The branched-chain amino acids (BCAA) accumulated in type 2 diabetes are independent contributors to insulin resistance. The activity of branched-chain α-keto acid dehydrogenase (BCKD) complex, rate-limiting enzyme in BCAA catabolism, is reduced in diabetic states, which contributes to elevated BCAA concentrations. However, the mechanisms underlying decreased BCKD activity remain poorly understood. Here, we demonstrate that mitochondrial phosphatase 2C (PP2Cm), a newly identified BCKD phosphatase that increases BCKD activity, was significantly downregulated in ob/ob and type 2 diabetic mice. Interestingly, in adiponectin (APN) knockout (APN−/−) mice fed with a high-fat diet (HD), PP2Cm expression and BCKD activity were significantly decreased, whereas BCKD kinase (BDK), which inhibits BCKD activity, was markedly increased. Concurrently, plasma BCAA and branched-chain α-keto acids (BCKA) were significantly elevated. APN treatment markedly reverted PP2Cm, BDK, BCKD activity, and BCAA and BCKA levels in HD-fed APN−/− and diabetic animals. Additionally, increased BCKD activity caused by APN administration was partially but significantly inhibited in PP2Cm knockout mice. Finally, APN-mediated upregulation of PP2Cm expression and BCKD activity were abolished when AMPK was inhibited. Collectively, we have provided the first direct evidence that APN is a novel regulator of PP2Cm and systematic BCAA levels, suggesting that targeting APN may be a pharmacological approach to ameliorating BCAA catabolism in the diabetic state.
Introduction
The branched-chain amino acids (BCAA) are essential amino acids such as leucine, isoleucine, and valine; their homeostasis is determined largely by catabolic activities in a number of organs including liver, muscle and adipose tissue (1–3). The first step of BCAA catabolism generates a set of corresponding branched-chain α-keto acids (BCKA), which are irreversibly decarboxylated by the branched-chain α-keto acid dehydrogenase (BCKD) complex (4). As with most nutrients, maintaining of the physiological level of BCAA is critical for cell metabolism and survival. However, many researchers have described increased BCAA and BCKA levels in diabetes and obesity (3,5–8). Furthermore, BCAA and their catabolites are strongly associated with insulin resistance (9–11), and elevated BCAA contributes to the development of insulin resistance (10,12). Mechanistically, elevated BCAA levels activate mTOR/p70S6 kinase, resulting in an increased I insulin receptor substrate-1 phosphorylation, thereby inhibiting phosphatidylinositol 3-kinase. This inhibition of phosphatidylinositol 3-kinase in turn leads to impaired insulin signaling (13,14). It is also reported that BCAA are independent predictors of insulin resistance, diabetes, and cardiovascular events (15–17). Therefore, it is necessary to determine the mechanisms of abnormal BCAA catabolism in order to better understand their association with metabolic-related pathogenesis.
The BCKD complex is the rate-limiting enzyme in BCAA catabolism (4,12); regulation of BCKD activity is therefore important for maintaining the homeostasis of systemic BCAA and BCKA. The complex consists of three catalytic components: a heterotetrameric (α2β2) branched-chain α-keto acid decarboxylase (E1), a homo-24 meric dihydrolipoyltransacylase (E2), and a homodimeric dihydrolipoamide dehydrogenase (E3). The activity of BCKD complex is controlled by the reversible phosphorylation of its E1a subunit (Ser293) by specific BCKD kinase (BDK) and phosphatase (BDP), respectively (4,18). Phosphorylation catalyzed by BDK inhibits the enzymatic activity of the BCKD complex, whereas it becomes activated when the Ser293 residue is dephosphorylated by BDP. A series of reports demonstrated that activation of BCKD was reduced in liver and adipose tissue, resulting in increased plasma BCAA and BCKA concentrations in diabetic and obese animals (6–8). Moreover, alterations of metabolism can influence BCKD activity partly through changes in BDK (3), suggesting that BDP might be suppressed. The mitochondrial phosphatase 2C (PP2Cm) is the only identified BDP (18,19), which specifically binds the BCKD complex and induces dephosphorylation of BCKD at Ser293 in the presence of BCKD substrates (18). Additionally, PP2Cm deficiency impairs BCAA catabolism, leading to elevated plasma BCAA and BCKA concentrations (18). However, the relationship between PP2Cm and reduced diabetic BCKD activity has not yet been investigated.
Adiponectin (APN) is an adipocytokine predominantly synthesized in and secreted from adipose tissue. APN helps regulating glucose and lipid metabolism (20,21) and has vascular/cardioprotective effects (22,23). More recently, Liu et al. (24) reported that APN corrects altered muscle BCAA metabolism induced by a high-fat diet (HD). In addition, several observations have revealed that obesity and type 2 diabetes are associated with decreased plasma APN levels (20,25,26). However, the correlation between decreased APN levels and reduced BCKD activity has never been investigated. More importantly, the underlying molecular mechanisms by which APN mediates disturbed BCAA catabolism in diabetes are completely unknown.
In the current study, we used both in vitro and in vivo experiments to identify hypoadiponectinemia as a contributing factor to reduced BCKD activity in diabetes. In diabetic mice, BCKD activity was reduced, and BCAA and BCKA levels were significantly elevated. APN treatment effectively reversed these pathological alterations, which were completely abolished by inhibition of AMPK and partially but significantly attenuated by knockout of PP2Cm expression. These findings lead us to conclude that impaired APN signaling is an important part of the underlying mechanism for disturbed BCAA catabolism in type 2 diabetes.
