ABSTRACT
An important regulator of fatty acid oxidation (FAO) is the allosteric inhibition of CPT-1 by malonyl-CoA produced by the enzyme acetyl-CoA carboxylase 2 (ACC2). Initial studies suggested that deletion of Acc2 (Acacb) increased fat oxidation and reduced adipose tissue mass but in an independently generated strain of Acc2 knockout mice we observed increased whole-body and skeletal muscle FAO and a compensatory increase in muscle glycogen stores without changes in glucose tolerance, energy expenditure or fat mass in young mice (12-16 weeks). The aim of the present study was to determine whether there was any effect of age or housing at thermoneutrality (29â°C; which reduces total energy expenditure) on the phenotype of Acc2 knockout mice. At 42-54 weeks of age, male WT and Acc2(-/-) mice had similar body weight, fat mass, muscle triglyceride content and glucose tolerance. Consistent with younger Acc2(-/-) mice, aged Acc2(-/-) mice showed increased whole-body FAO (24âh average respiratory exchange ratio=0.95±0.02 and 0.92±0.02 for WT and Acc2(-/-) mice respectively, P<0.05) and skeletal muscle glycogen content (+60%, P<0.05) without any detectable change in whole-body energy expenditure. Hyperinsulinaemic-euglycaemic clamp studies revealed no difference in insulin action between groups with similar glucose infusion rates and tissue glucose uptake. Housing Acc2(-/-) mice at 29â°C did not alter body composition, glucose tolerance or the effects of fat feeding compared with WT mice. These results confirm that manipulation of Acc2 may alter FAO in mice, but this has little impact on body composition or insulin action.
Subject(s)
Acetyl-CoA Carboxylase/physiology , Aging/physiology , Housing, Animal , Temperature , Acetyl-CoA Carboxylase/deficiency , Animals , Body Composition , Body Weight , Energy Metabolism , Fatty Acids/metabolism , Glucose Clamp Technique , Glucose Tolerance Test , Glycogen/analysis , Insulin/blood , Male , Mice , Mice, Knockout , Muscle, Skeletal/chemistry , Muscle, Skeletal/metabolism , Oxidation-Reduction , Phenotype , Triglycerides/analysisABSTRACT
We have previously shown that incubation for 1h with excess glucose or leucine causes insulin resistance in rat extensor digitorum longus (EDL) muscle by inhibiting AMP-activated protein kinase (AMPK). To examine the events that precede and follow these changes, studies were performed in rat EDL incubated with elevated levels of glucose or leucine for 30min-2h. Incubation in high glucose (25mM) or leucine (100µM) significantly diminished AMPK activity by 50% within 30min, with further decreases occurring at 1 and 2h. The initial decrease in activity at 30min coincided with a significant increase in muscle glycogen. The subsequent decreases at 1h were accompanied by phosphorylation of αAMPK at Ser485/491, and at 2h by decreased SIRT1 expression and increased PP2A activity, all of which have previously been shown to diminish AMPK activity. Glucose infusion in vivo, which caused several fold increases in plasma glucose and insulin, produced similar changes but with different timing. Thus, the initial decrease in AMPK activity observed at 3h was associated with changes in Ser485/491 phosphorylation and SIRT1 expression and increased PP2A activity was a later event. These findings suggest that both ex vivo and in vivo, multiple factors contribute to fuel-induced decreases in AMPK activity in skeletal muscle and the insulin resistance that accompanies it.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Animal Nutritional Physiological Phenomena , Glucose/metabolism , Muscle, Skeletal/metabolism , Acetyl-CoA Carboxylase/metabolism , Animals , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Gene Expression , Glucose/administration & dosage , Glycogen/metabolism , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Male , Nicotinamide Phosphoribosyltransferase/metabolism , Oxidation-Reduction , Phosphorylation , Rats , Sirtuin 1/genetics , Sirtuin 1/metabolismABSTRACT
SIRT1 is a NAD+-dependent deacetylase thought to regulate cellular metabolic pathways in response to alterations in nutrient flux. In the current study we investigated whether acute changes in SIRT1 expression affect markers of muscle mitochondrial content and also determined whether SIRT1 influenced muscle insulin resistance induced by acute glucose oversupply. In male Wistar rats either SIRT1 or a deacetylase inactive mutant form (H363Y) was electroprated into the tibialis cranialis (TC) muscle. The other leg was electroporated with an empty control vector. One week later, glucose was infused and hyperglycaemia was maintained at ~11mM. After 5 hours, 11mM glucose induced significant insulin resistance in skeletal muscle. Interestingly, overexpression of either SIRT1 or SIRT1 (H363Y) for 1 week did not change markers of mitochondrial content or function. SIRT1 or SIRT1 (H363Y) overexpression had no effect on the reduction in glucose uptake and glycogen synthesis in muscle in response to hyperglycemia. Therefore we conclude that acute increases in SIRT1 protein have little impact on mitochondrial content and that overexpressing SIRT1 does not prevent the development of insulin resistance during hyperglycaemia.
