Monocyte-astrocyte networks and the regulation of chemokine secretion in neurocysticercosis

Neurocysticercosis, caused by infection with larval Taenia solium, is a major cause of epilepsy worldwide. Larval degeneration, which is symptomatic, results in inflammatory cell influx. Astrocytes, the most abundant cell type and major cytokine-producing cell within the CNS, may be important in orchestrating inflammatory responses after larval degeneration. We investigated the effects of direct stimulation and of conditioned medium from T. solium larval Ag (TsAg)-stimulated monocytes (CoMTsAg) on neutrophil and astrocyte chemokine release. CoMTsAg, but not control conditioned medium, stimulated astrocyte CCL2/MCP-1 (161.5 +/- 16 ng/ml), CXCL8/IL-8 (416 +/- 6.2 ng/ml), and CXCL10/IFN-gamma-inducible protein (9.07 +/- 0.6 ng/ml) secretion after 24 h, whereas direct astrocyte or neutrophil stimulation with TsAg had no effect. There was rapid accumulation of CCL2 and CXCL8 mRNA within 1 h, with somewhat delayed expression of CXCL10 mRNA initially detected 8 h poststimulation. Neutralizing anti-TNF-alpha inhibited CoMTsAg-induced CCL2 mRNA accumulation by up to 99%, causing total abolition of CXCL10 and up to 77% reduction in CXCL8 mRNA. CoMTsAg induced maximal nuclear binding of NF-kappaB p65 and p50 by 1 h, with IkappaBalpha and IkappaBbeta decay within 15 min. In addition, CoMTsAg induced transient nuclear binding of AP-1, which peaked 4 h poststimulation. In NF-kappaB blocking experiments using pyrrolidine dithiocarbamate, CoMTsAg-induced CCL2 secretion was reduced by up to 80% (p = 0.0006), whereas CXCL8 was inhibited by up to 75% (p = 0.0003). In summary, the data show that astrocytes are an important source of chemokines following larval Ag stimulation. Such chemokine secretion is NF-kappaB dependent, likely to involve AP-1, and is regulated in a paracrine loop by monocyte-derived TNF-alpha.     

Leucine reduces the duration of insulin-induced PI 3-kinase activity in rat skeletal muscle

Leucine (Leu) is known to stimulate translation initiation of protein synthesis at mammalian target of rapamycin (mTOR) in the insulin signaling pathway. However, potential feedback from mTOR to upstream aspects of the insulin signaling pathway remains controversial. This study evaluates the impact of a physiological oral dose of Leu and/or carbohydrate (CHO) on upstream elements of the insulin signaling pathway using phosphatidylinositol 3-kinase (PI 3-kinase) activity and glucose uptake as markers for insulin sensitivity and glucose homeostasis. Rats (approximately 200 g) were fasted 12 h and administered oral doses of CHO (1.31 g glucose, 1.31 g sucrose), Leu (270 mg), or CHO plus Leu. Animals were killed at 15, 30, 60, and 90 min after treatment. Plasma and gastrocnemius muscles were collected for analyses. Treatments were designed to produce elevated blood glucose and insulin with basal levels of Leu (CHO); elevated Leu with basal levels of glucose and insulin (Leu); or a combined increase of glucose, insulin, and Leu (CHO + Leu). The CHO treatment stimulated PI 3-kinase activity and glucose uptake with no effect on the downstream translation initiation factor eIF4E. Leu alone stimulated the release of the translation initiation factor eIF4E from 4E-BP1 with no effects on PI 3-kinase activity or glucose uptake. The CHO + Leu treatment reduced the magnitude and duration of the PI 3-kinase response but maintained glucose uptake similar to the CHO treatment and eIF4E levels similar to the Leu treatment. These findings demonstrate that Leu reduces insulin-stimulated PI 3-kinase activity while increasing downstream translation initiation and with no effect on net glucose transport in skeletal muscle.     