Research Design and Methods
Animal Care and Drug Treatment
All experiments were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals and were approved by the Fourth Military Medical University Committee on Animal Care. Male ob/ob and wild-type (WT) C57BL/6 control mice were purchased from the Department of Pathology, the Fourth Military Medical University. Male APN knockout (APN−/−) mice (22) and WT C57BL/6 mice on the same background have previously been described. The whole-body PP2Cm knockout (PP2Cm−/−) mice (18,27) were a gift from Yibin Wang of University of California, Los Angeles (Los Angeles, CA).
Mice were rendered type 2 diabetic by the following procedures. Four-week-old WT C57BL/6 mice were fed with an HD (60% of kcal from fat; Research Diets, New Brunswick, NJ) for 6 weeks and injected with a low dose of streptozotocin (STZ) twice (28) (25 μg/g body wt i.p.; STZ in 0.05 mol/L sodium citrate, pH 4.5, once daily; Sigma, St. Louis, MO). Blood glucose and body weight were measured daily, and a diabetic condition was confirmed at 4 weeks after STZ injection by a nonfasting blood glucose level of ≥200 mg/dL. Fasting blood insulin was measured, and intraperitoneal glucose tolerance test (IPGTT) and insulin tolerance test (ITT) were carried out in each successful model group.
In some experiments, 7-week-old APN−/− and WT mice were randomized to receive a normal chow diet (ND) (12% of kcal from fat; control) or HD (45% of kcal from lard; Research Diets) for 4 weeks. Additionally, animals received different treatment as follows: 1) ob/ob, HD-fed APN−/− and PP2Cm−/− mice received vehicle (PBS) or APN (5 μg/g body wt; PeproTech, Rocky Hill, NJ) and 2) type 2 diabetic mice received vehicle, APN (5 μg/g body wt), AMPK activator AICAR (150 μg/g body wt; Sigma, St. Louis, MO), or APN in conjunction with the AMPK inhibitor compound C (CC) (20 μg/g body wt; Sigma) once daily for an additional 3 days. After 12 h of fasting, animals were killed for collection of tissues (liver, epididymal fat pad, and gastrocnemius muscle) and 0.5 mL aortic blood at 10–12 weeks of age.
Cell Culture and BCKA Challenge
AML12 mouse hepatocytes (American Type Culture Collection, Manassas, VA) were cultured in DMEM medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine plasma (HyClone, Waltham, MA). Experiments were carried out at three or four cell passages. The hepatocytes were transfected by small interfering RNA (siRNA) and incubated in DMEM medium (Invitrogen) with an additional mixture of all of the BCKA (Sigma) at 2.5 mmol/L. Each set of cells was randomized to receive treatment with 10 μg/mL APN or control vehicle. After 1 h of treatment, cells and supernatants were collected for Western blotting and BCKA analysis.
RNA Interference
siRNA constructs against AMPK α1 or AMPK α2 mRNA were designed and purchased from Gene Pharma (Shanghai, China). The siRNA sequences are as follows: AMPKα1, sense 5′-GCCGACCCAAUGAUAUCAUTT-3′, antisense 5′-AUGAUAUCAUUGGGUCGGCTT-3′; AMPKα2, sense 5′-GGACAGGGAAGCCUUAAAUTT-3′, antisense 5′-AUUUAAGGCUUCCCUGUCCTT-3′; and scrambled siRNA, sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT-3′.
Mouse hepatocytes were transfected with siRNA by using Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions. Efficiency of gene knockdown was confirmed using Western blotting 48 h after siRNA transfection.
BCAA and BCKA Analysis
Plasma and supernatant of cultured cells were collected and subsequently stored at −80°C. Determination of BCAA concentrations was performed in triplicate using a commercially available BCAA detection kit (Biovision, Milpitas, CA) per the manufacturer's instructions. BCKA concentrations were determined by HPLC as described by Loï et al. (29).
BCKD Enzyme Activity Assays
Tissue extraction and assessment of BCKD activity were performed as previously described (30). BCKD complex was concentrated from whole tissue extracts using 9% polyethylene glycol. BCKD activity was determined spectrophotometrically by measuring the rate of NADH production resulting from the conversion of α-keto-isovalerate to isobutyryl-CoA. A unit of enzyme activity was defined as 1 μmol NADH formed per minute at 30°C.
Western Blot Analysis
Proteins were separated on SDS-PAGE gels, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA), and incubated overnight at 4°C with antibodies directed against AMPKα (1:1,000; CST, Danvers, MA), AMPKα1 (1:1,000; CST), AMPKα2 (1:1,000; CST), phospho-AMPKα (Thr172, 1:1,000; CST), PP2Cm (1:1,000; a gift from Yibin Wang, University of California, Los Angeles), BDK (1:2,000; Abcam, Cambridge, MA), phospho-BCKD E1α (1:1,000; Bethyl Laboratories, Montgomery, TX), BCKD E1α (1:500; Santa Cruz Biotechnology, Dallas, TX), and GAPDH (1:5,000; Zhong Shan Golden Bridge Biotechnology, Beijing, China). After washing to remove excess primary antibody, blots were incubated for 1 h with horseradish peroxidase–conjugated secondary antibody. Binding was detected via enhanced chemiluminescence (Millipore). Films were scanned with ChemiDocXRS (Bio-Rad Laboratory, Hercules, CA). Densitometry was performed using Laboratory Image software.
