Decreased adiponectin links elevated adipose tissue autophagy with adipocyte endocrine dysfunction in obesity.

BACKGROUND/OBJECTIVES: Adipose tissue (AT) autophagy gene expression is elevated in human obesity, correlating with increased metabolic risk, but mechanistic links between the two remain unclear. Thus, the objective of this study was to assess whether elevated autophagy may cause AT endocrine dysfunction, emphasizing the putative role of adiponectin in fat-liver endocrine communication. SUBJECTS/METHODS: We utilized a large (N=186) human AT biobank to assess clinical associations between human visceral AT autophagy genes, adiponectin and leptin, by multivariate models. A broader view of adipocytokines association with elevated autophagy was assessed using adipocytokine array. Finally, to establish causality, ex vivo studies utilizing a murine AT-hepatocyte cell line co-culture system was used. RESULTS: Circulating high-molecular-weight adiponectin and leptin levels were associated with human omental-AT expression of ATG5 mRNA, associations that remained significant (ß=-0.197, P=0.011; ß=0.267, P<0.001, respectively) in a multivariate model adjusted for age, sex, body mass index and interleukin-6 (IL-6). A similar association was observed with omental-AT LC3A mRNA levels. Bafilomycin-A1 (Baf A) pretreatment of AT explants from high-fat-fed (HFF) mice had no effect on the secretion of some AT-derived endocrine factors, but partially or fully reversed obesity-related changes in secretion of a subset of adipocytokines by >30%, including the obesity-associated upregulation of IL-6, vascular endothelial growth factor, tumor necrosis factor alpha (TNFα) and certain insulin-like growth factor-binding proteins, and the HFF-induced downregulated secretion of IL-10 and adiponectin. Similarly, decreased adiponectin and increased leptin secretion from cultured adipocytes stimulated with TNFα+IL-1ß was partially reversed by small interfering RNA-mediated knockdown of ATG7. AT explants from HFF mice co-cultured with Hepa1c hepatoma cells impaired insulin-induced Akt and GSK3 phosphorylation. This effect was significantly reversed by pretreating explants with Baf A, but not if adiponectin was immunodepleted from the conditioned media. CONCLUSIONS: Reduced secretion of adiponectin may link obesity-associated elevated AT autophagy/lysosomal activity with adipose endocrine dysfunction.

Phenotypic and genetic variation in leptin as determinants of weight regain.

AIMS: Over 75% of obese subjects fail to maintain their weight following weight loss interventions. We aimed to identify phenotypic and genetic markers associated with weight maintenance/regain following a dietary intervention. SUBJECTS AND METHODS: In the 2-year Dietary Intervention Randomized Controlled Trial, we assessed potential predictors for weight changes during the 'weight loss phase' (0-6 months) and the 'weight maintenance/regain phase' (7-24 months). Genetic variation between study participants was studied using single-nucleotide polymorphisms in the leptin gene (LEP). RESULTS: Mean weight reduction was -5.5% after 6 months, with a mean weight regain of 1.2% of baseline weight during the subsequent 7-24 months. In a multivariate regression model, higher baseline high-molecular-weight adiponectin was the only biomarker predictor of greater success in 0- to 6-month weight loss (ß = -0.222, P-value = 0.044). In a multivariate regression model adjusted for 6-month changes in weight and various biomarkers, 6-month plasma leptin reduction exhibited the strongest positive association with 6-month weight loss (ß = 0.505, P-value < 0.001). Conversely, 6-month plasma leptin reduction independently predicted weight regain during the following 18 months (ß = -0.131, P-value < 0.013). Weight regain was higher among participants who had a greater (top tertiles) 6-month decrease in both weight and leptin (+3.4% (95% confidence interval 2.1-4.8)) as compared with those in the lowest combined tertiles (+0.2% (95% confidence interval -1.1 to 1.4)); P-value < 0.001. Weight regain was further significantly and independently associated with genetic variations in LEP (P = 0.006 for both rs4731426 and rs2071045). Adding genetic data to the phenotypic multivariate model increased its predictive value for weight regain by 34%. CONCLUSION: Although greater reduction in leptin concentrations during the initial phase of a dietary intervention is associated with greater weight loss in the short term, plasma leptin reduction, combined with the degree of initial weight loss and with genetic variations in the LEP gene, constitutes a significant predictor of subsequent long-term weight regain.

