Kyle L. Hoehn

Excess Lipid Availability Increases Mitochondrial Fatty Acid Oxidative Capacity in Muscle: Evidence Against a Role for Reduced Fatty Acid Oxidation in Lipid-Induced Insulin Resistance in Rodents

A reduced capacity for mitochondrial fatty acid oxidation in skeletal muscle has been proposed as a major factor leading to the accumulation of intramuscular lipids and their subsequent deleterious effects on insulin action. Here, we examine markers of mitochondrial fatty acid oxidative capacity in rodent models of insulin resistance associated with an oversupply of lipids. C57BL/6J mice were fed a high-fat diet for either 5 or 20 weeks. Several markers of muscle mitochondrial fatty acid oxidative capacity were measured, including 14C-palmitate oxidation, palmitoyl-CoA oxidation in isolated mitochondria, oxidative enzyme activity (citrate synthase, β-hydroxyacyl CoA dehydrogenase, medium-chain acyl-CoA dehydrogenase, and carnitine palmitoyl-transferase 1), and expression of proteins involved in mitochondrial metabolism. Enzyme activity and mitochondrial protein expression were also examined in muscle from other rodent models of insulin resistance. Compared with standard diet–fed controls, muscle from fat-fed mice displayed elevated palmitate oxidation rate (5 weeks +23%, P < 0.05, and 20 weeks +29%, P < 0.05) and increased palmitoyl-CoA oxidation in isolated mitochondria (20 weeks +49%, P < 0.01). Furthermore, oxidative enzyme activity and protein expression of peroxisome proliferator–activated receptor γ coactivator (PGC)-1α, uncoupling protein (UCP) 3, and mitochondrial respiratory chain subunits were significantly elevated in fat-fed animals. A similar pattern was present in muscle of fat-fed rats, obese Zucker rats, and db/db mice, with increases observed for oxidative enzyme activity and expression of PGC-1α, UCP3, and subunits of the mitochondrial respiratory chain. These findings suggest that high lipid availability does not lead to intramuscular lipid accumulation and insulin resistance in rodents by decreasing muscle mitochondrial fatty acid oxidative capacity.

Insulin resistance is a cellular antioxidant defense mechanism

We know a great deal about the cellular response to starvation via AMPK, but less is known about the reaction to nutrient excess. Insulin resistance may be an appropriate response to nutrient excess, but the cellular sensors that link these parameters remain poorly defined. In the present study we provide evidence that mitochondrial superoxide production is a common feature of many different models of insulin resistance in adipocytes, myotubes, and mice. In particular, insulin resistance was rapidly reversible upon exposure to agents that act as mitochondrial uncouplers, ETC inhibitors, or mitochondrial superoxide dismutase (MnSOD) mimetics. Similar effects were observed with overexpression of mitochondrial MnSOD. Furthermore, acute induction of mitochondrial superoxide production using the complex III antagonist antimycin A caused rapid attenuation of insulin action independently of changes in the canonical PI3K/Akt pathway. These results were validated in vivo in that MnSOD transgenic mice were partially protected against HFD induced insulin resistance and MnSOD+/− mice were glucose intolerant on a standard chow diet. These data place mitochondrial superoxide at the nexus between intracellular metabolism and the control of insulin action potentially defining this as a metabolic sensor of energy excess.

IRS1-Independent Defects Define Major Nodes of Insulin Resistance

Insulin resistance is a common disorder caused by a wide variety of physiological insults, some of which include poor diet, inflammation, anti-inflammatory steroids, hyperinsulinemia, and dyslipidemia. The common link between these diverse insults and insulin resistance is widely considered to involve impaired insulin signaling, particularly at the level of the insulin receptor substrate (IRS). To test this model, we utilized a heterologous system involving the platelet-derived growth factor (PDGF) pathway that recapitulates many aspects of insulin action independently of IRS. We comprehensively analyzed six models of insulin resistance in three experimental systems and consistently observed defects in both insulin and PDGF action despite a range of insult-specific defects within the IRS-Akt nexus. These findings indicate that while insulin resistance is associated with multiple deficiencies, the most deleterious defects and the origin of insulin resistance occur independently of IRS.

Pigment Epithelium-Derived Factor Contributes to Insulin Resistance in Obesity

Obesity is a major risk factor for insulin resistance; however, the factors linking these disorders are not well defined. Herein, we show that the noninhibitory serine protease inhibitor, pigment epithelium-derived factor (PEDF), plays a causal role in insulin resistance. Adipocyte PEDF expression and serum levels are elevated in several rodent models of obesity and reduced upon weight loss and insulin sensitization. Lean mice injected with recombinant PEDF exhibited reduced insulin sensitivity during hyperinsulinemic-euglycemic clamps. Acute PEDF administration activated the proinflammatory serine/threonine kinases c-Jun terminal kinase and extracellular regulated kinase in both muscle and liver, which corresponded with reduced insulin signal transduction. Prolonged PEDF administration stimulated adipose tissue lipolysis, resulted in ectopic lipid deposition, and reduced insulin sensitivity, while neutralizing PEDF in obese mice enhanced insulin sensitivity. Overall, these results identify a causal role for PEDF in obesity-induced insulin resistance.

