Michael J. Muehlbauer

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.

Gut microbiota from twins discordant for obesity modulate metabolism in mice.

Introduction Establishing whether specific structural and functional configurations of a human gut microbiota are causally related to a given physiologic or disease phenotype is challenging. Twins discordant for obesity provide an opportunity to examine interrelations between obesity and its associated metabolic disorders, diet, and the gut microbiota. Transplanting the intact uncultured or cultured human fecal microbiota from each member of a discordant twin pair into separate groups of recipient germfree mice permits the donors’ communities to be replicated, differences between their properties to be identified, the impact of these differences on body composition and metabolic phenotypes to be discerned, and the effects of diet-by-microbiota interactions to be analyzed. In addition, cohousing coprophagic mice harboring transplanted microbiota from discordant pairs provides an opportunity to determine which bacterial taxa invade the gut communities of cage mates, how invasion correlates with host phenotypes, and how invasion and microbial niche are affected by human diets. Cohousing Ln and Ob mice prevents increased adiposity in Ob cage mates (Ob). (A) Adiposity change after 10 days of cohousing. *P < 0.05 versus Ob controls (Student’s t test). (B) Bacteroidales from Ln microbiota invade Ob microbiota. Columns show individual mice. Methods Separate groups of germfree mice were colonized with uncultured fecal microbiota from each member of four twin pairs discordant for obesity or with culture collections from an obese (Ob) or lean (Ln) co-twin. Animals were fed a mouse chow low in fat and rich in plant polysaccharides, or one of two diets reflecting the upper or lower tertiles of consumption of saturated fats and fruits and vegetables based on the U.S. National Health and Nutrition Examination Survey (NHANES). Ln or Ob mice were cohoused 5 days after colonization. Body composition changes were defined by quantitative magnetic resonance. Microbiota or microbiome structure, gene expression, and metabolism were assayed by 16S ribosomal RNA profiling, whole-community shotgun sequencing, RNA-sequencing, and mass spectrometry. Host gene expression and metabolism were also characterized. Results and Discussion The intact uncultured and culturable bacterial component of Ob co-twins’ fecal microbiota conveyed significantly greater increases in body mass and adiposity than those of Ln communities. Differences in body composition were correlated with differences in fermentation of short-chain fatty acids (increased in Ln), metabolism of branched-chain amino acids (increased in Ob), and microbial transformation of bile acid species (increased in Ln and correlated with down-regulation of host farnesoid X receptor signaling). Cohousing Ln and Ob mice prevented development of increased adiposity and body mass in Ob cage mates and transformed their microbiota’s metabolic profile to a leanlike state. Transformation correlated with invasion of members of Bacteroidales from Ln into Ob microbiota. Invasion and phenotypic rescue were diet-dependent and occurred with the diet representing the lower tertile of U.S. consumption of saturated fats, and upper tertile of fruits and vegetables, but not with the diet representing the upper tertile of saturated fats, and lower tertile of fruit and vegetable consumption. These results reveal that transmissible and modifiable interactions between diet and microbiota influence host biology. Transforming Fat to Thin How much does the microbiota influence the hosts phenotype? Ridaura et al. (1241214 ; see the Perspective by Walker and Parkhill) obtained uncultured fecal microbiota from twin pairs discordant for body mass and transplanted them into adult germ-free mice. It was discovered that adiposity is transmissible from human to mouse and that it was associated with changes in serum levels of branched-chain amino acids. Moreover, obese-phenotype mice were invaded by members of the Bacteroidales from the lean mice, but, happily, the lean animals resisted invasion by the obese microbiota. Mice carrying gut bacteria from lean humans protect their cage mates from the effects of gut bacteria from fat humans. [Also see Perspective by Walker and Parkhill] The role of specific gut microbes in shaping body composition remains unclear. We transplanted fecal microbiota from adult female twin pairs discordant for obesity into germ-free mice fed low-fat mouse chow, as well as diets representing different levels of saturated fat and fruit and vegetable consumption typical of the U.S. diet. Increased total body and fat mass, as well as obesity-associated metabolic phenotypes, were transmissible with uncultured fecal communities and with their corresponding fecal bacterial culture collections. Cohousing mice harboring an obese twin’s microbiota (Ob) with mice containing the lean co-twin’s microbiota (Ln) prevented the development of increased body mass and obesity-associated metabolic phenotypes in Ob cage mates. Rescue correlated with invasion of specific members of Bacteroidetes from the Ln microbiota into Ob microbiota and was diet-dependent. These findings reveal transmissible, rapid, and modifiable effects of diet-by-microbiota interactions.

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.

