Increased plasma free fatty acid (FFA) concentrations are typically associated with many insulin-resistant states including obesity and type 2 diabetes mellitus (1-3). Furthermore, raising plasma FFA levels in healthy humans, by triglyceride/heparin infusions, can also acutely induce insulin resistance (4-11). Over thirty years ago, Randle et al. (12,13) demonstrated that FFAs compete with glucose for oxidation in isolated rat heart and diaphragmatic muscle preparations, and they speculated that increased fat oxidation may cause the insulin resistance associated with diabetes and obesity. They proposed that increased FFA oxidation leads to an increase in the intramitochondrial acetyl-coenzyme A (acetyl-CoA) and reduced/oxidized nicotinamide adenine dinucleotide (NADH/NAD + ) ratios, resulting in inactivation of pyruvate dehydrogenase activity. The consequent increase in intracellular citrate concentration causes inhibition of phosphofructokinase resulting in an increase in glucose-6-phosphate levels. The elevated glucose-6-phosphate levels would inhibit hexokinase II activity and then lead to decreased glucose uptake. However, recent studies by our group (14) and others (15,16) have called this mechanism into question. Boden and coworkers have shown that a reduction in carbohydrate oxidation was responsible for only one-third of the fatty acid-dependent decrease in glucose uptake, while impaired non-oxidative glucose metabolism accounted for the remainder (16). These workers suggested that two different defects might contribute to the impairment in nonoxidative glucose metabolism. At FFA concentrations of ∼0.75 mM, they found an increase in glucose-6-phosphate concentrations in muscle biopsies, suggesting an inhibitory effect of FFA on glycogen synthase activity, whereas at lower FFA concentrations (∼0.50 mM) they observed no difference in intramuscular glucose-6-phosphate concentration. In contrast, using carbon-13/phosphorous-31 nuclear magnetic resonance (NMR) spectroscopy under increased plasma FFA concentrations (∼1.8 mM), we observed a decrease in intramuscular glucose-6-phosphate concentration associated with a 50% reduction in insulin-stimulated muscle glycogen synthesis (14). These data suggest that acute elevations in plasma FFA levels in humans cause insulin resistance by initial inhibition of glucose transport and/or phosphorylation activity that is concurrently followed by a reduction in the rate of both muscle glycogen synthesis and glucose oxidation. Because glucose-6-phosphate (and not intracellular glucose) concentration was measured, it was not possible to distinguish between To examine the mechanism by which free fatty acids (FFA) induce insulin resistance in human skeletal muscle, glycogen, glucose-6-phosphate, and intracellular glucose concentrations were measured using carbon-13 and phosphorous-31 nuclear magnetic resonance spectroscopy in seven healthy subjects before and after a hyperinsulinemic-euglycemic clamp following a five-hour infusion of either lipid/heparin or glycerol/heparin. I...

To examine the mechanism by which free fatty acids (FFAs) induce insulin resistance in vivo, awake chronically catheterized rats underwent a hyperinsulinemic-euglycemic clamp with or without a 5-h preinfusion of lipid/heparin to raise plasma FFA concentrations. Increased plasma FFAs resulted in insulin resistance as reflected by a approximately 35% reduction in the glucose infusion rate (P < 0.05 vs. control). The insulin resistance was associated with a 40-50% reduction in 13C nuclear magnetic resonance (NMR)-determined rates of muscle glycogen synthesis (P < 0.01 vs. control) and muscle glucose oxidation (P < 0.01 vs. control), which in turn could be attributed to a approximately 25% reduction in glucose transport activity as assessed by 2-[1,2-3H]deoxyglucose uptake in vivo (P < 0.05 vs. control). This lipid-induced decrease in insulin-stimulated muscle glucose metabolism was associated with 1) a approximately 50% reduction in insulin-stimulated insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity (P < 0.05 vs. control), 2) a blunting in insulin-stimulated IRS-1 tyrosine phosphorylation (P < 0.05, lipid-infused versus glycerol-infused), and 3) a four-fold increase in membrane-bound, or active, protein kinase C (PKC) theta (P < 0.05 vs. control). We conclude that acute elevations of plasma FFA levels for 5 h induce skeletal muscle insulin resistance in vivo via a reduction in insulin-stimulated muscle glycogen synthesis and glucose oxidation that can be attributed to reduced glucose transport activity. These changes are associated with abnormalities in the insulin signaling cascade and may be mediated by FFA activation of PKC theta.

