AMP-activated protein kinase (AMPK) contributes to the anti-obesity effects and adipose tissue browning of alpha-lipoic acid in ovariectomized rats

Background Bilateral ovariectomy is an experimental model used to analyze the conditions of menopause and develop strategies for alleviation of the deleterious effects during estrogen deciency. Brown and beige adipocytes exert thermogenesis capacities and are promising therapeutic strategy for obesity. This study aims to investigate the adipose tissue browning potentials of antioxidant α-lipoic acid (ALA) and underlying mechanisms involved in ovariectomized (Ovx) rats. Methods: Results: Ovx group signicantly increased boy weight (BW) and fat pad mass as compared to Sham group, while ALA supplementation reversed these changes. Lipid proles including serum triglycerides (TG), total (TC) and low-density lipoprotein cholesterol (LDL-C) levels were signicantly elevated in the Ovx group, whereas the ALA treatment showed a signicant decrease in these levels. Furthermore, high density lipoprotein cholesterol (HDL-C) and myokine irisin secretion were increased by ALA as well. Morphology results showed ALA treatment reduced Ovx-induced adipocyte hypertrophy and enhanced UCP1 expression by immunohistochemical staining in inguinal WAT. Protein expression of brown fat-specic markers UCP1, PRDM16 and CIDEA was markedly reduced in Ovx rats, whereas ALA treatment reversed these changes. ALA signicantly increased liver kinase B1 (LKB1) and phosphorylation of AMP-activated protein kinase (AMPK) and the downstream acetyl-CoA carboxylase (ACC) that were decreased by Ovx, suggesting the browning effects were mediated by AMPK signaling. Conclusions: menopause white to beige adipocytes


Introduction
During menopause, women experience weight gain and re-deposition of body fat into the waist [1]. Obesity and fat accumulation increase the risk of metabolic syndromes and cardiovascular diseases, such as diabetes, dyslipidemia and hypertension [2]. Despite estrogen exerts bene cial effects against menopause associated metabolic abnormalities [3], long-term hormonal replacement therapy retains the limitations of adverse effect including breast and cervical cancer and is not primarily strategy for menopausal obesity and metabolic dysfunctions [4]. Seeking alternative therapy for menopauseassociated metabolic syndrome acquires further investigation.
In mammals, three types of adipose tissue have been identi ed based on the function and characteristics: white adipose tissue (WAT), brown adipose tissue (BAT), and beige adipose tissue [5]. WAT stores energy in the form of triglycerides and regulates whole-body energy homeostasis, while BAT dissipates energy as heat through enhanced mitochondrial content and uncoupling protein 1 (UCP1) [6].
Recently, recruitment of beige adipocytes has been observed in WAT in response to various stimuli such as cold exposure and pharmacological agents [7,8].
AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis and activated by ATPdepleting conditions such as physical exercise, ischemia, and glucose deprivation [9]. AMPK is regulated via phosphorylation of Thr 172 by upstream kinases, liver kinase B1 (LKB1) or Ca 2+ /calmodulin-dependent protein kinase kinases [10]. Activation of AMPK resulted in phosphorylation and inhibition of acetyl-CoA carboxylase (ACC), which plays an important role in fatty acid synthesis, and therefore promotes fatty acid oxidation as well as reduces fat accumulation [11]. In addition to the metabolic pathway, AMPK also regulates in ammatory cascades for improving chronic low-grade in ammatory diseases such as insulin resistance atherosclerosis [12]. Thus, therapeutics aimed at activating AMPK pathway unravels a promising approach in obesity and metabolic dysfunction.
Alpha-Lipoic acid (ALA) is a naturally occurring antioxidant which acts as a cofactor of mitochondrial respiratory chain enzymes and plays a crucial role in Krebs cycle phase of the mitochondrial energy metabolism [13]. ALA dose-dependently reduced body weight by increasing energy expenditure accompanied with enhanced Ucp1 mRNA expression in BAT, which might result from the suppression of hypothalamic AMPK activity [14].Recent investigation suggests that ALA induced mitochondria biogenesis and promoted browning of WAT in human subcutaneous adipocytes [15] ; however, its effect in vivo has not been fully established. The aim of the present study is to investigate the anti-obesity effects of ALA in Ovx rats and whether AMPK is involved in the browning capacities.
Animal Handling was in accordance with the Guide for the Care and Use of Laboratory Animals published by the US 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). To induce estrogen-insu ciency condition, rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneal injection) and subjected to bilateral ovariectomy. Small incisions were proceeded bilaterally on the back sides to expose the retroperitoneal ovaries. The ovaries were clamped and discarded, and the uterine tubes were ligated, following the suture of muscle and skin. The procedure of sham operation consisted of anesthesia, visualization of the ovaries through incisions into the abdominal cavity, and closure of the wounds as described previously [16].

