👤 Christopher B Newgard

The transcription factor carbohydrate response element binding protein (ChREBP) activates genes of glucose, fructose, and lipid metabolism in response to carbohydrate feeding. Integrated transcriptomic and metabolomic analyses in rats with GalNac-siRNA-mediated suppression of ChREBP expression in liver reveal other ChREBP functions. GalNac-siChREBP treatment reduces expression of genes involved in coenzyme A (CoA) biosynthesis, with lowering of CoA and short-chain acyl-CoA levels. Despite suppression of pyruvate kinase, pyruvate levels are maintained, possibly via increased expression of pyruvate and amino acid transporters. In addition, expression of multiple anaplerotic enzymes is decreased by GalNac-siChREBP treatment, affecting TCA cycle intermediates. Finally, GalNAc-siChREBP treatment suppresses late steps in purine and NAD synthesis, with increases in precursors and lowering of end products in both pathways. In sum, our study reveals functions of ChREBP beyond its canonical roles in carbohydrate and lipid metabolism to include regulation of substrate transport, mitochondrial function, and energy balance. Show less

All forms of diabetes result from insufficient functional β-cell mass. Thus, achieving the therapeutic goal of expanding β-cell mass requires a better mechanistic understanding of how β-cells proliferate. Glucose is a natural β-cell mitogen that mediates its effects in part through the glucose-responsive transcription factor, carbohydrate response element binding protein (ChREBP) and the anabolic transcription factor, MYC. However, mechanistic details by which glucose activates Myc at the transcriptional level are poorly understood. Here, siRNA was used to test the role of ChREBP in the glucose response of MYC, ChIP and ChIPseq to identify potential regulatory binding sites, chromatin conformation capture to identify DNA/DNA interactions, and an adenovirus was constructed to expresses x-dCas9 and an sgRNA that specifically disrupts the recruitment of ChREBP to a specific targeted ChoRE. We found that ChREBP is essential for glucose-mediated transcriptional induction of Myc, and for increases in Myc mRNA and protein abundance. Further, ChIPseq revealed that the carbohydrate response element (ChoRE) nearest to the Myc transcriptional start site (TSS) is immediately upstream of the gene encoding the lncRNA, Pvt1, 60,000 bp downstream of the Myc gene. Chromatin Conformation Capture (3C) confirmed a glucose-dependent interaction between these two sites. Transduction with an adenovirus expressing x-dCas9 and an sgRNA specifically targeting the highly conserved Pvt1 ChoRE, attenuates ChREBP recruitment, decreases Myc-Pvt1 DNA/DNA interaction, and decreases expression of the Pvt1 and Myc genes in response to glucose. Importantly, isolated and dispersed rat islet cells transduced with the ChoRE-disrupting adenovirus also display specific decreases in ChREBP-dependent, glucose-mediated expression of Pvt1 and Myc, as well as decreased glucose-stimulated β-cell proliferation. The mitogenic glucose response of Myc is mediated via glucose-dependent recruitment of ChREBP to the promoter of the Pvt1 gene and subsequent DNA looping with the Myc promoter. Show less

Tirzepatide, a dual GIP and GLP-1 receptor agonist, delivered superior glycemic control and weight loss compared to selective GLP-1 receptor (GLP-1R) agonism in patients with type 2 diabetes (T2D). These results have fueled mechanistic studies focused on understanding how tirzepatide achieves its therapeutic efficacy. Recently, we found that treatment with tirzepatide improves insulin sensitivity in humans with T2D and obese mice in concert with a reduction in circulating levels of branched-chain amino (BCAAs) and keto (BCKAs) acids, metabolites associated with development of systemic insulin resistance (IR) and T2D. Importantly, these systemic effects were found to be coupled to increased expression of BCAA catabolic genes in thermogenic brown adipose tissue (BAT) in mice. These findings led us to hypothesize that tirzepatide may lower circulating BCAAs/BCKAs by promoting their catabolism in BAT. To address this question, we utilized a murine model of diet-induced obesity and employed stable-isotope tracer studies in combination with metabolomic analyses in BAT and other tissues. Treatment with tirzepatide stimulated catabolism of BCAAs/BCKAs in BAT, as demonstrated by increased labeling of BCKA-derived metabolites, and increases in levels of byproducts of BCAA breakdown, including glutamate, alanine, and 3-hydroxyisobutyric acid (3-HIB). Further, chronic administration of tirzepatide increased levels of multiple amino acids in BAT that have previously been shown to be elevated in response to cold exposure. Finally, chronic treatment with tirzepatide led to a substantial increase in several TCA cycle intermediates (α-ketoglutarate, fumarate, and malate) in BAT. These findings suggest that tirzepatide induces a thermogenic-like amino acid profile in BAT, an effect that may account for reduced systemic levels of BCAAs in obese IR mice. Show less