Subject(s)
Glucose/pharmacology , Insulin Resistance , Muscle, Skeletal/metabolism , Sirtuin 1/physiology , Animals , Blood Glucose/metabolism , Cell Line , Electroporation , Hyperglycemia/metabolism , Male , Mice , Muscle, Skeletal/drug effects , Rats , Rats, Wistar , Signal TransductionABSTRACT
Fatty acids (FAs) are essential elements of all cells and have significant roles as energy substrates, components of cellular structure and signalling molecules. The storage of excess energy intake as fat in adipose tissue is an evolutionary advantage aimed at protecting against starvation, but in much of today's world, humans are faced with an unlimited availability of food, and the excessive accumulation of fat is now a major risk for human health, especially the development of type 2 diabetes (T2D). Since the first recognition of the association between fat accumulation, reduced insulin action and increased risk of T2D, several mechanisms have been proposed to link excess FA availability to reduced insulin action, with some of them being competing or contradictory. This review summarises the evidence for these mechanisms in the context of excess dietary FAs generating insulin resistance in muscle, the major tissue involved in insulin-stimulated disposal of blood glucose. It also outlines potential problems with models and measurements that may hinder as well as help improve our understanding of the links between FAs and insulin action.
Subject(s)
Energy Metabolism/physiology , Fatty Acids/metabolism , Insulin Resistance/physiology , Lipid Metabolism/physiology , Muscle, Skeletal/metabolism , Animals , Body Composition/physiology , Exercise/physiology , Humans , Oxidation-ReductionABSTRACT
We recently showed that bitter melon-derived triterpenoids (BMTs) activate AMPK and increase GLUT4 translocation to the plasma membrane in vitro, and improve glucose disposal in insulin resistant models in vivo. Here we interrogated the mechanism by which these novel compounds activate AMPK, a leading anti-diabetic drug target. BMTs did not activate AMPK directly in an allosteric manner as AMP or the Abbott compound (A-769662) does, nor did they activate AMPK by inhibiting cellular respiration like many commonly used anti-diabetic medications. BMTs increased AMPK activity in both L6 myotubes and LKB1-deficient HeLa cells by 20-35%. Incubation with the CaMKKß inhibitor, STO-609, completely attenuated this effect suggesting a key role for CaMKKß in this activation. Incubation of L6 myotubes with the calcium chelator EGTA-AM did not alter this activation suggesting that the BMT-dependent activation was Ca(2+)-independent. We therefore propose that CaMKKß is a key upstream kinase for BMT-induced activation of AMPK.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism , Hypoglycemic Agents/pharmacology , Momordica charantia/chemistry , Terpenes/pharmacology , AMP-Activated Protein Kinase Kinases , Calcium/metabolism , Enzyme Activation/drug effects , HeLa Cells , Humans , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/deficiency , Signal Transduction/drug effectsABSTRACT
The present study investigated the chronic efficacy of oleanolic acid (OA), a triterpenoid selected from our recent screening, on hyperglycemia in type-2 diabetic mice. C57BL/6J mice were fed a high-fat diet followed by low doses of streptozotocin to generate a type-2 diabetic model. OA (100 mg/kg/day) was administered orally for 2 weeks with its effects monitored for 6 weeks. High-fat feeding and streptozotocin generated a steady hyperglycemia (21.2 ± 1.1 mM) but OA administration reversed the hyperglycemia by ~60%. Interestingly, after the cessation of OA administration, the reversed hyperglycemia was sustained for the entire post-treatment period of the study (4 weeks) despite the reoccurrence of dyslipidemia. Examination of insulin secretion and pancreas morphology did not indicate improved ß-cell function as a likely mechanism. Urine glucose loss was decreased with substantial improvement of diabetic nephropathy after the OA treatment. Pair-feeding the OA-treated mice to an untreated group ruled out food intake as a main factor attributable for this sustained reduction in hyperglycemia. Studies with the use of glucose tracers revealed no increase in glucose influx into muscle, adipose tissue or liver in the OA-treated mice. Finally, we analyzed key regulators of gluconeogenesis in the liver and found significant increases in the phosphorylation of both Akt and FoxO1 after treatment with OA. Importantly, these increases were significantly correlated with a down-regulation of glucose-6-phosphatase expression. Our findings suggest triterpenoids are a potential source of new efficacious drugs for sustained control of hyperglycemia. The liver appears to be a major site of action, possibly by the suppression of hepatic glucose production via the Akt/FoxO1 axis.