The Impact of a Consortium of Fermented Milk Strains on the Gut Microbiome of Gnotobiotic Mice and Monozygotic Twins

Metagenomic analyses of gnotobiotic mice and monozygotic twins reveal the effects of eating a popular fermented milk product on their microbiomes. A Yogurt a Day… We all enjoy a tasty yogurt and believe that the bacterial species contained in this type of fermented milk product will keep us healthy. But how much influence do the microbes in these products have on our gut microbiomes and consequently our health, and are these effects generalizable to different human populations consuming different diets? These questions are of concern to regulatory agencies who are increasing pressure on manufacturers to validate the health claims of various foods, including yogurts. McNulty and his colleagues, in an exciting new study, describe a way to evaluate their effects on the human gut microbiome. First, they studied the effects of consuming a popular yogurt on the gut microbiomes of seven healthy adult female identical twin pairs. The bacterial and gene composition, as well as the gene expression patterns, of their gut microbial communities were analyzed before, during, and after consumption of the yogurt. These results were compared to those obtained in gnotobiotic mice that were first reared under conditions where the only microbes they harbored were 15 prominent, sequenced human gut bacterial symbionts, after which time they were exposed to the same 5 bacterial strains as those contained in the yogurt. McNulty and colleagues found during repeated sampling of the gut microbiomes of the twins over a 4-month period that the species and gene content of their gut microbial communities remained stable and were not appreciably perturbed by consuming the yogurt. After exposure of the humanized mice to the five bacterial strains in the fermented milk product, the researchers showed that the mice did not exhibit marked changes in the proportional representation of their human symbiotic bacterial species or genes, mirroring the results seen in the twins. However, analysis of gut bacterial gene expression profiles and of urinary metabolites in these mice disclosed that introducing the fermented milk product strains resulted in marked changes in a number of metabolic pathways, most prominently those related to carbohydrate processing. These latter findings helped direct follow-up studies of the twins’ gut samples where they found similar changes in metabolism as those observed in mice. These findings show that mice containing a sequenced model human gut microbiome can serve as part of a preclinical discovery pipeline designed to identify the effects of existing or new bacterial species with purported health benefits on the properties of the human gut microbiome. Although it remains unclear whether eating a yogurt a day will keep the doctor away, the study by McNulty and his colleagues paves the way for future work to analyze in more detail the direct effects of consuming foods containing bacterial species with potential health benefits on the gut microbiomes of various human populations. Understanding how the human gut microbiota and host are affected by probiotic bacterial strains requires carefully controlled studies in humans and in mouse models of the gut ecosystem where potentially confounding variables that are difficult to control in humans can be constrained. Therefore, we characterized the fecal microbiomes and metatranscriptomes of adult female monozygotic twin pairs through repeated sampling 4 weeks before, 7 weeks during, and 4 weeks after consumption of a commercially available fermented milk product (FMP) containing a consortium of Bifidobacterium animalis subsp. lactis, two strains of Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis subsp. cremoris, and Streptococcus thermophilus. In addition, gnotobiotic mice harboring a 15-species model human gut microbiota whose genomes contain 58,399 known or predicted protein-coding genes were studied before and after gavage with all five sequenced FMP strains. No significant changes in bacterial species composition or in the proportional representation of genes encoding known enzymes were observed in the feces of humans consuming the FMP. Only minimal changes in microbiota configuration were noted in mice after single or repeated gavage with the FMP consortium. However, RNA-Seq analysis of fecal samples and follow-up mass spectrometry of urinary metabolites disclosed that introducing the FMP strains into mice results in significant changes in expression of microbiome-encoded enzymes involved in numerous metabolic pathways, most prominently those related to carbohydrate metabolism. B. animalis subsp. lactis, the dominant persistent member of the FMP consortium in gnotobiotic mice, up-regulates a locus in vivo that is involved in the catabolism of xylooligosaccharides, a class of glycans widely distributed in fruits, vegetables, and other foods, underscoring the importance of these sugars to this bacterial species. The human fecal metatranscriptome exhibited significant changes, confined to the period of FMP consumption, that mirror changes in gnotobiotic mice, including those related to plant polysaccharide metabolism. These experiments illustrate a translational research pipeline for characterizing the effects of FMPs on the human gut microbiome.

Cafeteria diet is a robust model of human metabolic syndrome with liver and adipose inflammation: comparison to high-fat diet.

