Menopause, the loss of ovary function, results in the increased prevalence of obesity and metabolic syndromes including insulin resistance, type 2 diabetes mellitus, and cardiovascular diseases.¹ Despite estrogen exerting beneficial effects for menopause-induced metabolic abnormalities, long-term hormonal replacement therapy imparts risk of breast and cervical cancer and is not the primary strategy.² Dietary supplementation for counteracting obesity and metabolic risks requires further investigation for the management of postmenopausal health.

In mammals, three types of adipose tissue have been identified based on their functions and characteristics: white adipose tissue (WAT), brown adipose tissue (BAT), and beige adipose tissue.³ WAT stores energy in the form of triglyceride (TG) and regulates whole-body energy homeostasis, while BAT dissipates energy as heat through uncoupling protein 1 (UCP1) and produces thermogenesis capacity.⁴ Recently, the recruitment of beige adipocytes has been observed in WAT in response to various stimuli such as cold exposure and pharmacological interventions.⁵ Several transcription factors have been reported to induce beige fat formation, including PR domain containing 16 (PRDM16), peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), and cell death-inducing DNA fragmentation factor α-like effector A (CIDEA), as well as secretory mediators such as irisin and bone morphogenetic protein 7 (BMP7).⁶

AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and is regulated by adenosine triphosphate (ATP)-depleting conditions such as physical exercise, ischemia, and glucose deprivation.⁷ AMPK activation is induced via phosphorylation of Thr¹⁷² by liver kinase B1 (LKB1) or Ca²⁺/calmodulin-dependent protein kinase kinases (CAMKII).⁸ Activation of AMPK results in phosphorylation and inhibition of acetyl-CoA carboxylase (ACC), which plays an important role in fatty acid (FA) synthesis to promote FA oxidation and reduce fat accumulation.⁹ In addition to its metabolic effects, AMPK also regulates inflammatory cascades and alleviates chronic low-grade inflammatory diseases such as insulin resistance and atherosclerosis.¹⁰ It is reported that estradiol regulates BAT thermogenesis via hypothalamic AMPK to maintain energy homeostasis.¹¹ Thus, pharmacological and nutritional strategies to induce AMPK signaling and promote WAT browning are promising approaches in obesity management.

Alpha-lipoic acid (ALA) is a naturally occurring antioxidant that acts as a cofactor of mitochondrial respiratory chain enzymes and plays an important role in the Krebs cycle phase of mitochondrial energy metabolism.¹² It has been shown that ALA dose-dependently reduces body weight (BW) by increasing energy expenditure with the upregulation of Ucp1 mRNA in BAT, which might result from the suppression of hypothalamic AMPK activity.¹³ Recent investigation suggests that ALA induces mitochondria biogenesis and promotes the browning of WAT in human subcutaneous adipocytes.¹⁴ However, the beneficial effects of ALA in estrogen deficiency-induced obesity and its association with AMPK have not been fully established. Here, we investigated the anti-obesity effects of ALA in ovariectomy (Ovx) rats and explored whether AMPK signaling was involved in the regulation of WAT browning by ALA.

Ethical considerations

All experimental procedures conducted in the study complied with the accepted ethical guidelines of Animal Care and Use and the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and with the principles for laboratory animal use following the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised in 1996). This study was approved by the Institutional Animal Care and Use Committee of the National Defense Medical Center, Taiwan (Permission No. IACUC-16-089).

Animal preparation

Female Wistar rats (8 weeks old, 250 to 270 g) were obtained from BioLASCO Taiwan Co. Ltd. Ovx in rodents is a useful model to mimic human menopause and has been shown to cause weight gain and fat accumulation.¹⁵ In short, rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal injection) and subjected to bilateral Ovx. Small incisions were made bilaterally on the back sides to expose the ovaries. The ovaries were clamped and discarded and the uterine tubes were ligated, followed by the suturing of muscle and skin. The Sham operation consisted of anesthesia, visualization of the ovaries through incisions into the abdominal cavity, and closure of the wounds as described previously.¹⁶

Experimental groups

Rats were randomly divided into four groups: (1) Sham: rats were subjected to Sham operations (n=8); (2) Ovx: rats were ovariectomized bilaterally (n=11); (3) Ovx+ALA2: Ovx rats were administered ALA (200 mg/kg/day, gavage) (Sigma-Aldrich) beginning 1 week after operation (n=10). ALA was homogeneously suspended in 0.8% carboxymethylcellulose; and (4) Ovx+ALA3: rats were administered ALA (300 mg/kg/day, gavage) beginning 1 week after operation (n=6). After 8 weeks of treatment, rats were euthanized with pentobarbital (60 mg/kg, intraperitoneally); adipose tissues were dissected, weighed, immediately frozen in liquid nitrogen, and stored at −80 °C for further analysis.

