Effect of metformin on myotube BCAA catabolism

1 BACKGROUND

In recent years, the incidence of type 2 diabetes has dramatically increased in developed nations. In fact, the CDC reported an increase from 8.3% in 2010 to 9.4% in 2015,1 and an estimated 1.5 million people were newly diagnosed with diabetes.1 Similar trends have been reported in the United Kingdom.2 As a result of the rising prevalence of diabetes, use of antidiabetic medications has also increased. For example, the use of first-line pharmaceutical metformin rose from 55.4% of diabetics in 2000 to 83.6% in 2013.2 By 2013, metformin was prescribed as first-line therapy for 91% of newly diagnosed patients with type II diabetes mellitus and as an add-on for 79.9% of patients.2 Another study reported that 40.6% of outpatient visits with a diabetes diagnosis either initiated or continued metformin prescription.3 Thus, while estimates of the exact frequency of metformin use vary, it is clear that metformin is a commonly used and highly effective medication in the regulation of blood glucose.

Metformin acts as an antihyperglycemic agent through several potential mechanisms. First, metformin has been shown to activate 5′-AMP-activated protein kinase (AMPK) in the liver,4 which reduces gluconeogenesis. In peripheral tissues, metformin-mediated AMPK activation may increase mitochondrial biogenesis and muscle glucose uptake while reducing expression of lipogenic genes.4 For example, cultured C2C12 myotubes given metformin at 2 mM during days 3 to 8 of myotube differentiation exhibited strongly induced peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) expression,5 a master regulator of mitochondrial biogenesis.6-8 The same report demonstrated PGC-1α expression was also upregulated in skeletal muscle of mdx mice given 2 mg/mL in drinking water for 42 days.5 Similar findings have been observed in C2C12 myotubes treated with 2.5 mM metformin for 24 hours, which exhibited increased PGC-1α, nuclear respiratory factor 1 (NRF1), and mitochondrial transcription factor A (TFAM) protein expression, as well as elevated pAMPK expression.9 The same report also demonstrated that high-fat fed C5BL/6 mice given metformin for 16 weeks could reverse high-fat-induced suppression of gastrocnemius PGC-1α protein expression.9 Moreover, a report by Hasan et al10 showed that high-fat fed male albino rats given metformin at 320 mg/day via oral gavage for 4 weeks exhibited rescued levels of muscle PGC-1α mRNA expression vs high-fat only rats. Similarly, metformin (400 mg·kg⁻¹ day⁻¹ for 4 weeks) was shown to induce both messenger RNA (mRNA) and protein expression of PGC-1α, as well as activate both AMPK and extracellular signal–regulated kinases signaling in skeletal muscle in both wild-type and Ob/Ob mice.11 Similar findings have been obtained in rats given 300 mg/kg body weight which also induced PGC-1α expression in the soleus, white gastrocnemius, and red gastrocnemius vs saline-treated rats.12 Interestingly, a report using C2C12 cells treated with very low-dose (2 nM) metformin for 24 hours reported significant elevations in myotube PGC-1α expression,11 to a similar extent as those shown by Ljubicic et al.5

Along with the rising incidence of diabetes, an increasing interest in branched-chain amino acids (BCAAs) and their relationship with the severity of insulin resistance has developed. Several population studies have correlated circulating BCAAs with the severity of insulin resistance.13-20 Reduced BCAA catabolism has been suggested as a likely contributor to the accumulation of circulating BCAAs in obese and diabetic populations. Interestingly, the expressional regulation of BCAA catabolic enzymes appears to be regulated by PGC-1α.21 In fact, the activation of either PGC-1α or peroxisome proliferator-activator receptor α (PPARα) induces BCAA catabolic enzymes branched-chain aminotransferase (BCAT2) and branched-chain-alpha-keto acid dehydrogenase (BCKDH).21-23 BCKDH represents the committed and rate-limiting step of BCAA catabolism and is negatively regulated by branched-chain-alpha-keto acid dehydrogenase kinase (BCKDK) by phosphorylation.24 Other PPAR family members (PPARδ and PPARγ) have also been implicated in the regulation of BCAA catabolism.21 Not surprisingly, activities which promote PGC-1α such as exercise or fasting also promote BCAA metabolism.25, 26