MMP-12 induces IL-8/CXCL8 secretion through EGFR and ERK1/2 activation in epithelial cells

Macrophage metalloelastase (MMP-12) is described to be involved in pulmonary inflammatory response. To determine the mechanisms linking MMP-12 and inflammation, we examined the effect of recombinant human MMP-12 (rhMMP-12) catalytic domain on IL-8/CXCL8 production in cultured human airway epithelial (A549) cells. Stimulation with rhMMP-12 resulted in a concentration-dependent IL-8/CXCL8 synthesis 6 h later. Similar results were also observed in cultured BEAS-2B bronchial epithelial cells. In A549 cells, synthetic matrix metalloproteinase (MMP) inhibitors prevented rhMMP-12-induced IL-8/CXCL8 release. We further demonstrated that in A549 cells, rhMMP-12 induced transient, peaking at 5 min, activation of ERK1/2. Selective MEK inhibitors (U0126 and PD-98059) blocked both IL-8/CXCL8 release and ERK1/2 phosphorylation. IL-8/CXCL8 induction and ERK1/2 activation were preceded by EGF receptor (EGFR) tyrosine phosphorylation, within 2 min, and reduced by selective EGFR tyrosine kinase inhibitors (AG-1478 and PD168393) by a neutralizing EGFR antibody and by small interfering RNA oligonucleotides directed against EGFR, implicating EGFR activation. In addition, we observed an activation of c-Fos in A549 cells stimulated by rhMMP-12, dependent on ERK1/2. Using small interfering technique, we showed that c-Fos is involved in rhMMP-12-induced IL-8/CXCL8 production. From these results, we conclude that one mechanism, by which MMP-12 induces IL-8/CXCL8 release from the alveolar epithelium, is the EGFR/ERK1/2/activating protein-1 pathway.     

Adiponectin upregulates monocytic activin A but systemic levels are not altered in obesity or type 2 diabetes

Adiponectin is an adipocyte-derived protein with atheroprotective and immunoregulatory function. Adiponectin and activin A reduce foam cell formation and adiponectin activates the p38 MAPK pathway that is well described to induce activin A. Therefore, it was analyzed whether adiponectin alters activin A in primary human monocytes. Adiponectin dose- and time-dependently induced activin A in the supernatant, and the maximal amount was observed after 12 h of incubation. Adiponectin-stimulated release of activin A was blocked by a p38 MAPK inhibitor. Metformin and pioglitazone are drugs frequently used to treat diabetic patients and metformin slightly reduced monocytic activin A release whereas pioglitazone had no effect. Type 2 diabetes is associated with elevated inflammatory systemic cytokines but activin A serum levels were similar in slim probands, overweight controls and type 2 diabetic patients. Furthermore, activin A did not correlate to systemic adiponectin, body mass index, waist to hip ratio or C-reactive protein. These findings indicate that adiponectin upregulates monocytic activin A release via the p38 MAPK pathway, and this may in part explain the immunoregulatory and antiatherosclerotic effects of this adipokine.     

Isoleucine, a potent plasma glucose-lowering amino acid, stimulates glucose uptake in C2C12 myotubes

To examine which branched-chain amino acids affect the plasma glucose levels, we investigated the effects of leucine, isoleucine, and valine (0.3 g/kg body weight p.o.) in normal rats using the oral glucose tolerance test (OGTT, 2 g/kg). A single oral administration of isoleucine significantly reduced plasma glucose levels 30 and 60 min after the glucose bolus, whereas administration of leucine and valine did not produce a significant decrease. Oral administration of valine significantly enhanced the plasma glucose level at 30 min after the glucose administration and leucine had a similar effect at 120 min. At each measurement timepoint, the insulin levels of the treated groups were lower than that of the control group. We then investigated the effects of leucine, isoleucine or valine at the same concentration (1 mM) on glucose metabolism in C(2)C(12) myotubes in the absence of insulin. Glucose consumption was elevated by 16.8% in the presence of 1 mM isoleucine compared with the control. Conversely, 1 mM leucine or valine caused no significant changes in glucose consumption in the C(2)C(12) myotubes. The 2-deoxyglucose uptake of C(2)C(12) myotubes significantly increased upon exposure to 1-10 mM isoleucine and 5-10 mM leucine. However, isoleucine caused no significant difference in glycogen synthesis in C(2)C(12) myotubes, although leucine and valine caused a significant increase in intracellular glycogen compared with the control. The isoleucine effect on glucose uptake was mediated by phosphatidylinositol 3-kinase (PI3K), but was independent of mammalian target of rapamycin (mTOR). These results suggest that isoleucine stimulates the insulin-independent glucose uptake in skeletal muscle cells, which may contribute to the plasma glucose-lowering effect of isoleucine in normal rats.     

