Robert D. Stevens

A Branched-Chain Amino Acid-Related Metabolic Signature that Differentiates Obese and Lean Humans and Contributes to Insulin Resistance

Metabolomic profiling of obese versus lean humans reveals a branched-chain amino acid (BCAA)-related metabolite signature that is suggestive of increased catabolism of BCAA and correlated with insulin resistance. To test its impact on metabolic homeostasis, we fed rats on high-fat (HF), HF with supplemented BCAA (HF/BCAA), or standard chow (SC) diets. Despite having reduced food intake and a low rate of weight gain equivalent to the SC group, HF/BCAA rats were as insulin resistant as HF rats. Pair-feeding of HF diet to match the HF/BCAA animals or BCAA addition to SC diet did not cause insulin resistance. Insulin resistance induced by HF/BCAA feeding was accompanied by chronic phosphorylation of mTOR, JNK, and IRS1Ser307 and by accumulation of multiple acylcarnitines in muscle, and it was reversed by the mTOR inhibitor, rapamycin. Our findings show that in the context of a dietary pattern that includes high fat consumption, BCAA contributes to development of obesity-associated insulin resistance.

Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance.

Previous studies have suggested that insulin resistance develops secondary to diminished fat oxidation and resultant accumulation of cytosolic lipid molecules that impair insulin signaling. Contrary to this model, the present study used targeted metabolomics to find that obesity-related insulin resistance in skeletal muscle is characterized by excessive beta-oxidation, impaired switching to carbohydrate substrate during the fasted-to-fed transition, and coincident depletion of organic acid intermediates of the tricarboxylic acid cycle. In cultured myotubes, lipid-induced insulin resistance was prevented by manipulations that restrict fatty acid uptake into mitochondria. These results were recapitulated in mice lacking malonyl-CoA decarboxylase (MCD), an enzyme that promotes mitochondrial beta-oxidation by relieving malonyl-CoA-mediated inhibition of carnitine palmitoyltransferase 1. Thus, mcd(-/-) mice exhibit reduced rates of fat catabolism and resist diet-induced glucose intolerance despite high intramuscular levels of long-chain acyl-CoAs. These findings reveal a strong connection between skeletal muscle insulin resistance and lipid-induced mitochondrial stress.

SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation

Sirtuins are NAD+-dependent protein deacetylases. They mediate adaptive responses to a variety of stresses, including calorie restriction and metabolic stress. Sirtuin 3 (SIRT3) is localized in the mitochondrial matrix, where it regulates the acetylation levels of metabolic enzymes, including acetyl coenzyme A synthetase 2 (refs 1, 2). Mice lacking both Sirt3 alleles appear phenotypically normal under basal conditions, but show marked hyperacetylation of several mitochondrial proteins. Here we report that SIRT3 expression is upregulated during fasting in liver and brown adipose tissues. During fasting, livers from mice lacking SIRT3 had higher levels of fatty-acid oxidation intermediate products and triglycerides, associated with decreased levels of fatty-acid oxidation, compared to livers from wild-type mice. Mass spectrometry of mitochondrial proteins shows that long-chain acyl coenzyme A dehydrogenase (LCAD) is hyperacetylated at lysine 42 in the absence of SIRT3. LCAD is deacetylated in wild-type mice under fasted conditions and by SIRT3 in vitro and in vivo; and hyperacetylation of LCAD reduces its enzymatic activity. Mice lacking SIRT3 exhibit hallmarks of fatty-acid oxidation disorders during fasting, including reduced ATP levels and intolerance to cold exposure. These findings identify acetylation as a novel regulatory mechanism for mitochondrial fatty-acid oxidation and demonstrate that SIRT3 modulates mitochondrial intermediary metabolism and fatty-acid use during fasting.

