Olasunkanmi A. J. Adegoke

Elevations of plasma methylarginines in obesity and ageing are related to insulin sensitivity and rates of protein turnover

Aims/hypothesisIncreased circulating methylarginines (MA) have been linked to the metabolic syndrome to explain endothelial dysfunction and cardiovascular disease risk. Proteins that contain MA are regulatory and release them during catabolism. We hypothesised that increased protein turnover in insulin-resistant states contributes to an increase in circulating MA.Matwerials and methodsWe performed hyperinsulinaemic, euglycaemic, and isoaminoacidaemic experiments on 49 lean, obese and elderly subjects, with measurements of the kinetics of glucose and protein metabolism. Plasma MA, i.e. asymmetrical dimethylarginine (ADMA), symmetrical dimethylarginine (SDMA), and N -monomethyl-l-arginine (NMMA), lipids and body composition were measured.ResultsInsulin resistance of glucose and protein metabolism occurred in obese and elderly subjects. ADMA concentrations were 29 to 120% higher in obese and 34% higher in elderly than in lean subjects. SDMA were 34 and 20% higher in obese than in lean and than in elderly subjects, respectively. NMMA were 32% higher in obese than in lean subjects. ADMA differed by sex, being higher in men, namely by 1.75× in obese men and by 1.27× in elderly men. Postabsorptive ADMA (r=0.71), SDMA (r=0.46), and NMMA (r=0.31) correlated (all p<0.05) with rates of protein flux. All three MA correlated negatively with clamp glucose infusion rates and uptake (p<0.001). ADMA and SDMA correlated negatively with net protein synthesis and clamp amino acid infusion rates (p<0.05). All MA also correlated with adiposity indices and fasting insulin and triglycerides (p<0.05).Conclusions/interpretationObesity, sex and ageing affect MA. Elevations of the three MA in obese, and of ADMA in elderly men, are related to increased protein turnover and to lesser insulin sensitivity of protein metabolism. These interrelationships might amplify insulin resistance and endothelial dysfunction.

USP19 deubiquitinating enzyme supports cell proliferation by stabilizing KPC1, a ubiquitin ligase for p27Kip1.

ABSTRACT p27Kip1 is a cyclin-dependent kinase inhibitor that regulates the G1/S transition. Increased degradation of p27Kip1 is associated with cellular transformation. Previous work demonstrated that the ubiquitin ligases KPC1/KPC2 and SCFSkp2 ubiquitinate p27Kip1 in G1 and early S, respectively. The regulation of these ligases remains unclear. We report here that the USP19 deubiquitinating enzyme interacts with and stabilizes KPC1, thereby modulating p27Kip1 levels and cell proliferation. Cells depleted of USP19 by RNA interference exhibited an inhibition of cell proliferation, progressing more slowly from G0/G1 to S phase, and accumulated p27Kip1. This increase in p27Kip1 was associated with normal levels of Skp2 but reduced levels of KPC1. The overexpression of KPC1 or the use of p27−/− cells inhibited significantly the growth defect observed upon USP19 depletion. KPC1 was ubiquitinated in vivo and stabilized by proteasome inhibitors and by overexpression of USP19, and it also coimmunoprecipitated with USP19. Our results identify USP19 as the first deubiquitinating enzyme that regulates the stability of a cyclin-dependent kinase inhibitor and demonstrate that progression through G1 to S phase is, like the metaphase-anaphase transition, controlled in a hierarchical, multilayered fashion.