Statistical Analysis
All data are presented as means ± SEM of n independent experiments. All data (except densitometry) was subjected to ANOVA followed by a Bonferroni correction for a post hoc t test. Densitometry was analyzed using the Kruskal-Wallis test, followed by a Dunn post hoc test. Probabilities of ≤0.05 were considered statistically significant.
Results
PP2Cm Is Downregulated in ob/ob and Type 2 Diabetic Mice
Consistent with previous reports, plasma BCAA and BCKA levels were significantly increased in both ob/ob and type 2 diabetic mice compared with WT mice (Fig. 1A and B). Liver is the primary metabolic clearing house of BCKA; the reduction of BCKD activity causes increased circulating BCAA and BCKA concentrations (3). In addition, immunoreactivity of the BCKD pSer293 antibody is directly correlated with BCKD activity (18); we therefore examined the activity of BCKD and phosphorylation of BCKD at Ser293 in liver. We found that BCKD activity was significantly reduced and phosphorylation of BCKD was significantly increased in diabetic animals compared with WT mice (Fig. 1C–E and H). Interestingly, the newly identified BCKD phosphatase, PP2Cm, was markedly decreased in diabetic mice (Fig. 1F and H), indicating that downregulation of PP2Cm may be involved in reduced BCKD activity in diabetes.
PP2Cm is downregulated in ob/ob and type 2 diabetic mice. A and B: Plasma BCAA and BCKA concentrations in WT, ob/ob, and type 2 diabetic mice. C and D: Analysis of BCKD activity. E: Quantification of pSer293 BCKD E1α. PP2Cm (F) and BDK (G) protein levels in liver, adipose tissue, and skeletal muscle from WT, ob/ob, and type 2 diabetic mice. H: Representative immunoblots of pSer293 BCKD E1α, BCKD E1α, PP2Cm, BDK, and GAPDH in WT, ob/ob, and type 2 diabetic mice. All results are presented as mean ± SEM. *Significant difference between ob/ob or type 2 diabetic group versus WT group. *P < 0.05, **P < 0.01. n = 6–8. AT, adipose tissue; AU, arbitrary unit; L, liver; SM, skeletal muscle.
PP2Cm is downregulated in ob/ob and type 2 diabetic mice. A and B: Plasma BCAA and BCKA concentrations in WT, ob/ob, and type 2 diabetic mice. C and D: Analysis of BCKD activity. E: Quantification of pSer293 BCKD E1α. PP2Cm (F) and BDK (G) protein levels in liver, adipose tissue, and skeletal muscle from WT, ob/ob, and type 2 diabetic mice. H: Representative immunoblots of pSer293 BCKD E1α, BCKD E1α, PP2Cm, BDK, and GAPDH in WT, ob/ob, and type 2 diabetic mice. All results are presented as mean ± SEM. *Significant difference between ob/ob or type 2 diabetic group versus WT group. *P < 0.05, **P < 0.01. n = 6–8. AT, adipose tissue; AU, arbitrary unit; L, liver; SM, skeletal muscle.
Considerable evidence indicates that besides liver, adipose tissue, and skeletal muscle also play an important role in modulating circulating BCAA homeostasis (1,2); thus, the following experiments were performed. As shown in Fig. 1, BCKD activity was markedly downregulated in diabetic adipose tissue and skeletal muscle (Fig. 1C–E and H). Additionally, PP2Cm protein levels were significantly reduced in diabetic adipose tissue (Fig. 1F and H). To our surprise, changes of PP2Cm expression in skeletal muscle were not observed in diabetic animals (Fig. 1F and H), indicating that skeletal muscle may have a relatively low level of PP2Cm-dependent BCKD activation. Since the activity of BCKD complex is increased by PP2Cm and inhibited by BDK, we than analyzed expression of BDK. Our experimental results demonstrated that BDK expression was significantly increased in diabetic tissues, including liver and adipose tissue as well as skeletal muscle (Fig. 1G and H). These results indicate that decreased PP2Cm and increased BDK may both contribute to reduced BCKD activity in diabetic liver and adipose tissue, whereas increased BDK may be the primary cause of decreased BCKD activity in diabetic skeletal muscle.
APN Deficiency Contributes to Decreased BCKD Activity
It is known that APN reverts altered BCAA metabolism in muscle (24). In order to determine whether APN contributes to BCKD activity, 7-week-old WT and APN−/− mice were fed with either ND or 45% HD for a further 4 weeks, resulting in four subgroups: ND WT, HD WT, ND APN−/−, and HD APN−/−. As shown in Fig. 2, APN−/− mice revealed the significant increase of plasma BCAA, BCKA, and hepatic BCKD phosphorylation levels but reduction of BCKD activity in response to HD. No response was observed in WT mice with the same genetic background (Fig. 2A–E and Supplementary Fig. 2). More importantly, hepatic PP2Cm was markedly decreased in HD-fed APN−/− mice (Fig. 2F and Supplementary Fig. 2). We then sought to determine whether treatment with APN could significantly reverse altered BCKD activity. HD-fed APN−/− mice were given recombinant APN at 5 μg/g body wt once daily for an additional 3 days after the initial 4-week HD-feeding period. As we expected, treatment with APN completely corrected plasma BCAA and BCKA, hepatic BCKD activity, BCKD phosphorylation, and PP2Cm protein levels (Fig. 2L–Q and T). However, plasma glucose levels, glucose tolerance, insulin tolerance, and cholesterol concentrations did not show a difference between APN and vehicle-treated mice (Fig. 2H–K). Collectively, these findings demonstrate that APN deficiency is responsible for downregulated BCKD activity and PP2Cm expression.
