Eva Tomas

Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation.

gACRP30, the globular subunit of adipocyte complement-related protein of 30 kDa (ACRP30), improves insulin sensitivity and increases fatty acid oxidation. The mechanism by which gACRP30 exerts these effects is unknown. Here, we examined if gACRP30 activates AMP-activated protein kinase (AMPK), an enzyme that has been shown to increase muscle fatty acid oxidation and insulin sensitivity. Incubation of rat extensor digitorum longus (EDL), a predominantly fast twitch muscle, with gACRP30 (2.5 μg/ml) for 30 min led to 2-fold increases in AMPK activity and phosphorylation of both AMPK on Thr-172 and acetyl CoA carboxylase (ACC) on Ser-79. Accordingly, concentration of malonyl CoA was diminished by 30%. In addition, gACRP30 caused a 1.5-fold increase in 2-deoxyglucose uptake. Similar changes in malonyl CoA and ACC were observed in soleus muscle incubated with gACRP30 (2.5 μg/ml), although no significant changes in AMPK activity or 2-deoxyglucose uptake were detected. When EDL was incubated with full-length hexameric ACRP30 (10 μg/ml), AMPK activity and ACC phosphorylation were not altered. Administration of gACRP30 (75 μg) to C57 BL/6J mice in vivo led to increased AMPK activity and ACC phosphorylation and decreased malonyl CoA concentration in gastrocnemius muscle within 15–30 min. Both in vivo and in vitro, activation of AMPK was the first effect of gACRP30 and was transient, whereas alterations in malonyl CoA and ACC occurred later and were more sustained. Thus, gACRP30 most likely exerts its actions on muscle fatty acid oxidation by inactivating ACC via activation of AMPK and perhaps other signal transduction proteins.

Mice Lacking Adiponectin Show Decreased Hepatic Insulin Sensitivity and Reduced Responsiveness to Peroxisome Proliferator-activated Receptor γ Agonists

The adipose tissue-derived hormone adiponectin improves insulin sensitivity and its circulating levels are decreased in obesity-induced insulin resistance. Here, we report the generation of a mouse line with a genomic disruption of the adiponectin locus. We aimed to identify whether these mice develop insulin resistance and which are the primary target tissues affected in this model. Using euglycemic/insulin clamp studies, we demonstrate that these mice display severe hepatic but not peripheral insulin resistance. Furthermore, we wanted to test whether the lack of adiponectin magnifies the impairments of glucose homeostasis in the context of a dietary challenge. When exposed to high fat diet, adiponectin null mice rapidly develop glucose intolerance. Specific PPARγ agonists such as thiazolidinediones (TZDs) improve insulin sensitivity by mechanisms largely unknown. Circulating adiponectin levels are significantly up-regulated in vivo upon activation of PPARγ. Both TZDs and adiponectin have been shown to activate AMP-activated protein kinase (AMPK) in the same target tissues. We wanted to address whether the ability of TZDs to improve glucose tolerance is dependent on adiponectin and whether this improvement involved AMPK activation. We demonstrate that the ability of PPARγ agonists to improve glucose tolerance in ob/ob mice lacking adiponectin is diminished. Adiponectin is required for the activation of AMPK upon TZD administration in both liver and muscle. In summary, adiponectin is an important contributor to PPARγ-mediated improvements in glucose tolerance through mechanisms that involve the activation of the AMPK pathway.

Role of disulfide bonds in Acrp30/adiponectin structure and signaling specificity. Different oligomers activate different signal transduction pathways.

Acrp30/adiponectin is an adipocyte-derived serum protein with important roles in regulation of lipid and glucose metabolism, but which of its isoforms are biologically active remains controversial. We addressed this issue by first characterizing the structure of each individual Acrp30 oligomer and the determinants responsible for multimer formation. Freeze etch electron microscopy showed the trimer to exhibit a ball-and- stick-like structure containing a large globular sphere, an extended collagen stalk, and a smaller sphere on the opposite end of the stalk. The hexamer consists of two adjacent trimeric globular domains and a single stalk composed of collagen domains from two trimers. Although not necessary for trimer formation or stability, two of the three monomers in an Acrp30 trimer are covalently linked by a disulfide bond between cysteine residues at position 22. In contrast, assembly of hexameric and higher molecular weight (HMW) forms of Acrp30 depends upon formation of Cys22-mediated disulfide bonds because their reduction with dithiothreitol or substitution of Cys22 with alanine led exclusively to trimers. HMW and hexamer isoforms of Acrp30 activated NF-κB in C2C12 cells, but trimers, either natural, formed by reduction of Acrp30 hexamer, or formed by the C22A mutant, did not. In contrast, incubation of isolated rat extensor digitorum longus with naturally formed Acrp30 trimers or trimeric C22A Acrp30 led to increased phosphorylation of AMP-activated protein kinase-α at Thr172 and its activation. Hexameric and HMW Acrp30 could not activate AMP-activated protein kinase. Thus, trimeric and HMW/hexameric Acrp30 activate different signal transduction pathways, and Acrp30 represents a novel example of the control of ligand signaling via changes in its oligomerization state.

Hyperglycemia and insulin resistance: possible mechanisms.

