Anti-hyperglycemic action of metformin

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Metformin is currently the drug of first choice for the treatment of T2D, being prescribed to at least 120 million people worldwide. Metformin is regarded as an antihyperglycemic agent because it lowers blood glucose concentrations in T2D without causing overt hypoglycemia. Metformin is also frequently described as an insulin sensitizer leading to reduction in insulin resistance and significant reduction of plasma fasting insulin level. The improvement in insulin sensitivity by metformin could be ascribed to its positive effects on insulin receptor expression and tyrosine kinase activity [6]. Metformin may also exert its beneficial metabolic actions in part through the modulation of multiple components of the incretin axis. Maida et al. have indeed recently reported that metformin acutely increases plasma levels of glucagon-like peptide 1 (GLP-1) and induces islet incretin receptor gene expression through a mechanism that is dependent on peroxisome proliferator-activated receptor (PPAR)-α [7]. However, a growing body of evidence from clinical studies and animal models suggests that the primary function of metformin is to decrease hepatic glucose production [8], mainly by inhibiting gluconeogenesis [9, 10]. Several mechanisms have been proposed to explain this inhibitory action on hepatic gluconeogenesis, including changes in enzyme activities [11-13] or reduction in hepatic uptake of gluconeogenic substrates [14]. The preferential action of metformin in hepatocytes is due to the predominant expression of the organic cation transporter 1 (OCT1), which has been shown to facilitate cellular uptake of metformin [15]. Consistent with this, accumulation of metformin in the liver has been shown to be higher than in other tissues, reaching hundreds of mM in the periportal area [16]. Furtermore, deletion of the OCT1 gene in mouse dramatically reduces metformin uptake in hepatocytes and human individuals carrying polymorphisms of the gene (SLC22A1) display an impaired effect of metformin in lowering blood glucose levels [15].

Although the molecular target of metformin was elusive for several years, Zhou et al. reported that the activation of AMP-activated protein kinase (AMPK) was intimately associated with the pleiotropic actions of metformin [17]. AMPK is a phylogenetically conserved serine/threonine protein kinase viewed as a fuel gauge monitoring systemic and cellular energy status and which plays a crucial role in protecting cellular functions under energy-restricted conditions. AMPK is a heterotrimeric protein consisting of a catalytic a-subunit and two regulatory subunits b and g and each subunit has at least two isoforms. AMPK is activated by increase in the intracellular AMP-on-ATP ratio resulting from imbalance between ATP production and consumption. Activation of AMPK involves AMP binding to regulatory sites on the g subunits. This causes conformational changes that allosterically activate the enzyme and inhibit dephosphorylation of Thr172 within the activation loop of the catalytic a subunit. AMPK activation requires phosphorylation on Thr172 by upstream kinases, identified as the tumor suppressor serine/threonine kinase 11 (STK11/LKB1) and CaMKKb, which is further stimulated by the allosteric activator AMP [18]. Moreover, it has been recently shown that ADP, and therefore the ADP-on-ATP ratio, could also play a regulatory role on AMPK by binding to specific domains on the g subunit [19, 20]. Activated AMPK switches cells from an anabolic to a catabolic state, shutting down the ATP-consuming synthetic pathways and restoring energy balance. This regulation involves phosphorylation by AMPK of key metabolic enzymes and transcription factors/co-activators modulating gene expression [18]. As a result, glucose, lipid and protein synthesis as well as cell growth are inhibited whereas fatty acid oxidation and glucose uptake are stimulated.

Metformin most likely does not directly activate either LKB1 or AMPK as the drug does not influence the phosphorylation of AMPK by LKB1 in cell-free assay [21]. Rather, there is evidence that AMPK activation by metformin is secondary to its effect on the mitochondria, the primary target of the drug. One of the most significant breakthroughs in the understanding of the cellular mechanism of metformin was indeed made in the early 2000s by two independent research groups reporting for the first time that this member of the biguanides family induced mild and specific inhibition of the mitochondrial respiratory-chain complex 1 (Figure 1). The initial observation was made in both perfused livers and isolated hepatocytes from rodents [22, 23] but later expanded to other tissues, including skeletal muscle [24], endothelial cells [25], pancreatic beta cells [26], and neurons [27]. Although the exact mechanism(s) by which metformin inhibits the respiratory-chain complex 1 remains unknown, we have recently shown that this unique drug effect does not necessitate AMPK and is also found in human primary hepatocytes [28]. In addition, it seems that mitochondrial action of metformin requires intact cells [22, 29, 30] and is prevented, at least in hepatocytes, neither by inhibition of nitric oxide synthase nor by various reactive oxygen species (ROS) scavengers [22]. In addition, the maximal inhibitory effect of metformin on complex 1 activity is also lower than the reference inhibitor rotenone (~40% with metformin compared with 80% with rotenone), suggesting that, owing to different physico-chemical properties, their respective site of action differ on one or several of the subunits of the respiratory-chain complex 1. For instance, the positive charge of metformin was proposed to account for its accumulation within the matrix of energized mitochondria, driven by the membrane potential (Dy)[23]. Furthermore, the apolar hydrocarbon side-chain of the drug could also promote its binding to hydrophobic structures, especially the phospholipids of mitochondrial membranes [31]. Interestingly, it has been recently shown that metformin, by contrast to rotenone, also exerts an inhibitory effect on mitochondrial ROS production by selectively blocking the reverse electron flow through the respiratory-chain complex 1 [32, 33]. Further investigations are still required to clarify the mechanism(s) by which metformin modulates the respiratory-chain complex 1 by such a unique way. It is also worth mentioning that metformin probably also exerts some non-mitochondrial effects since it has been shown to affect erythrocyte metabolism, a cell-lacking mitochondria, by modulating membrane fluidity [34, 35].