APN deficiency contributes to decreased BCKD activity. Plasma BCAA and BCKA (A and B) and BCKD (C and D) activity and quantification of pSer293 BCKD E1α (E), PP2Cm (F), and BDK (G) protein levels in liver, adipose tissue, and skeletal muscle from WT and APN−/− mice fed with ND or HD. H: Analysis of blood glucose concentrations. I: Glucose tolerance tested by IPGTT. Insulin tolerance tested by ITT (J), blood cholesterol levels (K), and plasma BCAA and BCKA concentrations (L and M) in HD-fed APN−/− mice after vehicle or APN treatment. Detection of BCKD activity (N and O) and quantitative results of pSer293 BCKD E1α (P), PP2Cm (Q), and BDK protein level (R) testing in liver, adipose tissue, and skeletal muscle from HD-fed APN−/− mice injected with APN. S: Representative Western blots of pSer293 BCKD E1α, BCKD E1α, PP2Cm, BDK, and GAPDH in ND or HD-fed WT and APN−/− mice. T: Representative immunoblots of pSer293 BCKD E1α, BCKD E1α, PP2Cm, BDK, and GAPDH in HD-fed APN−/− mice injected with APN or vehicle. All results are presented as mean ± SEM. *Significant difference between APN−/− and WT mice fed with HD. %Significant difference between APN and vehicle treatment. * and %P < 0.05, ** and %%P < 0.01. n = 6–8. AT, adipose tissue; AU, arbitrary unit; L, liver; SM, skeletal muscle.
APN deficiency contributes to decreased BCKD activity. Plasma BCAA and BCKA (A and B) and BCKD (C and D) activity and quantification of pSer293 BCKD E1α (E), PP2Cm (F), and BDK (G) protein levels in liver, adipose tissue, and skeletal muscle from WT and APN−/− mice fed with ND or HD. H: Analysis of blood glucose concentrations. I: Glucose tolerance tested by IPGTT. Insulin tolerance tested by ITT (J), blood cholesterol levels (K), and plasma BCAA and BCKA concentrations (L and M) in HD-fed APN−/− mice after vehicle or APN treatment. Detection of BCKD activity (N and O) and quantitative results of pSer293 BCKD E1α (P), PP2Cm (Q), and BDK protein level (R) testing in liver, adipose tissue, and skeletal muscle from HD-fed APN−/− mice injected with APN. S: Representative Western blots of pSer293 BCKD E1α, BCKD E1α, PP2Cm, BDK, and GAPDH in ND or HD-fed WT and APN−/− mice. T: Representative immunoblots of pSer293 BCKD E1α, BCKD E1α, PP2Cm, BDK, and GAPDH in HD-fed APN−/− mice injected with APN or vehicle. All results are presented as mean ± SEM. *Significant difference between APN−/− and WT mice fed with HD. %Significant difference between APN and vehicle treatment. * and %P < 0.05, ** and %%P < 0.01. n = 6–8. AT, adipose tissue; AU, arbitrary unit; L, liver; SM, skeletal muscle.
Since BCKD activity is also significantly reduced in adipose tissue and skeletal muscle, we thus determined the effect of APN knockout upon BCKD activity in these tissues. Compared with HD-fed WT mice, HD-fed APN−/− mice revealed greater reduction of BCKD activity in adipose tissue and skeletal muscle (Fig. 2C–E and Supplementary Fig. 2), which were completely corrected by APN treatment (Fig. 2N–P and T). Interestingly, there was no change of PP2Cm level in skeletal muscle but not in adipose tissue from HD-fed APN−/− mice (Fig. 2F and Supplementary Fig. 2). APN treatment markedly increased the downregulated PP2Cm expression in adipose tissue (Fig. 2Q and T). In addition, BDK expression was significantly increased in liver, skeletal muscle, and adipose tissue from HD-fed APN−/− mice (Fig. 2G and Supplementary Fig. 2), which was reverted by APN treatment (Fig. 2R and T). Collectively, these findings demonstrate for the first time that APN deficiency causes downregulated BCKD activity and PP2Cm expression and upregulated BDK levels.
PP2Cm Deficiency Partially Inhibits APN-Activated BCKD
Data have shown that plasma APN concentration is significantly reduced in diabetic patients and animals (20,26). Consequently, we sought to determine whether exogenous APN administration could change BCKD activity in diabetic mice. Diabetic animals were injected with vehicle or APN (5 μg/g body wt, daily) for 3 days. As illustrated in Fig. 3, plasma glucose levels, glucose tolerance, and insulin tolerance did not show a difference between APN and vehicle-treated mice (Fig. 3A–C). As we expected, there was significant decrease in BCAA and BCKA levels but increase in BCKD activity in APN-treated mice compared with those only treated with vehicle (Fig. 3D–H). In addition, APN treatment significantly increased PP2Cm protein expression in diabetic liver and adipose tissue but not in skeletal muscle (Fig. 3I). Injection with APN markedly reduced BDK levels in diabetic animals (Fig. 3J). Taken together, these results indicate that APN can increase downregulated BCKD activity and ameliorate disturbed BCAA catabolism during diabetes.
