Examination of 'lipotoxicity' in skeletal muscle of high-fat fed and ob/ob mice.

Excess lipid accumulation resulting from an elevated supply of plasma fatty acids is linked to the pathogenesis of the metabolic syndrome and heart disease. The term 'lipotoxicity' was coined to describe how lipid accumulation leads to cellular dysfunction and death in non-adipose tissues including the heart, pancreas and liver. While lipotoxicity has been shown in cultured skeletal muscle cells, the degree of lipotoxicity in vivo and the functional consequences are unresolved. We studied three models of fatty acid overload in male mice: 5 h Intralipid((R)) and heparin infusion, prolonged high fat feeding (HFF) and genetic obesity induced by leptin deficiency (ob/ob mice). Markers of apoptosis, proteolysis and autophagy were assessed as readouts of lipotoxicity. The Intralipid((R)) infusion increased caspase 3 activity in skeletal muscle, demonstrating that enhancing fatty acid flux activates pro-apoptotic pathways. HFF and genetic obesity increased tissue lipid content but did not influence apoptosis. Gene array analysis revealed that HFF reduced the expression of 31 pro-apoptotic genes. Markers of autophagy (LC3beta and beclin-1 expression) were unaffected by HFF and were associated with enhanced Bcl(2) protein expression. Proteolytic activity was similarly unaffected by HFF or in ob/ob mice. Thus, contrary to our previous findings in muscle culture in vitro and in other non-adipose tissues in vivo, lipid overload did not induce apoptosis, autophagy or proteolysis in skeletal muscle. A broad transcriptional suppression of pro-apoptotic proteins may explain this resistance to lipid-induced cell death in skeletal muscle.

Exercise and thiazolidinedione therapy normalize insulin action in the obese Zucker fatty rat.

Thiazolidinediones and exercise are both known to improve insulin action independently. Therefore, we determined whether combined therapy could normalize insulin action in the Zucker fatty (ZF) rat. Rats were fed troglitazone as a 0.2% food admixture over a 3-week exercise training period (treadmill running 5 days/week, 20 m/min, 0% grade, 60 min/day). Subsequent to drug and/or exercise therapy, animals were chronically cannulated in the carotid artery (sampling) and jugular vein (infusion). After a 4-day recovery from surgery, animals were exposed to a hyperinsulinemic (40 mU x kg(-1) x min(-1)) euglycemic clamp (8.5 +/- 0.12 mmol/l; P = 0.45 between groups). Independently, exercise (n = 7) and troglitazone (n = 7) improved the glucose disposal rate 20% (P = 0.04) and 76% (P = 0.001), respectively, when compared with untreated ZF controls (n = 11). In combination, exercise and troglitazone therapy (n = 6) produced significant increments in the following: tracer-determined glucose disposal rate (combined therapy, 52.4 +/- 2.9 mg x kg(-1) x min(-1), vs. untreated ZF, 25.8 +/- 0.8 mg x kg(-1) x min(-1); P = 0.0001), total GLUT4 protein (twofold increase; P = 0.001), insulin receptor substrate (IRS)-1 protein (fourfold increase; P = 0.0001), and Akt phosphorylation (2.9-fold increase; P = 0.002). In conclusion, 1) exercise and troglitazone therapy each improved insulin action in the ZF rat, whereas the combination of the two led to complete normalization of insulin sensitivity, and 2) combination treatment also resulted in normalization of GLUT4 total protein, IRS-1 protein, and Akt phosphorylation compared with lean littermates.

Novel glucosensor for hypoglycemic detection localized to the portal vein.

In this investigation, we sought to constrain the locus of essential portohepatic glucosensors and test the hypothesis that they reside strictly in the portal vein and not the liver. Male Wistar rats were chronically cannulated in the carotid artery (sampling), jugular vein (infusion), and portal vein, either adjacent to (POR[ADJ], 0.6 +/- 0.1 cm, n = 6) or upstream from (POR[UPS], 2.7 +/- 0.1 cm, n = 8) the liver. Animals were exposed to one of three protocols distinguished by the site of glucose infusion: POR(UPS), POR(ADJ), or peripheral (PER). Systemic hypoglycemia (2.4 +/- 0.1 mmol/l) was induced via jugular vein insulin infusion (50 mU x kg(-1) x min[-1]). Arterial plasma catecholamines were assessed at basal (-30 and 0 min) and during sustained hypoglycemia (60, 75, 90, 105 min). By design, hepatic glucose was significantly elevated during POR(UPS) and POR(ADJ) versus PER (4.3 +/- 0.1 vs. 2.4 +/- 0.1 mmol/l, respectively; P < 0.05). There were no significant differences between protocols in arterial glucose or insulin concentrations (9,372 +/- 1,798 pmol/l). When liver and systemic glucose concentrations were allowed to fall concomitantly (PER), epinephrine was elevated 16-fold above basal levels (3.0 +/- 0.6 vs. 46.4 +/- 4.3 nmol/l, P < 0.001). When portohepatic normoglycemia was maintained during POR(UPS), a 67% suppression in the epinephrine response versus that during PER was observed (P < 0.001). However, when the cannula was advanced adjacent to the liver, by comparison with PER, there was no suppression in the sympathoadrenal response (P = 0.73). While both POR(UPS) and POR(ADJ) yielded elevated liver glycemia in the face of systemic hypoglycemia, only POR(UPS) yielded an elevated portal vein glucose concentration. That only POR(UPS) resulted in a significant suppression of the sympathoadrenal response is consistent with the localization of the glucosensors to the portal vein.