In striated muscle, the plasma membrane forms tubular invaginations (transverse tubules or T-tubules) that function in depolarization-contraction coupling. Caveolin-3 and amphiphysin were implicated in their biogenesis. Amphiphysin isoforms have a putative role in membrane deformation at endocytic sites. An isoform of amphiphysin 2 concentrated at T-tubules induced tubular plasma membrane invaginations when expressed in nonmuscle cells. This property required exon 10, a phosphoinositide-binding module. In developing myotubes, amphiphysin 2 and caveolin-3 segregated in tubular and vesicular portions of the T-tubule system, respectively. These findings support a role of the bilayer-deforming properties of amphiphysin at T-tubules and, more generally, a physiological role of amphiphysin in membrane deformation.

The effect of AMP-activated protein kinase (AMPK) activation on skeletal muscle glucose metabolism was examined in awake rats by infusing them with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR; 40 mg/kg bolus and 7.5 mg ⋅ kg−1 ⋅ min−1constant infusion) along with a variable infusion of glucose (49.1 ± 2.4 μmol ⋅ kg−1 ⋅ min−1) to maintain euglycemia. Activation of AMPK by AICAR caused 2-deoxy-d-[1,2-3H]glucose (2-DG) uptake to increase more than twofold in the soleus and the lateral and medial gastrocnemius compared with saline infusion and occurred without phosphatidylinositol 3-kinase activation. Glucose uptake was also assessed in vitro by use of the epitrochlearis muscle incubated either with AICAR (0.5 mM) or insulin (20 mU/ml) or both in the presence or absence of wortmannin (1.0 μM). AICAR and insulin increased muscle 2-DG uptake rates by ∼2- and 2.7-fold, respectively, compared with basal rates. Combining AICAR and insulin led to a fully additive effect on muscle glucose transport activity. Wortmannin inhibited insulin-stimulated glucose uptake. However, neither wortmannin nor 8-(p-sulfophenyl)-theophylline (10 μM), an adenosine receptor antagonist, inhibited the AICAR-induced activation of glucose uptake. Electrical stimulation led to an about threefold increase in glucose uptake over basal rates, whereas no additive effect was found when AICAR and contractions were combined. In conclusion, the activation of AMPK by AICAR increases skeletal muscle glucose transport activity both in vivo and in vitro. This cellular pathway may play an important role in exercise-induced increase in glucose transport activity.

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Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.

Stiff-Man syndrome (SMS) is a rare disease of the central nervous system characterized by chronic muscle rigidity and autoimmunity directed against synaptic antigens. In a subset of patients, generally positive for antiamphiphysin autoantibodies, SMS has an autoimmune paraneoplastic origin. Amphiphysin isoforms are expressed at high levels in brain and skeletal muscle and often are overexpressed in breast cancer. We report here the occurrence of rhabdomyolysis in a patient with SMS, breast cancer, and antibodies that recognize both brain and muscle amphiphysin isoforms. Immunotherapy induced a remission of both rhabdomyolysis and SMS symptoms. Autoimmune rhabdomyolysis may represent a paraneoplastic complication of cancer patients with amphiphysin autoimmunity.

High fat diets can impact the expression of bioactive peptide signaling molecules known as adipokines. Adipocyte cells synthesize adipokines and these molecules regulate energy metabolism, neuroendocrine function and immune function. One particular adipokine is the 12 kDa protein, resistin. Resistin was originally identified in a study that concluded that mice heterozygous for a null PPARγ allele exhibit adipocyte hypertrophy on a high fat diet and have an increased sensitivity to insulin. Additional research has shown resistin was linked to increased insulin resistance; however, the specific mechanism responsible for the change in insulin sensitivity has not yet been established. The goal of this research project is to provide a simplified and comprehensive model system for understanding adipokine expression by analyzing the expression of resistin using western blot analysis. Developing a simplified model framework will allow for subsequent investigations into adipokine expression that exhibit a clear understanding of the impact of excess adipose tissue on adipokine expression. The mice have been fed a high fat diet over a period of three weeks and the epididymal fat pads have been removed. western blot analysis will be utilized to compare resistin expression in the fat obtained from the low and high fat fed mice.