Measurement of plasma levels of lipid profiles

Whole blood samples (1 mL) were collected by cardiac puncture and centrifuged at 12,000 g for 5 minutes at 4 °C. Plasma levels of TG, total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were detected by the Fuji DRI-CHEM 3030 analyzer (Fuji Photo Film).

Enzyme-linked immunosorbent assay for plasma irisin levels

Determination of plasma levels of irisin was performed using a commercial enzyme-linked immunosorbent assay (ELISA) kit (BioVision Inc.) according to the manufacturer’s instructions.

Histological analysis and immunohistochemistry staining

Browning of WAT mostly occurs in subcutaneous WAT depots; in rodents, the inguinal fat depot is normally used.¹⁷ Thus, inguinal WAT (iWAT) was dissected and fixed in 10% paraformaldehyde, embedded in paraffin blocks, sectioned, and serially stained with hematoxylin & eosin for histological analysis. For immunohistochemistry (IHC) staining, sections of iWAT were deparaffinized, boiled in antigen-retrieval solution, and treated with anti-UCP1 primary antibody (1:500, Abcam). The secondary antibody (1:1,000, anti-rabbit horseradish peroxidase [HRP]-conjugated) was incubated for 1 hour at room temperature. An enzymatic reaction was performed with 3,3'-diaminobenzidine tetrahydrochloride (HRP-DAB). The images were captured under a light microscope (Nikon) and photographed at ×200 magnification. Cell morphology and size were analyzed in 10 random fields per section using ImageJ software version 1.53b (National Institutes of Health).

Western blot analysis

Frozen iWAT was prepared in 200 μL of radioimmunoprecipitation assay buffer (Millipore), supplemented with 5% Protease Inhibitor Cocktail (Millipore) at 4 °C by bead homogenizer. Cellular debris was removed by centrifugation at 14,000 ×g for 20 minutes at 4 °C. The protein concentration of the supernatant was determined using a bicinchoninic acid assay kit (Thermo Scientific) following the manufacturer’s instructions. Aliquots (20 μg) of protein extract were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin in Tris‐buffered saline containing 0.1% Tween 20 (TBST) for 1 hour at room temperature. Primary antibodies against phosphorylated AMPK (p-AMPK), AMPK, phosphorylated ACC (p-ACC), ACC, LKB1 (1:1,000, Cell Signaling Technology), and β-actin (1:5,000, Cell Signaling Technology) and UCP1, PRDM16, and CIDEA (1:1,000, Abcam) were incubated at 4 °C overnight. The membranes were then probed with corresponding secondary antibodies conjugated with HRP (1:3,000, Cell Signaling Technology). The density of the individual protein bands was quantified by densitometric scanning using ImageJ software version 1.53b.

Cell culture and differentiation

3T3-L1 mouse embryo fibroblasts (American Type Culture Collection [ATCC]) were cultured as previously described.¹⁶ Cell differentiation was induced by the addition of differentiation cocktail, including 10 μg/mL insulin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX). After incubation for 3 days, the culture medium was replaced with fresh Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum and insulin (10 μg/mL) every 3 days. The cells were fully differentiated into mature adipocytes on day 9. During the differentiation process (day 0 to 9), cells were treated with ALA (100 μM) in the presence or absence of AMPK inhibitor dorsomorphin (DOR; 5 μM).

Cell viability

Cell viability was measured by a cell counting kit-8 (CCK-8) assay following the manufacturer’s instructions. Briefly, 10⁴ cells/well were placed into 96-well plates and treated with various concentrations of ALA (0, 50, 100, 250, and 400 μM) for 9 days. Cell viability was determined by colorimetry using the CCK-8 assay. Insoluble formazan crystals were dissolved in dimethyl sulfoxide, and the absorbance was measured at 490 nm.

Oil Red O staining

At the end of differentiation, cells were stained by Oil Red O (Sigma-Aldrich) for determination of lipid accumulation and visualized using bright-field microscopy. Oil droplets were eluted by administration with 100% isopropanol, and optical density was measured using a spectrophotometer at 520 nm.

