Pioglitazone has direct effects on insulin sensitivity and intracellular lipid content in L6 skeletal muscle cells.

Using L6 skeletal muscle cell line, rendered insulin resistant by incubation with triglyceride-rich lipoproteins (TGRLs), we sought to answer the question whether pioglitazone has direct effects on this cell line. Incubation of L6 cells with TGRLs led to an increase in the intramyocellular triglyceride content. Moreover, TGRLs led to a reduction in insulin-stimulated glycogen content and GSK-3 phosphorylation. All these changes induced by TGRLs could be antagonized by incubation of L6 cells with pioglitazone. The PPAR-γ antagonist GW9662 reversed the pioglitazone effects. We conclude that pioglitazone has direct insulin-sensitizing effects on the L6 skeletal muscle cell line, which are paralleled by a reduction in intramyocellular triglyceride accumulation.

Effect of postprandial lipemia on plasma concentrations of A-FABP, RBP-4 and visfatin.

BACKGROUND AND AIMS: Several studies indicate that changes in the plasma concentrations of adipocyte-fatty acid binding protein (A-FABP), retinol binding protein-4 (RBP-4) and visfatin are associated with chronic states of insulin resistance. Recent studies have shown that postprandial lipemia induces an acute state of insulin resistance. The aim of this study was to investigate the effect of postprandial lipemia on the plasma concentrations of A-FABP, RBP-4 and visfatin. METHODS AND RESULTS: In a within-subject crossover study, we administered a standardized high-fat meal to 24 healthy subjects (12 males and 12 females). Plasma concentrations of adipocytokines were measured in the morning after an overnight fast and during postprandial lipemia, i.e. 2, 4 and 6 hours after meal ingestion (postprandial experiment). To exclude potential confounding factors affecting the adipocytokine plasma concentrations, a control experiment without meal ingestion was performed over the same time period (postabsorptive control experiment). Comparing plasma concentrations of A-FABP, RBP-4 and visfatin between the postprandial and the postabsorptive control experiments, we found no significant differences. Within either of the two experiments, a decrease of A-FABP was noted reaching, however, statistical significance only in the postprandial experiment, i.e. 2 and 4 hours after meal ingestion. CONCLUSION: Postprandial lipemia has no significant effect on the plasma concentrations of visfatin, A-FABP or RBP-4 in relation to their postabsorptive plasma profiles. We conclude that prolonged states of insulin resistance are required to affect plasma concentrations of these adipocytokines.

Postprandial lipaemia induces an acute decrease of insulin sensitivity in healthy men independently of plasma NEFA levels.

AIMS/HYPOTHESIS: Typical Western diets cause postprandial lipaemia for 18 h per day. We tested the hypothesis that postprandial lipaemia decreases insulin sensitivity. SUBJECTS, MATERIALS AND METHODS: Employing a randomised crossover design, we administered two types of virtually isocaloric meals to ten healthy volunteers on two separate occasions. The meals (Meals 1 and 2) were both designed to produce a rise in triglycerides, but only Meal 1 generated a rise in NEFA, too. Insulin sensitivity, as quantified by an IVGTT with minimal model analysis, was calculated postabsorptively at 08.00 h and postprandially at 13.00 h, i.e. 3 h after meal ingestion. RESULTS: Triglycerides rose from 0.91+/-0.31 mmol/l postabsorptively to 2.08+/-0.70 mmol/l postprandially with Meal 1 (p=0.005) and from 0.92+/-0.41 to 1.71+/-0.79 mmol/l with Meal 2 (p=0.005). Neither the triglyceride levels at 13.00 h, nor the post-meal AUCs for triglycerides were statistically different between Meal 1 and Meal 2. NEFA rose from 0.44+/-0.17 mmol/l postabsorptively to 0.69+/-0.16 mmol/l postprandially with Meal 1 (p=0.005) and showed no significant change with Meal 2 (0.46+/-0.31 mmol/l postabsorptively vs 0.36+/-0.32 mmol/l postprandially, p=0.09). Both the NEFA level at 13.00 h and the post-meal AUC for NEFA were significantly higher after Meal 1 than Meal 2. Compared with the postabsorptive state, insulin sensitivity decreased postprandially after each of the two meals to a comparable degree (Meal 1: -53%, p=0.02; Meal 2: -45%, p=0.005). CONCLUSIONS/INTERPRETATION: Our study reveals a drop in insulin sensitivity during postprandial lipaemia and strongly suggests that decreased insulin sensitivity is brought about by elevated plasma levels of triglyceride-rich lipoproteins independently of plasma NEFA levels.