Subject(s)
Diabetes Mellitus, Type 2/metabolism , Forkhead Transcription Factors/physiology , Gluconeogenesis/physiology , Hyperglycemia/prevention & control , Liver/metabolism , Oleanolic Acid/pharmacology , Proto-Oncogene Proteins c-akt/physiology , Animals , Base Sequence , Blood Glucose/metabolism , DNA Primers , Forkhead Box Protein O1 , Glucose Tolerance Test , Insulin/metabolism , Insulin Secretion , Male , Mice , Mice, Inbred C57BL , Triglycerides/blood , Triglycerides/metabolismABSTRACT
Elevated mitochondrial reactive oxygen species have been suggested to play a causative role in some forms of muscle insulin resistance. However, the extent of their involvement in the development of diet-induced insulin resistance remains unclear. To investigate, manganese superoxide dismutase (MnSOD), a key mitochondrial-specific enzyme with antioxidant modality, was overexpressed, and the effect on in vivo muscle insulin resistance induced by a high-fat (HF) diet in rats was evaluated. Male Wistar rats were maintained on chow or HF diet. After 3 wk, in vivo electroporation (IVE) of MnSOD expression and empty vectors was undertaken in right and left tibialis cranialis (TC) muscles, respectively. After one more week, insulin action was evaluated using hyperinsulinemic euglycemic clamp, and tissues were subsequently analyzed for antioxidant enzyme capacity and markers of oxidative stress. MnSOD mRNA was overexpressed 4.5-fold, and protein levels were increased by 70%, with protein detected primarily in the mitochondrial fraction of muscle fibers. This was associated with elevated MnSOD and glutathione peroxidase activity, indicating that the overexpressed MnSOD was functionally active. The HF diet significantly reduced whole body and TC muscle insulin action, whereas overexpression of MnSOD in HF diet animals ameliorated this reduction in TC muscle glucose uptake by 50% (P < 0.05). Decreased protein carbonylation was seen in MnSOD overexpressing TC muscle in HF-treated animals (20% vs. contralateral control leg, P < 0.05), suggesting that this effect was mediated through an altered redox state. Thus interventions causing elevation of mitochondrial antioxidant activity may offer protection against diet-induced insulin resistance in skeletal muscle.
Subject(s)
Diet, High-Fat/adverse effects , Insulin Resistance , Muscle, Skeletal/enzymology , Oxidative Stress , Superoxide Dismutase/metabolism , Up-Regulation , Animals , Electroporation , Gene Transfer Techniques , Glutathione Peroxidase/metabolism , Humans , Lower Extremity , Male , Mitochondria, Muscle/enzymology , Mitochondria, Muscle/metabolism , Muscle Fibers, Skeletal/enzymology , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolism , Protein Carbonylation , RNA, Messenger/metabolism , Rats , Rats, Wistar , Recombinant Fusion Proteins/metabolism , Superoxide Dismutase/geneticsABSTRACT
Mitochondrial dysfunction and endoplasmic reticulum (ER) stress have been implicated in hepatic steatosis and insulin resistance. The present study investigated their roles in the development of hepatic steatosis and insulin resistance during de novo lipogenesis (DNL) compared to extrahepatic lipid oversupply. Male C57BL/6J mice were fed either a high fructose (HFru) or high fat (HFat) diet to induce DNL or lipid oversupply in/to the liver. Both HFru and HFat feeding increased hepatic triglyceride within 3 days (by 3.5 and 2.4 fold) and the steatosis remained persistent from 1 week onwards (p<0.01 vs Con). Glucose intolerance (iAUC increased by â¼60%) and blunted insulin-stimulated hepatic Akt and GSK3ß phosphorylation (â¼40-60%) were found in both feeding conditions (p<0.01 vs Con, assessed after 1 week). No impairment of mitochondrial function was found (oxidation capacity, expression of PGC1α, CPT1, respiratory complexes, enzymatic activity of citrate synthase & ß-HAD). As expected, DNL was increased (â¼60%) in HFru-fed mice and decreased (32%) in HFat-fed mice (all p<0.05). Interestingly, associated with the upregulated lipogenic enzymes (ACC, FAS and SCD1), two (PERK/eIF2α and IRE1/XBP1) of three ER stress pathways were significantly activated in HFru-fed mice. However, no significant ER stress was observed in HFat-fed mice during the development of hepatic steatosis. Our findings indicate that HFru and HFat diets can result in hepatic steatosis and insulin resistance without obvious mitochondrial defects via different lipid metabolic pathways. The fact that ER stress is apparent only with HFru feeding suggests that ER stress is involved in DNL per se rather than resulting from hepatic steatosis or insulin resistance.
