Katsuhiko Funai

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.

Increased AS160 phosphorylation, but not TBC1D1 phosphorylation, with increased postexercise insulin sensitivity in rat skeletal muscle

A single exercise bout can increase insulin-independent glucose transport immediately postexercise and insulin-dependent glucose transport (GT) for several hours postexercise. Akt substrate of 160 kDa (AS160) and TBC1D1 are paralog Rab GTPase-activating proteins that have been proposed to contribute to these exercise effects. Previous research demonstrated greater AS160 and Akt threonine phosphorylation in rat skeletal muscle at 3-4 h postexercise concomitant with enhanced insulin-stimulated GT. To further probe whether these signaling events or TBC1D1 phosphorylation were important for the enhanced postexercise insulin-stimulated GT, male Wistar rats were studied using four experimental protocols (2-h swim exercise, differing with regard to timing of muscle sampling and whether food was provided postexercise) that were known to vary in their influence of insulin-independent and insulin-dependent GT postexercise. The results indicated that, in isolated rat epitrochlearis muscle, 1) elevated phosphorylation of AS160 (measured using anti-phospho-Akt substrate, PAS-AS160, and phosphospecific anti-Thr(642)-AS160, pThr(642)-AS160) consistently tracked with elevated insulin-stimulated GT; 2) PAS-TBC1D1 was not different from sedentary values at 3 or 27 h postexercise, when insulin sensitivity was increased; 3) insulin-stimulated Akt activity was not increased postexercise in muscles with increased insulin sensitivity; 4) PAS-TBC1D1 was increased immediately postexercise, when insulin-independent GT was elevated, and reversed at 3 and 27 h postexercise, when insulin-independent GT was also reversed; and 5) there was no significant effect of exercise or insulin on total abundance of AS160, TBC1D1, Akt, or GLUT4 protein with any of the protocols. The results are consistent with increased AS160 phosphorylation (PAS-AS160 or pThr(642)-AS160) but not increased PAS-TBC1D1 or Akt activity, which is important for increased postexercise insulin-stimulated GT in rat skeletal muscle. They also support the idea that increased TBC1D1 phosphorylation may play a role in the insulin-independent increase in GT postexercise.

Inhibition of contraction-stimulated AMP-activated protein kinase inhibits contraction-stimulated increases in PAS-TBC1D1 and glucose transport without altering PAS-AS160 in rat skeletal muscle.

OBJECTIVE Phosphorylation of two members of the TBC1 domain family of proteins, Akt substrate of 160 kDa (AS160, also known as TBC1D4) and TBC1D1, has been implicated in the regulation of glucose transport in skeletal muscle. Insulin-stimulated phosphorylation (measured using the phospho-Akt substrate [PAS] antibody) of AS160 and TBC1D1 appears to occur in an Akt-dependent manner, but the kinases responsible for contraction-stimulated PAS-AS160 and PAS-TBC1D1 remain unclear. AMP-activated protein kinase (AMPK) and Akt, both activated by contraction, can each phosphorylate AS160 and TBC1D1 in cell-free assays. RESEARCH DESIGN AND METHODS To evaluate the roles of AMPK and Akt on insulin- or contraction-stimulated PAS-AS160, PAS-TBC1D1, and glucose transport, rat epitrochlearis was incubated with and without compound C (inhibitor of AMPK) or Wortmannin (inhibitor of phosphatidylinositol [PI] 3-kinase, which is upstream of Akt) before and during insulin stimulation or contraction. RESULTS Insulin-stimulated glucose transport and phosphorylation of both AS160 and TBC1D1 were completely inhibited by Wortmannin. Wortmannin eliminated contraction stimulation of phospho-Ser21/9glycogen synthase kinase 3α/β (pGSK3; Akt substrate) and PAS-AS160 but did not significantly alter pAMPK, phospho-Ser79acetyl CoA carboxylase (pACC; AMPK substrate), PAS-TBC1D1, or glucose transport in contraction-stimulated muscle. Compound C completely inhibited contraction-stimulated pACC and PAS-TBC1D1 and partially blocked glucose transport, but it did not significantly alter pAkt, pGSK3, or PAS-AS160. CONCLUSIONS These data suggest that 1) insulin stimulates glucose transport and phosphorylation of AS160 and TBC1D1 in a PI 3-kinase/Akt–dependent manner, 2) contraction stimulates PAS-AS160 (but not PAS-TBC1D1 or glucose transport) in a PI 3-kinase/Akt–dependent manner, and 3) contraction stimulates PAS-TBC1D1 and glucose transport (but not PAS-AS160) in an AMPK-dependent manner.

Exercise and insulin: Convergence or divergence at AS160 and TBC1D1?

Akt substrate of 160 kDa (called AS160 or TBC1D4) and TBC1D1, Rab GTPase-activating proteins that regulate glucose transport, become phosphorylated with exercise or insulin stimulation. Evidence suggests that this convergence may prove to be imperfect, and each stimulus will produce a unique phosphosignature, providing a plausible mechanism for their apparently unique and overlapping roles in exercise- and insulin-stimulated glucose transport.