Abstract: Sustained hyperglycemia impairs insulin‐stimulated glucose utilization and glycogen synthesis in human and rat skeletal muscles, a phenomenon referred to clinically as glucose toxicity. In rat extensor digitorum longus (EDL) muscle preparations preincubated for 2‐4 h in a hyperglycemic medium (25 mM vs. 0 mM glucose), we have shown that the ability of insulin to stimulate glucose incorporation into glycogen is impaired. Interestingly, this was associated with a decreased activation of Akt/PKB, but not its upstream regulator, PI3‐kinase. A similar pattern of signaling abnormalities has been observed in adipocytes, L6 muscle cells, C2C12 cells, and (as reported here) EDL incubated with C2‐ceramide. On the other hand, no increase was observed in ceramide mass in EDL incubated with 25 mM glucose. Hyperglycemia‐induced insulin resistance also has been described in adipocytes, where it has been linked to activation of novel and conventional protein kinase C isoforms that phosphorylate the insulin receptor and IRS. In addition, we have recently shown that hyperglycemia causes insulin resistance in cultured human umbilical vein endothelial cells (HUVEC). Here, it was associated with an increased propensity to apoptosis and, as in muscle, with an impaired ability of insulin to activate Akt. Interestingly, these effects of hyperglycemia and an increase in diacylglycerol synthesis, which is also caused, were prevented by adding AICAR, an activator of AMP‐activated protein kinase (AMPK), to the incubation medium. These results suggest that hyperglycemia causes insulin resistance in cells other than those in classic insulin target tissues. Whether AMPK activation can reverse or prevent insulin resistance in all of these cells remains to be determined.

The Endosomal Compartment Is an Insulin-Sensitive Recruitment Site for GLUT4 and GLUT1 Glucose Transporters in Cardiac Myocytes

In nonstimulated cardiomyocytes, the glucose transporter GLUT4 is confined to intracellular vesicles forming at least two populations: a storage pool enriched in GLUT4 (pool 1) and an endosomal pool containing both GLUT4 and GLUT1 (pool 2). We have now studied the dynamics of these pools in response to insulin or the mitochondrial inhibitor rotenone in rat cardiomyocytes. Rotenone recruited GLUT4 and GLUT1 to the cell surface from endosomal pool 2 without affecting pool 1. Kinetic experiments were consistent with rotenone acting on an intracellular compartment that is in close connection with the plasma membrane. In contrast, insulin caused rapid, complete depletion of GLUT4 from pool 1 and reduced the GLUT1 content of pool 2 by approximately 50%, whereas, surprisingly, no net decrease in GLUT4 occurred in this pool. Subsequent insulin withdrawal resulted in slow replenishment of pool 2 with GLUT1 and of pool 1 with GLUT4. When pool 1 was still largely depleted of GLUT4, a second insulin challenge did red...

Metabolic and hormonal interactions between muscle and adipose tissue.

From the perspective of a muscle physiologist, adipose tissue has long been perceived predominantly as a fuel reservoir that provides muscle and other tissues with NEFA when exogenous nutrients are insufficient for their energy needs. Recently, studies have established that adipose tissue is also an endocrine organ. Among the hormones it releases are adiponectin and leptin, both of which can activate AMP-activated protein kinase and increase fatty acid oxidation in skeletal muscle and probably other tissues. Deficiencies of leptin or leptin receptor, adiponectin and IL-6 are associated with obesity, insulin resistance and a propensity to type 2 diabetes. In addition, a lack of adiponectin has been linked to atherosclerosis. Whether this pathology reflects a deficient activation of AMP-activated protein kinase in peripheral tissues remains to be determined. Finally, recent studies have suggested that skeletal muscle may also function as an endocrine organ when it releases the cytokine IL-6 into the circulation during sustained exercise. Interestingly, one of the apparent effects of IL-6 is to stimulate lipolysis, causing the release of NEFA from the adipocyte. Thus, hormonal communications exist between the adipocyte and muscle that could enable them to talk to each other. The physiological relevance of this cross talk clearly warrants further study.

CHARACTERIZATION OF TWO DISTINCT INTRACELLULAR GLUT4 MEMBRANE POPULATIONS IN MUSCLE FIBER. DIFFERENTIAL PROTEIN COMPOSITION AND SENSITIVITY TO INSULIN

A major objective for the understanding of muscle glucose disposal is the elucidation of the intracellular trafficking pathway of GLUT4 glucose carriers in the muscle fiber. In this report, we provide functional and biochemical characterization of two distinct intracellular GLUT4 vesicle pools obtained from rat skeletal muscle. The two pools showed a differential response to insulin; thus, one showed a marked decrease in GLUT4 levels but the other did not. They also showed a markedly different protein composition as detected by quantitative vesicle immunoisolation analysis. The GLUT4 pool showing no response to insulin contained SCAMP proteins and the vSNARE proteins VAMP2 and cellubrevin, whereas only VAMP2 was found in the insulin-recruitable GLUT4 pool. SDS-PAGE and further silver staining of the immunoprecipitates revealed discrete polypeptide bands associated to the insulin-sensitive pool, and all these polypeptide bands were found in the insulin-insensitive population. Furthermore, some polypeptide ...