Introduction An association of elevated branched chain amino

Introduction An association of elevated branched-chain CHIR-124 australia (BCAA—Leu, Val, Ile) with obesity and insulin resistance was first reported nearly 50 years ago (Felig et al., 1969). With the advent of metabolomics technologies, it has since become apparent that the association of BCAA and related metabolites (glutamate/glutamine, phenylalanine, tyrosine, C3 and C5 acylcarnitines) with metabolic diseases is among the strongest reported for any biomarker (Newgard, 2017, Newgard et al., 2009, Wang et al., 2011). BCAA and their metabolites are also prognostic at baseline for incident type 2 diabetes (T2D) and obesity intervention outcomes (Palmer et al., 2015, Shah et al., 2012, Wang et al., 2011). Moreover, in meta-analysis of 16,596 individuals, BCAA levels were strongly associated with a SNP near the PPM1K (also referred to as PP2Cm) gene, which encodes the phosphatase that dephosphorylates and activates the branched-chain ketoacid dehydrogenase (BCKDH) complex, the first committed step in BCAA catabolism (Lotta et al., 2016). Further analysis revealed that a change of 1 SD in Ile, Leu, or Val levels was associated with significant increases in odds ratios for T2D. Together, these findings demonstrate strong genetic and biochemical associations of perturbed BCAA metabolism with human metabolic diseases and conditions. In parallel, evidence has mounted that elevated BCAA levels in obesity result at least in part from lower rates of catabolism in liver and adipose tissue (Herman et al., 2010, Hsiao et al., 2011, Lian et al., 2015, Pietiläinen et al., 2008, She et al., 2007, White et al., 2016). Coordinated downregulation of multiple BCAA metabolizing enzymes appears to account for reduced BCAA catabolism in adipose tissue in obese rodents and humans (Herman et al., 2010, Hsiao et al., 2011, Pietiläinen et al., 2008, She et al., 2007). In contrast, in liver, where BCKDH activity is substantially higher than other tissues, obesity is associated with hyper-phosphorylation of the regulatory Ser293 of the e1a subunit (also designated as E1α) of BCKDH to suppress its activity (Lian et al., 2015, She et al., 2007, White et al., 2016). The large, multi-subunit BCKDH enzyme complex consists of three components (e1, e2, and e3) that carry out different phases of a reaction in which branched-chain α-keto acids (BCKAs) are converted to branched-chain acyl coenzyme A (CoA). The BCKDH kinase (BDK) and the PPM1K phosphatase are tightly associated with the e2 component, and carry out phosphorylation and dephosphorylation of Ser293 of the adjoining e1a subunit, respectively. Increased phosphorylation of BCKDH on Ser293 in liver of rodent models of obesity occurs secondary to elevated expression of BDK, and lower expression of PPM1K (Lian et al., 2015, She et al., 2007). The balance of BDK and PPM1K expression in liver is influenced by adiponectin (Lian et al., 2015) and may also be partly controlled through a brain-liver axis (Shin et al., 2014). However, while these studies and others involving transgenic knockout of BDK or PPM1K in mice (Joshi et al., 2006, Lu et al., 2009) show that modulation of BDK and PPM1K expression can affect circulating BCAA levels, mechanisms by which BDK or PPM1K might regulate glucose and lipid homeostasis have not been established in animal models or humans.
Results
Discussion In the current study, we demonstrate that BDK and PPM1K, the kinase and phosphatase pair that control BCKDH activity and BCAA levels, also modulate hepatic lipid metabolism by regulating reversible phosphorylation of ACL on Ser454 (Figure 4G). ACL is an important enzyme in DNL and regulation of fatty acid oxidation due to its contributions to production of cytosolic acetyl CoA and malonyl CoA from citrate. In contrast to phosphorylation of BCKDH e1a on Ser293, which results in inhibition of enzyme activity, phosphorylation of ACL on Ser454 is activating, leading to increased generation of acetyl-CoA and malonyl CoA, the latter serving as the immediate substrate for lipogenesis (Potapova et al., 2000). Increased malonyl CoA levels also inhibit fatty acid oxidation via allosteric inhibition of carnitine palmitoyltransferase-1 (McGarry, 2002). Consistent with this construct, we demonstrate that modulation of the ratio of BDK:PPM1K activities in favor of PPM1K by two distinct experimental approaches not only activates BCKDH to lower BCAA and BCKA levels, but also results in marked reduction in hepatic steatosis, lowering of RER, and increased hepatic even-chain acylcarnitines, all consistent with reduced lipogenesis and increased fatty acid oxidation in the liver. We also observed improved glucose tolerance in response to those maneuvers, possibly secondary to the marked lowering of hepatic TG content (Samuel and Shulman, 2017). These improvements in metabolic health mirror those observed in response to ACL knockout in genetically obese mice (Wang et al., 2009), and suggest that the BDK:PPM1K axis serves as a metabolic regulatory node that integrates BCAA, glucose, and lipid metabolism via two distinct phosphoprotein targets.