Portal vein afferents are critical for the sympathoadrenal response to hypoglycemia.

We sought to elucidate the role of the portal vein afferents in the sympathetic response to hypoglycemia. Laparotomy was performed on 27 male Wistar rats. Portal veins were painted with either 90% phenol (denervation group [PDN]) or 0.9% saline solution (sham-operated group [SHAM]). Rats were chronically cannulated in the carotid artery (sampling), jugular vein (infusion), and portal vein (infusion). After a recovery period of 5 days, animals were exposed to a hyperinsulinemic-hypoglycemic clamp, with glucose infused either portally (POR) or peripherally (PER). In all animals, systemic hypoglycemia (2.48+/-0.09 mmol/l) was induced via jugular vein insulin infusion (50 mU x kg(-1) x min(-1)). Arterial plasma catecholamines were assessed at basal (-30 and 0 min) and during sustained hypoglycemia (60, 75, 90, and 105 min). By design, portal vein glucose concentrations were significantly elevated during POR versus PER (4.4+/-0.14 vs. 2.5+/-0.07 mmol/l; P<0.01, respectively) for both PDN and SHAM. There were no significant differences in arterial glucose or insulin concentration between the four experimental conditions at any point in time. When portal glycemia and systemic glycemia fell concomitantly (SHAM-PER), epinephrine increased 12-fold above basal (3.75+/-0.34 and 44.56+/-6.1 nmol/l; P<0.001). However, maintenance of portal normoglycemia (SHAM-POR) caused a 50% suppression of the epinephrine response, despite cerebral hypoglycemia (22.2+/-3.1 nmol/l, P<0.001). Portal denervation resulted in a significant blunting of the sympathoadrenal response to whole-body hypoglycemia (PDN-PER 27.6+/-3.8 nmol/l vs. SHAM-PER; P<0.002). In contrast to the sham experiments, there was no further suppression in arterial epinephrine concentrations observed during PDN-POR versus PDN-PER (P = 0.8). These findings indicate that portal vein afferent innervation is critical for hypoglycemic detection and normal sympathoadrenal counterregulation.

Hypoglycemic detection does not occur in the hepatic artery or liver: findings consistent with a portal vein glucosensor locus.

Our laboratory has previously demonstrated that hypoglycemic detection occurs in the portal vein, not the liver. To ascertain whether hypoglycemic detection may also occur in the hepatic artery, normoglycemia was established across the liver via a localized hepatic artery glucose infusion. Male mongrel dogs (n = 7) were infused with insulin (5.0 mU x kg(-1) x min(-1)) via the jugular vein to induce systemic hypoglycemia. Animals participated in two hyperinsulinemic-hypoglycemic clamp experiments distinguished by the site of glucose infusion. During the liver irrigation protocol, glucose was infused via the hepatic artery (HA protocol) to maintain liver normoglycemia as systemic glucose concentrations were systematically lowered over 260 min (nadir = 2.2 +/- 0.01 mmol/l). During control experiments, glucose was infused peripherally (PER protocol) to control reductions in blood glucose. Arterial glucose concentrations were not significantly different at any time between the two protocols (P = 0.73). Hepatic artery and liver glucose concentrations were significantly elevated in the HA versus PER protocol throughout the duration of the progressive hyperinsulinemic-hypoglycemic clamp. During the PER protocol, epinephrine and norepinephrine concentrations increased significantly above basal values (0.53 +/- 0.06 and 0.85 +/- 0.2 nmol/l, respectively) to plateaus of 4.4 +/- 0.86 (P = 0.0001) and 3.6 +/- 0.69 nmol/l (P = 0.001), respectively. There were no significant differences between the two protocols in the epinephrine (P = 0.81) and the norepinephrine (P = 0.68) response to hypoglycemia. The current findings indicate that glucosensors important to hypoglycemic detection do not reside in the hepatic artery. Furthermore, these data confirm our previous findings that glucosensors important to hypoglycemic detection are not present in the liver, but are in fact localized to the portal vein.

Thiazolidinedione treatment prevents free fatty acid-induced insulin resistance in male wistar rats.