Suppression of Oxidative Stress by β-Hydroxybutyrate, an Endogenous Histone Deacetylase Inhibitor

Stress Protector During prolonged fasting, the oxidation of fatty acids leads to increased accumulation of d-β-hydroxybutyrate (βOHB) in the bloodstream. Such increased concentrations of βOHB inhibit class I histone deacetylases. Histone acetylation in turn influences transcriptional activity at various genes. Shimazu et al. (p. 211, published online 6 December; see the Perspective by Sassone-Corsi) found that among the genes showing increased transcription in animals treated with high concentrations of βOHB were two genes implicated in cellular responses to oxidative stress. When treated ahead of time with βOHB, mice were protected from the toxic effects of the oxidative stress causing poison paraquat. Ketone bodies, metabolites that accumulate during fasting, change gene expression by inhibiting histone deacetylases. [Also see Perspective by Sassone-Corsi] Concentrations of acetyl–coenzyme A and nicotinamide adenine dinucleotide (NAD+) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-β-hydroxybutyrate (βOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous βOHB, or fasting or calorie restriction, two conditions associated with increased βOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by βOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with βOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with βOHB conferred substantial protection against oxidative stress.

SIRT3 Deficiency and Mitochondrial Protein Hyperacetylation Accelerate the Development of the Metabolic Syndrome

Acetylation is increasingly recognized as an important metabolic regulatory posttranslational protein modification, yet the metabolic consequence of mitochondrial protein hyperacetylation is unknown. We find that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. Mice lacking SIRT3 (SIRT3KO) placed on a HFD show accelerated obesity, insulin resistance, hyperlipidemia, and steatohepatitis compared to wild-type (WT) mice. The lipogenic enzyme stearoyl-CoA desaturase 1 is highly induced in SIRT3KO mice, and its deletion rescues both WT and SIRT3KO mice from HFD-induced hepatic steatosis and insulin resistance. We further identify a single nucleotide polymorphism in the human SIRT3 gene that is suggestive of a genetic association with the metabolic syndrome. This polymorphism encodes a point mutation in the SIRT3 protein, which reduces its overall enzymatic efficiency. Our findings show that loss of SIRT3 and dysregulation of mitochondrial protein acetylation contribute to the metabolic syndrome.

Hepatic expression of malonyl-CoA decarboxylase reverses muscle, liver and whole-animal insulin resistance

Lipid infusion or ingestion of a high-fat diet results in insulin resistance, but the mechanism underlying this phenomenon remains unclear. Here we show that, in rats fed a high-fat diet, whole-animal, muscle and liver insulin resistance is ameliorated following hepatic overexpression of malonyl–coenzyme A (CoA) decarboxylase (MCD), an enzyme that affects lipid partitioning. MCD overexpression decreased circulating free fatty acid (FFA) and liver triglyceride content. In skeletal muscle, levels of triglyceride and long-chain acyl-CoA (LC-CoA)—two candidate mediators of insulin resistance—were either increased or unchanged. Metabolic profiling of 36 acylcarnitine species by tandem mass spectrometry revealed a unique decrease in the concentration of one lipid-derived metabolite, β-OH-butyrate, in muscle of MCD-overexpressing animals. The best explanation for our findings is that hepatic expression of MCD lowered circulating FFA levels, which led to lowering of muscle β-OH-butyrate levels and improvement of insulin sensitivity.

Relationships between circulating metabolic intermediates and insulin action in overweight to obese, inactive men and women