Subject(s)
Endoplasmic Reticulum Stress , Fatty Liver/physiopathology , Insulin Resistance , Lipid Metabolism/physiology , Lipogenesis , Liver/pathology , Adipogenesis , Animals , Blotting, Western , Dietary Fats/administration & dosage , Fatty Liver/etiology , Fructose/administration & dosage , Glucose Intolerance/etiology , Glucose Intolerance/physiopathology , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Mitochondria/pathology , Oxidative StressABSTRACT
It has long been known that excesses of glucose and branched chain amino acids, such as leucine, lead to insulin resistance in skeletal muscle. A recent study in incubated rat muscle suggests that both molecules may do so by virtue of their ability to downregulate the fuel sensing and signaling enzyme AMP-activated protein kinase (AMPK) and activate mTOR/p70S6 kinase (p70S6K) signaling. The results also demonstrated that inhibition of mTOR/p70S6K with rapamycin prevented the development of insulin resistance but had no effect on AMPK activity (Thr172 phosphorylation of its catalytic subunit). In contrast, activation of AMPK by both AICAR and α-lipoic acid led to the phosphorylation of specific molecules that diminished both mTOR/p70S6K signaling and insulin resistance. These findings suggest that downregulation of AMPK precedes mTOR/p70S6K activation in mediating glucose and leucine-induced insulin resistance, although the mechanism by which it does so remains to be determined. Also requiring study is how an excess of the two nutrients leads to AMPK downregulation.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Amino Acids, Branched-Chain/metabolism , Gene Expression Regulation, Enzymologic/physiology , Glucose/metabolism , Insulin Resistance/physiology , Muscle, Skeletal/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Animals , Mice , Models, Biological , Muscle, Skeletal/physiology , Rats , Signal Transduction/physiologyABSTRACT
Weight loss inhibits thyrotropic function and reduces metabolic rate, thereby contributing to weight regain. Under negative energy balance there is an increase in the hypothalamic expression of both neuropeptide Y (NPY) and agouti related peptide (AgRP), the endogenous antagonist of melanocortin 4 (MC4) receptors. Both NPY and MC4 receptor antagonism reduce thyrotropic function centrally, but it is not known whether these pathways operate by similar or distinct mechanisms. We compared the time-course of effects of acute or chronic intracerebroventricular (ICV) administration of NPY (1.2 nmol acute bolus, or 3.5 nmol/day for 6 days) or the MC4 receptor antagonist HS014 (1.5 nmol bolus, or 4.8 nmol/day) on plasma concentrations of thyroid stimulating hormone (TSH) or free thyroxine (T4) in male rats pair-fed with vehicle-infused controls. These doses equipotently induced hyperphagia in acute studies, reduced latency to feed, and increased white adipose tissue mass after 6 days of infusion. Acute central NPY but not HS014 administration significantly reduced plasma TSH concentrations within 30-60 min and plasma free T4 levels within 90-120 min. These inhibitory effects were sustained for up to 5-6 days of continuous NPY infusion. HS014 induced a transient decrease in plasma free T4 levels that was observed only after 1-2 days of continuous ICV infusion. While both NPY and HS014 significantly increased corticosteronemia within an hour after ICV injection, the effect of NPY was significantly more pronounced and was sustained for up to 4 days of administration. Both NPY and HS014 significantly decreased the brown adipose tissue protein levels of uncoupling protein-3. We conclude that central NPY and MC4 antagonism decrease thyrotropic function via partially distinct mechanisms with different time courses, possibly involving glucocorticoid effects of NPY. MC4 receptor antagonism increases adiposity via pathways independent of increased food intake or changes in circulating concentrations of TSH, free T4 or corticosterone.