Taken together, the activation of AMPK by metformin in the liver, and probably in other tissues, is the direct consequence of a transient reduction in cellular energy status induced by the mild and specific inhibition of the respiratory-chain complex 1 by the drug [28] (Figure 1). In line with this, methyl succinate, a substrate of the respiratory-chain complex 2 bypassing the inhibition of complex 1, has been shown to antagonize the metformin-induced AMPK activation in pancreatic beta cell line [26]. Furthermore, Hawley et al.. have recently reported that AMPK activation by metformin is abolished in cell line stably expressing AMPK complexes containing AMP-insensitive g2 mutant, demonstrating that increased cytosolic AMP indeed triggers the activation of the kinase by the drug [36].

AMPK involvement in the antidiabetic effect of metformin was initially supported by a study showing that the glucose-lowering effect of the drug is greatly decreased in mice lacking hepatic LKB1 [37]. LKB1/AMPK signaling has been reported to regulate the phosphorylation and nuclear exclusion of CREB-regulated transcription coactivator 2 (CRTC2, also referred to as TORC2) [37]. CRTC2 has been identified as a pivotal regulator of hepatic glucose output in response to fasting by directing transcriptional activation of the gluconeogenic program [38]. Non-phosphorylated CRTC2 translocates to the nucleus, where it associates with phosphorylated CREB to drive the expression of peroxisome proliferator-activated receptor-g coactivator-1a (PGC-1a) and its subsequent gluconeogenic target genes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase). Phosphorylation on the Ser 171 residue of CRTC2 by AMPK and/or AMPK-related kinases, including the salt-inducible kinases (SIK), is critical for determining the activity, cellular localization and degradation of CRTC2, thereby inhibiting the gluconeogenic program [37, 38]. However, since CRTC2 is O-glycolysated at Ser171 in insulin resistance state, making phosphorylation impossible [39], it is unlikely that metformin regulates gluconeogenesis through CTRC2 phosphorylation. A possible alternative mechanism for metformin inhibitory action on TORC2-mediated gluconeogenesis has been recently proposed (Figure 2), involving increase of hepatic SIRT1 activity, an NAD⁺-dependent protein deacetylase, through AMPK-mediated induction of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD biosynthesis [40]. SIRT1 has been reported to deacetylate CRTC2, resulting in the loss of protection from COP1-mediated ubiquitination and subsequent degradation [41]. This likely occurs in parallel with another mechanism for metformin action which involves the disassembly of the CREB-CBP (CREB binding protein)-CRTC2 complex at PGC-1a and PEPCK promoters [42]. The regulation of gluconeogenic gene expression by metformin appears to be dependent on the phosphorylation of CBP at Ser436, but not CRTC2, through AMPK-induced atypical PKCi/l activation [42]. In addition, a variety of transcription factors have been shown to participate in the metformin-induced inhibition of gluconeogenic genes in the liver (Figure 2). Kim et al. demonstrated that metformin regulates hepatic gluconeogenesis through an AMPK-mediated upregulation of the orphan nuclear receptor small heterodimer partner (SHP), which operates as a transcriptional repressor [43]. SHP inhibits CREB-dependent hepatic gluconeogenic gene expression via direct interaction with CREB and competition with CRTC2 binding in the CREB-CBP complex [44]. Takashima et al. have proposed a role for Krüppel-like factor 15 (KLF15) in the metformin-induced inhibition of genes coding for both gluconeogenic and amino acid catabolic enzymes, these later being potentially implicated in the regulation of gluconeogenesis through the control of gluconeogenic substrate availability [45]. Metformin suppressed KLF15 gene expression and promoted its ubiquitination for proteasomal degradation. Restoration of KLF15 expression only partially rescued the inhibitory effect of metformin on hepatic glucose production, indicating that other factors also contribute to metformin action [45].