Measurement of oxygen consumption

The rate of oxygen consumption (OCR) was determined using the CLARIOstar Plus microplate reader (BMG Labtech). Cells were plated in culture plates at a density of 2×10³ cells. OCR was measured after the addition of a phosphorescent oxygen probe and mineral oil (Cayman Chemical).

Statistical analysis

Statistical analysis was performed with GraphPad Prism version 8 Software (GraphPad). These data are presented as mean±standard error of the mean. A two-way analysis of variance (ANOVA) and least significant difference post hoc comparison test were used to compare the means of BW and food intake by group and time. Statistical evaluation of other data was performed with one-way ANOVA followed by the Newman-Keuls comparison method. Normality of the data was analyzed by the Shapiro-Wilk method. Differences with P<0.05 were considered statistically significant.

Availability of data and materials

The datasets generated and/or analyzed during the current study are available in the figshare repository (https://doi.org/10.6084/m9.figshare.14233997).

Effects of ALA on BW, adiposity, and food intake in Ovx rats

Nine weeks after Ovx, the BW of the Ovx group (374.3±8.2 g) was significantly increased compared to that of the Sham group (296.6±1.21 g) (P<0.05). Interestingly, the Ovx+ALA2 (333.1±7.4 g) and Ovx+ALA3 (309.3±3.7 g) groups showed significantly lower BW than those of the Ovx group (P<0.05) (Fig. 1A). A similar result was observed in BW gain (Fig. 1B). Fig. 1C shows that food intake was not significantly affected during 8 weeks of ALA treatment. Total visceral fat pad mass consists of peri-renal, retroperitoneal, and mesenteric WAT weight. Fig. 1D shows a significant increase in total visceral fat pad in the Ovx group (19.0±1.1 g) compared with the Sham group (11.2±1.4 g) (P<0.05). The increase in visceral fat pad mass in Ovx rats was not observed in the Ovx+ALA2 (16.0±1.7 g) and Ovx+ALA3 (7.9±0.6 g) groups (P<0.05), indicating that ALA drastically attenuated fat pad mass in a dose-dependent manner.

Effects of ALA treatment in plasma lipid profiles of Ovx rats

We next evaluated the effects of ALA on the lipid profiles of Ovx rats. As shown in Fig. 1E, plasma levels of TG in the Ovx group were significantly increased compared to the Sham group (P<0.05). The increase in plasma TG in Ovx rats was markedly attenuated by the administration of ALA 300 mg/kg (P<0.05) in a dose-dependent manner. Moreover, ALA supplementation markedly decreased Ovx-induced elevation of TC and LDL-C (P<0.05) (Fig. 1F and G). However, HDL-C was drastically elevated by ALA treatment in Ovx rats (P<0.05) (Fig. 1H). These results indicate that ALA has beneficial effects on lipid profiles.

Effects of ALA treatment on adipocyte size and UCP1 protein expression in iWAT of Ovx rats

Assessment of adipocyte cross-sectional area in iWAT showed that Ovx resulted in an increase in adipocyte size, and the administration of ALA reduced adipocyte hypertrophy and increased the appearance of small multilocular adipocytes (P<0.05) (Fig. 2A and B). Subsequently, IHC staining of UCP1 showed enhanced UCP1 expression in both ALA2- and ALA3-treated Ovx rats (Fig. 2C), suggesting that ALA-induced browning of iWAT produces thermogenic beige adipocytes.

ALA increased biomarkers of beige fat development in iWAT of Ovx rats

To further examine whether ALA-induced beige fat formation, we assessed the protein expression of beige fat-specific biomarkers. Western blot analysis showed that UCP1 protein expression was significantly lower in iWAT of the Ovx group than that of the Sham group (Fig. 3A). ALA treatment significantly upregulated UCP1 protein expression compared to the Ovx group (P<0.05). In addition, transcription factors that regulate the trans-differentiation of white into beige adipocytes, including PRDM16 (Fig. 3B) and CIDEA (Fig. 3C), were significantly upregulated in the Ovx+ALA2 and Ovx+ALA3 groups compared to the Ovx group. Collectively, these results indicate the potential of ALA in the conversion of white to beige fat in iWAT of Ovx rats.