Hepatic de novo lipogenesis is influenced by the branched-chain α-keto acid dehydrogenase (BCKDH) kinase (BCKDK). Here, we aimed to determine whether circulating levels of the immediate substrates of BCKDH, the branched-chain α-keto acids (BCKAs), and hepatic BCKDK expression are associated with the presence and severity of nonalcoholic fatty liver disease (NAFLD). Eighty metabolites (3 BCKAs, 14 amino acids, 43 acylcarnitines, 20 ceramides) were quantified in plasma from 288 patients with bariatric surgery with severe obesity and scored liver biopsy samples. Metabolite principal component analysis factors, BCKAs, branched-chain amino acids (BCAAs), and the BCKA/BCAA ratio were tested for associations with steatosis grade and presence of nonalcoholic steatohepatitis (NASH). Of all analytes tested, only the Val-derived BCKA, α-keto-isovalerate, and the BCKA/BCAA ratio were associated with both steatosis grade and NASH. Gene expression analysis in liver samples from 2 independent bariatric surgery cohorts showed that hepatic BCKDK mRNA expression correlates with steatosis, ballooning, and levels of the lipogenic transcription factor SREBP1. Experiments in AML12 hepatocytes showed that SREBP1 inhibition lowered BCKDK mRNA expression. These findings demonstrate that higher plasma levels of BCKA and hepatic expression of BCKDK are features of human NAFLD/NASH and identify SREBP1 as a transcriptional regulator of BCKDK. Show less

A strong association of obesity and insulin resistance with increased circulating levels of branched-chain and aromatic amino acids and decreased glycine levels has been recognized in human subjects for decades. More recently, human metabolomics and genetic studies have confirmed and expanded upon these observations, accompanied by a surge in preclinical studies that have identified mechanisms involved in the perturbation of amino acid homeostasis- how these events are connected to dysregulated glucose and lipid metabolism, and how elevations in branched-chain amino acids (BCAA) may participate in the development of insulin resistance, type 2 diabetes (T2D), and other cardiometabolic diseases and conditions. In human cohorts, BCAA and related metabolites are now well established as among the strongest biomarkers of obesity, insulin resistance, T2D, and cardiovascular diseases. Lowering of BCAA and branched-chain ketoacid (BCKA) levels by feeding BCAA-restricted diet or by the activation of the rate-limiting enzyme in BCAA catabolism, branched-chain ketoacid dehydrogenase (BCKDH), in rodent models of obesity have clear salutary effects on glucose and lipid homeostasis, but BCAA restriction has more modest effects in short-term studies in human T2D subjects. Feeding of rats with diets enriched in sucrose or fructose result in the induction of the ChREBP transcription factor in the liver to increase expression of the BCKDH kinase (BDK) and suppress the expression of its phosphatase (PPM1K) resulting in the inactivation of BCKDH and activation of the key lipogenic enzyme ATP-citrate lyase (ACLY). These and other emergent links between BCAA, glucose, and lipid metabolism motivate ongoing studies of possible causal actions of BCAA and related metabolites in the development of cardiometabolic diseases. Show less

Branched-chain amino acids (BCAA) are strongly associated with dysregulated glucose and lipid metabolism, but the underlying mechanisms are poorly understood. We report that inhibition of the kinase (BDK) or overexpression of the phosphatase (PPM1K) that regulates branched-chain ketoacid dehydrogenase (BCKDH), the committed step of BCAA catabolism, lowers circulating BCAA, reduces hepatic steatosis, and improves glucose tolerance in the absence of weight loss in Zucker fatty rats. Phosphoproteomics analysis identified ATP-citrate lyase (ACL) as an alternate substrate of BDK and PPM1K. Hepatic overexpression of BDK increased ACL phosphorylation and activated de novo lipogenesis. BDK and PPM1K transcript levels were increased and repressed, respectively, in response to fructose feeding or expression of the ChREBP-β transcription factor. These studies identify BDK and PPM1K as a ChREBP-regulated node that integrates BCAA and lipid metabolism. Moreover, manipulation of the BDK:PPM1K ratio relieves key metabolic disease phenotypes in a genetic model of severe obesity. Show less