C2C12 细胞胰岛素增敏的机制

目的:观察口服葡萄糖负荷对小鼠小肠组织网膜素基因表达的影响及网膜素对C2C12肌管细胞胰岛素敏感性的影响,并进一步探讨其机制。方法:半定量逆转录聚合酶链反应(RT-PCR)技术检测小鼠小肠组织网膜素mRNA的表达;葡萄糖转运实验观察网膜素对C2C12肌管细胞胰岛素敏感性的影响;Western blot检测Akt(Ser473)的磷酸化水平。结果:口服葡萄糖负荷30min后小鼠小肠组织网膜素基因表达显著减低(P<0.05),而60min后恢复至负荷前水平;葡萄糖转运实验发现:网膜素作用10min对C2C12肌管细胞基础葡萄糖转运无影响,但显著增加了胰岛素刺激的葡萄糖转运(P<0.05);Western Blot发现:网膜素和AICAR均显著增加了C2C12肌管细胞Ak(tSer473)的磷酸化水平(P<0.05)。结论:网膜素作为一种胃肠激素还受到葡萄糖负荷的调节,在C2C12肌管细胞,网膜素可通过增加Akt的磷酸化发挥胰岛素增敏作用。     

Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake

The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.     

Leucine facilitates the insulin-stimulated glucose uptake and insulin signaling in skeletal muscle cells: involving mTORC1 and mTORC2

Leucine, a branched-chain amino acid, has been shown to promote glucose uptake and increase insulin sensitivity in skeletal muscle, but the exact mechanism remains unestablished. We addressed this issue in cultured skeletal muscle cells in this study. Our results showed that leucine alone did not have an effect on glucose uptake or phosphorylation of protein kinase B (AKT), but facilitated the insulin-induced glucose uptake and AKT phosphorylation. The insulin-stimulated glucose uptake and AKT phosphorylation were inhibited by the phosphatidylinositol 3-kinase inhibitor, wortmannin, but the inhibition was partially reversed by leucine. The inhibitor of mammalian target of rapamycin complex 1 (mTORC1), rapamycin, had no effect on the insulin-stimulated glucose uptake, but eliminated the facilitating effect of leucine in the insulin-stimulated glucose uptake and AKT phosphorylation. In addition, leucine facilitation of the insulin-induced AKT phosphorylation was neutralized by knocking down the core component of the mammalian target of rapamycin complex 2 (mTORC2) with specific siRNA. Together, these findings show that leucine can facilitate the insulin-induced insulin signaling and glucose uptake in skeletal muscle cells through both mTORC1 and mTORC2, implicating the potential importance of this amino acid in glucose homeostasis and providing new mechanistic insights.     

Glucose metabolism in isolated brown adipocytes under beta-adrenergic stimulation. Quantitative contribution of glucose to total thermogenesis.