Effects of ALA treatment on plasma levels of irisin in Ovx rats

Irisin is a myokine secreted from skeletal muscle to promote energy expenditure through UCP1-mediated thermogenesis and induction of WAT browning.^18,19 We investigated whether ALA treatment affected the level of circulating irisin in Ovx rats. As shown in Fig. 4A, plasma irisin levels were significantly decreased in the Ovx group compared to the Sham group (P<0.05) Additionally, ALA treatment brought about a significant increase in plasma irisin levels in Ovx rats, suggesting that ALA-induced myokine secretion promotes WAT browning.

ALA-induced the activation of AMPK signaling in iWAT of Ovx rats

AMPK is a master energy switch regulator and plays an important role in mediating the browning of WAT.²⁰ We examined whether ALA treatment affected phosphorylation levels of AMPK in iWAT of Ovx rats. As shown in Fig. 4B, the expression of p-AMPK/AMPK was significantly lower in the Ovx group than in the Sham group. ALA supplementation markedly increased the p-AMPK/AMPK ratio in Ovx rats (P<0.05). It is well established that AMPK activation phosphorylates and inhibits ACC, an essential enzyme in promoting FA oxidation and reducing fat accumulation.²¹ Fig. 4C further indicates that the p-ACC/ACC ratio was dramatically increased by ALA administration in Ovx rats. Moreover, AMPK is mainly activated by phosphorylation of the α subunit at Thr¹⁷² by LKB1 under energy stress.⁸ Intriguingly, ALA markedly increased the expression of LKB1 in Ovx rats (Fig. 4D). These results suggest that ALA induces activation of the LKB1-AMPK-ACC axis to mediate the browning of WAT.

Effects of ALA administration on lipid accumulation, OCR, and UCP1 protein expression in 3T3-L1 cells

Cell viability of ALA is shown in Fig. 5A. No significant cytotoxicity of ALA was observed below the concentration of 250 μM. Thus, 100 and 250 μM of ALA were assessed on lipid droplet accumulation using Oil Red O stain. Results indicated that both ALA 100 and ALA 250 groups had significantly reduced lipid accumulation in adipocytes (Fig. 5B). Furthermore, ALA (100 μM) treatment markedly increased OCR, and the addition of AMPK inhibitor DOR (5 μM) significantly reversed the increase of OCR by ALA (Fig. 5C). As shown in Fig. 5D, UCP1 protein expression in the ALA group was significantly higher than that of the control (CTL) group. In the ALA+DOR group, the increase of UCP1 expression was significantly reduced compared to that of ALA, indicating that AMPK plays an important role in mediating the metabolic effects of ALA.

In the present study, we showed that administration of ALA significantly suppresses BW gain and fat pad mass in Ovx rats. We also found that plasma levels of TG, TC, and LDL-C were higher and HDL-C was lower in Ovx rats, but these detrimental effects were improved by ALA supplementation. In addition, ALA treatment reduced adipocyte size and exhibited unique staining of UCP1 of iWAT. Myokine irisin secretion as well as expression of thermogenic proteins such as UCP1, PRDM16, and CIDEA were upregulated by ALA, suggesting that ALA exerts browning capacities. We further demonstrated that ALA treatment increased LKB1 and stimulated the phosphorylation of AMPK and its downstream target ACC, revealing that the beneficial effects were mediated at least in part by activation of the LKB1/AMPK pathway. Intriguingly, inhibition of AMPK reversed the induction of UCP1 caused by ALA in 3T3-L1 cells. To the best of our knowledge, this is the first study to examine the effects of ALA on estrogen deficiency-induced metabolic alterations and WAT browning via activation of LKB1/AMPK.