Subject(s)
Neuropeptide Y/pharmacology , Peptides, Cyclic/pharmacology , Receptor, Melanocortin, Type 4/antagonists & inhibitors , Thyrotrophs/metabolism , Adipose Tissue, Brown/anatomy & histology , Adipose Tissue, Brown/metabolism , Agouti-Related Protein/metabolism , Animals , Body Weight , Corticosterone/blood , Eating/drug effects , Energy Metabolism , Humans , Hyperphagia/metabolism , Male , Neuropeptide Y/metabolism , Peptides, Cyclic/metabolism , Rats , Receptor, Melanocortin, Type 4/metabolism , Thyrotropin/blood , Thyroxine/bloodABSTRACT
Glucose infusion into rats causes skeletal muscle insulin resistance that initially occurs without changes in insulin signaling. The aim of the current study was to prolong glucose infusion and evaluate other events associated with the transition to muscle insulin resistance. Hyperglycemia was produced in rats by glucose infusion for 3, 5 and 8 h. The rate of infusion required to maintain hyperglycemia was reduced at 5 and 8 h. Glucose uptake into red quadriceps (RQ) and its incorporation into glycogen decreased between 3 and 5 h, further decreasing at 8 h. The earliest observed change in RQ was decreased AMPKα2 activity associated with large increases in muscle glycogen content at 3 h. Activation of the mTOR pathway occurred at 5 h. Akt phosphorylation (Ser(473)) was decreased at 8 h compared to 3 and 5, although no decrease in phosphorylation of downstream GSK-3ß (Ser(9)) and AS160 (Thr(642)) was observed. White quadriceps showed a similar but delayed pattern, with insulin resistance developing by 8 h and decreased AMPKα2 activity at 5 h. These results indicate that, in the presence of a nutrient overload, alterations in muscle insulin signaling occur, but after insulin resistance develops and appropriate changes in energy/nutrient sensing pathways occur.
Subject(s)
Glucose/metabolism , Insulin Resistance , Muscles/metabolism , AMP-Activated Protein Kinases/metabolism , Animals , GTPase-Activating Proteins/metabolism , Glucose/administration & dosage , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Male , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, WistarABSTRACT
BACKGROUND AND PURPOSE: The peroxisome proliferator-activated receptor (PPAR)δ has been considered a therapeutic target for diabetes and obesity through enhancement of fatty acid oxidation. The present study aimed to characterize the effects of PPARδ agonists during insulin resistance of the whole body, muscle and liver. EXPERIMENTAL APPROACH: Wistar rats and C57BL/J6 mice were fed a high fat diet (HF) and then treated with PPARδ agonists NNC61-5920 and GW501516. The effects on insulin resistance were evaluated by hyperinsulinaemic clamp or glucose tolerance tests combined with glucose tracers. KEY RESULTS: In HF rats, 3 weeks of treatment with NNC61-5920 reduced the glucose infusion rate (by 14%, P < 0.05) and glucose disposal into muscle (by 20-30%, P < 0.01) during hyperinsulinaemic clamp. Despite increased mRNA expression of carnitine palmitoyltransferase-1, pyruvate dehydrogenase kinase 4 and uncoupling protein 3 in muscle, plasma and muscle triglyceride levels were raised (P < 0.01). Similar metabolic effects were observed after extended treatment with NNC61-5920 and GW501516 to 6 weeks. However, HF mice treated with NNC61-5920 improved their plasma lipid profile, glucose tolerance and insulin action in muscle. In both HF rats and mice, NNC61-5920 treatment attenuated hepatic insulin resistance and decreased expression of stearoyl-CoA desaturase 1, fatty acid translocase protein CD36 and lipoprotein lipase in liver. CONCLUSIONS AND IMPLICATIONS: PPARδ agonists exacerbated insulin resistance in HF rats in contrast to their beneficial effects on metabolic syndrome in HF mice. These opposing metabolic consequences result from their different effects on lipid metabolism and insulin sensitivity in skeletal muscle of these two species.
