Metformin and thiazolidinediones (TZDs) are believed to exert their antidiabetic effects via different mechanisms. As evidence suggests that both impair cell respiration in vitro, this study compared their effects on mitochondrial functions. The activity of complex I of the respiratory chain, which is known to be affected by metformin, was measured in tissue homogenates that contained disrupted mitochondria. In homogenates of skeletal muscle, metformin and TZDs reduced the activity of complex I (30 mmol/l metformin, ؊15 ؎ 2%; 100 mol/l rosiglitazone, ؊54 ؎ 7; and 100 mol/l pioglitazone, ؊12 ؎ 4; P < 0.05 each). Inhibition of complex I was confirmed by reduced state 3 respiration of isolated mitochondria consuming glutamate ؉ malate as substrates for complex I (30 mmol/l metformin, ؊77 ؎ 1%; 100 mol/l rosiglitazone, ؊24 ؎ 4; and 100 mol/l pioglitazone, ؊18 ؎ 5; P < 0.05 each), whereas respiration with succinate feeding into complex II was unaffected. In line with inhibition of complex I, 24-h exposure of isolated rat soleus muscle to metformin or TZDs reduced cell respiration and increased anaerobic glycolysis (glucose oxidation: 270 mol/l metformin, ؊30 ؎ 9%; 9 mol/l rosiglitazone, ؊25 ؎ 8; and 9 mol/l pioglitazone, ؊45 ؎ 3; lactate release: 270 mol/l metformin, ؉84 ؎ 12; 9 mol/l rosiglitazone, ؉38 ؎ 6; and 9 mol/l pioglitazone, ؉64 ؎ 11; P < 0.05 each). As both metformin and TZDs inhibit complex I activity and cell respiration in vitro, similar mitochondrial actions could contribute to their antidiabetic effects. Diabetes

To examine the molecular mechanisms by which plasma amino acid elevation impairs insulin action, we studied seven healthy men twice in random order during infusion of an amino acid mixture or saline (total plasma amino acid ϳ6 vs. ϳ2 mmol/l). Somatostatin-insulinglucose clamps created conditions of low peripheral hyperinsulinemia (ϳ100 pmol/l, 0 -180 min) and prandial-like peripheral hyperinsulinemia (ϳ430 pmol/l, 180 -360 min). At low peripheral hyperinsulinemia, endogenous glucose production (EGP) did not change during amino acid infusion but decreased by ϳ70% during saline infusion (EGP 150 -180 min 11 ؎ 1 vs. 3 ؎ 1 mol ⅐ kg ؊1 ⅐ min ؊1 , P ‫؍‬ 0.001). Prandial-like peripheral hyperinsulinemia completely suppressed EGP during both protocols, whereas whole-body rate of glucose disappearance (R d ) was ϳ33% lower during amino acid infusion (R d 330 -360 min 50 ؎ 4 vs. 75 ؎ 6 mol ⅐ kg ؊1 ⅐ min ؊1 , P ‫؍‬ 0.002) indicating insulin resistance. In skeletal muscle biopsies taken before and after prandiallike peripheral hyperinsulinemia, plasma amino acid elevation markedly increased the ability of insulin to activate S6 kinase 1 compared with saline infusion (ϳ3.7-vs. ϳ1.9-fold over baseline). Furthermore, amino acid infusion increased the inhibitory insulin receptor substrate-1 phosphorylation at Ser312 and Ser636/639 and decreased insulin-induced phosphoinositide 3-kinase activity. However, plasma amino acid elevation failed to reduce insulin-induced Akt/protein kinase B and glycogen synthase kinase 3␣ phosphorylation. In conclusion, amino acids impair 1) insulin-mediated suppression of glucose production and 2) insulin-stimulated glucose disposal in skeletal muscle. Our results suggest that overactivation of the mammalian target of rapamycin/S6 kinase 1 pathway and inhibitory serine phosphorylation of insulin receptor substrate-1 underlie the impairment of insulin action in amino acidinfused humans. Diabetes 54:2674 -2684, 2005

BackgroundMuscular insulin resistance is frequently characterized by blunted increases in glucose-6-phosphate (G-6-P) reflecting impaired glucose transport/phosphorylation. These abnormalities likely relate to excessive intramyocellular lipids and mitochondrial dysfunction. We hypothesized that alterations in insulin action and mitochondrial function should be present even in nonobese patients with well-controlled type 2 diabetes mellitus (T2DM).Methods and FindingsWe measured G-6-P, ATP synthetic flux (i.e., synthesis) and lipid contents of skeletal muscle with 31P/1H magnetic resonance spectroscopy in ten patients with T2DM and in two control groups: ten sex-, age-, and body mass-matched elderly people; and 11 younger healthy individuals. Although insulin sensitivity was lower in patients with T2DM, muscle lipid contents were comparable and hyperinsulinemia increased G-6-P by 50% (95% confidence interval [CI] 39%–99%) in all groups. Patients with diabetes had 27% lower fasting ATP synthetic flux compared to younger controls (p = 0.031). Insulin stimulation increased ATP synthetic flux only in controls (younger: 26%, 95% CI 13%–42%; older: 11%, 95% CI 2%–25%), but failed to increase even during hyperglycemic hyperinsulinemia in patients with T2DM. Fasting free fatty acids and waist-to-hip ratios explained 44% of basal ATP synthetic flux. Insulin sensitivity explained 30% of insulin-stimulated ATP synthetic flux.ConclusionsPatients with well-controlled T2DM feature slightly lower flux through muscle ATP synthesis, which occurs independently of glucose transport /phosphorylation and lipid deposition but is determined by lipid availability and insulin sensitivity. Furthermore, the reduction in insulin-stimulated glucose disposal despite normal glucose transport/phosphorylation suggests further abnormalities mainly in glycogen synthesis in these patients.