Muscle lipogenesis balances insulin sensitivity and strength through calcium signaling

Exogenous dietary fat can induce obesity and promote diabetes, but endogenous fat production is not thought to affect skeletal muscle insulin resistance, an antecedent of metabolic disease. Unexpectedly, the lipogenic enzyme fatty acid synthase (FAS) was increased in the skeletal muscle of mice with diet-induced obesity and insulin resistance. Skeletal muscle-specific inactivation of FAS protected mice from insulin resistance without altering adiposity, specific inflammatory mediators of insulin signaling, or skeletal muscle levels of diacylglycerol or ceramide. Increased insulin sensitivity despite high-fat feeding was driven by activation of AMPK without affecting AMP content or the AMP/ATP ratio in resting skeletal muscle. AMPK was induced by elevated cytosolic calcium caused by impaired sarco/endoplasmic reticulum calcium ATPase (SERCA) activity due to altered phospholipid composition of the sarcoplasmic reticulum (SR), but came at the expense of decreased muscle strength. Thus, inhibition of skeletal muscle FAS prevents obesity-associated diabetes in mice, but also causes muscle weakness, which suggests that mammals have retained the capacity for lipogenesis in muscle to preserve physical performance in the setting of disrupted metabolic homeostasis.

In vivo exercise followed by in vitro contraction additively elevates subsequent insulin-stimulated glucose transport by rat skeletal muscle

The cellular mechanisms whereby prior exercise enhances insulin-stimulated glucose transport (GT) are not well understood. Previous studies suggested that a prolonged increase in phosphorylation of Akt substrate of 160 kDa (AS160) may be important for the postexercise increase in insulin sensitivity. In the current study, the effects of in vivo exercise and in vitro contraction on subsequent insulin-stimulated GT were studied separately and together. Consistent with results from previous studies, prior exercise resulted in an increase in AS160 (642)Thr phosphorylation immediately after exercise in rat epitrochlearis muscles, and this increase remained 3 h postexercise concomitant with enhanced insulin-stimulated GT. For experiments with in vitro contraction, isolated rat epitrochlearis muscles were electrically stimulated to contract in the presence or absence of rat serum. As expected, insulin-stimulated GT measured 3 h after electrical stimulation in serum, but not after electrical stimulation without serum, exceeded resting controls. Immediately after electrical stimulation with or without serum, phosphorylation of both AS160 (detected by phospho-Akt substrate, PAS, antibody, or phospho-(642)Thr antibody) and its paralog TBC1D1 (detected by phospho-(237)Ser antibody) was increased. However, both AS160 and TBC1D1 phosphorylation had reversed to resting values at 3 h poststimulation with or without serum. Increasing the amount of exercise (from 1 to 2 h) or electrical stimulation (from 5 to 10 tetani) did not further elevate insulin-stimulated GT. In contrast, the combination of prior exercise and electrical stimulation had an additive effect on the subsequent increase in insulin-stimulated GT, suggesting that these exercise and electrical stimulation protocols may amplify insulin-stimulated GT through distinct mechanisms, with a persistent increase in AS160 phosphorylation potentially important for increased insulin sensitivity after exercise, but not after in vitro contraction.

Contraction-stimulated glucose transport in rat skeletal muscle is sustained despite reversal of increased PAS-phosphorylation of AS160 and TBC1D1

Akt substrate of 160 kDa (AS160), the most distal insulin signaling protein known to be important for insulin-stimulated glucose transport, becomes phosphorylated with skeletal muscle contraction. Akt, AMP-activated protein kinase (AMPK), and Ca(2+)/calmodulin-dependent kinase II (CaMKII) have been implicated in regulating AS160 and/or glucose transport. Our primary aim was to assess time courses for contractions effects on glucose transport and phosphorylation of Akt, AMPK, CaMKII, and AS160. Isolated rat epitrochlearis muscles were studied without or with contraction (5, 10, 20, 40, 60 min). Phospho-Akt substrate (PAS) antibody was used to measure AS160 PAS phosphorylation by quantifying the approximately 160-kDa band on PAS immunoblots (PAS-160); a separate band at 150 kDa (PAS-150) that responded similarly to contraction was also identified. Using specific antibodies for AS160 or TBC1D1 on immunoblots, the molecular mass of PAS-160 was found to correspond with that of AS160 and not TBC1D1, whereas PAS-150 corresponded with TBC1D1 and not AS160. Furthermore, supernatant of sample immunodepleted with anti-AS160 had greatly reduced PAS-160, whereas supernatant of sample immunodepleted with anti-TBC1D1 had greatly reduced PAS-150, providing further evidence that PAS-160 and PAS-150 correspond with PAS-AS160 and PAS-TBC1D1, respectively. Contraction induced transient increases in PAS-160, PAS-150, phospho-glycogen synthase kinase 3 (an Akt substrate) and phospho-CaMKII; glucose transport and phospho-AMPK increases were maintained for 60 min of contraction. These data suggest the following: 1) PAS-160 (AS160) and PAS-150 (TBC1D1) respond to contraction transiently, despite sustained stimulation; 2) continual AMPK activation was insufficient for sustained increase in PAS-160 or PAS-150; and 3) sustained elevation of PAS-160 or PAS-150 was unnecessary to maintain contraction-stimulated glucose transport for up to 60 min.