Subject(s)
Dietary Fats/administration & dosage , Insulin Resistance , Muscle, Skeletal/drug effects , PPAR delta/agonists , Animals , Biomarkers/metabolism , Glucose/metabolism , Glucose Tolerance Test , Lipid Metabolism/drug effects , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/metabolism , Organ Specificity , RNA, Messenger/metabolism , Rats , Rats, Wistar , Species Specificity , Thiazoles/pharmacology , Triglycerides/metabolismABSTRACT
OBJECTIVE: Branched-chain amino acids, such as leucine and glucose, stimulate protein synthesis and increase the phosphorylation and activity of the mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase (p70S6K). We examined in skeletal muscle whether the effects of leucine and glucose on these parameters and on insulin resistance are mediated by the fuel-sensing enzyme AMP-activated protein kinase (AMPK). RESEARCH DESIGN AND METHODS: Rat extensor digitorum longus (EDL) muscle was incubated with different concentrations of leucine and glucose with or without AMPK activators. Muscle obtained from glucose-infused rats was also used as a model. RESULTS: In the EDL, incubation with 100 or 200 µmol/l leucine versus no added leucine suppressed the activity of the α2 isoform of AMPK by 50 and 70%, respectively, and caused concentration-dependent increases in protein synthesis and mTOR and p70S6K phosphorylation. Very similar changes were observed in EDL incubated with 5.5 or 25 mmol/l versus no added glucose and in muscle of rats infused with glucose in vivo. Incubation of the EDL with the higher concentrations of both leucine and glucose also caused insulin resistance, reflected by a decrease in insulin-stimulated Akt phosphorylation. Coincubation with the AMPK activators AICAR and α-lipoic acid substantially prevented all of those changes and increased the phosphorylation of specific sites of mTOR inhibitors raptor and tuberous sclerosis complex 2 (TSC2). In contrast, decreases in AMPK activity induced by leucine and glucose were not associated with a decrease in raptor or TSC2 phosphorylation. CONCLUSIONS: The results indicate that both leucine and glucose modulate protein synthesis and mTOR/p70S6 and insulin signaling in skeletal muscle by a common mechanism. They also suggest that the effects of both molecules are associated with a decrease in AMPK activity and that AMPK activation prevents them.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Adenylate Kinase/metabolism , Glucose/pharmacology , Leucine/pharmacology , Muscle, Skeletal/enzymology , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/metabolism , Animals , Carrier Proteins/drug effects , Carrier Proteins/metabolism , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Insulin Resistance/physiology , Intracellular Signaling Peptides and Proteins/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Kinetics , Lactates/metabolism , Muscle, Skeletal/drug effects , Phosphoproteins/drug effects , Phosphoproteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/drug effects , Protein Serine-Threonine Kinases/metabolism , Pyruvates/metabolism , Rats , Ribonucleotides/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/drug effects , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , TOR Serine-Threonine KinasesABSTRACT
Alpha-calcitonin gene-related peptide (alphaCGRP) is a neuropeptide that is expressed in motor and sensory neurons. It is a powerful vasodilator and has been implicated in diverse metabolic roles. However, its precise physiological function remains unclear. In this study, we investigated the role of alphaCGRP in lipid metabolism by chronically challenging alphaCGRP-specific knockout (alphaCGRP(-/-)) and control mice with high-fat diet regimens. At the start of the study, both animal groups displayed similar body weights, serum lipid markers, and insulin sensitivity. However, alphaCGRP(-/-) mice displayed higher core temperatures, increased energy expenditures, and a relative daytime (nonactive) depression in respiratory quotients, which indicated increased beta-oxidation. In response to fat feeding, alphaCGRP(-/-) mice were comparatively protected against diet-induced obesity with an attenuated body weight gain and an overall reduction in adiposity across all the three diets examined. AlphaCGRP(-/-) mice also displayed improved glucose handling and insulin sensitivity, lower im and hepatic lipid accumulation, and improved overall metabolic health. These findings define a new role for alphaCGRP as a mediator of energy metabolism and opens up therapeutic opportunities to target CGRP action in obesity.
Subject(s)
Body Temperature/physiology , Calcitonin Gene-Related Peptide/physiology , Dietary Fats/adverse effects , Obesity/physiopathology , AMP-Activated Protein Kinases/metabolism , Acetyl-CoA Carboxylase/metabolism , Adiposity/physiology , Animals , Blotting, Western , Body Weight/physiology , Calcitonin Gene-Related Peptide/deficiency , Calcitonin Gene-Related Peptide/genetics , Citrate (si)-Synthase/metabolism , DNA, Mitochondrial/genetics , Dietary Fats/administration & dosage , Energy Metabolism/physiology , Fatty Liver/metabolism , Fatty Liver/pathology , Fatty Liver/physiopathology , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitochondrial Proteins/metabolism , Obesity/etiology , Obesity/genetics , Oxygen Consumption/physiology , Triglycerides/metabolismABSTRACT
Activation of AMP-activated protein kinase (AMPK) is thought to convey many of the beneficial effects of exercise via its inhibitory effect on acetyl-CoA carboxylase 2 (ACC2) and promotion of fatty acid oxidation. Hence, AMPK and ACC have become major drug targets for weight loss and improved insulin action. However, it remains unclear whether or how activation of the fatty acid oxidation pathway without a concomitant increase in energy expenditure could be beneficial. Here, we have used either pharmacological (administration of the AMPK agonist 5(') aminoimidazole-4-carboxamide-riboside) or genetic means (mutation of the ACC2 gene in mice) to manipulate fatty acid oxidation to determine whether this is sufficient to promote leanness. Both of these strategies increased whole-body fatty acid oxidation without altering energy expenditure or adiposity. We conclude that negative energy balance is a prerequisite for weight reduction, and increased fatty acid oxidation per se has little, if any, effect to reduce adiposity.