Nck2, an unexpected regulator of adipogenesis.

The regulation of adipose tissue expansion by adipocyte hypertrophy and/or hyperplasia is the topic of extensive investigations given the potential differential contribution of the 2 processes to the development of numerous chronic diseases associated with obesity. We recently discovered that the loss-of-function of the Src homology domain-containing protein Nck2 in mice promotes adiposity accompanied with adipocyte hypertrophy and impaired function, and enhanced adipocyte differentiation in vitro. Moreover, in severely-obese human's adipose tissue, we found that Nck2 expression is markedly downregulated. In this commentary, our goal is to expand upon additional findings providing further evidence for a unique Nck2-dependent mechanism regulating adipogenesis. We propose that Nck2 should be further investigated as a regulator of the reliance of white adipose tissue on hyperplasia versus hypertrophy during adipose tissue expansion, and hence, as a potential novel molecular target in obesity.

Activation of mitogen-activated protein kinases in human heart during cardiopulmonary bypass.

Mitogen-activated protein kinases (MAPKs) have been shown to be activated in both in vitro and in vivo models of cardiac tissue in response to ischemia/reperfusion injury. We investigated whether MAPKs are activated in human heart during coronary artery bypass grafting (CABG) surgery. During elective CABG surgery of 8 patients, 3 right atrial appendage biopsies were obtained at baseline, at the end of cross-clamping, and after coronary reperfusion. The expression of the p38-MAPK, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases (ERK1/2) MAPKs was not altered during CABG. The phosphorylation and activation of both ERK1/2 and p38-MAPK were increased approximately 2-fold by ischemia and even more (8- and 4-fold, respectively) by reperfusion. Although the ischemic period did not result in a significant activation of JNK, an approximately 6-fold increase in JNK activity could be observed after reperfusion. In conclusion, distinct activation patterns of ERK1/2, p38, and JNK MAPKs can be observed in human heart during CABG.

Prolonged oxidative stress impairs insulin-induced GLUT4 translocation in 3T3-L1 adipocytes.

Prolonged exposure of 3T3-L1 adipocytes to micromolar concentrations of H2O2 results in an impaired response to the acute metabolic effects of insulin. In this study, we further characterized the mechanisms by which oxidative stress impairs insulin stimulation of glucose transport activity. Although insulin induced a 2.5-fold increase in plasma membrane GLUT4 content and a 50% reduction in its abundance in the low-density microsomal (LDM) fraction in control cells, oxidation completely prevented these responses. The net effect of insulin on 2-deoxyglucose uptake activity was reduced in oxidized cells and could be attributed to GLUT1 translocation. Insulin stimulation of insulin receptor substrate (IRS) 1 tyrosine phosphorylation and the association of IRS-1 with phosphatidylinositol (PI) 3-kinase were not impaired by oxidative stress. However, a 1.9-fold increase in the LDM content of the p85 subunit of PI 3-kinase after insulin stimulation was observed in control, but not in oxidized, cells. Moreover, although insulin induced an increase in IRS-1-associated PI 3-kinase activity in the LDM in control cells, this effect was prevented by oxidation. These findings suggest that prolonged low-grade oxidative stress impairs insulin-stimulated GLUT4 translocation, potentially by interfering with compartment-specific activation of PI 3-kinase.

The HIV protease inhibitor nelfinavir induces insulin resistance and increases basal lipolysis in 3T3-L1 adipocytes.