It is well established that oxidative stress during menopause can aggravate the inflammatory status that contributes to the detrimental effects of obesity and metabolic syndromes.¹⁵ Ovx suppresses expression of the mitochondrial proteins involved in ATP synthesis and thermogenic UCP1 in BAT and subcutaneous WAT. This contributes to impairment of mitochondrial function and heat generation, reducing energy expenditure.²² Estrogen exerts antioxidant and anti-inflammatory effects in the modulation of insulin action.²³ Moreover, reduced energy expenditure during the menopause transition may predispose to obesity and dyslipidemia.²⁴ Thus, antioxidant treatment has been proposed as a therapeutic strategy to prevent obesity and associated comorbidities.²⁵ In line with our report, Delgobo et al.²⁶ showed that ALA improved antioxidant defenses and alleviated oxidative stress, inflammation, and lipid profiles in estrogen-independent mechanisms. The enhancement of energy expenditure in BAT and the trans-differentiation of white to beige adipocytes are gaining attention for their potential to prevent the metabolic complications of obesity.²⁷ The development of beige adipocytes in WAT enhances energy expenditure and switches adipocytes from an energy storage state to an energy dissipation state via induction of UCP1⁴ to reduce the risk of metabolic diseases. We demonstrated that major transcriptional regulators of WAT browning such as PRDM16, PGC-1α, and CIDEA were upregulated by ALA (Fig. 3). PGC-1α promotes UCP1 to activate thermogenesis in these two distinct types of adipocytes,²⁸ while PRDM16 plays a critical role during the differentiation of BAT and trans-differentiation of white into beige adipocytes. Furthermore, irisin is secreted as a myokine and an adipokine into circulation following the cleavage of fibronectin type III domain-containing protein 5 (FNDC5) to induce the browning of WAT.²⁹ Emerging evidence supports the weak affect of irisin on classical BAT isolated from the interscapular depot, suggesting that activation of the thermogenic program in response to irisin is a selective characteristic of beige cells.³⁰ We found that Ovx decreased the browning of iWAT and irisin secretion, leading to weight gain after estrogen depletion (Fig. 4A), in line with our previous study.⁵ Sul et al.³¹ further indicated that exogenous administration of E₂ enhanced browning in vivo and in vitro, which might result from the antioxidant activity of E₂. ALA supplementation increased the plasma levels of irisin, suggesting that the browning effects of ALA are partly mediated via irisin production. The lower weight gain in ALA-treated Ovx rats was consistent with a previous report, which showed that the anti-obesity effects of ALA are due to the enhancement of energy expenditure.¹³ ALA supplementation reduced fat depots and the appearance of multilocular adipocytes within the iWAT, which may be attributed to the upregulation of lipolysis³² and the downregulation of adipogenesis.³³

Estrogen deficiency-induced oxidative stress alters lipid metabolism, resulting in excessive TG, TC, and LDL-C accumulation, which are common risk factors for the pathological progression of metabolic disorders.³⁴ The current study showed that ALA treatment reduced TG, TC, and LDL-C and increased HDL-C levels in Ovx rats. These results were in accordance with a previous report showing that ALA reduced TG accumulation in skeletal muscles, pancreatic islets, and adipose tissue in diabetes-prone obese rats and increased FA oxidation.³⁵ The improvement in lipid profiles by ALA may result from its antioxidative and anti-inflammatory properties.³⁶ Moreover, BAT and beige adipocytes generate heat using TG-derived FA from the circulation in a UCP1-dependent process, leading to improvement of lipid profiles and prevention of atherosclerosis progression.³⁷ Thus, the beneficial effects of ALA in lowering TG and cholesterol might be a consequence of the induction of browning in iWAT.

Activation of AMPK inhibits the synthesis of FA and enhances its oxidation by phosphorylation of its downstream target ACC,³⁸ which directs FA toward degradation. Inhibition of AMPK activity in WAT has been widely observed in obese and diabetic rodents as well as in human subjects with insulin resistance.³⁹ Deletion of AMPKα1 impaired the thermogenic program in BAT, possibly due to the defective unwinding of the DNA of the promoter region of the PRDM16,⁴⁰ suggesting that AMPK coordinates thermogenic BAT and beige adipocyte functions. In the present study, we showed that Ovx has inhibitory effects in AMPK signaling cascades, spanning from the upstream LKB1 to the downstream ACC, whereas ALA treatment reverses these changes.

As depicted in Fig. 6, ALA ameliorated E₂ deficiency-induced obesity and metabolic disorders via the reprogramming of white to thermogenic beige adipocytes. Activation of AMPK may contribute to the beneficial effects of ALA. Thus, ALA provides a potential preventive strategy for the improvement of metabolic dysfunctions after menopause.

The authors declare no conflict of interest.

Study concept and design: YWC; acquisition of data: YWC and TP; analysis and interpretation of data: YWC and TP; drafting of the manuscript: SYH and MTC; critical revision of the manuscript: HHS and YML; statistical analysis: YWC; obtained funding: SYH and MTC; administrative, technical, or material support: CWK, SYC, and PYC; and study supervision: HHS and YML.