Olanzapine impairs glycogen synthesis and insulin signaling in L6 skeletal muscle cells.

Second-generation antipsychotic agents (SGAs) are increasingly replacing first-generation antipsychotic agents due to their superior activity against the negative symptoms of schizophrenia, decreased extrapyramidal symptoms and better tolerability. However, some SGAs are associated with adverse metabolic effects as significant weight gain, lipid disorders and diabetes mellitus. The pathogenesis of SGA-induced disturbances of glucose homeostasis is unclear. In vivo studies suggest a direct influence of SGAs on peripheral insulin resistance. To this end, we analyzed whether olanzapine might alter glycogen synthesis and the insulin-signaling cascade in L6 myotubes. Glycogen content was diminished in a dose- and time-dependent manner. Within the insulin-signaling cascade IRS-1 tyrosine phosphorylation was induced several fold by insulin and was diminished by preincubation with olanzapine. IRS-1-associated PI3K activity was stimulated by insulin three-fold in L6 myotubes. Olanzapine inhibited insulin-stimulated IRS-1-associated PI3K activity in a dose-dependent manner. Protein mass of AKT, GSK-3 and GS was unaltered, whereas phosphorylation of AKT and GSK-3 was diminished, and pGS was increased. Finally, we compared olanzapine with amisulpride, an SGA clinically not associated with the induction of diabetes mellitus. Glycogen content was diminished in olanzapine-preincubated L6 cells, whereas this effect was not observed under the amisulpride conditions. We conclude that olanzapine impairs glycogen synthesis via inhibition of the classical insulin-signaling cascade and that this inhibitory effect may lead to the induction of insulin resistance in olanzapine-treated patients.

Human triglyceride-rich lipoproteins impair glucose metabolism and insulin signalling in L6 skeletal muscle cells independently of non-esterified fatty acid levels.

AIMS/HYPOTHESIS: Elevated fasting and postprandial plasma levels of triglyceride-rich lipoproteins (TGRLs), i.e. VLDL/remnants and chylomicrons/remnants, are a characteristic feature of insulin resistance and are considered a consequence of this state. The aim of this study was to investigate whether intact TGRL particles are capable of inducing insulin resistance. METHODS: We studied the effect of highly purified TGRLs on glycogen synthesis, glycogen synthase activity, glucose uptake, insulin signalling and intramyocellular lipid (IMCL) content using fully differentiated L6 skeletal muscle cells. RESULTS: Incubation with TGRLs diminished insulin-stimulated glycogen synthesis, glycogen synthase activity, glucose uptake and insulin-stimulated phosphorylation of Akt and glycogen synthase kinase 3. Insulin-stimulated tyrosine phosphorylation of IRS-1, and IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase (PI3K) activity were not impaired by TGRLs, suggesting that these steps were not involved in the lipoprotein-induced effects on glucose metabolism. The overall observed effects were time- and dose-dependent and paralleled IMCL accumulation. NEFA concentration in the incubation media did not increase in the presence of TGRLs indicating that the effects observed were solely due to intact lipoprotein particles. Moreover, co-incubation of TGRLs with orlistat, a potent active-site inhibitor of various lipases, did not alter TGRL-induced effects, whereas co-incubation with receptor-associated protein (RAP), which inhibits interaction of TGRL particles with members of the LDL receptor family, reversed the TGRL-induced effects on glycogen synthesis and insulin signalling. CONCLUSIONS/INTERPRETATION: Our data suggest that the accumulation of TGRLs in the blood stream of insulin-resistant patients may not only be a consequence of insulin resistance but could also be a cause for it.

