OBJECTIVE-Obesity is associated with endocrine abnormalities that predict the progression of insulin resistance to type 2 diabetes. Because skeletal muscle has been shown to secrete proteins that could be used as biomarkers, we characterized the secreted protein profile of muscle cells derived from extremely obese (BMI 48.8 Ϯ 14.8 kg/m 2 ; homeostasis model assessment [HOMA] 3.6 Ϯ 1.0) relative to lean healthy subjects (BMI 25.7 Ϯ 3.2 kg/m 2 ; HOMA 0.8 Ϯ 0.2).RESEARCH DESIGN AND METHODS-We hypothesized that skeletal muscle would secrete proteins that predict the severity of obesity. To test this hypothesis, we used a "bottom-up" experimental design using stable isotope labeling by amino acids in culture (SILAC) and liquid chromatography/mass spectometry/ mass spectometry (LC-MS/MS) to both identify and quantify proteins secreted from cultured myotubes derived from extremely obese compared with healthy nonobese women.RESULTS-Using SILAC, we discovered a 2.9-fold increase in the secretion of myostatin from extremely obese human myotubes. The increased secretion and biological activity of myostatin were validated by immunoblot (3.16 Ϯ 0.18, P Ͻ 0.01) and a myoblast proliferation assay using conditioned growth medium. Myostatin was subsequently shown to increase in skeletal muscle (23%, P Ͻ 0.05) and plasma (35%, P Ͻ 0.05) and to correlate (r 2 ϭ 0.6, P Ͻ 0.05) with the severity of insulin resistance.CONCLUSIONS-Myostatin is a potent antianabolic regulator of muscle mass that may also play a role in energy metabolism. These findings show that increased expression of myostatin in skeletal muscle with obesity and insulin resistance results in elevated circulating myostatin. This may contribute to systemic metabolic deterioration of skeletal muscle with the progression of insulin resistance to type 2 diabetes. Diabetes 58: [30][31][32][33][34][35][36][37][38] 2009 O besity and type 2 diabetes are associated with endocrine abnormalities that are either precipitated by or precede the onset of peripheral insulin resistance (1). These include changes in circulating proteins and peptides that produce endothelial dysfunction, low-grade inflammation, and a prothrombotic state, all of which contribute to increased cardiovascular risk (2-4). Secreted proteins or the "secretome" constitute an important class of biologically active molecules that are released into circulation where they facilitate cross-talk between organ systems. Because secreted proteins are also involved in the progression of cardiovascular disease and cancer, there is significant interest in mining the secretome for novel biological markers (5). Whereas endocrine organs specialize in the secretion of proteins into circulation, there is mounting evidence that adipose tissue and skeletal muscle constitutively or intermittently secrete bioactive proteins (6,7). In this study, we hypothesized that skeletal muscle of extremely obese and insulinresistant women would secrete proteins into circulation that act as prognostic or diagnostic biomarkers of obesityass...

Aspartame consumption is implicated in the development of obesity and metabolic disease despite the intention of limiting caloric intake. The mechanisms responsible for this association remain unclear, but may involve circulating metabolites and the gut microbiota. Aims were to examine the impact of chronic low-dose aspartame consumption on anthropometric, metabolic and microbial parameters in a diet-induced obese model. Male Sprague-Dawley rats were randomized into a standard chow diet (CH, 12% kcal fat) or high fat (HF, 60% kcal fat) and further into ad libitum water control (W) or low-dose aspartame (A, 5–7 mg/kg/d in drinking water) treatments for 8 week (n = 10–12 animals/treatment). Animals on aspartame consumed fewer calories, gained less weight and had a more favorable body composition when challenged with HF compared to animals consuming water. Despite this, aspartame elevated fasting glucose levels and an insulin tolerance test showed aspartame to impair insulin-stimulated glucose disposal in both CH and HF, independently of body composition. Fecal analysis of gut bacterial composition showed aspartame to increase total bacteria, the abundance of Enterobacteriaceae and Clostridium leptum. An interaction between HF and aspartame was also observed for Roseburia ssp wherein HF-A was higher than HF-W (P<0.05). Within HF, aspartame attenuated the typical HF-induced increase in the Firmicutes:Bacteroidetes ratio. Serum metabolomics analysis revealed aspartame to be rapidly metabolized and to be associated with elevations in the short chain fatty acid propionate, a bacterial end product and highly gluconeogenic substrate, potentially explaining its negative affects on insulin tolerance. How aspartame influences gut microbial composition and the implications of these changes on the development of metabolic disease require further investigation.