Despite several studies demonstrating metformin's ability to induce PGC-1α, to our knowledge the effect of metformin on indicators of BCAA catabolism remains relatively unexplored. Given metformin is known to activate PGC-1α, which as described above is a positive regulator of BCAA catabolic gene expression,21-23 one might expect metformin to induce BCAA catabolic gene expression. However, one report demonstrated metformin suppressed BCAT2 expression in both C2C12 myotubes and in a mouse model of maple syrup urine disease, however, the effects of metformin on BCKDH expression remains unknown.27 One possible mechanism that explains an upregulation of PGC-1α by metformin, with a concurrent decrease in BCAA metabolism, is the simultaneous downregulation of Kruppel-like factor 15 (KLF15), which is recognized as a coordinating transcription factor in the metabolism of BCAA (including the expression of muscle BCAT2).28 Interestingly, metformin was previously shown to significantly increase PGC-1α in primary mouse hepatocytes, and reduced PGC-1α acetylation (the indication of PGC-1α activation and Sirt1 activity), while suppressing KLF15 expression in HepG2 cells.29

Thus, given metformin is such a commonly used antidiabetic therapy, and the strong link between elevated circulating BCAA and severity of insulin resistance, it seems important to investigate the effect of metformin on BCAA metabolism.21-23 Additionally, it has been suggested that the dose of metformin used in basic experimental research likely plays a vital role in determining the mechanisms by which metformin functions.30 In fact, several in vitro studies have used concentrations of metformin that far exceed physiological concentrations in the blood.30 Large variability in metformin pharmacokinetics have been observed, however steady-state metformin plasma levels likely range from 54 to 4133 ng/mL (400 nM-31 µM).4 Therefore, the aim of this study is to examine the effect of metformin (both physiological and supraphysiological) on indicators of BCAA catabolism. Our hypothesis is that metformin will inhibit mitochondrial metabolism thereby increasing AMPK activation and PGC-1α expression leading to increased expression of regulators of mitochondrial biogenesis and BCAA catabolism. However, it is unclear if these expressional changes will alter cell metabolism or BCAA catabolism.

2 METHODS

3 RESULTS

3.1 Supraphysiological metformin treatment induces regulators of mitochondrial biogenesis while suppressing indicators of BCAA catabolism

To investigate the effect of metformin on cell energetics, indicators of mitochondrial biogenesis and BCAA catabolism were measured following supraphysiological metformin (2 mM) treatment. Metformin significantly increased PGC-1α mRNA expression by 129.7% ± 78.1% compared to control (Figure 1A). Several downstream targets of PGC-1α were also upregulated. Specifically, NRF1 increased by 37.1% ± 28.8%, TFAM increased by 91.5% ± 30.8%, and citrate synthase (CS) increased by 72.0% ± 39.7% (Figure 1A). And although slightly elevated, a significant induction of PGC-1α was not observed at the protein level (Figure 1B). Interestingly, BCAA catabolic gene expression was shown to be depressed, despite PGC-1α mRNA upregulation. Specifically, BCAT2 mRNA decreased by 35.4% ± 25.9% and BCKDHa mRNA decreased by 90.0% ± 6.8% vs control cells (Figure 1B). BCAT2 also was significantly suppressed (decreased by 24.3% ± 21.0%) at the protein level (Figure 1C). Because we observed an induction of several metabolic genes following 24-hour metformin treatment, we next explored the effect of 2 mM metformin at earlier time points. Similar to 24-hour treatment, 2 mM metformin significantly induced mRNA expression of PGC-1α, TFAM, and CS (Figure 1D). We also investigated the effect of metformin at earlier time points on both BCAT2 and BCKDHa expression. Interestingly, BCAT2 was subtly but significantly induced (increased 32.1% ± 20.6%), while BCKDHa expression remained unaltered (Figure 1E). Additionally, we explored the temporal effect of metformin on BCKDHa and AMPK phosphorylation in a 4-hour time course. Treatment with metformin at 2 mM led to the suppression of BCKDHa activity at both 3 and 4 hours (Figure 2A). This inactivation of BCKDHa activity was paralleled by concurrent increases in AMPK activation, which was significantly elevated at the 4-hour time point (Figure 2B).

3.2 Physiological metformin does not alter regulators of mitochondrial biogenesis or BCAA catabolism

To explore the effect of physiological metformin on myotube BCAA catabolism, these experiments were repeated following treatment of metformin at a physiological concentration (30 µM). No changes were seen in indicators of mitochondrial biogenesis at either the mRNA (Figure 3A) or protein level (Figure 3C) following 24-hour treatment, or following shorter treatment durations (Figure 3D). Similarly, 24-hour treatment resulted in no change in mRNA or protein expression of BCAA catabolic enzymes (Figure 3B and 3C, respectively), however, BCKDHa (but not BCAT2) mRNA was suppressed by 30 µM metformin following shorter treatment durations (4, 8, and 12 hours; Figure 3E). We also assessed BCKDHa and AMPK activation following up to 4-hour treatment, and activities of both enzymes remained unaltered by 30 µM metformin across a 4-hour time course (Figure 4).