Specific increase in p85alpha expression in response to dexamethasone is associated with inhibition of insulin-like growth factor-I stimulated phosphatidylinositol 3-kinase activity in cultured muscle cells.

The stimulation of phosphatidylinositol (PI) 3-kinase by insulin-like growth factor I (IGF-I) in L6 cultured skeletal muscle cells is inhibited by the glucocorticoid dexamethasone. The objective of this study was to investigate the mechanism of dexamethasone action by determining its effects on the expression of the p85alpha and p85beta regulatory subunit isoforms of PI 3-kinase, their coupling with the p110 catalytic subunit, and their association with insulin receptor substrate 1 (IRS-1) in response to IGF-I stimulation. Dexamethasone induced a 300% increase in p85alpha protein content in the L6 cultured myoblast cell line, whereas it increased p110 content by only 38% and had no effect on p85beta. The increase in p85alpha protein was associated with a coordinate increase in p85alpha mRNA. Stimulation with IGF-I induced the association of p85alpha and p85beta with IRS-1, and this was accompanied by increased amounts of the p110 catalytic subunit and markedly increased PI 3-kinase activity in IRS-1 immunoprecipitates. In cells treated with dexamethasone, greater amounts of p85alpha and lower amounts of p85beta, respectively, were found in IRS-1 immunoprecipitates, such that the alpha/beta ratio was markedly higher than in control cells. In spite of the increase in both total and IRS-1-associated p85alpha following dexamethasone treatment, IRS-1-associated p110 catalytic subunit and PI 3-kinase activity were decreased by approximately 50%. Thus, dexamethasone induces a specific increase in expression of the p85alpha regulatory subunit that is not associated with a coordinate increase in the p110 catalytic subunit of PI 3-kinase. As a consequence, in dexamethasone-treated cells, p85alpha that is not coupled with p110 competes with both p85alpha.p110 and p85beta.p110 complexes for association with IRS-1, leading to increased p85alpha but decreased p85beta, p110, and PI 3-kinase activity in IRS-1 immunoprecipitates.

The effect of dietary protein restriction on the progression of diabetic and nondiabetic renal diseases: a meta-analysis.

BACKGROUND: Dietary protein has long been thought to play a role in the progression of chronic renal disease, but clinical trials to date have not consistently shown that dietary protein restriction is beneficial. PURPOSE: To use meta-analysis to assess the efficacy of dietary protein restriction in previously published studies of diabetic and nondiabetic renal diseases, including the recently completed Modification of Diet in Renal Disease Study. DATA SOURCES: The English-language medical literature published from January 1966 through December 1994 was searched for studies examining the effect of low-protein diets in humans with chronic renal disease. A total of 1413 patients in five studies on nondiabetic renal disease (mean length of follow-up, 18 to 36 months) and 108 patients in five studies of type I diabetes mellitus (mean length of follow-up, 9 to 35 months) were included. STUDY SELECTION: Randomized, controlled studies were selected for nondiabetic renal disease; randomized, controlled studies or time-controlled studies with nonrandomized crossover design were selected for diabetic nephropathy. DATA EXTRACTION: Data in tables, figures, or text were independently extracted by two of the authors. DATA SYNTHESIS: The relative risk for progression of renal disease in patients receiving a low-protein diet compared with patients receiving a usual-protein diet was calculated by using a random-effects model. In five studies of nondiabetic renal disease, a low-protein diet significantly reduced the risk for renal failure or death (relative risk, 0.67 [95% Cl, 0.50 to 0.89]). In five studies of insulin-dependent diabetes mellitus, a low-protein diet significantly slowed the increase in urinary albumin level or the decline in glomerular filtration rate or creatinine clearance (relative risk, 0.56 [Cl, 0.40 to 0.77]). Tests for heterogeneity showed no significant differences in relative risk among studies of either diabetic or nondiabetic renal disease. No significant differences were seen between diet groups in pooled mean arterial blood pressure (diabetic and nondiabetic patients) or glycosylated hemoglobin level (diabetic patients only). CONCLUSION: Dietary protein restriction effectively slows the progression of both diabetic and nondiabetic renal diseases.