OBJECTIVE To determine whether circulating metabolic intermediates are related to insulin resistance and β-cell dysfunction in individuals at risk for type 2 diabetes. RESEARCH DESIGN AND METHODS In 73 sedentary, overweight to obese, dyslipidemic individuals, insulin action was derived from a frequently sampled intravenous glucose tolerance test. Plasma concentrations of 75 amino acids, acylcarnitines, free fatty acids, and conventional metabolites were measured with a targeted, mass spectrometry–based platform. Principal components analysis followed by backward stepwise linear regression was used to explore relationships between measures of insulin action and metabolic intermediates. RESULTS The 75 metabolic intermediates clustered into 19 factors comprising biologically related intermediates. A factor containing large neutral amino acids was inversely related to insulin sensitivity (SI) (R2 = 0.26). A factor containing fatty acids was inversely related to the acute insulin response to glucose (R2 = 0.12). Both of these factors, age, and a factor containing medium-chain acylcarnitines and glucose were inversely and independently related to the disposition index (DI) (R2 = 0.39). Sex differences were found for metabolic predictors of SI and DI. CONCLUSIONS In addition to the well-recognized risks for insulin resistance, elevated concentrations of large, neutral amino acids were independently associated with insulin resistance. Fatty acids were inversely related to the pancreatic response to glucose. Both large neutral amino acids and fatty acids were related to an appropriate pancreatic response, suggesting that these metabolic intermediates might play a role in the progression to type 2 diabetes, one by contributing to insulin resistance and the other to pancreatic failure. These intermediates might exert sex-specific effects on insulin action.

Metabolomics Applied to Diabetes Research: Moving From Information to Knowledge

Type 2 diabetes is caused by a complex set of interactions between genetic and environmental factors. Recent work has shown that human type 2 diabetes is a constellation of disorders associated with polymorphisms in a wide array of genes, with each individual gene accounting for <1% of disease risk (1). Moreover, type 2 diabetes involves dysfunction of multiple organ systems, including impaired insulin action in muscle and adipose, defective control of hepatic glucose production, and insulin deficiency caused by loss of β-cell mass and function (2). This complexity presents challenges for a full understanding of the molecular pathways that contribute to the development of this major disease. Progress in this area may be aided by the recent advent of technologies for comprehensive metabolic analysis, sometimes termed “metabolomics.” Herein, we summarize key metabolomics methodologies, including nuclear magnetic resonance (NMR) and mass spectrometry (MS)-based metabolic profiling technologies, and discuss “nontargeted” versus “targeted” approaches. Examples of the application of these tools to diabetes and metabolic disease research at the cellular, animal model, and human disease levels are summarized, with a particular focus on insights gained from the more quantitative targeted methodologies. We also provide early examples of integrated analysis of genomic, transcriptomic, and metabolomic datasets for gaining knowledge about metabolic regulatory networks and diabetes mechanisms and conclude by discussing prospects for future insights. In principal, metabolomics can provide certain advantages relative to other “omics” technologies (genomics, transcriptomics, proteomics) in diabetes research: 1 ) Estimates vary, but one current source, the Human Metabolome Database (HMDB)-Canada (3), currently lists ∼6,500 discrete small molecule metabolites, significantly less than the estimate of 25,000 genes, 100,000 transcripts, and 1,000,000 proteins. 2 ) Metabolomics measures chemical phenotypes that are the net result of genomic, transcriptomic, and proteomic variability, therefore providing the most integrated profile of biological status. 3 ) Metabolomics is in …

Leptin therapy in insulin-deficient type I diabetes

In nonobese diabetic mice with uncontrolled type 1 diabetes, leptin therapy alone or combined with low-dose insulin reverses the catabolic state through suppression of hyperglucagonemia. Additionally, it mimics the anabolic actions of insulin monotherapy and normalizes hemoglobin A1c with far less glucose variability. We show that leptin therapy, like insulin, normalizes the levels of a wide array of hepatic intermediary metabolites in multiple chemical classes, including acylcarnitines, organic acids (tricarboxylic acid cycle intermediates), amino acids, and acyl CoAs. In contrast to insulin monotherapy, however, leptin lowers both lipogenic and cholesterologenic transcription factors and enzymes and reduces plasma and tissue lipids. The results imply that leptin administration may have multiple short- and long-term advantages over insulin monotherapy for type 1 diabetes.