3.3 Supraphysiological but not physiological metformin suppresses myotube metabolism

Next, we investigated the effect of metformin on myotube metabolism at both physiological and supraphysiological concentrations. Basal and peak mitochondrial metabolism were suppressed by 2 mM metformin. Specifically, basal oxygen consumption decreased by 55.7% ± 3.6% and peak metabolic capacity decreased by 45.1% ± 7.6% vs control cells (Figure 5A). Similarly, glycolytic metabolism during peak ECAR was decreased (by 30.0% ± 5.9%) following 2 mM metformin treatment (Figure 5B). Two millimolar treatment also led to a significant depression in basal (P < .001) and peak (P < .001) cell metabolism (Figure 5C). Conversely, physiological metformin concentrations did not alter any type or level of myotube metabolism (Figure 5).

In alignment with the observed suppression in cell metabolism, 24-hour 2 mM metformin also caused significant reduction in the mRNA expression of key electron transport chain components including cytochrome c oxidase (COX; decreased by 77.1% ± 23.6%) and ATP Syn (decreased by 44.6% ± 6.5%) vs control cells (Figure 6B). Shorter treatment periods only resulted in subtle, insignificant changes in electron transport chain component expression (Figure 6A). Interestingly, 30 uM metformin reduced mRNA expression of COX at 4, 8, and 12 hours, but did not alter ATP Syn expression (Figure 6C). And following 24-hour treatment, gene expression of these respiratory components had returned to control levels (Figure 6D).

3.4 Supraphysiological metformin suppresses myotube KLF15 protein expression

Because we saw decreases in BCAT2 and BCKDHa despite also observing significant elevation of PGC-1α mRNA expression, we measured KLF15 mRNA and protein expression (as KLF15 is a recognized regulator of BCAA catabolism28). Interestingly, while KLF15 mRNA expression was increased at each time point following 2 mM metformin treatment (Figure 7A), KLF15 protein expression was decreased by 28.9% ± 12.0% (P = .08) following 24-hour treatment (Figure 7C). And consistent with previous experiments using physiological levels of metformin, 30 µM did not alter KLF15 mRNA expression (Figure 7B).

4 DISCUSSION

Metformin represents a highly effective and commonly used pharmacological tool in the regulation of blood glucose levels among diabetic populations. In this report, we investigated the effects of metformin on indicators of BCAA catabolism in cultured myotubes as dysregulated BCAA catabolism has been previously reported in diabetics. While past reports have investigated the effect of metformin on similar indicators, to our knowledge, a comprehensive investigation on the effects of metformin on enzymatic mRNA and protein expression, as well as activation of the BCKDH complex has yet to be performed. In our report, we identify several interesting effects of metformin on myotube metabolism. First, we observed a significant induction of PGC-1α at the mRNA (but not protein, P = .10) level by metformin at 2 mM. Other reports have demonstrated a significant induction of PGC-1α at the protein level using cultured C2C12 myotubes.5 While our report failed to find a significant induction of PGC-1α at the protein level, we suspect differences in treatment duration may have been responsible for discrepancies between findings by Ljubicic et al5 and those in the present report. We did however observed a significant induction in PGC-1α at the mRNA level at several time points which corresponded with the upregulation of several downstream targets of PGC-1α which regulate mitochondrial biogenesis. Interestingly, we observed suppressed mRNA expression of both BCAT2 and BCKDHa by metformin despite upregulation of PGC-1α expression. This is surprising given ectopic PGC-1α overexpression has previously been linked with upregulation of both BCAT2 and BCKDHa.21 We next investigated the protein expression of BCAT2 and BCKDHa following 2 mM metformin treatment and observed reduced BCAT2, but not BCKDHa levels in the metformin group. Because the BCKDH complex is negatively regulated by BCKDK, we next temporally measured the phosphorylation status of both AMPK (as an indicator of cell energetics) and BCKDHa. Not surprisingly, metformin activated AMPK following 4 hours. More interestingly, metformin treatment led to significantly greater phosphorylation of BCKDHa, suggesting metformin suppresses BCKDH complex activity. Similarly, reductions in mRNA expression of electron transport components, mitochondrial metabolism (both basal and peak), and cellular metabolic rate and capacity were observed in cells treated with 2 mM metformin. Interestingly, physiological levels of metformin (30 µM) also resulted in reduced mRNA expression of BCKDHa and COX, suggesting physiological levels of metformin may stimulate some, but not all of the same adaptations as 2 mM metformin, and may do so at a different magnitude and timeline.