Fed-state clamp stimulates cellular mechanisms of muscle protein anabolism and modulates glucose disposal in normal men

Since maximum anabolism occurs postprandially, we developed a simulated fed state with clamped hyperinsulinemia, physiological hyperglycemia, and hyperaminoacidemia (Hyper-3) and explored muscle cellular mechanisms. Whole body [1-(13)C]leucine and [3-(3)H]glucose kinetics in healthy men were compared between hyperinsulinemic, euglycemic, isoaminoacidemic (Hyper-1, n = 10) and Hyper-3 (n = 9) clamps. In Hyper-3 vs. Hyper-1, nonoxidative leucine R(d) [rate of disappearance (synthesis)] was stimulated more (45 +/- 4 vs. 24 +/- 4 micromol/min, P < 0.01) and endogenous R(a) [rate of appearance (breakdown)] was inhibited similarly; hence net balance increased more (86 +/- 6 vs. 49 +/- 2 micromol/min, P < 0.001). Glucose R(d) was similar; thus Hyper-3 metabolic clearance rate (331 +/- 23 vs. 557 +/- 41 ml/min, P < 0.0005) and R(d)/insulin (M, 0.65 +/- 0.10 vs. 1.25 +/- 0.10 mg.min(-1).pmol(-1).l, P < 0.001) were less, despite higher insulin (798 +/- 74 vs. 450 +/- 24 pmol/l, P < 0.005). In vastus lateralis muscle biopsies, phosphorylation of Akt (P = 0.025), mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70(S6K1); P = 0.008), S6 (P = 0.049), and 4E-binding protein 1 (4E-BP1; P = 0.001) increased. With decreased eukaryotic initiation factor-4E (eIF4E).4E-BP1 complex (P = 0.01), these are consistent with increased mTOR complex 1 (mTORC1) signaling and translation initiation of protein synthesis. Although mRNA expression of ubiquitin, MAFbx 1, and MuRF-1 was unchanged, total ubiquitinated proteins decreased 20% (P < 0.01), consistent with proteolysis suppression. The Hyper-3 clamp increases whole body protein synthesis, net anabolism, and muscle protein translation initiation pathways and decreases protein ubiquitination. The main contribution of hyperaminoacidemia is stimulation of synthesis rather than inhibition of proteolysis, and it attenuates the expected increment of glucose disposal.

mTORC1 and the regulation of skeletal muscle anabolism and mass.

The mass and integrity of skeletal muscle is vital to whole-body substrate metabolism and health. Indeed, defects in muscle metabolism and functions underlie or exacerbate diseases like diabetes, rheumatoid arthritis, and cancer. Physical activity and nutrition are the 2 most important environmental factors that can affect muscle health. At the molecular level, the mammalian target of rapamycin complex 1 (mTORC1) is a critical signalling complex that regulates muscle mass. In response to nutrition and resistance exercise, increased muscle mass and activation of mTORC1 occur in parallel. In this review, we summarize recent findings on mTORC1 and its regulation in skeletal muscle in response to resistance exercise, alone or in combination with intake of protein or amino acids. Because increased activity of the complex is implicated in the development of muscle insulin resistance, obesity, and some cancers (e.g., ovarian, breast), drugs that target mTORC1 are being developed or are in clinical trials. However, various cancers are associated with extensive muscle wasting, due in part to tumour burden and malnutrition. This muscle wasting may also be a side effect of anticancer drugs. Because loss of muscle mass is associated not only with metabolic abnormalities but also dose limiting toxicity, we review the possible implications for skeletal muscle of long-term inhibition of mTORC1, especially in muscle wasting conditions.

Impaired growth and force production in skeletal muscles of young partially pancreatectomized rats: a model of adolescent type 1 diabetic myopathy?