PP2Cm deficiency partially inhibits APN-activated BCKD. Analysis of blood glucose concentrations (A), glucose tolerance tested by IPGTT (B), and insulin tolerance tested by ITT in type 2 diabetic animals administered with APN or vehicle (C). BCAA and BCKA concentrations in plasma (D and E) and BCKD activity (F and G) in liver, adipose tissue, and skeletal muscle from ob/ob, type 2 diabetic, and PP2Cm−/− mice, each injected with APN or vehicle. H: Western blot for hepatic BCKD E1α phosphorylation in ob/ob, type 2 diabetic, and PP2Cm−/− mice, each administered with APN. PP2Cm (I) and BDK (J) protein levels were detected by Western blot in liver, adipose tissue, and skeletal muscle from ob/ob and type 2 diabetic mice injected with APN. K: BDK levels were assessed by Western blot in liver, adipose tissue, and skeletal muscle from PP2Cm−/− mice treated with APN. All results are presented as mean ± SEM. %Significant difference between APN- and vehicle-injected group. &Significant difference between APN treated ob/ob and PP2Cm−/− group. % and &P < 0.05, %% and &&P < 0.01. n = 5–8. A, APN; AT, adipose tissue; AU, arbitrary unit; L, liver; SM, skeletal muscle; V, vehicle.
PP2Cm deficiency partially inhibits APN-activated BCKD. Analysis of blood glucose concentrations (A), glucose tolerance tested by IPGTT (B), and insulin tolerance tested by ITT in type 2 diabetic animals administered with APN or vehicle (C). BCAA and BCKA concentrations in plasma (D and E) and BCKD activity (F and G) in liver, adipose tissue, and skeletal muscle from ob/ob, type 2 diabetic, and PP2Cm−/− mice, each injected with APN or vehicle. H: Western blot for hepatic BCKD E1α phosphorylation in ob/ob, type 2 diabetic, and PP2Cm−/− mice, each administered with APN. PP2Cm (I) and BDK (J) protein levels were detected by Western blot in liver, adipose tissue, and skeletal muscle from ob/ob and type 2 diabetic mice injected with APN. K: BDK levels were assessed by Western blot in liver, adipose tissue, and skeletal muscle from PP2Cm−/− mice treated with APN. All results are presented as mean ± SEM. %Significant difference between APN- and vehicle-injected group. &Significant difference between APN treated ob/ob and PP2Cm−/− group. % and &P < 0.05, %% and &&P < 0.01. n = 5–8. A, APN; AT, adipose tissue; AU, arbitrary unit; L, liver; SM, skeletal muscle; V, vehicle.
To further determine whether PP2Cm is required by APN to mediate its effects on BCKD activity, we treated PP2Cm−/− mice with vehicle or APN. Interestingly, the positive effects of APN on BCKD were reduced but not completely lost in PP2Cm−/− mice. Specifically, plasma BCAA and BCKA concentrations were significantly higher, but BCKD activity was markedly lower in PP2Cm−/− mice treated with APN compared with those in APN-treated diabetic and WT control groups (Fig. 3D–H and Supplementary Fig. 3A–C). These data suggested that other factors may also contribute the stimulatory effect of APN upon BCKD. Indeed, APN treatment markedly attenuated the BDK expression in PP2Cm−/− mice compared with vehicle-treated ones (Fig. 3K). Taken together, these observations support the hypothesis that APN can regulate PP2Cm (upregulating) and BDK (downregulating) in the opposite ways, thus increasing BCKD activity and ameliorating disturbed BCAA catabolism in diabetic disease.
AMPK Is Necessary for APN-Mediated BCKD Activation
Considerable evidence exists that AMPK acts as an integrator of nutritional and hormonal signals that monitor systemic and cellular energy status (31). We then investigated whether AMPK contributes to decreased BCKD activity in diabetes. Consistent with previous reports, phosphorylated AMPK was markedly downregulated in type 2 diabetic mice (Fig. 4A). We treated these mice with AICAR (21) (a cell-permeable activator of AMPK [150 μg/g body wt daily]) for 3 days. As shown in Fig. 4, AICAR treatment resulted in a significant decrease in BCAA and BCKA concentrations while augmenting BCKD activity and PP2Cm in diabetic mice liver (Fig. 4B–G). To determine whether adipose tissue and skeletal muscle contribute to system BCAA homeostasis in response to AICAR treatment, the following experiments were conducted. Treatment with AICAR significantly increased the activity of BCKD in diabetic adipose tissue and skeletal muscle (Fig. 4D and E). Moreover, AICAR injection significantly upregulated PP2Cm protein levels in adipose tissue but did not influence PP2Cm expression in skeletal muscle from diabetic animals (Fig. 4G). However, AICAR caused a significant reduction of BDK in diabetic skeletal muscle, as well as in liver and adipose tissue (Fig. 4H). Altogether, these results suggest that AMPK may be an endogenous regulator of BCKD activity and PP2Cm and BDK expression.