To quantify the potential of brown adipose tissue as a target organ for glucose oxidation, O2 consumption and glucose metabolism in isolated rat brown adipocytes were measured in the presence and absence of insulin, by using the beta-agonists isoprenaline or Ro 16-8714 to stimulate thermogenesis. Basal metabolic rate (278 mumol of O2/h per g of lipid) was maximally stimulated with isoprenaline (20 nm) and Ro 16-8714 (20 microM) to 1633 and 1024 mumol of O2/h per g respectively, whereas insulin had no effect on O2 consumption. Total glucose uptake, derived from the sum of [U-14C]glucose incorporation into CO2 and total lipids and lactate release, was enhanced with insulin. Isoprenaline and Ro 16-8714 had no effect on insulin-induced glucose uptake, but promoted glucose oxidation while inhibiting insulin-dependent lipogenesis and lactate production. A maximal value for glucose oxidation was obtained under the combined action of Ro 16-8714 and insulin, which corresponded to an equivalent of 165 mumol of O2/h per g of lipid. This makes it clear that glucose is a minor substrate for isolated brown adipocytes, fuelling thermogenesis by a maximum of 16%.     

Zinc-mediated inhibition of cyclic nucleotide phosphodiesterase activity and expression suppresses TNF-alpha and IL-1 beta production in monocytes by elevation of guanosine 3',5'-cyclic monophosphate

The trace element zinc affects several aspects of immune function, such as the release of proinflammatory cytokines from monocytes. We investigated the role of cyclic nucleotide signaling in zinc inhibition of LPS-induced TNF-alpha and IL-1beta release from primary human monocytes and the monocytic cell line Mono Mac1. Zinc reversibly inhibited enzyme activity of phosphodiesterase-1 (PDE-1), PDE-3, and PDE-4 in cellular lysate. It additionally reduced mRNA expression of PDE-1C, PDE-4A, and PDE-4B in intact cells. Although these PDE can also hydrolyze cAMP, only the cellular level of cGMP was increased after incubation with zinc, whereas cAMP was found to be even slightly reduced due to inhibition of its synthesis. To investigate whether an increase in cGMP alone is sufficient to inhibit cytokine release, the cGMP analogues 8-bromo-cGMP and dibutyryl cGMP as well as the NO donor S-nitrosocysteine were used. All three treatments inhibited TNF-alpha and IL-1beta release after stimulation with LPS. Inhibition of soluble guanylate cyclase-mediated cGMP synthesis with LY83583 reversed the inhibitory effect of zinc on LPS-induced cytokine release. In conclusion, inhibition of PDE by zinc abrogates the LPS-induced release of TNF-alpha and IL-1beta by increasing intracellular cGMP levels.     

Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin-8), in human adult mesenchymal stem cells via the extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and NF-kappaB pathways

Blood vessel injury results in limited oxygen tension and diffusion leading to hypoxia, increased anaerobic metabolism, and elevated production of acidic metabolites that cannot be easily removed due to the reduced blood flow. Therefore, an acidic extracellular pH occurs in the local microenvironment of disrupted bone. The potential role of acidic pH and glu-leu-arg (ELR(+)) CXC chemokines in early events in bone repair was studied in human mesenchymal stem cells (hMSCs) treated with medium of decreasing pH (7.4, 7.0, 6.7, and 6.4). The cells showed a reciprocal increase in CXCL8 (interleukin-8, IL-8) mRNA levels as extracellular pH decreased. At pH 6.4, CXCL8 mRNA was induced >60x in comparison to levels at pH 7.4. hMSCs treated with osteogenic medium (OGM) also showed an increase in CXCL8 mRNA with decreasing pH; although, at a lower level than that seen in cells grown in non-OGM. CXCL8 protein was secreted into the medium at all pHs with maximal induction at pH 6.7. Inhibition of the G-protein-coupled receptor alpha, G(alphai), suppressed CXCL8 levels in response to acidic pH; whereas phospholipase C inhibition had no effect on CXCL8. The use of specific mitogen-activated protein kinase (MAPK) signal transduction inhibitors indicated that the pH-dependent increase in CXCL8 mRNA is due to activation of ERK and p38 pathways. The JNK pathway was not involved. NF-kappaB inhibition resulted in a decrease in CXCL8 levels in hMSCs grown in non-OGM. However, OGM-differentiated hMSCs showed an increase in CXCL8 levels when treated with the NF-kappaB inhibitor PDTC, a pyrrolidine derivative of dithiocarbamate.     