Differential Metabolic Impact of Gastric Bypass Surgery Versus Dietary Intervention in Obese Diabetic Subjects Despite Identical Weight Loss

The enhanced decrease in circulating branched-chain amino acids and their metabolites after gastric bypass occurs by mechanisms other than weight loss. Dissecting the Quick Fix In the Wizard of Oz, when Dorothy encounters a split in the yellow brick road, the Scarecrow assures her that all paths lead to the land of Oz. We’ve witnessed the perils Dorothy met along the path she chose; however, we don’t know what she would have encountered had she followed another route to Oz. Similarly, obese patients with type 2 diabetes can take one of two paths to weight loss—dietary intervention or gastric bypass surgery (GBP). Although the end result—weight loss—is the same, the metabolic shifts that occur en route appear to differ. Now, Laferrère et al. show that in patients with equivalent weight loss, those who underwent GBP displayed a larger decrease in certain circulating amino acids than did subjects who pursued the dietary intervention path. This difference may help to explain why patients who opted for the surgical intervention boasted better improvement in glucose homeostasis—including enhanced insulin sensitivity—than did those who lost weight by controlling their dietary intake. Obese patients with type 2 diabetes strive to lose weight for reasons more momentous than an approaching swimsuit season. Weight loss can improve the body’s ability to metabolize glucose and thus stems the serious complications of diabetes. Patients often can reduce or forgo their diabetes medications. However, in such patients, glycemic control is improved to a greater extent within days after GBP—before weight loss occurs—than after diet-induced shedding of pounds and inches. Precisely why remains a mystery, but research in animal models has revealed that higher-than-normal blood concentrations of branched-chain amino acids (BCAAs) and their metabolites play a role in the loss of insulin sensitivity. Furthermore, recent studies in human patients show a robust positive correlation between insulin resistance and blood levels of BCAAs and their by-products. Finally, obese people have higher circulating concentrations of these amino acids compared to their lean counterparts; the same goes for individuals with versus without diabetes. These observations imply that the rapid reversal of diabetes symptoms in GBP patients may have something to do with BCAA metabolism. Here, the authors measured circulating amounts of a variety of amino acids and acylcarnitines—some of which are produced primarily from BCAA metabolism—to characterize the differential metabolic responses to weight loss induced by GBP versus dietary intervention in obese type 2 diabetes patients. Circulating concentrations of total amino acids, BCAAs, and BCAA metabolites all decreased significantly after GBP but not after dietary intervention, despite equivalent weight loss. These findings were consistent in two patient cohorts, one from the New York Obesity Nutrition Research Center and one from Duke University; in the latter group, the effects were shown to persist for months. These data support the notion that the surgical intervention promoted enhanced BCAA metabolism by mechanisms separate from weight loss and suggest that changes in circulating amino acids pave the road to the correction of glycemic control observed after GBP. Glycemic control is improved more after gastric bypass surgery (GBP) than after equivalent diet-induced weight loss in patients with morbid obesity and type 2 diabetes mellitus. We applied metabolomic profiling to understand the mechanisms of this better metabolic response after GBP. Circulating amino acids (AAs) and acylcarnitines (ACs) were measured in plasma from fasted subjects by targeted tandem mass spectrometry before and after a matched 10-kilogram weight loss induced by GBP or diet. Total AAs and branched-chain AAs (BCAAs) decreased after GBP, but not after dietary intervention. Metabolites derived from BCAA oxidation also decreased only after GBP. Principal components (PC) analysis identified two major PCs, one composed almost exclusively of ACs (PC1) and another with BCAAs and their metabolites as major contributors (PC2). PC1 and PC2 were inversely correlated with pro-insulin concentrations, the C-peptide response to oral glucose, and the insulin sensitivity index after weight loss, whereas PC2 was uniquely correlated with levels of insulin resistance (HOMA-IR). These data suggest that the enhanced decrease in circulating AAs after GBP occurs by mechanisms other than weight loss and may contribute to the better improvement in glucose homeostasis observed with the surgical intervention.