AMPK is necessary for APN-mediated BCKD activation. A: Expression of phospho-AMPKα at Thr172 and AMPKα levels in liver, adipose tissue, and skeletal muscle from type 2 diabetic mice. Left: Phospho-AMPKα, AMPKα, and GAPDH protein levels were assessed by Western blot analysis. Right: Quantification of Western blot data. BCAA and BCKA concentrations in plasma (B and C) and BCKD activity (D and E) in liver, adipose tissue, and skeletal muscle from type 2 diabetic mice treated with AICAR. F: Immunoblotting of hepatic BCKD E1α phosphorylation levels in type 2 diabetic mice treated with AICAR. PP2Cm (G) and BDK (H) protein levels were measured by Western blot in liver, adipose tissue, and skeletal muscle from type 2 diabetic mice treated with AICAR or vehicle. Analysis of BCAA and BCKA in plasma (I and J) and BCKD activity (K and L) in liver, adipose tissue, and skeletal muscle from type 2 diabetic animals treated with APN or APN plus CC. M: Hepatic BCKD E1α phosphorylation levels in type 2 diabetic mice injected with APN or APN plus CC. PP2Cm (N) and BDK (O) expression was detected in type 2 diabetic animals treated with APN or APN plus CC. All results are presented as mean ± SEM. *Significant difference between type 2 diabetic and normal groups. %Significant difference between AICAR and vehicle treatment. &Significant difference between APN and APN plus CC treatment. % and &P < 0.05; **, %%, and &&P < 0.01. n = 5–8. A, APN; AT, adipose tissue; AU, arbitrary unit; CC, compound C; L, liver; SM, skeletal muscle; V, vehicle.
AMPK is necessary for APN-mediated BCKD activation. A: Expression of phospho-AMPKα at Thr172 and AMPKα levels in liver, adipose tissue, and skeletal muscle from type 2 diabetic mice. Left: Phospho-AMPKα, AMPKα, and GAPDH protein levels were assessed by Western blot analysis. Right: Quantification of Western blot data. BCAA and BCKA concentrations in plasma (B and C) and BCKD activity (D and E) in liver, adipose tissue, and skeletal muscle from type 2 diabetic mice treated with AICAR. F: Immunoblotting of hepatic BCKD E1α phosphorylation levels in type 2 diabetic mice treated with AICAR. PP2Cm (G) and BDK (H) protein levels were measured by Western blot in liver, adipose tissue, and skeletal muscle from type 2 diabetic mice treated with AICAR or vehicle. Analysis of BCAA and BCKA in plasma (I and J) and BCKD activity (K and L) in liver, adipose tissue, and skeletal muscle from type 2 diabetic animals treated with APN or APN plus CC. M: Hepatic BCKD E1α phosphorylation levels in type 2 diabetic mice injected with APN or APN plus CC. PP2Cm (N) and BDK (O) expression was detected in type 2 diabetic animals treated with APN or APN plus CC. All results are presented as mean ± SEM. *Significant difference between type 2 diabetic and normal groups. %Significant difference between AICAR and vehicle treatment. &Significant difference between APN and APN plus CC treatment. % and &P < 0.05; **, %%, and &&P < 0.01. n = 5–8. A, APN; AT, adipose tissue; AU, arbitrary unit; CC, compound C; L, liver; SM, skeletal muscle; V, vehicle.
Since AMPK activation has been recognized as a mechanism of action for APN (20,32), we therefore examined whether AMPK contributes to APN-activated BCKD in diabetic mice. To do so, type 2 diabetic mice were treated in three groups: vehicle, APN (5 μg/g body wt daily), and APN plus CC (a potent AMPK inhibitor [30 min before APN injection; 20 μg/g body wt daily]) for 3 days. As per our expectations, compound C (CC) completely blocked the effect of APN on diabetic BCAA, BCKA, and BCKD activity and PP2Cm and BDK expression (Fig. 4I–O). These findings indicate that AMPK is necessary for APN-activated BCKD in vivo.
The remote actions of AMPK could potentially have secondary effects on BCKD. Moreover, liver has an extremely high BCKD activity compared with other tissues (3,33). Thus, in vitro experiments were performed in mouse hepatocytes. BCKA-challenged mouse hepatocytes were incubated with vehicle, APN (10 μg/mL), APN (10 μg/mL) + CC (added 30 min before APN treatment [20 pmol/mL]), or AICAR (2 pmol/mL) for 1 h. As shown in Supplementary Fig. 4, AICAR resulted in significantly reduced BCKA and phosphorylated BCKD levels and increased PP2Cm protein expression; in contrast, coincubation with APN and CC had no effect on BCKA catabolism (Supplementary Fig. 4E–H). Taken together, these data directly demonstrate that AMPK contributes to APN-activated BCKD and BCAA catabolism in mouse hepatocytes.
AMPKα2 Contributes to Total AMPKα Activity in APN-Stimulated BCKD Activation
AMPK exists as a heterotrimeric complex consisting of a catalytic α subunit and two regulatory β and γ subunits. Phosphorylation of Thr172 in AMPKα is associated with activation of both the α1 and α2 subunits of AMPK (34). Thus, we investigated the relative contributions of AMPKα2 and AMPKα1 to total AMPKα activity in BCKD of mouse hepatocytes challenged with BCKA. Mouse hepatocytes were transfected with AMPKα2- or AMPKα1-specific siRNA, and AMPKα2 or AMPKα1 levels were examined by Western blot. As depicted in Fig. 5A, siRNA transfection reduced the levels of AMPKα2 and AMPKα1 protein by 81% and 83%, respectively. Importantly, PP2Cm expression and BCKD activity were reduced, and BCKA accumulated in the AMPKα2 siRNA knockdown group (Fig. 5A–D).