Sixteen hours of fasting differentially affects hepatic and muscle insulin sensitivity in mice

Fasting readily induces hepatic steatosis. Hepatic steatosis is associated with hepatic insulin resistance. The purpose of the present study was to document the effects of 16 h of fasting in wild-type mice on insulin sensitivity in liver and skeletal muscle in relation to 1) tissue accumulation of triglycerides (TGs) and 2) changes in mRNA expression of metabolically relevant genes. Sixteen hours of fasting did not show an effect on hepatic insulin sensitivity in terms of glucose production in the presence of increased hepatic TG content. In muscle, however, fasting resulted in increased insulin sensitivity, with increased muscle glucose uptake without changes in muscle TG content. In liver, fasting resulted in increased mRNA expression of genes promoting gluconeogenesis and TG synthesis but in decreased mRNA expression of genes involved in glycogenolysis and fatty acid synthesis. In muscle, increased mRNA expression of genes promoting glucose uptake, as well as lipogenesis and beta-oxidation, was found. In conclusion, 16 h of fasting does not induce hepatic insulin resistance, although it causes liver steatosis, whereas muscle insulin sensitivity increases without changes in muscle TG content. Therefore, fasting induces differential changes in tissue-specific insulin sensitivity, and liver and muscle TG contents are unlikely to be involved in these changes.     

Levonorgestrel and norethindrone alter insulin action on skeletal muscle of the female rat

Prospective studies of women receiving oral contraceptives suggest that the progestin component may induce insulin resistance and variable deterioration of glucose tolerance. Because the tissue sites and nature of this insulin antagonism are not well-defined, we studied the effects of two parenterally administered progestins, levonorgestrel (NG) and norethindrone (NE), on insulin-regulated glucose uptake and phenylalanine release by the perfused rat hindquarter. Female rats were injected sc for 14 days with NG or NE (10 or 30 micrograms/kg/day). Low-dose NG and high-dose NE approximate the per kg dose received by women taking a high-dose progestin oral contraceptive. Phenylalanine release and glucose uptake (nmole/min/g) by the perfused hindquarters were calculated from the A-V difference for each. Progestin treatment (30 micrograms/kg/d) significantly reduced phenylalanine release from hindquarters perfused without exogenous insulin. Hindquarters from the high dose NG and low and high dose NE rats perfused with insulin (100 microU/ml) released 22% less phenylalanine than control rats perfused with the same insulin concentration (P less than 0.01) but the net suppression below baseline was similar in the control and steroid-treated groups. High-dose progestin treatment did not alter glucose uptake by hindquarters perfused without exogenous insulin. Insulin (100 microU/ml) increased glucose uptake by hindquarters of control and progestin-treated rats as compared to animals in the same treatment group perfused without exogenous insulin (P less than 0.01). High dose NE impaired insulin-stimulated glucose uptake 24% below values of the control group (P less than 0.01). The other NE and NG doses had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Significance of Ca2+, Rb+ fluxes, of cAMP and cGMP for the CCK8-modulated insulin release

In rat pancreatic islets the effects of cholecystokinin-8 (CCK8) on glucose-mediated insulin release, 45Ca2+ net uptake, 45Ca2+ efflux, 86Rb+ efflux, cAMP- and cGMP levels were studied. In the presence of a substimulatory glucose concentration (3 mM) CCK8 concentrations of up to 1 microM had no effect on insulin release, but CCK8 at 10 nM potentiated the stimulatory effect of glucose (11.1 mM). 10 nM CCK8 enhanced glucose-stimulated 45Ca2+ net uptake but was ineffective at substimulatory glucose levels. CCK8 had no effect on cAMP and cGMP levels in the presence of 11.1 mM glucose, CCK8 increased 86Rb+ (a measure of K+) in the presence of both 3 and 11.1 mM glucose. This effect was abolished when Ca2+ was omitted from the perifusion medium. CCK8 did not alter glucose (11.1 mM)-stimulated 45Ca2+ efflux rate. These data indicate that (1) CCK8 potentiates glucose-stimulated insulin secretion possibly via an effect on Ca2+ uptake, 2) by affecting Ca2+ uptake, CCK8 enhances K+ efflux, and 3) CCK8 does not mediate its effect via cAMP or cGMP. With respect to 86Rb+ efflux the mechanism of CCK8 action appears to be different from that of glucose. When the mechanism of CCK action on islets is compared with that on exocrine pancreas (data from others) there are similarities (importance of Ca2+ uptake and non-importance of cAMP and cGMP).     