HIV protease inhibitors (HPIs) are potent antiretroviral agents clinically used in the management of HIV infection. Recently, HPI therapy has been linked to the development of a metabolic syndrome in which adipocyte insulin resistance appears to play a major role. In this study, we assessed the effect of nelfinavir on glucose uptake and lipolysis in differentiated 3T3-L1 adipocytes. An 18-h exposure to nelfinavir resulted in an impaired insulin-stimulated glucose uptake and activation of basal lipolysis. Impaired insulin stimulation of glucose up take occurred at nelfinavir concentrations >10 micromol/l (EC(50) = 20 micromol/l) and could be attributed to impaired GLUT4 translocation. Basal glycerol and free fatty acid (FFA) release were significantly enhanced with as low as 5 micromol/l nelfinavir, displaying fivefold stimulation of FFA release at 10 micromol/l. Yet, the antilipolytic action of insulin was preserved at this concentration. Potential underlying mechanisms for these metabolic effects included both impaired insulin stimulation of protein kinase B Ser 473 phosphorylation with preserved insulin receptor substrate tyrosine phosphorylation and decreased expression of the lipolysis regulator perilipin. Troglitazone pre- and cotreatment with nelfinavir partly protected the cells from the increase in basal lipolysis, but it had no effect on the impairment in insulin-stimulated glucose uptake induced by this HPI. This study demonstrates that nelfinavir induces insulin resistance and activates basal lipolysis in differentiated 3T3-L1 adipocytes, providing potential cellular mechanisms that may contribute to altered adipocyte metabolism in treated HIV patients.

Autophagy in adipose tissue of patients with obesity and type 2 diabetes.

BACKGROUND: Pathophysiology of obesity is closely associated with enhanced autophagy in adipose tissue (AT). Autophagic process can promote survival or activate cell death. Therefore, we examine the occurrence of autophagy in AT of type 2 diabetes (T2D) patients in comparison to obese and lean individuals without diabetes. METHODOLOGY/PRINCIPAL FINDINGS: Numerous autophagosomes accumulated within adipocytes were visualized by electron transmission microscopy and by immunofluorescence staining for autophagy marker LC3 in obese and T2D patients. Increased autophagy was demonstrated by higher LC3-II/LC3-I ratio, up-regulated expression of LC3 and Atg5 mRNA, along with decreased p62 and mTOR protein levels. Increased autophagy occurred together with AT inflammation. CONCLUSIONS: Our data suggest fat depot-related differences in autophagy regulation. In subcutaneous AT, increased autophagy is accompanied by increased markers of apoptosis in patients with obesity independently of T2D. In contrast, in visceral AT only in T2D patients increased autophagy was related to higher markers of apoptosis.

Reactive oxygen species activate glucose transport in L6 myotubes.

Under oxidative stress, increased energy requirements are needed To induce repair mechanisms. As glucose is a major energy source in L6 myotubes, we evaluated glucose metabolism and transport, following exposure to glucose oxidase (H2O2 generating system), or xanthine oxidase (O2. and H2O2 generating system), added to the medium. Exposure for 24 h to 5 mM glucose and 50 mU/ml glucose oxidase, or to 50 microM xanthine and 20 mU/ml xanthine oxidase resulted in significant oxidant stress indicated by increased DNA binding activity of NF-kappa B. Under these conditions, approximately 2-fold increase in glucose consumption, lactate production and CO2 release were observed. 2-deoxyglucose uptake into myotubes increased time and dose dependently, reaching a 2.6 +/- 0.4-fold and 2.2 +/- 0.7-fold after 24 h exposure to glucose oxidase and xanthine oxidase, respectively. Peroxidase prevented this effect, indicating the role of H2O2 in mediating glucose uptake activation. The elevation in glucose uptake under oxidative stress was associated with increased expression of GLUT1 mRNA and protein. The observed 2-deoxyglucose uptake activation by oxidants was not limited to the L6 cell line and was observed in 3T3-L1 adipocytes as well.

Metabolic effects of gamma-linolenic acid-alpha-lipoic acid conjugate in streptozotocin diabetic rats.

Data suggesting the involvement of increased oxidative stress in the pathophysiology of diabetes has raised interest in the potential therapeutic benefit of antioxidants. Although beneficial metabolic effects of antioxidant supplementation have been suggested, an antioxidant mode of action, particularly in skeletal muscle, has not been documented. In the present study, we evaluate the metabolic effects of a gamma-linolenic acid-alpha-lipoic acid conjugate (GLA-LA) in streptozotocin-induced diabetic rats, and assess its potential mode of action by comparing its effects with equimolar administration of LA and GLA alone. Ten days of oral supplementation of 20 mg/kg body weight GLA-LA, but not LA or GLA alone, caused a mild reduction in fasting blood glucose concentration as compared with vehicle-treated diabetic rats (375 +/- 11 vs. 416 +/- 16 mg/dl, p = 0.03), with no change in fasting plasma insulin levels. A peripheral insulin-sensitizing effect could be observed with GLA-LA, LA, and GLA treatments, as demonstrated by a significant (p < 0.04) 23%, 13%, and 10% reduction, respectively, in the area under the glucose curve following an intravenous insulin tolerance test. This effect was associated with a 67% and 50% increase in GLUT4 protein content in the membranes of gastrocnemius muscle of GLA-LA and LA-treated animals, respectively; however, no change was observed with GLA treatment alone. Interestingly, both GLA-LA and LA treatments corrected a diabetes-related decrease in the gastrocnemius muscle low-molecular-weight reduced thiols content. These data demonstrate insulin-sensitizing properties of the GLA-LA conjugate by distinct mechanisms attributable to each of its components, which are associated with antioxidant effects.