AMPKα2 contributes to total AMPKα activity in APN-stimulated BCKD activation. A: Representative immunoblots of hepatocyte lysate for pSer293 BCKD E1α, BCKD E1α, AMPKα1, AMPKα2, PP2Cm, and GAPDH after transfection with scramble, AMPKα1, or AMPKα2 siRNA. The relative levels of PP2Cm (B) and pSer293 BCKD E1α (C) were quantified, and BCKA concentrations (D) were analyzed in hepatocytes transfected with scramble, AMPKα1, or AMPKα2 siRNA. PP2Cm protein (E), BCKD E1α phosphorylation (F), and BCKA levels (G) in scramble, AMPKα1, or AMPKα2 siRNA transfected hepatocytes treated with or without APN for 1 h. All results are presented as mean ± SEM. **Significant difference between scramble siRNA and AMPKα2 siRNA group. %Significant difference between APN- and vehicle-treated group. %P < 0.05, ** and %%P < 0.01. n = 6–12 wells. AU, arbitrary unit.
AMPKα2 contributes to total AMPKα activity in APN-stimulated BCKD activation. A: Representative immunoblots of hepatocyte lysate for pSer293 BCKD E1α, BCKD E1α, AMPKα1, AMPKα2, PP2Cm, and GAPDH after transfection with scramble, AMPKα1, or AMPKα2 siRNA. The relative levels of PP2Cm (B) and pSer293 BCKD E1α (C) were quantified, and BCKA concentrations (D) were analyzed in hepatocytes transfected with scramble, AMPKα1, or AMPKα2 siRNA. PP2Cm protein (E), BCKD E1α phosphorylation (F), and BCKA levels (G) in scramble, AMPKα1, or AMPKα2 siRNA transfected hepatocytes treated with or without APN for 1 h. All results are presented as mean ± SEM. **Significant difference between scramble siRNA and AMPKα2 siRNA group. %Significant difference between APN- and vehicle-treated group. %P < 0.05, ** and %%P < 0.01. n = 6–12 wells. AU, arbitrary unit.
We further examined whether AMPKα2 is involved in APN-induced BCKD activation and PP2Cm expression. Mouse hepatocytes transfected with either AMPKα2 or AMPKα1 siRNA were challenged with BCKA and then treated with vehicle or APN (10 μg/mL) for 1 h. As shown in Fig. 5, AMPKα2 siRNA completely blocked the effects of APN on PP2Cm expression, BCKD activity, and BCKA levels. This effect was not seen with scrambled siRNA or AMPKα1 siRNA transfection (Fig. 5E, F, and G). Overall, these results directly demonstrate that AMPKα2 deletion/inactivation modulates BCKD activity and that the PP2Cm regulatory function of APN is dependent on AMPKα2 in vitro.
Discussion
Here, we have made several important observations. First, we validated that PP2Cm was downregulated in diabetic mice, which may, at least in part, contribute to reduced BCKD activity potentially caused by an APN deficiency. Second, APN treatment reverted downregulated PP2Cm expression and BCKD activity, as well as elevated plasma BCAA and BCKA levels in diabetic mice. However, the increased BCKD activity mediated by APN treatment was partially abolished in PP2Cm−/− mice. Third, the downregulated BDK level was involved in APN-activated BCKD complex, especially in skeletal muscle. Last, AMPK in diabetic mice was significantly impaired, which results in reduced BCKD activity and accumulation of BCAA and BCKA. These effects could be reversed by APN injection. Importantly, APN upregulated PP2Cm expression in hepatocytes, which depends on AMPKα2. Collectively, our studies have established a novel mechanism that impaired APN signaling contributes to diabetic BCAA catabolism.
BCKD complex is the most important regulatory enzyme in BCAA catabolism; the activity of BCKD is regulated by a phosphorylation-dephosphorylation cycle and responsive to alterations in various metabolic conditions (35). Studies have reported that decreased BCKD activity causes downregulation of BCAA catabolism in diabetes (6,7,36). It also has demonstrated that BDK is responsible for phosphorylation and inactivation of BCKD complex and considered a primary regulator of BCKD activation (6). Diabetic and obese animals showed increased BDK expression (6,8). Moreover, alteration of metabolic status can influence BCKD activity partly via changes in BDK (3), indicating that alteration of BDP may also be important. But no information about BDP is available in diabetes. Identification of a specific BDP proved elusive for many years; PP2Cm was identified as the only endogenous BDP until recently (18,19,27). In the current study, we demonstrated for the first time that PP2Cm was significantly decreased in liver and adipose tissue from diabetic animals, which may at least partially contribute to reduction of BCKD activity and elevated plasma BCKA levels. Altogether, these findings provide a new explanation for the disturbed regulation of BCAA catabolism in diabetes.
Hypoadiponectinemia is commonly seen in diabetes (20,26), and APN is a critical regulator in lipid and glucose metabolism (20). Moreover, Liu et al. (24) recently demonstrated that HD-fed APN−/− mice have significantly increased levels of muscle BCAA, which can be corrected with APN supplementation for 2 weeks. However, the role of APN in systemic BCAA catabolism remains unclear. In our study, APN−/− mice fed with 45% HD for 4 weeks showed mild insulin resistance (Supplementary Fig. 2C and E) but normal blood glucose levels (Supplementary Fig. 2A).
In addition, we demonstrated that HD-fed APN−/− mice had significant reduced BCKD activity and PP2Cm levels and increased BDK expression, along with high plasma BCAA and BCKA concentrations. Treatment with APN for 3 days significantly reversed these trends, but insulin resistance remained (Fig. 2H–J). More importantly, APN treatment completely reverted the reduction of BCKD activity and PP2Cm expression and the elevation of BDK level in diabetic models. These results suggest that hypoadiponectinemia contributes to downregulated BCKD activity and PP2Cm expression and upregulated BDK level, as well as abnormal BCAA catabolism. This supports the notion that APN has a wide range of impacts on metabolism (24).