Plasma concentrations of amino acids are frequently elevated in insulin-resistant states, and a proteinenriched diet can impair glucose metabolism. This study examined effects of short-term plasma amino acid (AA) elevation on whole-body glucose disposal and cellular insulin action in skeletal muscle. Seven healthy men were studied for 5.5 h during euglycemic (5.5 mmol/l), hyperinsulinemic (430 pmol/l), fasting glucagon (65 ng/ l), and growth hormone (0.4 g/l) somatostatin clamp tests in the presence of low (ϳ1.6 mmol/l) and increased (ϳ4.6 mmol/l) plasma AA concentrations. Glucose turnover was measured with D-[6,6-2 H 2 ]glucose. Intramuscular concentrations of glycogen and glucose-6-phosphate (G6P) were monitored using 13 C and 31 P nuclear magnetic resonance spectroscopy, respectively. A ϳ2.1-fold elevation of plasma AAs reduced whole-body glucose disposal by 25% (P < 0.01). Rates of muscle glycogen synthesis decreased by 64% (180 -315 min, 24 ؎ 3; control, 67 ؎ 10 mol ⅐ l ؊1 ⅐ min ؊1 ; P < 0.01), which was accompanied by a reduction in G6P starting at 130 min (⌬G6P 260 -300 min , 18 ؎ 19; control, 103 ؎ 33 mol/l; P < 0.05). In conclusion, plasma amino acid elevation induces skeletal muscle insulin resistance in humans by inhibition of glucose transport/phosphorylation, resulting in marked reduction of glycogen synthesis. Diabetes 51:599 -605, 2002 P lasma concentrations of alanine and particularly branched-chain amino acids (AAs) are elevated in insulin-resistant states such as obesity (1,2), and high dietary protein intake impairs glucose metabolism mainly by changing the utilization of gluconeogenic precursors (3-6).The mechanisms by which AAs could reduce skeletal muscle glucose uptake are as yet unclear. At the cellular level, availability of substrates for energy production, such as AAs and free fatty acids (FFAs), may play an important role in modulating the response to insulin (7). In vitro studies demonstrated that AAs may inhibit glucose utilization in skeletal muscle at various levels. AAs could decrease glucose oxidation by substrate competition with glucose (8,9) and/or reduce glucose uptake (10) by interaction with early steps of insulin signaling (11). Studies in humans, however, revealed controversial results. Infusion of AAs decreased forearm and whole-body glucose disposal in some (12-15), but not all (16,17), studies. Moreover, endogenous release of insulin (18) and glucagon (19) induced by plasma AA elevation might have obscured possible direct effects of AAs in those studies. Taking together all these factors, it is uncertain whether AAs directly induce skeletal muscle insulin resistance in vivo and if so, which mechanism (glucose uptake versus substrate competition) is responsible for such an effect.This study was therefore designed to examine effects of plasma AA elevation on skeletal muscle glucose metabolism by combining isotope dilution technique with in vivo nuclear magnetic resonance (NMR) spectroscopy of gastrocnemius muscle from healthy young humans. In vivo [ 13 C]NMR spectroscop...

We have cloned a novel pancreatic beta cell and neuroendocrine cell-specific calcium-binding protein termed secretagogin. The cDNA obtained by immunoscreening a human pancreatic cDNA library using the recently described murine monoclonal antibody D24 contains an open reading frame of 828 base pairs. This codes for a cytoplasmic protein with six putative EF finger hand calcium-binding motifs. The gene could be localized to chromosome 6 by alignment with GenBank genomic sequence data. Northern blot analysis demonstrated abundant expression of this protein in the pancreas and to a lesser extent in the thyroid, adrenal medulla, and cortex. In addition it was expressed in scant quantity in the gastrointestinal tract (stomach, small intestine, and colon). Thyroid tissue expression of secretagogin was restricted to C-cells. Using a sandwich capture enzyme-linked immunosorbent assay with a detection limit of 6.5 pg/ml, considerable amounts of constitutively secreted protein could be measured in tissue culture supernatants of stably transfected RIN-5F and dog insulinoma (INS-H1) cell clones; however, in stably transfected Jurkat cells, the protein was only secreted upon CD3 stimulation. Functional analysis of transfected cell lines expressing secretagogin revealed an influence on calcium flux and cell proliferation. In RIN-5F cells, the antiproliferative effect is possibly due to secretagogin-triggered down-regulation of substance P transcription.

Polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5 (n-3)) inhibit T lymphocyte activation probably by displacing acylated signaling proteins from membrane lipid rafts. Under physiological conditions, saturated fatty acyl residues of such proteins partition into the cytoplasmic membrane lipid leaflet with high affinity for rafts that are enriched in saturated fatty acyl-containing lipids. However, the biochemical alteration causing displacement of acylated proteins from rafts in PUFA-treated T cells is still under debate but could principally be attributed to altered protein acylation or changes in raft lipid composition. We show that treatment of Jurkat T cells with polyunsaturated eicosapentaenoic acid (20:5 (n-3)) results in marked enrichment of PUFAs (20:5; 22:5) in lipids from isolated rafts. Moreover, PUFAs were significantly incorporated into phosphatidylethanolamine that predominantly resides in the cytoplasmic membrane lipid leaflet. Notably, palmitate-labeled Src family kinase Lck and the linker for activation of T cells (LAT) were both displaced from lipid rafts indicating that acylation by PUFAs is not required for protein displacement from rafts in PUFA-treated T cells. In conclusion, these data provide strong evidence that displacement of acylated proteins from rafts in PUFA-treated T cells is predominantly due to altered raft lipid composition.

Polyunsaturated fatty acids (PUFAs) exert immunosuppressive effects, but the molecular alterations leading to T cell inhibition are not yet elucidated. Signal transduction seems to involve detergent-resistant membrane domains (DRMs) acting as functional rafts within the plasma membrane bilayer with Src family protein tyrosine kinases being attached to their cytoplasmic leaflet. Since DRMs include predominantly saturated fatty acyl moieties, we investigated whether PUFAs could affect T cell signaling by remodeling of DRMs. Jurkat T cells cultured in PUFA-supplemented medium showed a markedly diminished calcium response when stimulated via the transmembrane CD3 complex or glycosyl phosphatidylinositol (GPI)- anchored CD59. Immunofluorescence studies indicated that CD59 but not Src family protein tyrosine kinase Lck remained in a punctate pattern after PUFA enrichment. Analysis of DRMs revealed a marked displacement of Src family kinases (Lck, Fyn) from DRMs derived from PUFA-enriched T cells compared with controls, and the presence of Lck in DRMs strictly correlated with calcium signaling. In contrast, GPI-anchored proteins (CD59, CD48) and ganglioside GM1, both residing in the outer membrane leaflet, remained in the DRM fraction. In conclusion, PUFA enrichment selectively modifies the cytoplasmic layer of DRMs and this alteration could underlie the inhibition of T cell signal transduction by PUFAs.

Decreased skeletal muscle glucose disposal and increased endogenous glucose production (EGP) contribute to postprandial hyperglycemia in type 2 diabetes, but the contribution of hepatic glycogen metabolism remains uncertain. Hepatic glycogen metabolism and EGP were monitored in type 2 diabetic patients and nondiabetic volunteer control subjects (CON) after mixed meal ingestion and during hyperglycemic-hyperinsulinemic-somatostatin clamps applying 13 C nuclear magnetic resonance spectroscopy (NMRS) and variable infusion dual-tracer technique. Hepatocellular lipid (HCL) content was quantified by 1 H NMRS. Before dinner, hepatic glycogen was lower in type 2 diabetic patients (227 ؎ 6 vs. CON: 275 ؎ 10 mmol/l liver, P < 0.001). After meal ingestion, net synthetic rates were 0.76 ؎ 0.16 (type 2 diabetic patients) and 1.36 ؎ 0.15 mg ⅐ kg ؊1 ⅐ min ؊1 (CON, P < 0.02), resulting in peak concentrations of 283 ؎ 15 and 360 ؎ 11 mmol/l liver. Postprandial rates of EGP were ϳ0.3 mg ⅐ kg ؊1 ⅐ min ؊1(30 -170 min; P < 0.05 vs. CON) higher in type 2 diabetic patients. Under clamp conditions, type 2 diabetic patients featured ϳ54% lower (P < 0.03) net hepatic glycogen synthesis and ϳ0.5 mg ⅐ kg ؊1 ⅐ min ؊1 higher (P < 0.02) EGP. Hepatic glucose storage negatively correlated with HCL content (R ‫؍‬ ؊0.602, P < 0.05). Type 2 diabetic patients exhibit 1) reduction of postprandial hepatic glycogen synthesis, 2) temporarily impaired suppression of EGP, and 3) no normalization of these defects by controlled hyperglycemic hyperinsulinemia. Thus, impaired insulin sensitivity and/or chronic glucolipotoxicity in addition to the effects of an altered insulin-to-glucagon ratio or increased free fatty acids accounts for defective hepatic glycogen metabolism in type 2 diabetic patients. Diabetes 53: 3048 -3056, 2004