Lipoic acid reduces glycemia and increases muscle GLUT4 content in streptozotocin-diabetic rats.

Alpha lipoic acid (lipoate [LA]), a cofactor of alpha-ketodehydrogenase, exhibits unique antioxidant properties. Recent studies suggest a direct effect of LA on glucose metabolism in both human and experimental diabetes. This study examines the possibility that LA positively affects glucose homeostasis in streptozotocin (STZ)-induced diabetic rats by altering skeletal muscle glucose utilization. Blood glucose concentration in STZ-diabetic rats following 10 days of intraperitoneal (i.p.) injection of LA 30 mg/kg was reduced compared with that in vehicle-treated diabetic rats (495 +/- 131 v 641 +/- 125 mg/dL in fed state, P = .003, and 189 +/- 48 v 341 +/- 36 mg/dL after 12-hour fast, P = .001). No effect of LA on plasma insulin was observed. Gastrocnemius muscle crude membrane GLUT4 protein was elevated both in control and in diabetic rats treated with LA by 1.5- and 2.8-fold, respectively, without significant changes in GLUT4 mRNA levels. Gastrocnemius lactic acid was increased in diabetic rats (19.9 +/- 5.5 v 10.4 +/- 2.8 mumol/g muscle, P < .05 v nondiabetic rats), and was normal in LA-treated diabetic rats (9.1 +/- 5.0 mumol/g muscle). Insulin-stimulated 2-deoxyglucose (2 DG) uptake into isolated soleus muscle was reduced in diabetic rats compared with the control group (474 +/- 15 v 568 +/- 52 pmol/mg muscle 30 min, respectively, P = .05). LA treatment prevented this reduction, resulting in insulin-stimulated glucose uptake comparable to that of nondiabetic animals. These results suggest that daily LA treatment may reduce blood glucose concentrations in STZ-diabetic rats by enhancing muscle GLUT4 protein content and by increasing muscle glucose utilization.

Lipoic acid acutely induces hypoglycemia in fasting nondiabetic and diabetic rats.

Lipoic acid (LA) is a unique antioxidant that increases peripheral glucose utilization in diabetic patients. This study was conducted to investigate whether the inhibition of glucose production could be an additional mechanism for the action of LA. Intravenous (i.v.) LA injection (100 or 60 mg/kg body weight) to fasting nondiabetic or streptozotocin (STZ)-induced diabetic rats caused a rapid reduction in blood glucose with no effect on circulating insulin levels. In vivo conversion of fructose to glucose was not inhibited by LA, whereas the gluconeogenesis flux from alanine was completely prevented. Reduced liver pyruvate carboxylase (PC) activity in vivo is suggested by the finding that LA induced a decrease in liver coenzyme A (CoA) content (44% and 28% reduction in nondiabetic and diabetic rats, respectively, compared with vehicle-treated animals) and liver acetyl CoA content (80% and 67% reduction in nondiabetic and diabetic rats, respectively). A reduction in plasma free carnitine (42% and 22% in nondiabetic and diabetic rats, respectively) was observed in LA-treated animals, and acylcarnitine levels were increased twofold. This could be attributed to elevated levels of C16 and C18 acylcarnitine, without a detectable accumulation of lipoylcarnitine. Under such conditions, a significant increase in the plasma free fatty acid (FFA) concentration (204% in nondiabetic and 151% in diabetic animals) with no elevation in beta-hydroxybutyrate levels was noted. In conclusion, this study suggests that short-term administration of LA at high dosage to normal and diabetic rats causes an inhibition of gluconeogenesis secondary to an interference with hepatic fatty acid oxidation. This may render LA an antihyperglycemic agent for the treatment of diabetic subjects, who display glucose overproduction as a major metabolic abnormality.