Additionally, evidence by Newgard et al. (10) indicates that changes in BCAA levels contribute to the development of insulin resistance. An interesting point that warrants further investigation is that the positive effect of APN on BCAA may subsequently contribute to the insulin-sensitizing effect of APN. Lastly, BCAA levels changed acutely upon short-time APN administration, whereas plasma glucose, glucose tolerance, insulin tolerance, and plasma cholesterol levels were unchanged (Figs. 2H–K and 3A–C). Although the precise mechanism causing the rapid BCAA regulatory response after short-period APN administration remains unclear, this result further supports that BCAAs may be useful biomarkers for monitoring the early response to therapeutic interventions for metabolic disease.
In the current study, we observed that APN deficiency in the APN−/− mice did not result in elevated BCAA levels under basal physiological condition. However, in reviewing numerous previous publications using this model, we found that our observation is very consistent with previous publications, showing that the APN−/− mouse does not have phenotypic changes under physiological condition. However, when pathologically challenged, such as by exposure to HD (37) or myocardial ischemia (22), these animals show much severer tissue injury than WT controls. These results indicate that although other molecules present in APN−/− animals are sufficient to compensate the effect of APN under normal physiological conditions, APN plays an essential role in counteracting pathological stress, such as HD. Additionally, APN partly upregulated BCKD activity and markedly downregulated BDK expression in PP2Cm-deficient mice, suggesting that BDK was involved in APN-activated BCKD. It has been shown in literature that thyroid hormone and sex hormones regulate expression of BDK (38); our present data also revealed that BDK was the downstream molecular of APN and contributed to APN-mediated BCAA catabolism. Thus, APN may signal both BDK and PP2Cm in opposite ways to increase BCKD activity and improve BCAA catabolism in diabetes.
AMPK has been considered a master switch in regulating glucose and lipid metabolism (39) and plays an essential role in the actions of APN (40). Nevertheless, AMPK can also modulate transcription of specific genes involved in energy metabolism (31). In this study, we observed that BCKD activity was elevated by activation of AMPK both in vitro and in vivo by pharmacological or genetic methods. Moreover, when the AMPK inhibitor CC or siRNA was applied, the effect of APN on BCKD was virtually abolished. These results might provide us with a better understanding of the role of AMPK in the regulation of metabolism.
The possibility remains that the remote role of APN-AMPK signaling in glucose and lipid metabolism could show secondary effects on BCKD activity in vivo; we thus performed in vitro experiments. Because BCKD capacity mostly resides in liver (3,33), mouse hepatocytes were used in present study. Here, mouse hepatocytes challenged with BCKA and incubated with APN or AICAR helped to address the fact that APN-AMPK directly upregulated BCKD activity (Supplementary Fig. 4A–H). Interestingly, effect of APN upon BCKA occurred prior to the significant upregulation of PP2Cm levels, suggesting that APN may improve BCKA catabolism via a signaling system in addition to upregulation of PP2Cm. Although the detailed molecular mechanisms cannot be addressed in the current study, it is possible that APN may enhance BCKD/PP2Cm interaction, thus increasing BCKD activity–independent PP2Cm expression. This interesting possibility will be directly investigated in our future study. To date, it remains unclear how PP2Cm expression is regulated. Previous studies (27,38) and our unpublished data have revealed that PP2Cm expression can be regulated by the availability of nutrients as well as stress (i.e., ischemia and heart failure). Here, we demonstrated that APN was the first identified endogenous molecule that can upregulate PP2Cm level.
In summary, this study provides evidence that reductions of APN signaling could underlie the decreased BCKD activity observed in type 2 diabetes. Our study also reveals direct evidence that APN can modulate expression of hepatic PP2Cm via an AMPKα2-dependent pathway. These new insights provide a better understanding of the underlying regulatory mechanisms involved in diabetic BCKD activity and identify potential therapeutic targets to mitigate BCAA catabolism in metabolic diseases.
Article Information
Acknowledgments. The authors thank Dr. Xinliang Ma, Thomas Jefferson University, for help with revising the manuscript. The authors also thank Dr. Yibin Wang and Dr. Haipeng Sun, University of California, Los Angeles, for the generous supply of the antibody to PP2Cm and PP2Cm−/− mice.
Funding. This work was supported by the Program for National Science Fund for Distinguished Young Scholars of China (grant no. 81225001), National Key Basic Research Program of China (973 Program, 2013CB531204), New Century Excellent Talents in University (grant no. NCET-11-0870), National Science Funds of China (grant nos. 81070676 and 81170186), Program for Changjiang Scholars and Innovative Research Team in University (no. PCSIRT1053), and Major Science and Technology Project of China “Significant New Drug Development” (grant no. 2012ZX09J12108-06B).
Duality of Interest. No potential conflicts of interests relevant to this article were reported.
Author Contributions. K.L. designed methods and experiments, carried out the laboratory experiments, and wrote the paper. C.D. performed RT-PCR analysis and analyzed data. Y.L. analyzed data and interpreted the results. D.Z. contributed to the data of Fig. 5. W.Y. reviewed and edited the manuscript. H.Z. and Z.H. researched data and contributed to discussion. P.L., L.Z., and H.P. performed diabetic animal model and collected the samples. J.Z., C.G., and C.X. performed BCAA and BCKA analysis. H.C. and L.X. contributed to discussion. L.T. defined the research theme and revised the manuscript critically. L.T. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.