The association of hepatic mitochondrial function with insulin resistance and non-alcoholic fatty liver (NAFL) or steatohepatitis (NASH) remains unclear. This study applied high-resolution respirometry to directly quantify mitochondrial respiration in liver biopsies of obese insulin-resistant humans without (n = 18) or with (n = 16) histologically proven NAFL or with NASH (n = 7) compared to lean individuals (n = 12). Despite similar mitochondrial content, obese humans with or without NAFL had 4.3- to 5.0-fold higher maximal respiration rates in isolated mitochondria than lean persons. NASH patients featured higher mitochondrial mass, but 31%-40% lower maximal respiration, which associated with greater hepatic insulin resistance, mitochondrial uncoupling, and leaking activity. In NASH, augmented hepatic oxidative stress (H2O2, lipid peroxides) and oxidative DNA damage (8-OH-deoxyguanosine) was paralleled by reduced anti-oxidant defense capacity and increased inflammatory response. These data suggest adaptation of the liver ("hepatic mitochondrial flexibility") at early stages of obesity-related insulin resistance, which is subsequently lost in NASH.

Muscle insulin resistance is a key feature of obesity and type 2 diabetes and is strongly associated with increased intramyocellular lipid content and inflammation. However, the cellular and molecular mechanisms responsible for causing muscle insulin resistance in humans are still unclear. To address this question, we performed serial muscle biopsies in healthy, lean subjects before and during a lipid infusion to induce acute muscle insulin resistance and assessed lipid and inflammatory parameters that have been previously implicated in causing muscle insulin resistance. We found that acute induction of muscle insulin resistance was associated with a transient increase in total and cytosolic diacylglycerol (DAG) content that was temporally associated with protein kinase (PKC)θ activation, increased insulin receptor substrate (IRS)-1 serine 1101 phosphorylation, and inhibition of insulin-stimulated IRS-1 tyrosine phosphorylation and AKT2 phosphorylation. In contrast, there were no associations between insulin resistance and alterations in muscle ceramide, acylcarnitine content, or adipocytokines (interleukin-6, adiponectin, retinol-binding protein 4) or soluble intercellular adhesion molecule-1. Similar associations between muscle DAG content, PKCθ activation, and muscle insulin resistance were observed in healthy insulin-resistant obese subjects and obese type 2 diabetic subjects. Taken together, these data support a key role for DAG activation of PKCθ in the pathogenesis of lipid-induced muscle insulin resistance in obese and type 2 diabetic individuals.lipotoxicity | insulin signaling

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.

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

Greater changes in insulin sensitivity after intake of an isoenergetic HCF than after intake of an HP diet might help to explain the diverse effects of these diets on diabetes risk. This trial is registered at clinicaltrials.gov as NCT00579657.