
8.75 ± 2.78*
20.54 ± 2.80
18.93 ± 1.30
8.60 ± 1.03
6.43 ± 0.86
70.49 ± 11.21
46.72 ± 9.39
Obesity, dyslipidaemia and metabolic syndrome are increasingly prevalent due to excess energy intake and nutrient availability. Energy restriction and exercise may prevent metabolic disorders, but are seldom successful. Thus there is an unmet medical need to develop therapeutic agents that mimic energy restriction and exercise to combat metabolic disorders. SIRT1 is thought to play a pivotal role in metabolic adaptation to energy restriction, and is proposed as a potential target for treating metabolic disorders. This study provides the first evidence that SIRT1 is a target of ALA. ALA activated SIRT1 in a cell-free system, and ALA also increased the NAD+/NADH ratio and upregulated SIRT1 in C2C12 myotubes. Furthermore, we demonstrated that ALA enhanced fatty acid β-oxidation, increased ATGL production, and decreased FAS production through the SIRT1/AMPK signalling pathway.
ALA is a strong antioxidant which can be reduced to dihydrolipoase by either mitochondrial lipoamide dehydrogenase or the thioredoxin/thioredoxin reductase system to increase the NAD+/NADH ratio. The ability of ALA to increase NAD+ and the NAD+/NADH ratio may explain, at least in part, how ALA treatment increases SIRT1 activity. On the other hand, activation of AMPK was linked to SIRT1 protein production in skeletal muscle cells . As ALA activates AMPK, another possibility is that ALA induces SIRT1 production through AMPK activation. ALA-stimulated AMPK phosphorylation was inhibited by the SIRT1 inhibitor, suggesting that SIRT1 acts as an upstream activator of AMPK. These observations were further confirmed by the knockdown of SIRT1, which also reduced AMPK phosphorylation in C2C12 myotubes. Our results agree with those of Hou et al , who showed that polyphenols mediate lipid-lowering effects through the SIRT1/LKB1/AMPK signalling pathway in HepG2 cells. Our results are in line with those of Lan et al , who demonstrated that SIRT1 leads to deacetylation of a lysine residue on LKB-1 to activate AMPK. However, these results differ from the findings of Canto et al , who showed that AMPK acted upstream of SIRT1 by modulating NAD+ metabolism. AMPK has been shown to increase SIRT1 activity by regulating NAMPT, an NAD+-biosynthetic enzyme . On the other hand, SIRT1 was upregulated by the p53/FOXO3a complex , and AMPK has been shown to regulate FOXO1 activity through phosphorylation . Although AMPK and SIRT1 can activate each other in different orders, the two molecules have similar effects on energy metabolism. These findings support the notion that cellular metabolism is regulated in a concerted manner.
We demonstrated that oral administration of ALA decreased body weight and visceral fat mass in diabetic mice fed on a high-fat diet and in db/db mice. The lipid-lowering effect of ALA was associated with increased ATGL and suppressed FAS protein production in liver and skeletal muscle of both STZ/NA-induced and db/db diabetic mice. These responses were attenuated by inhibition of SIRT1 or AMPK, suggesting that these lipid metabolism-related proteins were regulated through a SIRT1/AMPK-dependent pathway. These results agree with those reported by Hou et al, who showed that activation of SIRT1 by resveratrol exerted a lipid-lowering effect though AMPK . These data also agree with those of Gaidhu et al , who reported that AICAR induces AMPK activation, which promotes energy dissipation through ATGL induction. On the other hand, it is well known that AMPK phosphorylates and inhibits ACC activity, which in turn increases fatty acid oxidation. Inhibition of FAS, a lipogenic enzyme, is another important consequence of AMPK activation by ALA. These findings are in accordance with results of previous studies showing that FAS gene expression was abrogated by treatment with AICAR in hepatocytes . These results define a novel mechanism by which ALA has a lipid-lowering effect by activating SIRT1 and AMPK and by regulating lipid-related proteins of ACC, ATGL and FAS. These observations are consistent with results showing amelioration of insulin sensitivity and triacylglycerol clearance by ALA in skeletal muscle and liver . However, the impaired glucose tolerance was not improved by ALA treatment in either STZ/NA-induced or db/db diabetic mice (ESM Figs 1 and 2). Although HbA1c was slightly reduced, the differences between treated and untreated groups were not statistically significant (Table 2). These data suggest that treatment with ALA alone may have limitations for exerting an anti-glycaemic effect.
Taken together, ALA exerts an anti-obesity effect by activating the SIRT1/AMPK signalling pathway. This study demonstrates novel pharmacological actions of ALA, including activation of SIRT1 in muscle cells and regulation of lipid-related proteins. ALA not only enhances fatty acid oxidation but also promotes a lipid-lowering effect by regulating FAS and ATGL expression. Oral administration of ALA decreased body weight and the visceral fat content of both db/db and STZ/NA-induced diabetic mice.
This study was supported by grants NSC 99-3112-B-166-001 and NSC 98-3112-B-038-001 from the National Science Council, Taipei, Taiwan.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
H-ML, S-GW and W-LC contributed to conception and design of the study; W-LC and C-HK contributed to analysis and interpretation of data. W-LC and H-ML drafted the article and S-GW, W-LC and C-HK revised the article for intellectual content. All authors gave final approval of the version to be published.
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