An interdisciplinary course in the basic sciences for senior medical and PhD students.

Integrating clinical and basic sciences throughout the medical school curriculum has become a major objective of various innovations in medical education. While early clinical exposure has evolved as an efficient means of introducing clinical studies in the preclinical years, interdisciplinary integration of basic sciences during the clinical years remains a challenge. The authors describe their three years of experience with an interdisciplinary course designed to demonstrate the continuum of medical information from the clinic to the basic sciences. In this course, sixth-year medical students are required to choose one of three to four different one-week programs, each of which requires them to conduct an in-depth investigation of a defined clinical topic. Program coordinators are encouraged to work in clinician-basic scientist teams and to use a variety of teaching methods, with an emphasis on tutored individual and group learning based on critical readings of original papers. Coordinators are also encouraged to enable graduate research students to participate. From 1998 to 2000, students participated in nine programs, seven of which were coordinated by interdisciplinary teams. Several clinical and basic science disciplines were represented in each program, and various teaching methods were used. Graduate students participated in two of the programs. Evaluation of the programs (a debriefing discussion as well as short written evaluations) indicated moderate to good achievement of the course objectives.

Regulation of glucose transporters--implications for insulin resistance states.

Altered glucose homeostasis in the different diabetic states often results from a combination of insulin deficiency (absolute or relative), and impaired hormone action. The latter involves alterations in the expression and/or function of glucose transporters in insulin responsive peripheral tissues - skeletal muscle and adipose tissue. Since whole body glucose utilization depends mainly on controlled changes in glucose transport in these tissues, this review focuses on the role of glucose transporters in the regulation of insulin-stimulated glucose transport activity. The molecular mechanisms by which several inducers of insulin resistance inhibit insulin action on glucose uptake are also discussed. Better understanding of the complex regulation of glucose transport and transporters will hopefully shed light on potential sites for new pharmaceutical interventions. Several excellent reviews have been published in the past 2 years detailing various aspects which are discussed only briefly in this review. They are mentioned in the text to allow further reading.

Peripheral blood leucocyte subclasses as potential biomarkers of adipose tissue inflammation and obesity subphenotypes in humans.

While obesity is clearly accepted as a major risk factor for cardio-metabolic morbidity, it is also apparent that some obese patients largely escape this association, forming a unique obese subphenotype(s). Current approaches to define such subphenotypes include clinical biomarkers that largely reflect already manifested comorbidities, such as markers of dyslipidaemia, hyperglycaemia and impaired regulation of vascular tone, and anthropometric or imaging-based assessment of adipose tissue distribution. Low-grade inflammation, evident both systemically and within adipose tissue (particularly intra-abdominal fat depots), seems to characterize the more cardio-metabolically morbid forms of obesity. Indeed, several systemic inflammatory markers (C-reactive protein), adipokines (retinol-binding protein 4, adiponectin) and cytokines have been shown to correlate in humans with adipose tissue inflammation and with obesity-associated health risks. Circulating leucocytes constitute a diverse group of cells that form a major arm of the immune system. They are both major sources of cytokines and likely also of infiltrating adipose tissue immune cells in obesity. In the present review, we summarize currently available literature on 'classical' blood white cell classes and on more specific leucocyte subclasses present in the circulation in human obesity. We critically raise the possibility that leucocytes may constitute clinically available markers for the more morbidity-associated obesity subphenotype(s), and when available, for intra-abdominal adipose tissue inflammation.

Improved glucose tolerance in mice receiving intraperitoneal transplantation of normal fat tissue.