Statistical analysis
These data are presented as means ± standard errors of the mean (SEM). A Two-Way ANOVA and LSD post-hoc comparison test was used to compare the means of body weight and food intake between the groups and time. Statistical evaluation of others was performed with one-way analysis of variance (ANOVA) followed by the Newman-Keuls comparison method. Differences with P < 0.05 was considered as statistical signi cance.

Results
Effects of ALA on body weight (BW) and food intake in Ovx rats As shown in Fig. 1A, nine weeks after Ovx, body weight (BW) of Ovx (374.3 ± 8.2 g) group was signi cantly increased as compared to Sham group (296.6 ± 1.21 g) (P < 0.05). Ovx + ALA2 (333.1 ± 7.4 g) and Ovx + ALA3 (309.3 ± 3.7 g) groups showed signi cantly lower levels of BW than that of Ovx group (P < 0.05). As shown in Fig. 1B, there was no signi cant difference in food intake among the groups at the time of 8-week treatment.

Effects of ALA on visceral and inguinal fat pad mass in Ovx rats
Total visceral fat pad mass was consisted of peri-renal, retroperitoneal and mesentery adipose tissue weight. Figure 1C showed Ovx groups (19.0 ± 1.1 g) exhibited signi cant increases in total visceral fat pad mass when compared with the Sham group (11.2 ± 1.4 g) (P < 0.05). The increase of visceral fat pad mass in Ovx rats was signi cantly reduced in Ovx + ALA2 (16.0 ± 1.7 g) and Ovx + ALA3 (7.9 ± 0.6 g) (P < 0.05). Similar results were observed in the inguinal fat pat mass, showing that ALA treatment drastically attenuated inguinal fat pad mass in a dose dependent manner (Fig. 1D).
ALA treatment improved lipid pro les of TG, TC, LDL-C, and HDL-C in Ovx rats We next evaluated the effects of ALA on lipid pro les of Ovx rats. As shown in Fig. 2A, serum levels of triglycerides (TG) in the Ovx group were signi cantly increased when compared to Sham group (P < 0.05).
High dose ALA treatment (300 mg/kg) markedly reduced the triglyceride levels as compared to the Ovx group (P < 0.05) Moreover, ALA supplement decreased Ovx-induced elevation of serum levels of cholesterol (TC) and LDL-C (P < 0.05) ( Fig. 2B and C). The 8-week ALA treatment in Ovx rats resulted in a signi cant increase in serum HDL-C levels (P < 0.05) (Fig. 2D).
Effects of ALA treatment on H&E staining for adipocyte sizes and immunohistochemistry for UCP1 expression in inguinal WAT in Ovx rats Adipocyte sizes of inguinal white adipose tissue (WAT) were assessed by H&E stain to examine the morphology changes. As shown in Fig. 3A & B, adipocyte sizes in the Ovx group were signi cantly greater than those of Sham group, which were dramatically diminished in the Ovx + ALA2 and Ovx + ALA3 groups (P < 0.05).
Meanwhile, by immunohistochemical (IHC) staining of UCP1, we found signi cant increase of UCP1positive stained area was observed in the Ovx + ALA2 and Ovx + ALA3 groups as compared with the Ovx group (P < 0.05) (Fig. 3C&D), suggesting that the thermogenic small multilocular beige adipocytes formation was triggered by ALA treatment.
ALA increased brown fat-regulated proteins expression in inguinal WAT of Ovx rats Consistent with IHC results, Western blot analysis in inguinal WAT showed that UCP1 protein expression was signi cantly lower in Ovx group than that of Sham group (Fig. 4A). ALA treatment signi cantly upregulated the protein expression of UCP1 as compared to Ovx group (P < 0.05). In addition, transcription factors that regulate the trans-differentiation of white into beige adipocyte including PR domain containing 16 (PRDM16) (Fig. 4B), and cell death-inducing DNA fragmentation factor alpha-like effector A (CIDEA) (Fig. 4C) were signi cantly upregulated in Ovx + ALA2 and Ovx + ALA3 groups.
Collectively, these results indicated ALAL induced browning of inguinal WAT.