Understanding the mechanism of action of metformin is further complicated by our recent study establishing that both LKB1 and AMPK activities are dispensable for metformin-induced inhibition of glucose output or gluconeogenesis [46]. We indeed reported that a reduction in hepatic energy status, but not AMPK activation, constitutes the critical factor underlying the effects of metformin on the regulation of hepatic glucose output [46]. Since the rate of hepatic glucose production is closely linked to hepatic energy metabolism (6 ATP equivalents are required per molecule of glucose synthesized), disruption of the main energy supply in hepatocytes (mitochondrial oxidative phosphorylation) through inhibition of the respiratory-chain complex 1 would have a profound effect on the flux through gluconeogenesis (Figure 2). In addition, as AMP tends to rise whenever ATP falls, this could also provide an alternative explanation for the acute inhibition of gluconeogenesis by metformin via allosteric regulation of key enzymes in this pathway, such as fructose-1,6-bisphosphatase [47]. Of particular note is the metformin-induced inhibition of glucose production independently of transcriptional repression of gluconeogenic genes. Interestingly, forced expression of key gluconeogenic genes through PGC-1a overexpression did not reduced metformin-induced reduction of glucose output, but was again associated with a significant depletion of energy stores [46]. These results indicate that metformin could acutely suppress gluconeogenesis via a transcription-independent process and that changes in gene expression are therefore not the exclusive determinant in the regulation of glucose output (Figure 2). Interestingly, suppression of hepatic glucose production by metformin in insulin-resistant high-fat fed rats is dependent on an inhibition of the substrate flux through G6Pase, and not of a decrease in the amount of the protein [13], supporting the notion that metformin action is related to a reduction of the gluconeogenic flux rather than a direct inhibition of gluconeogenic gene expression.

In addition to the suppression of endogenous glucose production, metformin has been shown to be beneficial in improving lipid metabolism. Evidence that metformin improved fatty liver disease by reversing hepatic steatosis in ob/ob mice [48, 49] and in rodents fed a high-fat diet [50] has been demonstrated but also reported in some clinical studies [51, 52]. Metformin-induced reduction in hepatic lipid content is consistent with an increase in both fatty acid oxidation and inhibition of lipogenesis, presumably mediated by AMPK activation [17, 48, 53]. Indeed, AMPK coordinates the changes in the hepatic lipid metabolism and, so, regulates the partitioning of fatty acids between oxidative and biosynthetic pathways [18]. Thus, AMPK activation by metformin induces the phosphorylation and inactivation of acetyl CoA carboxylase (ACC), an important rate-controlling enzyme for the synthesis of malonyl-CoA, which is both a critical precursor for the biosynthesis of fatty acids and a potent inhibitor of mitochondrial fatty acid oxidation [17]. In human hepatoma HepG2 cells, metformin enhances ACC phosphorylation and induces the reduction on triglycerides levels, which can be supported by increased fatty acid oxidation and/or decreased fatty acid synthesis [53]. In addition, AMPK suppresses expression of lipogenic genes such as fatty acid synthase, S14 and ACC by direct phosphorylation of transcription factors (carbohydrate response element binding protein (ChREBP) and hepatocyte nuclear factor 4 (HNF4)) and co-activators (p300) [54-59]. Metformin participates in the regulation of lipogenesis gene expression by down-regulating sterol regulatory element-binding protein-1c (SREBP-1c) gene expression [17] and by inhibiting its proteolytic processing and transcriptional activity upon AMPK-mediated phosphorylation at Ser372 [60]. A recent study by Kim et al. reported that metformin induces AMPK-mediated thyroid hormone receptor 4 (TR4) phosphorylation and repression of stearoyl-CoA desaturase 1 (SCD1) expression, a rate-limiting enzymes involved in the biosynthesis of monounsaturated fatty acids from saturated fatty acids [61].

Fatty liver disease is strongly associated with insulin resistance and the apparent inhibition of hepatic glucose production by metformin may be secondary to the primary improvement of hepatic steatosis and insulin resistance. This hypothesis may also offer a potential explanation for the loss of metformin effect on blood glucose levels in liver-specific LKB1 knockout mice fed a high-fat diet [37]. Thus, impaired metformin-induced AMPK phosphorylation could fail to reduce hepatic lipid levels and to improve insulin sensitivity in liver-specific LKB1-deficient mice, impeding normalization of blood glucose levels.