Obesity has reached epidemic proportions worldwide and reports estimate that American children consume up to 25% of calories from snacks. Several animal models of obesity exist, but studies are lacking that compare high‐fat diets (HFD) traditionally used in rodent models of diet‐induced obesity (DIO) to diets consisting of food regularly consumed by humans, including high‐salt, high‐fat, low‐fiber, energy dense foods such as cookies, chips, and processed meats. To investigate the obesogenic and inflammatory consequences of a cafeteria diet (CAF) compared to a lard‐based 45% HFD in rodent models, male Wistar rats were fed HFD, CAF or chow control diets for 15 weeks. Body weight increased dramatically and remained significantly elevated in CAF‐fed rats compared to all other diets. Glucose‐ and insulin‐tolerance tests revealed that hyperinsulinemia, hyperglycemia, and glucose intolerance were exaggerated in the CAF‐fed rats compared to controls and HFD‐fed rats. It is well‐established that macrophages infiltrate metabolic tissues at the onset of weight gain and directly contribute to inflammation, insulin resistance, and obesity. Although both high fat diets resulted in increased adiposity and hepatosteatosis, CAF‐fed rats displayed remarkable inflammation in white fat, brown fat and liver compared to HFD and controls. In sum, the CAF provided a robust model of human metabolic syndrome compared to traditional lard‐based HFD, creating a phenotype of exaggerated obesity with glucose intolerance and inflammation. This model provides a unique platform to study the biochemical, genomic and physiological mechanisms of obesity and obesity‐related disease states that are pandemic in western civilization today.

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.

Lysine Glutarylation Is a Protein Posttranslational Modification Regulated by SIRT5

We report the identification and characterization of a five-carbon protein posttranslational modification (PTM) called lysine glutarylation (Kglu). This protein modification was detected by immunoblot and mass spectrometry (MS), and then comprehensively validated by chemical and biochemical methods. We demonstrated that the previously annotated deacetylase, sirtuin 5 (SIRT5), is a lysine deglutarylase. Proteome-wide analysis identified 683 Kglu sites in 191 proteins and showed that Kglu is highly enriched on metabolic enzymes and mitochondrial proteins. We validated carbamoyl phosphate synthase 1 (CPS1), the rate-limiting enzyme in urea cycle, as a glutarylated protein and demonstrated that CPS1 is targeted by SIRT5 for deglutarylation. We further showed that glutarylation suppresses CPS1 enzymatic activity in cell lines, mice, and a model of glutaric acidemia type I disease, the last of which has elevated glutaric acid and glutaryl-CoA. This study expands the landscape of lysine acyl modifications and increases our understanding of the deacylase SIRT5.

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.

Hypothalamic CaMKK2 Contributes to the Regulation of Energy Balance

Detailed knowledge of the pathways by which ghrelin and leptin signal to AMPK in hypothalamic neurons and lead to regulation of appetite and glucose homeostasis is central to the development of effective means to combat obesity. Here we identify CaMKK2 as a component of one of these pathways, show that it regulates hypothalamic production of the orexigenic hormone NPY, provide evidence that it functions as an AMPKalpha kinase in the hypothalamus, and demonstrate that it forms a unique signaling complex with AMPKalpha and beta. Acute pharmacologic inhibition of CaMKK2 in wild-type mice, but not CaMKK2 null mice, inhibits appetite and promotes weight loss consistent with decreased NPY and AgRP mRNAs. Moreover, the loss of CaMKK2 protects mice from high-fat diet-induced obesity, insulin resistance, and glucose intolerance. These data underscore the potential of targeting CaMKK2 as a therapeutic intervention.

Association of a peripheral blood metabolic profile with coronary artery disease and risk of subsequent cardiovascular events

Background—Molecular tools may provide insight into cardiovascular risk. We assessed whether metabolites discriminate coronary artery disease (CAD) and predict risk of cardiovascular events. Methods and Results—We performed mass–spectrometry–based profiling of 69 metabolites in subjects from the CATHGEN biorepository. To evaluate discriminative capabilities of metabolites for CAD, 2 groups were profiled: 174 CAD cases and 174 sex/race-matched controls (“initial”), and 140 CAD cases and 140 controls (“replication”). To evaluate the capability of metabolites to predict cardiovascular events, cases were combined (“event” group); of these, 74 experienced death/myocardial infarction during follow-up. A third independent group was profiled (“event-replication” group; n=63 cases with cardiovascular events, 66 controls). Analysis included principal-components analysis, linear regression, and Cox proportional hazards. Two principal components analysis–derived factors were associated with CAD: 1 comprising branched-chain amino acid metabolites (factor 4, initial P=0.002, replication P=0.01), and 1 comprising urea cycle metabolites (factor 9, initial P=0.0004, replication P=0.01). In multivariable regression, these factors were independently associated with CAD in initial (factor 4, odds ratio [OR], 1.36; 95% CI, 1.06 to 1.74; P=0.02; factor 9, OR, 0.67; 95% CI, 0.52 to 0.87; P=0.003) and replication (factor 4, OR, 1.43; 95% CI, 1.07 to 1.91; P=0.02; factor 9, OR, 0.66; 95% CI, 0.48 to 0.91; P=0.01) groups. A factor composed of dicarboxylacylcarnitines predicted death/myocardial infarction (event group hazard ratio 2.17; 95% CI, 1.23 to 3.84; P=0.007) and was associated with cardiovascular events in the event-replication group (OR, 1.52; 95% CI, 1.08 to 2.14; P=0.01). Conclusions—Metabolite profiles are associated with CAD and subsequent cardiovascular events.