Resistin modulates glucose uptake and glucose transporter-1 (GLUT-1) expression in trophoblast cells

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity. Here, we examine the effect of resistin on glucose uptake in human trophoblast cells and we demonstrate that transplacental glucose transport is mediated by glucose transporter (GLUT)-1. Furthermore, we evaluate the type of signal transduction induced by resistin in GLUT-1 regulation. BeWo choriocarcinoma cells and primary cytotrophoblast cells were cultured with increasing resistin concentrations for 24 hrs. The main outcome measures include glucose transport assay using [³H]-2-deoxy glucose, GLUT-1 protein expression by Western blot analysis and GLUT-1 mRNA detection by quantitative real-time RT-PCR. Quantitative determination of phospho(p)-ERK1/2 in cell lysates was performed by an Enzyme Immunometric Assay and Western blot analysis. Our data demonstrate a direct effect of resistin on normal cytotrophoblastic and on BeWo cells: resistin modulates glucose uptake, GLUT-1 messenger ribonucleic acid (mRNA) and protein expression in placental cells. We suggest that ERK1/2 phosphorylation is involved in the GLUT-1 regulation induced by resistin. In conclusion, resistin causes activation of both the ERK1 and 2 pathway in trophoblast cells. ERK1 and 2 activation stimulated GLUT-1 synthesis and resulted in increase of placental glucose uptake. High resistin levels (50–100 ng/ml) seem able to affect glucose-uptake, presumably by decreasing the cell surface glucose transporter.     

An amino acid mixture improves glucose tolerance and insulin signaling in Sprague-Dawley rats

The aims of this investigation were to evaluate the effect of an amino acid supplement on the glucose response to an oral glucose challenge (experiment 1) and to evaluate whether differences in blood glucose response were associated with increased skeletal muscle glucose uptake (experimental 2). Experiment 1 rats were gavaged with either glucose (CHO), glucose plus an amino acid mixture (CHO-AA-1), glucose plus an amino acid mixture with increased leucine concentration (CHO-AA-2), or water (PLA). CHO-AA-1 and CHO-AA-2 had reduced blood glucose responses compared with CHO, with no difference in insulin among these treatments. Experiment 2 rats were gavaged with either CHO or CHO-AA-1. Fifteen minutes after gavage, a bolus containing 2-[(3)H]deoxyglucose and [U-(14)C]mannitol was infused via a tail vein. Blood glucose was significantly lower in CHO-AA-1 than in CHO, whereas insulin responses were similar. Muscle glucose uptake was higher in CHO-AA-1 compared with CHO in both fast-twitch red (8.36 ± 1.3 vs. 5.27 ± 0.7 μmol·g(-1)·h(-1)) and white muscle (1.85 ± 0.3 vs. 1.11 ± 0.2 μmol·g(-1)·h(-1)). There was no difference in Akt/PKB phosphorylation between treatment groups; however, the amino acid treatment resulted in increased AS160 phosphorylation in both muscle fiber types. Glycogen synthase phosphorylation was reduced in fast-twitch red muscle of CHO-AA-1 compared with CHO, whereas mTOR phosphorylation was increased. These differences were not noted in fast-twitch white muscle. These findings suggest that amino acid supplementation can improve glucose tolerance by increasing skeletal muscle glucose uptake and intracellular disposal through enhanced intracellular signaling.