A comparison of rat small intestinal insulin and insulin-like growth factor I receptors during fasting and refeeding.

Insulin-like growth factor I (IGF-I) and insulin may be important regulators of intestinal growth. To investigate small intestinal IGF-I receptors (IGF-IR) and insulin receptors (IR) during intestinal cell atrophy and regeneration, we compared indexes of IGF-IR and IR expression in rat jejunum after 72 h of fasting and 24-72 h of enteral refeeding. Fasting induced intestinal atrophy, reduced plasma insulin and IGF-I concentrations, and markedly decreased jejunal IGF-I messenger RNA (mRNA) levels; these changes were reversed by refeeding. Fasting significantly increased jejunal specific insulin binding, IR content (to 230% of the fed control value), and the 9.6- and 7.4-kilobase IR mRNA transcript levels (to 202% and 218% of control values, respectively). These IR indexes rapidly decreased to control levels with refeeding. Levels of IGF-IR (by Scatchard analysis) and IGF-I-R mRNA were not significantly altered with fasting. The 11-kilobase IGF-IR mRNA transcript increased significantly during the first 24 h of refeeding (to 166% of the control value), and IGF-IR number rose 3-fold. We conclude that rat jejunal IR and IGF-IR are differentially regulated by nutrient availability. Up-regulation of jejunal IGF-I and IGF-IR expression during refeeding suggests a role for the IGF action pathway in gut trophic responses to enteral nutrients.

cDNA cloning of the rat IGF I receptors: structural analysis of rat and human IGF I and insulin receptors reveals differences in alternative splicing and receptor-specific domain conservation.

IGF I and insulin receptors are homologous proteins that function in distinct physiological pathways. To define domains that might contribute to differences between IGF I and insulin receptors, we cloned the rat IGF I receptor cDNA and performed a comparative sequence analysis of specific functional domains in the two receptor types of rats and humans. Since alternative splicing has been shown to alter the activities of both IGF I and insulin receptors, we also examined the mRNA splicing patterns of the two receptors. The C-terminal region exhibits the lowest degree of amino acid homology between rat and human IGF I receptors (85%) and the tyrosine kinase domain the highest homology (98%). In the region corresponding to the CAG+/-alternative splicing site of the human IGF I receptor, a nucleotide change in the rat eliminates the alternative acceptor splice site. The rat IGF I receptor has no equivalent to the alternatively spliced exon 11 of the insulin receptor. The IGF I and insulin receptors are highly homologous in the tyrosine kinase domain (84%), but differ markedly in other specific regions (e.g., 22-26% homology in the transmembrane domain, 45% homology in the C-terminal domain). We speculate that these regions of divergent sequence may have roles in determining distinct signaling properties of IGF I and insulin receptors.

Insulin-like growth factor I receptor gene structure.

The insulin-like growth factor I (IGF I) receptor is a tyrosine kinase-containing transmembrane protein that plays an important role in cell growth control. We have isolated and characterized human genomic DNA clones containing the entire coding sequence of the IGF I receptor. Results of restriction analysis and sequencing of multiple overlapping clones were consistent with the existence of a single IGF I receptor gene. The complete receptor coding sequence is contained in 21 exons. There is striking homology with the insulin receptor gene in overall size (approximately 100 kilobases) and in the number and size of individual exons. An exon analogous to the alternatively spliced exon 11 of the insulin receptor gene could not be detected. An alternative internal splice site corresponding to a known alternatively spliced mRNA transcript was shown to be located at the 5' end of exon 14. Knowledge of the structure of the IGF I receptor gene should facilitate further studies on the structural determinants of receptor function, the relationships between insulin and IGF I receptors, and the molecular basis for multiple IGF I receptor species.