Effects of ALA on serum irisin levels in Ovx rats
Irisin is an exercise-induced myokine secreted from skeletal muscle and associated with enhanced energy expenditure because of the ability to induce WAT browning [17]. We then determined whether ALA treatment stimulated the circulating levels of irisin in Ovx rats. As shown in Fig. 5, serum irisin levels were signi cantly reduced in the Ovx group as compared with the Sham group (P < 0.05), an effect that was drastically reversed by ALA treatment.
ALA induces the activation of AMPK pathway in inguinal WAT of Ovx rats AMPK activation is a master energy switch regulator and plays an important role in mediating the browning of WAT [18], we then measured changes in phosphorylation levels of AMPK in Ovx rats. As shown in Fig. 6A, the expression of p-AMPK/AMPK was signi cantly lower in Ovx group than that of Sham group. ALA supplementation markedly increased the p-AMPK/AMPK levels in Ovx rats (P < 0.05). AMPK is mainly activated by phosphorylation of the α subunit at Thr172 by liver kinase B1 (LKB1) under energy stress [10]. We found that ALA markedly increased the expression LKB1 in Ovx rats (Fig. 6B). AMPK activation phosphorylates and inhibits acetyl-CoA carboxylase (ACC), an important enzyme in promoting fatty acid oxidation and reduces fat accumulation. Figure 6C indicated p-ACC/ACC expression was dramatically increased by ALA administration in Ovx rats. These results suggested that LKB1-AMPK-ACC signaling is involved in the browning effect of ALA on iWAT.