Purpose There is mounting evidence that skeletal muscle produces and secretes biologically active proteins or “myokines” that facilitate metabolic cross talk between organ systems. The increased expression of myostatin, a secreted anabolic inhibitor of muscle growth and development, has been associated with obesity and insulin resistance. Despite these intriguing findings, there have been few studies linking myostatin and insulin resistance. Methods To explore this relationship in more detail, we quantified myostatin protein in muscle and plasma from 10 insulin-resistant, middle aged (53.1 ± 5.5 years) men before and after 6 months of moderate aerobic exercise training (1200 kcal/wk at 40–55% peak VO2). To establish a case-effect relationship we also injected C57/Bl6 male mice with high-physiologic levels of recombinant myostatin protein. Results Myostatin protein levels were shown to decrease in muscle (37%, P=0.042, n=10) and matching plasma samples (28.7 pre-training to 22.8 ng/ml post-training, P=0.003, n=9) with aerobic exercise. Furthermore, the strong correlation between plasma myostatin levels and insulin sensitivity (R2 = 0.82, P<0.001, n=9) suggested a cause-effect relationship that was subsequently confirmed by inducing insulin resistance in myostatin-injected mice. A modest increase (44%) in plasma myostatin levels was also associated with significant reductions in the insulin-stimulated phosphorylation of AKT (Thr308) in both muscle and liver of myostatin treated animals. Conclusions These findings indicate that both muscle and plasma myostatin protein levels are regulated by aerobic exercise and furthermore, that myostatin is in the causal pathway of acquired insulin resistance with physical inactivity.

BackgroundGastrointestinal dysfunction and gut microbial composition disturbances have been widely reported in autism spectrum disorder (ASD). This study examines whether gut microbiome disturbances are present in the BTBRT + tf/j (BTBR) mouse model of ASD and if the ketogenic diet, a diet previously shown to elicit therapeutic benefit in this mouse model, is capable of altering the profile.FindingsJuvenile male C57BL/6 (B6) and BTBR mice were fed a standard chow (CH, 13 % kcal fat) or ketogenic diet (KD, 75 % kcal fat) for 10–14 days. Following diets, fecal and cecal samples were collected for analysis. Main findings are as follows: (1) gut microbiota compositions of cecal and fecal samples were altered in BTBR compared to control mice, indicating that this model may be of utility in understanding gut-brain interactions in ASD; (2) KD consumption caused an anti-microbial-like effect by significantly decreasing total host bacterial abundance in cecal and fecal matter; (3) specific to BTBR animals, the KD counteracted the common ASD phenotype of a low Firmicutes to Bacteroidetes ratio in both sample types; and (4) the KD reversed elevated Akkermansia muciniphila content in the cecal and fecal matter of BTBR animals.ConclusionsResults indicate that consumption of a KD likely triggers reductions in total gut microbial counts and compositional remodeling in the BTBR mouse. These findings may explain, in part, the ability of a KD to mitigate some of the neurological symptoms associated with ASD in an animal model.Electronic supplementary materialThe online version of this article (doi:10.1186/s13229-016-0099-3) contains supplementary material, which is available to authorized users.

Cecal microbiota from type 2 diabetic (db/db) and control (db/(+)) mice was obtained following 6 weeks of sedentary or exercise activity. qPCR analysis revealed a main effect of exercise, with greater abundance of select Firmicutes species and lower Bacteroides/Prevotella spp. in both normal and diabetic exercised mice compared with sedentary counterparts. Conversely, Bifidobacterium spp. was greater in exercised normal but not diabetic mice (exercise × diabetes interaction). How exercise influences gut microbiota requires further investigation.