AIMS/HYPOTHESIS: The association between increased (visceral) fat mass, insulin resistance and type 2 diabetes mellitus is well known. Yet, it is unclear whether the mere increase in intra-abdominal fat mass, or rather functional alterations in fat tissue in obesity contribute to the development of insulin resistance in obese patients. Here we attempted to isolate the metabolic effect of increased fat mass by fat tissue transplantation. METHODS: Epididymal fat pads were removed from male C57Bl6/J mice and transplanted intraperitoneally into male littermates (recipients), increasing the combined perigonadal fat mass by 50% (p < 0.005). At 4 and 8 weeks post-transplantation, glucose and insulin tolerance tests were performed, and insulin, NEFA and adipokines measured. RESULTS: Circulating levels of NEFA, adiponectin and leptin were not significantly different between transplanted and sham-operated control mice, while results of the postprandial insulin tolerance test were similar between the two groups. In contrast, under fasting conditions, the mere increase in intra-abdominal fat mass resulted in decreased plasma glucose levels (6.9 +/- 0.4 vs 8.1 +/- 0.3 mmol/l, p = 0.03) and a approximately 20% lower AUC in the glucose tolerance test (p = 0.02) in transplanted mice. Homeostasis model assessment of insulin resistance (HOMA-IR) was 4.1 +/- 0.4 in transplanted mice (vs 6.2 +/- 0.7 in sham-operated controls) (p = 0.02), suggesting improved insulin sensitivity. Linear regression modelling revealed that while total body weight positively correlated, as expected, with HOMA-IR (beta: 0.728, p = 0.006), higher transplanted fat mass correlated with lower HOMA-IR (beta: -0.505, p = 0.031). CONCLUSIONS/INTERPRETATION: Increasing intra-abdominal fat mass by transplantation of fat from normal mice improved, rather than impaired, fasting glucose tolerance and insulin sensitivity, achieving an effect opposite to the expected metabolic consequence of increased visceral fat in obesity.

Differential effects of IRS1 phosphorylated on Ser307 or Ser632 in the induction of insulin resistance by oxidative stress.

AIMS/HYPOTHESIS: Induction of stress kinases leading to serine hyperphosphorylation of IRS1 may link oxidative stress to insulin resistance. The aim of this study was to investigate the roles of the phosphorylated serine residues Ser307 and Ser632, two sites implicated in the inhibition of IRS1 function in insulin signalling. MATERIALS AND METHODS: Fao hepatoma cells were exposed to an H(2)O(2)-generating system, and antibodies against the two phosphorylated serine residues were used for immunoprecipitation, immunoblot and immunofluorescence analyses. RESULTS: Exposure to approximately 50 mumol/l H(2)O(2) for 2 h resulted in IRS1 phosphorylation on both Ser307 and Ser632, concomitant with activation of inhibitor kappa kinase beta (IKKbeta) and c-Jun kinase (JNK). Immunoprecipitation studies revealed that the maximum overlap between phospho (p) Ser307-IRS1 and pSer632-IRS1 was 20%, and confocal microscopy suggested distinct localisations of IRS1 molecules phosphorylated on either site. Although pSer307-IRS1 showed decreased insulin-induced tyrosine phosphorylation and interaction with phosphatidylinositol 3-kinase (PI3K) in response to insulin, pSer632-IRS1 molecules were normally tyrosine-phosphorylated and exhibited typical associated PI3K activity. Salicylic acid and SP600125 partially inhibited IKKbeta and JNK, respectively, which indicated distinct roles for these two kinases in the phosphorylation of IRS1 at the two serine sites. Protection against oxidation-mediated impairment in insulin-induced phosphorylation of protein kinase B/Akt and in glycogen synthesis was achieved only by combining salicylic acid and SP600125. CONCLUSIONS/INTERPRETATION: These results suggest that pSer307-IRS1 and pSer632-IRS1 may define two minimally overlapping pools of IRS1 in response to oxidative stress, contributing differentially to insulin resistance. A combination of stress kinase inhibitors is required to protect against insulin resistance and IRS1 hyperphosphorylation induced by oxidative stress.

Cellular mechanisms of insulin resistance, lipodystrophy and atherosclerosis induced by HIV protease inhibitors.

Accumulating clinical evidence now links HIV protease inhibitors (HPIs) to the pathogenesis of insulin resistance, dyslipidaemia, lipodystrophy and atherosclerosis associated with highly active anti-retroviral therapy. Here we briefly describe the evidence for a distinct causative role for HPIs, and explore the cellular mechanisms proposed to underlie these side-effects. Acute inhibition of GLUT4-mediated glucose transport, and defective insulin signalling induced by chronic exposure to nelfinavir, are described as cellular mechanisms of insulin resistance. Interference with adipogenesis and adipocyte apoptosis and nelfinavir-induced activation of lipolysis are discussed as potential mechanisms of HPI-induced lipodystrophy. HPI-induced free radical production, apoptosis and increased glucose utilization in vascular smooth muscle cells are presented as possible novel mechanisms for atherosclerosis. Common pathways and cause-effect relationships between the various cellular mechanisms presented are then discussed, with emphasis on the role of insulin resistance, free radical production and enhanced lipolysis. Understanding the cellular mechanisms of HPI-induced side-effects will enhance the search for improved anti-retroviral therapy, and may also shed light on the pathogenesis of common forms of insulin resistance, dyslipidaemia and atherosclerosis.