This present study investigated the temporal effects of type 1 diabetes mellitus (T1DM) on adolescent skeletal muscle growth, morphology and contractile properties using a 90% partial pancreatecomy (Px) model of the disease. Four week-old male Sprague-Dawley rats were randomly assigned to Px (n = 25) or Sham (n = 24) surgery groups and euthanized at 4 or 8 weeks following an in situ assessment of muscle force production. Compared to Shams, Px were hyperglycemic (>15 mM) and displayed attenuated body mass gains by days 2 and 4, respectively (both P<0.05). Absolute maximal force production of the gastrocnemius plantaris soleus complex (GPS) was 30% and 50% lower in Px vs. Shams at 4 and 8 weeks, respectively (P<0.01). GP mass was 35% lower in Px vs Shams at 4 weeks (1.24±0.06 g vs. 1.93±0.03 g, P<0.05) and 45% lower at 8 weeks (1.57±0.12 vs. 2.80±0.06, P<0.05). GP fiber area was 15–20% lower in Px vs. Shams at 4 weeks in all fiber types. At 8 weeks, GP type I and II fiber areas were ∼25% and 40% less, respectively, in Px vs. Shams (group by fiber type interactions, P<0.05). Phosphorylation states of 4E-BP1 and S6K1 following leucine gavage increased 2.0- and 3.5-fold, respectively, in Shams but not in Px. Px rats also had impaired rates of muscle protein synthesis in the basal state and in response to gavage. Taken together, these data indicate that exposure of growing skeletal muscle to uncontrolled T1DM significantly impairs muscle growth and function largely as a result of impaired protein synthesis in type II fibers.

Jejunal mucosal protein synthesis: validation of luminal flooding dose method and effect of luminal osmolarity

To validate a system to study acute regulation of protein synthesis in intestinal mucosa by luminal nutrients, we compared the fractional rate of protein synthesis (Ks) in jejunal mucosa using the intravenous flooding dose technique with the administration of a comparable concentration and specific activity of tracer in a luminal perfusate. Routes of tracer administration and surgery and perfusion trauma had no effect on mucosal Ks. Furthermore, four 10-cm jejunal segments (within a piglet) simultaneously but separately perfused with a luminal flooding dose had similar Ks values (mean, 43 +/- 2%/day; P > 0.05). Nutrient solutions perfused through four intestinal segments within an animal did not affect plasma levels of most amino acids or glucose. Because cellular hydration is important in regulating metabolism, the effects of physiological variation in luminal osmolarity were studied. Luminal osmolarities between 250 and 380 mosM did not affect mucosal Ks. The system described allows multiple comparisons within an animal and provides a robust model to study acute modulation of protein synthesis in intestinal mucosa by luminal stimuli.

Anabolic and catabolic mediators of intestinal protein turnover: a new experimental approach.

Studies on regulation of protein turnover in skeletal muscle have revealed the important contributions of protein synthesis and catabolism to tissue protein balance, and have identified a host of specific anabolic and catabolic stimuli and biochemical mechanisms that regulate these processes. This knowledge is critical to current efforts designed to promote anabolism and limit atrophy. Of the tissues with a potentially large contribution to whole-body amino acid metabolism, protein turnover of the intestine stands out as being poorly understood. The intestine is subject to complexities in regulation of its metabolism that are not apparent for other tissues. The study of intestinal protein turnover also entails some important technical challenges. We recently developed an in-situ experimental system for study of intestinal mucosal protein synthesis with the following unique features: multiple observations within an animal; controlled delivery of nutritional stimuli to the apical side, basolateral side, or both; and luminal delivery of tracer in a flooding dose for determination of protein synthesis. We have begun to use the system to test the specific roles of individual luminal nutrients in regulation of mucosal protein synthesis. We have also used protease gene expression as an index of potential regulation of catabolic pathways.

Skeletal muscle protein synthesis and the abundance of the mRNA translation initiation repressor PDCD4 are inversely regulated by fasting and refeeding in rats.