Epidemiological studies show coffee consumption to be correlated to large risk reductions in the prevalence of type 2 diabetes (T2D). Such correlations are seen with decaffeinated and caffeinated coffee, and occur regardless of gender, method of brewing, or geography. They also exist despite clear evidence showing that caffeine causes acute postprandial hyperglycemia and lower whole-body insulin sensitivity. As the beneficial effects of coffee consumption exist for both decaffeinated and caffeinated coffee, a component of coffee other than caffeine must be responsible. This review examines the specific coffee compounds responsible for coffee's effects on T2D, and their potential physiological mechanisms of action. Being plant-derived, coffee contains many beneficial compounds found in fruits and vegetables, including antioxidants. In fact, coffee is the largest source of dietary antioxidants in industrialized nations. When green coffee is roasted at high temperatures, Maillard reactions create a number of unique compounds. Roasting causes a portion of the antioxidant, chlorogenic acid, to be transformed into quinides, compounds known to alter blood glucose levels. Coffee consumption may also mediate levels of gut peptides (glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1), hormones intimately involved in the regulation of satiety and insulin secretion. Finally, coffee may have prebiotic-like properties, altering gut flora and ultimately digestion. In summary, it is evident that a better understanding of the role of coffee in the development and prevention of T2D has the potential to uncover novel therapeutic targets and nutraceutical formulations for the disease.

Glycogen, the primary storage form of glucose, is a rapid and accessible form of energy that can be supplied to tissues on demand. Each glycogen granule, or "glycosome," is considered an independent metabolic unit composed of a highly branched polysaccharide and various proteins involved in its metabolism. In this Minireview, we review the literature to follow the dynamic life of a glycogen granule in a multicompartmentalized system, the cell, and how and where glycogen granules appear and the factors governing its degradation. A better understanding of the importance of cellular compartmentalization as a regulator of glycogen metabolism is needed to unravel its role in brain energetics.

Quantification of subcellular glycogen in resting human muscle: granule size, number, and location. J Appl Physiol 93: 1598-1607, 2002. First published July 12, 2002 10.1152/ japplphysiol.00585.2001.-A few qualitative investigations suggested that location of muscle glycogen (G) granules in specific sites may be associated with distinct metabolic roles. Similarly, it has been suggested that the acid-soluble and -insoluble G fractions (macro-and proglycogen, respectively) are different metabolic pools and also could exist as separate entities. We employed a transmission electron microscopic technique to quantify subcellular G particle size, number, and location in human vastus lateralis biopsies of 11 resting men. The intra-and interobserver variability for the various measures was generally Ͻ4%. Granule size and number were quantified in subcellular compartments (subsarcolemmal, intra-and intermyofibrillar). Subcellular location was critical: G was more densely concentrated in the subsarcolemmal than in the myofibrillar space, whereas the single-particle volume was greater in the latter. Single-particle diameter ranged from 10 to 44 m and followed a continuous, normal distribution. This implies that proglycogen is not a distinct entity, but rather that pro-and macroglycogen are divisions of smaller and larger molecules. These results demonstrate a compartmentalized pattern of subcellular G deposition in human skeletal muscle for both the size and density of granules.glycosome; metabolic compartments; electron microscopy; carbohydrate; glycogen regulation; proglycogen; macroglycogen SINCE THE DISCOVERY OF GLYCOGEN by Claude Bernard, numerous investigators have addressed many aspects of its metabolism. Although muscle glycogen concentration has been routinely quantified biochemically, the subcellular organization of glycogen particles has been studied much less frequently and only with qualitative, descriptive transmission electron microscopy (TEM) methods. Wanson and Drochmans (31) performed the first comprehensive description of rabbit skeletal muscle glycogen in its particulate ␤-form. Drochmans (5) had previously examined negatively stained liver glycogen by using TEM and described three glycogen structures in liver: ␣-, ␤-, and ␥-particles. The ␣-particles were the typical liver rosettes, and the ␤-particles were the 20-to 30-m spheroid units forming the ␣-rosettes. The ␥-particles were identified as 3-m subunits of both ␣-and ␤-structures. The single ␤-particles described in muscles by Wanson and Drochmans (31) corresponded in size and shape to the ␤-subunits that constituted the ␣-rosettes in liver.Scott and Still (28) proposed that particulate glycogen was not a molecule in the traditional static sense but rather a dynamic organelle. In 1970, Meyer et al. (21) were among the first to suggest that glycogen was complexed with proteins and represented a structural and functional unit of the muscle cells. Using TEM, they estimated the diameter of this glycogen particle to be 20-30 m. This value was in agree...