Troglitazone causes acute mitochondrial membrane depolarisation and an AMPK-mediated increase in glucose phosphorylation in muscle cells.

AIMS/HYPOTHESIS: Troglitazone was the first thiazolidinedione (TZD) approved for clinical use, exerting hypoglycaemic effects related to its action as a ligand of the peroxisome proliferator-activated receptor gamma receptor in adipocytes. However, emerging evidence suggests that mitochondrial function may be affected by troglitazone, and that skeletal muscle cells acutely respond to troglitazone by enhancing glucose uptake. The aim of the present study was to determine the cellular mechanisms by which troglitazone acutely stimulates glucose utilisation in skeletal muscle cells. METHODS: L6 cells overexpressing GLUT4myc were incubated with troglitazone. Glucose uptake, transport and phosphorylation as well as AMP-activated protein kinase (AMPK) signalling and insulin signalling were examined. Changes in mitochondrial membrane potential were measured using the J-aggregate-forming dye JC-1. AMPK signalling was interfered with using AMPK alpha1/alpha2 siRNA. RESULTS: Troglitazone acutely (in 10 min) reduced the mitochondrial membrane potential in L6GLUT4myc myotubes and robustly stimulated AMPK activity. Following 30 min of incubation with troglitazone or insulin, 2-deoxyglucose uptake was stimulated 1.5- and 2.1-fold respectively, and in cells treated with troglitazone, a 1.8-fold increase in the 2-deoxyglucose-6-phosphate:2-deoxyglucose ratio was observed. Moreover, contrary to insulin, troglitazone did not significantly stimulate 3-O-methylglucose uptake. Unlike insulin, troglitazone did not increase surface GLUT4myc content and did not increase IRS1-associated phosphatidylinositol 3-kinase activity or Akt phosphorylation on T308 and S473. Interestingly, interfering with troglitazone-induced activation of AMPK by decreasing the expression of the enzyme using siRNA inhibited the stimulation of 2-deoxyglucose uptake by the TZD. CONCLUSIONS/INTERPRETATION: We propose that troglitazone acutely increases glucose flux in muscle via an AMPK-mediated increase in glucose phosphorylation.

Push/pull mechanisms of GLUT4 traffic in muscle cells.

AIMS: Understanding the mechanisms by which insulin regulates glucose transporter 4 (GLUT4) traffic in skeletal muscle has been a major challenge since the discoveries of glucose transporter's translocation and the cloning of GLUT4. Here we summarize our work of the past 5 years on the regulation of GLUT4 traffic in skeletal muscle cells. METHODS: L6 cells overexpressing GLUT4 harbouring an exofacial myc epitope gave us the opportunity to perform dynamic assessments of GLUT4 exocytosis, endocytosis, as well as a means to follow GLUT4 molecules along their journey through intracellular compartments. RESULTS: We found that insulin stimulation, which results in the expected gain in surface GLUT4, is mostly attributed to enhanced GLUT4 exocytosis, and does not significantly affect the initial rate of internalization. Two mechanisms by which insulin enhances GLUT4 exocytosis are described: 'Pull' relates to actin remodelling-based segregation of the insulin signalling molecules and the directed recruitment of GLUT4/VAMP2 containing vesicles. 'Push' is the accelerated inter-endosomal transit of endocytosed GLUT4 molecules through the recycling endosome. The interface between the two types of regulatory input by insulin is suggested to be the budding of GLUT4 from the transferrin receptor (TfR)-containing, recycling endosome. CONCLUSIONS: We propose a model on the identity of the GLUT4 pools responsible for GLUT4 recruitment to the plasma membrane in the basal state, or following insulin or hyperosmolarity stimuli.