Subject(s)
Adiposity/physiology , Energy Metabolism/physiology , Fatty Acids/metabolism , Mitochondria/metabolism , AMP-Activated Protein Kinases/metabolism , Acetyl-CoA Carboxylase/genetics , Acetyl-CoA Carboxylase/metabolism , Animals , Mice , Oxidation-Reduction , Up-RegulationABSTRACT
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.
Subject(s)
Antioxidants/metabolism , Insulin Resistance/physiology , 3T3-L1 Cells , Adipocytes/drug effects , Adipocytes/metabolism , Animals , Antimycin A/pharmacology , Antioxidants/pharmacology , Cell Line , Insulin Resistance/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria/drug effects , Mitochondria/metabolism , Models, Biological , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Reactive Oxygen Species/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxides/metabolismABSTRACT
OBJECTIVE: Medium-chain fatty acids (MCFAs) have been reported to be less obesogenic than long-chain fatty acids (LCFAs); however, relatively little is known regarding their effect on insulin action. Here, we examined the tissue-specific effects of MCFAs on lipid metabolism and insulin action. RESEARCH DESIGN AND METHODS: C57BL6/J mice and Wistar rats were fed either a low-fat control diet or high-fat diets rich in MCFAs or LCFAs for 4-5 weeks, and markers of mitochondrial oxidative capacity, lipid levels, and insulin action were measured. RESULTS: Mice fed the MCFA diet displayed reduced adiposity and better glucose tolerance than LCFA-fed animals. In skeletal muscle, triglyceride levels were increased by the LCFA diet (77%, P < 0.01) but remained at low-fat diet control levels in the MCFA-fed animals. The LCFA diet increased (20-50%, P < 0.05) markers of mitochondrial metabolism in muscle compared with low-fat diet-fed controls; however; the increase in oxidative capacity was substantially greater in MCFA-fed animals (50-140% versus low-fat-fed controls, P < 0.01). The MCFA diet induced a greater accumulation of liver triglycerides than the LCFA diet, likely due to an upregulation of several lipogenic enzymes. In rats, isocaloric feeding of MCFA or LCFA high-fat diets induced hepatic insulin resistance to a similar degree; however, insulin action was preserved at the level of low-fat diet-fed controls in muscle and adipose from MCFA-fed animals. CONCLUSIONS: MCFAs reduce adiposity and preserve insulin action in muscle and adipose, despite inducing steatosis and insulin resistance in the liver. Dietary supplementation with MCFAs may therefore be beneficial for preventing obesity and peripheral insulin resistance.
Subject(s)
Dietary Fats/pharmacology , Fatty Acids, Nonesterified/pharmacology , Insulin Resistance/physiology , Insulin/pharmacology , Mitochondria, Muscle/metabolism , Adipose Tissue/anatomy & histology , Adipose Tissue/metabolism , Animals , Body Composition , Body Weight , Diet, Fat-Restricted , Energy Metabolism , Fatty Acids/metabolism , Glucose/metabolism , Insulin/metabolism , Mice , Mice, Inbred C57BL , Mitochondria, Muscle/drug effects , Organ Size , Oxidation-Reduction , Rats , Rats, Wistar , Triglycerides/metabolismABSTRACT
Hepatic insulin resistance is the major contributor to fasting hyperglycemia in type 2 diabetes. Here we report that the endosomal adaptor protein APPL1 increases hepatic insulin sensitivity by potentiating insulin-mediated suppression of the gluconeogenic program. Insulin-stimulated activation of Akt and suppression of gluconeogenesis in hepatocytes are enhanced by APPL1 overexpression, but are attenuated by APPL1 knockdown. APPL1 interacts with Akt and blocks the association of Akt with its endogenous inhibitor tribble 3 (TRB3) through direct competition, thereby promoting Akt translocation to the plasma membrane and the endosomes for further activation. In db/db diabetic mice, the blockage of the augmented interaction between Akt and TRB3 by hepatic overexpression of APPL1 is accompanied by a marked attenuation of hyperglycemia and insulin resistance. These results suggest that the potentiating effects of APPL1 on insulin-stimulated suppression of hepatic glucose production are attributed to its ability in counteracting the inhibition of Akt activation by TRB3.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carrier Proteins/metabolism , Diabetes Mellitus, Type 2/enzymology , Glucose/biosynthesis , Hepatocytes/enzymology , Insulin/metabolism , Nerve Tissue Proteins/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Animals , Binding, Competitive , Cell Cycle Proteins/metabolism , Cells, Cultured , Gene Knockdown Techniques , Gluconeogenesis , Glucose/metabolism , Mice , Mice, Inbred C57BL , Mice, Obese , RNA Interference , Rats , Signal TransductionABSTRACT