CaMKII-Mediated Phosphorylation of the Myosin Motor Myo1c Is Required for Insulin-Stimulated GLUT4 Translocation in Adipocytes

The unconventional myosin Myo1c has been implicated in insulin-regulated GLUT4 translocation to the plasma membrane in adipocytes. We show that Myo1c undergoes insulin-dependent phosphorylation at S701. Phosphorylation was accompanied by enhanced 14-3-3 binding and reduced calmodulin binding. Recombinant CaMKII phosphorylated Myo1c in vitro and siRNA knockdown of CaMKIIdelta abolished insulin-dependent Myo1c phosphorylation in vivo. CaMKII activity was increased upon insulin treatment and the CaMKII inhibitors CN21 and KN-62 or the Ca(2+) chelator BAPTA-AM blocked insulin-dependent Myo1c phosphorylation and insulin-stimulated glucose transport in adipocytes. Myo1c ATPase activity was increased after CaMKII phosphorylation in vitro and after insulin stimulation of CHO/IR/IRS-1 cells. Expression of wild-type Myo1c, but not S701A or ATPase dead mutant K111A, rescued the inhibition of GLUT4 translocation by siRNA-mediated Myo1c knockdown. These data suggest that insulin regulates Myo1c function via CaMKII-dependent phosphorylation, and these events play a role in insulin-regulated GLUT4 trafficking in adipocytes likely involving Myo1c motor activity.

Sialic Acid Modification of Adiponectin Is Not Required for Multimerization or Secretion but Determines Half-Life in Circulation

Adiponectin is an adipocyte-secreted, insulin-sensitizing hormone the circulating levels of which are reduced in conditions of insulin resistance and diabetes. Previous work has demonstrated the importance of posttranslational modifications, such as proline hydroxylation and lysine hydroxylation/glycosylation, in adiponectin oligomerization, secretion, and function. Here we describe the first functional characterization of adiponectin sialylation. Using a variety of biochemical approaches we demonstrated that sialylation occurs on previously unidentified O-linked glycans on Thr residues of the variable domain in human adiponectin. Enzymatic removal of sialic acid or its underlying O-linked sugars did not affect adiponectin multimer composition. Expression of mutant forms of adiponectin (lacking the modified Thr residues) or of wild-type adiponectin in cells defective in sialylation did not compromise multimer formation or secretion, arguing against a structural role for this modification. Activity of desialylated adiponectin was comparable to control adiponectin in L6 myotubes and acute assays in adiponectin(-/-) mice. In contrast, plasma clearance of desialylated adiponectin was accelerated compared with that of control adiponectin, implicating a role for this modification in determining the half-life of circulating adiponectin. Uptake of desialylated adiponectin by isolated primary rat hepatocytes was also accelerated, suggesting a role for the hepatic asialoglycoprotein receptor. Finally, after chronic administration in adiponectin(-/-) mice steady-state levels of desialylated adiponectin were lower than control adiponectin and failed to recapitulate the improvements in glucose and insulin tolerance tests observed with control adiponectin. These data suggest an important role for sialic acid content in the regulation of circulating adiponectin levels and highlight the importance of understanding mechanisms regulating adiponectin sialylation/desialylation.

Global Phosphoproteomics Identifies a Major Role for AKT and 14-3-3 in Regulating EDC3

Insulin plays an essential role in metabolic homeostasis in mammals, and many of the underlying biochemical pathways are regulated via the canonical phosphatidylinositol 3-kinase/AKT pathway. To identify novel metabolic actions of insulin, we conducted a quantitative proteomics analysis of insulin-regulated 14-3-3-binding proteins in muscle cells. These studies revealed a novel role for insulin in the post-transcriptional regulation of mRNA expression. EDC3, a component of the mRNA decay and translation repression pathway associated with mRNA processing bodies, was shown to be phosphorylated by AKT downstream of insulin signaling. The major insulin-regulated site was mapped to Ser-161, and phosphorylation at this site led to increased 14-3-3 binding. Functional studies indicated that induction of 14-3-3 binding to EDC3 causes morphological changes in processing body structures, inhibition of microRNA-mediated mRNA post-transcriptional regulation, and alterations in the protein- protein interactions of EDC3. These data highlight an important new arm of the insulin signaling cascade in the regulation of mRNA utilization.

Mitochondrial regulation of insulin action

Insulin resistance is the prodrome of many metabolic diseases and identifying ways to correct this pathological condition is a major goal for medical research. The foremost barrier to the development of new treatments is that the precise etiology of insulin resistance is uncertain. Recent studies suggest that changes in mitochondrial structure or function drive this condition, however much of this evidence is circumstantial. This Signaling Networks in Focus article provides a brief overview of known and speculative regulatory intersections whereby mitochondrial dysfunction at the levels of lipid oxidation, oxidative stress, calcium, adenine nucleotides, and protons may regulate insulin sensitivity. If mitochondrial dysfunction underlies the origins of metabolic disease then determining the precise molecular pathway will be essential for the development of new treatment and prevention strategies.