Optimal skeletal muscle mass is vital to human health, because defects in muscle protein metabolism underlie or exacerbate human diseases. The mammalian target of rapamycin complex 1 is critical in the regulation of mRNA translation and protein synthesis. These functions are mediated in part by the ribosomal protein S6 kinase 1 (S6K1) through mechanisms that are poorly understood. The tumor suppressor programmed cell death 4 (PDCD4) has been identified as a novel substrate of S6K1. Here, we examined 1) the expression of PDCD4 in skeletal muscle and 2) its regulation by feed deprivation (FD) and refeeding. Male rats (~100 g; n = 6) were subjected to FD for 48 h; some rats were refed for 2 h. FD suppressed muscle fractional rates of protein synthesis and Ser(67) phosphorylation of PDCD4 (-50%) but increased PDCD4 abundance (P < 0.05); refeeding reversed these changes (P < 0.05). Consistent with these effects being regulated by S6K1, activation of this kinase was suppressed by FD (-91%, P < 0.05) but was increased by refeeding. Gavaging rats subjected to FD with a mixture of amino acids partially restored muscle fractional rates of protein synthesis and reduced PDCD4 abundance relative to FD. Finally, when myoblasts were grown in amino acid- and serum-free medium, phenylalanine incorporation into proteins in cells depleted of PDCD4 more than doubled the values in cells with a normal level of PDCD4 (P < 0.0001). Thus feeding stimulates fractional protein synthesis in skeletal muscle in parallel with the reduction of the abundance of this mRNA translation inhibitor.

Maintaining adequate nutrition, not probiotic administration, prevents growth stunting and maintains skeletal muscle protein synthesis rates in a piglet model of colitis.

Malnutrition and cytokine-induced catabolism are pervasive in children with inflammatory bowel diseases (IBD), however, the benefits of aggressive nutrition support or of probiotics on nutrient and functional deficiencies and growth remain unclear. Piglets with dextran sulfate (DS)-induced colitis consuming a 50% macronutrient restricted diet (C-MR) were compared with those receiving probiotics (C-MRP) or adequate nutrition (C-WN) and with healthy well-nourished controls (REF). C-WN versus REF had reduced growth (−34% chest circumference and −22% snout-to-rump length gain) and a tendency toward lesser weight gain, but no differences in skeletal muscle protein fractional synthesis rates (FSR) or initiation of translation via the mTOR pathway were observed. Compared with C-WN, the C-MR and C-MRP piglets had lower weight gain, growth, and skeletal muscle FSR, and lower phosphorylated p70S6K1 with higher eIF4E*4E-BP1, indicative of reduced initiation of protein translation. Finally, plasma leucine concentrations were positively correlated with weight and phosphorylated p70S6K1, whereas negatively correlated with eIF4E*4E-BP1. In conclusion, reductions in weight gain, growth, protein turnover, skeletal muscle FSR, and initiation of protein translation with moderate macronutrient restriction in colitis are not ameliorated by probiotic supplementation. However, maintaining adequate nutrient intake during colitis preserves whole body protein metabolism, but growth remains compromised.

Ketoisocaproic acid, a metabolite of leucine, suppresses insulin-stimulated glucose transport in skeletal muscle cells in a BCAT2-dependent manner

Although leucine has many positive effects on metabolism in multiple tissues, elevated levels of this amino acid and the other branched-chain amino acids (BCAAs) and their metabolites are implicated in obesity and insulin resistance. While some controversies exist about the direct effect of leucine on insulin action in skeletal muscle, little is known about the direct effect of BCAA metabolites. Here, we first showed that the inhibitory effect of leucine on insulin-stimulated glucose transport in L6 myotubes was dampened when other amino acids were present, due in part to a 140% stimulation of basal glucose transport (P < 0.05). Importantly, we also showed that α-ketoisocaproic acid (KIC), an obligatory metabolite of leucine, stimulated mTORC1 signaling but suppressed insulin-stimulated glucose transport (-34%, P < 0.05) in an mTORC1-dependent manner. The effect of KIC on insulin-stimulated glucose transport was abrogated in cells depleted of branched-chain aminotransferase 2 (BCAT2), the enzyme that catalyzes the reversible transamination of KIC to leucine. We conclude that although KIC can modulate muscle glucose metabolism, this effect is likely a result of its transamination back to leucine. Therefore, limiting the availability of leucine, rather than those of its metabolites, to skeletal muscle may be more critical in the management of insulin resistance and its sequelae.