A myosin II ATPase inhibitor reduces force production, glucose transport, and phosphorylation of AMPK and TBC1D1 in electrically stimulated rat skeletal muscle

Contraction-stimulated glucose transport by skeletal muscle appears to be caused by the cumulative effects of multiple inputs [potentially including AMP-activated protein kinase (AMPK), Ca(2+) flux, and force production], making it challenging to isolate the roles of these putative regulatory factors. To distinguish the effects of force production from the direct consequences of Ca(2+) flux, the predominantly type II rat epitrochlearis muscle was incubated without (vehicle) or with N-benzyl-p-toluenesulfonamide (BTS), a highly specific myosin II ATPase inhibitor that prevents force production by electrically stimulated (ES) type II fibers without altering cytosolic Ca(2+). In ES muscles, BTS vs. vehicle had an 84% reduction in force production and a 57% decrement in contraction-stimulated 3-O-methylglucose transport (3MGT). BTS did not alter the ES increase in phosphorylation of CaMKII (indicative of cytosolic Ca(2+)) or the amount of glycogen depletion. ES caused significant reductions in ATP (48%) and phosphocreatine (67%) concentrations for vehicle-treated muscles. For BTS-treated muscles, ES did not reduce ATP and caused only a 42% decrease in phosphocreatine. There was an ES increase in phosphorylation of AMPK, acetyl-CoA carboxylase (an AMPK substrate), and TBC1D1 for vehicle-treated muscles but not for BTS-treated muscles. These results point toward an essential role for tension-related events, including AMPK activation, in the 57% contraction-stimulated increase in 3MGT that was inhibited by BTS and further suggest a possible role for TBC1D1 phosphorylation. Non-tension-related events (e.g., increased cytosolic Ca(2+) rather than increased AMPK and TBC1D1 phosphorylation) are implicated in the contraction-stimulated increase in 3MGT that persisted in the presence of BTS.

Skeletal Muscle Phospholipid Metabolism Regulates Insulin Sensitivity and Contractile Function

Skeletal muscle insulin resistance is an early defect in the development of type 2 diabetes. Lipid overload induces insulin resistance in muscle and alters the composition of the sarcoplasmic reticulum (SR). To test the hypothesis that skeletal muscle phospholipid metabolism regulates systemic glucose metabolism, we perturbed choline/ethanolamine phosphotransferase 1 (CEPT1), the terminal enzyme in the Kennedy pathway of phospholipid synthesis. In C2C12 cells, CEPT1 knockdown altered SR phospholipid composition and calcium flux. In mice, diet-induced obesity, which decreases insulin sensitivity, increased muscle CEPT1 expression. In high-fat diet–fed mice with skeletal muscle–specific knockout of CEPT1, systemic and muscle-based approaches demonstrated increased muscle insulin sensitivity. In CEPT1-deficient muscles, an altered SR phospholipid milieu decreased sarco/endoplasmic reticulum Ca2+ ATPase–dependent calcium uptake, activating calcium-signaling pathways known to improve insulin sensitivity. Altered muscle SR calcium handling also rendered these mice exercise intolerant. In obese humans, surgery-induced weight loss increased insulin sensitivity and decreased skeletal muscle CEPT1 protein. In obese humans spanning a spectrum of metabolic health, muscle CEPT1 mRNA was inversely correlated with insulin sensitivity. These results suggest that high-fat feeding and obesity induce CEPT1, which remodels the SR to preserve contractile function at the expense of insulin sensitivity.

Nutrient-dependent phosphorylation channels lipid synthesis to regulate PPARα

Peroxisome proliferator-activated receptor (PPAR)α is a nuclear receptor that coordinates liver metabolism during fasting. Fatty acid synthase (FAS) is an enzyme that stores excess calories as fat during feeding, but it also activates hepatic PPARα by promoting synthesis of an endogenous ligand. Here we show that the mechanism underlying this paradoxical relationship involves the differential regulation of FAS in at least two distinct subcellular pools: cytoplasmic and membrane-associated. In mouse liver and cultured hepatoma cells, the ratio of cytoplasmic to membrane FAS-specific activity was increased with fasting, indicating higher cytoplasmic FAS activity under conditions associated with PPARα activation. This effect was due to a nutrient-dependent and compartment-selective covalent modification of FAS. Cytoplasmic FAS was preferentially phosphorylated during feeding or insulin treatment at Thr-1029 and Thr-1033, which flank a dehydratase domain catalytic residue. Mutating these sites to alanines promoted PPARα target gene expression. Rapamycin-induced inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), a mediator of the feeding/insulin signal to induce lipogenesis, reduced FAS phosphorylation, increased cytoplasmic FAS enzyme activity, and increased PPARα target gene expression. Rapamycin-mediated induction of the same gene was abrogated with FAS knockdown. These findings suggest that hepatic FAS channels lipid synthesis through specific subcellular compartments that allow differential gene expression based on nutritional status.