Discussion
In the present study, we showed that administration of ALA signi cantly suppressed the elevation in body weight and fat pad mass after ovariectomy. We further indicated that serum concentration of TG, TC and LDL-C were higher and that of HDL-C was lower in Ovx rats, but these deleterious effects were ameliorated by ALA supplementation. In addition, ALA treatment reduced adipocyte size and exhibited unique staining of UCP1 in inguinal WAT. Myokine irisin secretion as well as expression of thermogenic proteins such as UCP1, PRDM16 and CIDEA were upregulated by ALA, suggesting that ALA exerts the browning capacities in Ovx rats. We further demonstrated that ALA treatment increased LKB1 and stimulated the phosphorylation of AMPK-Thr 172 and its downstream target ACC, revealing the bene cial effects are mediated at least in part by activation of the LKB1/AMPK pathway. To the best of our knowledge, this is the rst study to examine the effects of ALA on estrogen-de ciency induced metabolic alterations and WAT browning via activation of LKB1/AMPK.
Several studies have addressed that oxidative stress during menopause might aggravate the in ammatory state associated with obesity and metabolic syndromes [19]. Estrogen exerts antioxidant and anti-in ammatory effects in modulation insulin action [20]. Moreover, reduced energy expenditure during menopause transition may predispose to obesity and dyslipidemia [21]. Thus, antioxidant treatment has been proposed as a therapy strategy to prevent obesity and associated comorbidities. In line with our report, Delgobo et al., showed ALA improved antioxidant defenses and alleviated oxidative stress, in ammation and serum lipid in estrogen-independent mechanism [22]. The enhancement of energy expenditure in BAT or inducing the trans-differentiation of white to beige adipocytes because these strategies have the potential to prevent the metabolic complications of obesity [23]. Development of beige adipocytes in WAT enhances energy expenditure and switches adipocytes from an energy storage state to an energy dissipation state via expressing UCP1 [6], which reduces the risk of obesity and metabolic diseases. We demonstrated that major transcriptional regulators of WAT browning such as PRDM16, PGC-1α and CIDEA were upregulated by ALA (Fig. 4). PGC1α promotes UCP1 to activate thermogenesis in these two distinct types of adipocytes [46], 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 hormone from muscle into the circulation following cleavage of bronectin type 3 domain containing protein 5 (FNDC5) to induce browning of WAT [24]. Emerging evidence supports that irisin has little effect on classical BAT isolated from the interscapular depot, suggesting that the activation of the thermogenic program in response to irisin is a selective characteristic of beige cells [25]. We found that Ovx decreased browning of iWAT and irisin secretion, leading to weight gain after estrogen depletion (Fig. 5), this nding is in line with our previous literature [7]. Sul et al further indicated exogenous administration of E 2 enhanced browning in vivo and invitro, which might result from the anti-oxidant activity of E 2 [26]. ALA supplementation increased the serum levels of irisin, suggesting the browning effects of ALA is partly mediated via irisin production. The lower weight gain in ALA-treated Ovx rats was in accordance with previous report, which showed the anti-obesity effects by ALA is due to enhancement of energy expenditure [14]. ALA supplementation displayed the reduction of fat depots and appearance of multiocular adipocytes within the iWAT, which may be attributed to an increase in lipolysis [27] and decrease in adipogenesis [28].
Estrogen de ciency-induced augmentation of oxidative stress alters lipid metabolism, resulting in excessive TG, TC, and LDL-C accumulation, which is a common risk factor for the development metabolic dysfunction [29,30]. The current study showed ALA treatment reduced TG, TC, and LDL-C as well as increased HDL-C levels in Ovx rats (Fig. 2). These results are in accordance with previous report showing that ALA reduced TG accumulation in skeletal muscles, pancreatic islets, and adipose tissue in diabetesprone obese rats and increased fatty acid oxidation [31]. Evidence has also been shown that the improvement in lipid pro le of ALA may be associated with the antioxidative and anti-in ammatory properties [32]. Moreover, BAT and beige adipocytes generate heat by utilizing TG-derived fatty acids from the circulation in a UCP1-dependent process, leading to the improvement of lipid pro les and prevention of atherosclerosis progression [33]. Thus, the bene cial effects of ALA in lowering TG and cholesterol might act as a consequence of the induction of browning in inguinal WAT.
Activation of AMPK inhibits fatty acid synthesis and enhances fatty acid oxidation by phosphorylation of its downstream target ACC [34], which directs FAs towards degradation. Previous studies showed that reduction of pAMPK in WAT is widely observed in obese and diabetic rodents as well as in human subjects accompanied with insulin resistance [35]. In addition, deletion of AMPKα1 impaired the thermogenic program in BAT [36], which may result from the defective unwinding of the DNA of the promoter region of the PRDM16 [37]. These data suggest that activation of AMPK plays an important role in thermogenic function. In the present study, we showed that Ovx rats have reduction at several steps of the AMPK pathway, spanning from the upstream LKB1 to the downstream ACC whereas ALA treatment reversed these deleterious changes during estrogen de ciency (Fig. 6). ALA promoted beige adipocytes development in rats with Ovx and activation of AMPK signaling plays an essential role in mediating the browning effect of ALA.

Conclusion
In conclusion, as shown in Fig. 7, our present study indicated that ALA effectively reduced E 2 de ciencyinduced obesity via reprogramming of white to theromogenic beige adipocytes concomitant with the activation of AMPK signaling. Thus, ALA provides a potential preventive strategy for improving postmenopausal obesity.

Consent for publication: Not applicable
Availability of data and materials: The datasets generated and/or analyzed during the current study are available in the gshare repository, 10.6084/m9. gshare.14233997. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.