Because we observed a significant increase in PGC-1α expression, yet a perplexing reduction of BCAT2 and BCKDHa expression/activity, we next measured the effect of metformin on KLF15 which was previously shown to play an important role in metabolic flexibility in the muscle (including the upregulation of BCAA catabolic enzymes).28 Given previous observations have shown metformin (and other AMPK activators such as AICAR) suppress KLF15 mRNA expression, we expected to find similar results.29 To our surprise, KLF15 mRNA was induced in cells treated with metformin at 2 mM, which is in opposition to similar observations in hepatocytes treated with metformin at 1 mM for 12 or 24 hours.29 However, at the protein level, 2 mM metformin reduced KLF15 protein expression following 24-hour treatment, which is in line with our initial hypothesis. These results suggest a disconnection between the mRNA induction and protein expression of KLF15, which may be a byproduct of the difference in mRNA and protein response at a single time point (ie, mRNA levels may reflect current cell response while protein levels may be slightly delayed). Taken together, our observations demonstrate that metformin may suppress BCAA catabolic enzyme expression in cultured myotubes, despite an upregulation in PGC-1α. Our current working hypothesis based on the presented data (and past observations) is shown in Figure 8. Briefly, metformin inhibits mitochondrial metabolism, promoting an activation of AMPK and subsequently PGC-1α. PGC-1α activation leads to the increased expression of numerous metabolic targets including PGC-1α and markers of mitochondrial biogenesis. Despite upregulation in these genes, the expression of electron transport chain components is suppressed in metformin-treated cells. Additionally, metformin appears to reduce KLF15 protein levels which may contribute to reduced expression of BCAA catabolic enzymes. Although speculation, this process may also contribute to metformin's ability to reduce blood glucose by reducing circulating hepatic gluconeogenic substrates.

Despite these interesting findings, our report is not without limitations. First, the in vitro nature of our study limits several aspects of the study design, which may reduce the generalizability of our findings in vivo. For example, while we tested both physiological and supraphysiological levels of metformin, cells were exposed to a constant dose for up to 24 hours which does not account for physiological fluctuations in circulating levels of metformin that occur when metformin is taken orally in vivo. Second, our study did not determine if alterations in BCAA catabolic enzyme expression following metformin treatment result in a diminished capacity to oxidize BCAA. However, based on the metabolic assay results from our report, we believe that metformin-suppression of mitochondrial respiration is a strong indicator that metabolism (including that of BCAA) is lower in metformin-treated cells. Importantly, others have shown KLF15 is vital for the maintenance of blood glucose (using KLF15 KO mice), especially during times of low carbohydrate consumption.32 The importance of KLF15 was attributed to its role in the production of carbon skeletons from amino acid catabolism as gluconeogenic substrates.32 The report also demonstrates that KLF15 KO mice display reduced skeletal muscle BCAT2 expression as well as elevated circulating BCAA.32 For this reason, it is curious if metformin-mediated suppression of BCAT2 and amino acid catabolism in skeletal muscle results in reduced hepatic gluconeogenic substrates, further contributing to metformin's inhibition of hepatic glucose production (speculation which requires further investigation). Additionally, during our experiments, we cannot exclude the possibility that metformin was cytotoxic to myotubes. Although metformin reduced cell viability at 2 mM (Figure S1), the viability assay utilized in this report is based on the activity of mitochondrial dehydrogenases, which could conceivably be dampened by metformin treatment (through reduction of cell metabolism) without causing significant cell death. Thus, it is unclear if 2 mM metformin is cytotoxic or simply slowing cell metabolism. We selected 2 mM metformin because it has been used during previous experiments,5, 27 while other experiments used metformin concentrations ranging from 1-2.5 mM for various durations (up to 5 days) for in vitro experiments.9, 29 It is also worthy of discussion that the majority of changes in metabolic gene/protein expression and changes in metabolism occurred following treatment with 2 mM metformin, which far exceeds physiologically attainable levels when metformin is consumed orally.4, 30 Thus, it is unclear if these observations are of significance in vivo given the exceedingly high concentrations used during our experiments to produce the observed effects. Furthermore, it is also difficult to distinguish if the effects of high-concentration metformin are a product of only the specific molecular targets measured in the present study, or if additional targets that we did not measure played a role in these observations. Finally, the in vitro nature of our study lacks the measurement of chronic metformin consumption, which is relevant because many people rely on metformin continually to regulate blood glucose. Therefore, the effect of chronic metformin on regulators of BCAA metabolism in skeletal muscle (and other tissues) remains unclear.

These limitations aside, our report demonstrates that high levels of metformin can upregulate transcription factors in skeletal muscle that govern mitochondrial biogenesis while suppressing mitochondrial metabolism. The report also describes how supraphysiological levels of metformin suppress the expression and activity of BCAA catabolic enzymes in skeletal muscle, possibly through suppression of KLF15. However, it is unclear if KLF15 suppression is directly responsible for suppressed BCAA catabolic gene expression. It is also unclear if metformin alters circulating levels of BCAA in insulin-resistant populations, which requires further investigation.

CONFLICT OF INTERESTS

The authors declare that there are no conflict of interests.