Type 2 diabetes is characterized by hyperlipidemia, hyperinsulinemia, and insulin resistance. The aim of this study was to investigate whether acute hyperlipidemia-induced insulin resistance in the presence of hyperinsulinemia was due to defective insulin signaling. Hyperinsulinemia (approximately 300 mU/l) with hyperlipidemia or glycerol (control) was produced in cannulated male Wistar rats for 0.5, 1 h, 3 h, or 5 h. The glucose infusion rate required to maintain euglycemia was significantly reduced by 3 h with lipid infusion and was further reduced after 5 h of infusion, with no difference in plasma insulin levels, indicating development of insulin resistance. Consistent with this finding, in vivo skeletal muscle glucose uptake (31%, P < 0.05) and glycogen synthesis rate (38%, P < 0.02) were significantly reduced after 5 h compared with 3 h of lipid infusion. Despite the development of insulin resistance, there was no difference in the phosphorylation state of multiple insulin-signaling intermediates or muscle diacylglyceride and ceramide content over the same time course. However, there was an increase in cumulative exposure to long-chain acyl-CoA (70%) with lipid infusion. Interestingly, although muscle pyruvate dehydrogenase kinase 4 protein content was decreased in hyperinsulinemic glycerol-infused rats, this decrease was blunted in muscle from hyperinsulinemic lipid-infused rats. Decreased pyruvate dehydrogenase complex activity was also observed in lipid- and insulin-infused animals (43%). Overall, these results suggest that acute reductions in muscle glucose metabolism in rats with hyperlipidemia and hyperinsulinemia are more likely a result of substrate competition than a significant early defect in insulin action or signaling.
Subject(s)
Feedback, Physiological/physiology , GTPase-Activating Proteins/metabolism , Insulin Receptor Substrate Proteins/metabolism , Insulin Resistance , Insulin/pharmacology , Lipids/pharmacology , Muscle, Skeletal/drug effects , Oncogene Protein v-akt/metabolism , Animals , Blood Glucose/analysis , Blood Glucose/drug effects , Blood Glucose/metabolism , Feedback, Physiological/drug effects , Glycogen/metabolism , Infusions, Intravenous , Insulin/administration & dosage , Insulin/blood , Insulin Resistance/physiology , Lipids/administration & dosage , Lipids/blood , Male , Metabolic Networks and Pathways/drug effects , Muscle, Skeletal/metabolism , Phosphorylation/drug effects , Rats , Rats, WistarABSTRACT
Major urinary protein-1 (MUP-1) is a low molecular weight secreted protein produced predominantly from the liver. Structurally it belongs to the lipocalin family, which carries small hydrophobic ligands such as pheromones. However, the physiological functions of MUP-1 remain poorly understood. Here we provide evidence demonstrating that MUP-1 is an important player in regulating energy expenditure and metabolism in mice. Both microarray and real-time PCR analysis demonstrated that the MUP-1 mRNA abundance in the liver of db/db obese mice was reduced by approximately 30-fold compared with their lean littermates, whereas this change was partially reversed by treatment with the insulin-sensitizing drug rosiglitazone. In both dietary and genetic obese mice, the circulating concentrations of MUP-1 were markedly decreased compared with the lean controls. Chronic elevation of circulating MUP-1 in db/db mice, using an osmotic pump-based protein delivery system, increased energy expenditure and locomotor activity, raised core body temperature, and decreased glucose intolerance as well as insulin resistance. At the molecular level, MUP-1-mediated improvement in metabolic profiles was accompanied by increased expression of genes involved in mitochondrial biogenesis, elevated mitochondrial oxidative capacity, decreased triglyceride accumulation, and enhanced insulin-evoked Akt signaling in skeletal muscle but not in liver. Altogether, these findings raise the possibility that MUP-1 deficiency might contribute to the metabolic dysregulation in obese/diabetic mice, and suggest that the beneficial metabolic effects of MUP-1 are attributed in part to its ability in increasing mitochondrial function in skeletal muscle.