We sought to ascertain whether pretreatment with troglitazone (20 days) could prevent acute free fatty acid (FFA)-induced insulin resistance in male Wistar rats. Animals were divided into three groups: 1) control, 2) FFA infusion alone (FFA1), and 3) thiazolidinedione (TZD)-treated + FFA infusion (FFA1). Days before a hyperinsulinemic-euglycemic clamp, all animals were cannulated in the jugular vein (infusion) and carotid artery (sampling). Animals were allowed 5 days to recover from surgery and fasted 12 h before the experiment. Glucose (variable), insulin (40 mU. kg(-1). min(-1)), and Liposyn (heparinized 10% lipid emulsion) infusions were initiated simultaneously and continued from 0-120 min. Steady-state glucose, 8.3 +/- 0.14 mmol/l, and insulin concentrations, 7.3 +/- 2.45 nmol/l, were the same between groups. Interestingly, steady-state FFA levels were significantly lower in animals pretreated with TZD compared with FFA alone (1.83 +/- 0.26 vs. 2.96 +/- 0.25 mmol/l; P = 0.009), despite matched intralipid infusion rates. A second group of TZD-treated animals (TZD + FFA2) were infused with intralipid at a higher infusion rate (44%) to match the arterial concentrations of FFA1. The glucose infusion and insulin-stimulated glucose disposal rates (GDRs) were significantly decreased (40%) for untreated Liposyn infused (FFA1) compared with control rats. In addition, insulin receptor substrate-1 (IRS-1) phosphorylation and IRS-1-associated phosphatidylinositol (PI) 3-kinase activity was significantly reduced, 30-50%, in FFA1 rats. TZD pretreatment prevented the FFA-induced decrement in insulin signaling. Fatty acid translocase (FAT/CD36) also was significantly reduced (56%) in untreated FFA1 rats after the clamp but remained identical to control values for TZD-treated rats. In conclusion, acutely elevated FFA levels 1) induced a significant reduction in tracer-determined GDR paralleled by impaired tyrosine phosphorylation of IRS-1 and reduced IRS-1-associated PI 3-kinase activity and 2) induced a significant reduction in FAT/CD36 total protein. TZD pretreatment prevented FFA-induced decrements in insulin action and prevented the reduction in FAT/CD36 protein.

The 2009 stock conference report: inflammation, obesity and metabolic disease.

Obesity is linked with many deleterious health consequences and is associated with increased risk of chronic disease including type 2 diabetes, atherosclerosis and certain forms of cancer. Recent work has highlighted the impact of obesity to activate inflammatory gene networks and suggests a causal function of inflammation in the pathogenesis of the metabolic syndrome. Since 2005, when Dr Gokhan Hotamisligil chaired the fourth Stock Conference in Istanbul, Turkey, entitled 'Obesity and Inflammation', there has been an explosion of studies investigating the relationship between obesity, inflammation and substrate metabolism. The exuberance surrounding this field of research is exemplified by the body of work that has been published in these past 4 years, including over 1400 publications. During this time, several novel mechanisms relating to cellular inflammation have been uncovered including the role of the hematopoietic system, toll-like receptor activation, endoplasmic reticulum stress and very recently T-cell activation in obesity-induced insulin resistance. These discoveries have led us to rethink cellular nutrient sensing and its role in inflammation and metabolic disease. Despite burgeoning investigation in this field, there still remain a number of unanswered questions. This review that evolved from the 2009 Stock Conference summarizes current research and identifies the deficiencies in our understanding of this topic. The overall goal of this Stock Conference was to bring together leading investigators in the field of inflammation and obesity research in the hope of fostering new ideas, thus advancing the pursuit of novel therapeutic strategies to reduce disease risk and or better treat chronic disease including type 2 diabetes, cardiovascular disease and cancer.

Evidence of a role for insulin-like growth factor binding protein (IGFBP)-3 in metabolic regulation.

IGF-binding protein (IGFBP)-3 is a metabolic regulator that has been shown to inhibit insulin-stimulated glucose uptake in murine models. This finding contrasts with epidemiological evidence of decreased serum IGFBP-3 in patients with type 2 diabetes. The purpose of this study was to clarify the role of IGFBP-3 in metabolism. Four-week-old male IGFBP-3(-/-) and control mice were subjected to a high-fat diet (HFD) for 12 wk. IGFBP-3(-/-) mice were heavier before the initiation of HFD and at the end of the study period. Resting metabolic rate was significantly decreased in knockout mice; however, respiratory exchange ratio was not significantly different. Fasting blood glucose and insulin levels were significantly elevated in IGFBP-3(-/-) mice. However, IGFBP-3(-/-) mice had relatively normal glucose tolerance because the relative glucose excursion over time was not different between the groups. During hyperinsulinemic clamps, IGFBP-3(-/-) mice had increased basal hepatic glucose production, but after insulin stimulation, no differences in hepatic glucose production were observed. A second cohort of older IGFBP-3(-/-) mice on HFD displayed unexpected evidence of hepatic steatosis. In summary, glucose tolerance and clamp testing indicate that IGFBP-3(-/-) mice preserve insulin sensitivity despite evidence of increased basal glucose turnover and hepatic steatosis. We provide evidence that genetic deletion of IGFBP-3 modulates hepatic carbohydrate and lipid metabolism.