High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice.

OBJECTIVE: Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. METHODS: We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. RESULTS: Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. CONCLUSIONS: We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and during changes in hepatic insulin action in liver alter membrane properties - in particular those of mitochondria which are highly abundant in hepatocytes. In turn, a progressive decrease in the abundance of mitochondrial membrane proteins throughout HF-exposure likely impacts on mitochondrial energy metabolism, substrate exchange across mitochondrial membranes, contributes to oxidative stress, mitochondrial damage, and the development of insulin resistance in liver.

Osteopontin deficiency protects against obesity-induced hepatic steatosis and attenuates glucose production in mice.

AIMS/HYPOTHESIS: Obesity is strongly associated with the development of non-alcoholic fatty liver disease (NAFLD). The cytokine osteopontin (OPN) was recently shown to be involved in obesity-induced adipose tissue inflammation and reduced insulin response. Accumulating evidence links OPN to the pathogenesis of NAFLD. Here we aimed to identify the role of OPN in obesity-associated hepatic steatosis and impaired hepatic glucose metabolism. METHODS: Wild-type (WT) and Opn (also known as Spp1) knockout (Opn (-/-)) mice were fed a high-fat or low-fat diet to study OPN effects in obesity-driven hepatic alterations. RESULTS: We show that genetic OPN deficiency protected from obesity-induced hepatic steatosis, at least in part, by downregulating hepatic triacylglycerol synthesis. Conversely, absence of OPN promoted fat storage in adipose tissue thereby preventing the obesity-induced shift to ectopic fat accumulation in the liver. Euglycaemic-hyperinsulinaemic clamp studies revealed that insulin resistance and excess hepatic glucose production in obesity were significantly attenuated in Opn (-/-) mice. OPN deficiency markedly improved hepatic insulin signalling as shown by enhanced insulin receptor substrate-2 phosphorylation and prevented upregulation of the major hepatic transcription factor Forkhead box O1 and its gluconeogenic target genes. In addition, obesity-driven hepatic inflammation and macrophage accumulation was blocked by OPN deficiency. CONCLUSIONS/INTERPRETATION: Our data strongly emphasise OPN as mediator of obesity-associated hepatic alterations including steatosis, inflammation, insulin resistance and excess gluconeogenesis. Targeting OPN action could therefore provide a novel therapeutic strategy to prevent obesity-related complications such as NAFLD and type 2 diabetes.

Diet dependence of diabetes in the New Zealand Obese (NZO) mouse: total fat, but not fat quality or sucrose accelerates and aggravates diabetes.

BACKGROUND: Obesity and diabetes in mice can be modified by dietary variables. Here we systematically analysed the effect of the sucrose and fat content and of the fat quality in New Zealand Obese mice, a mouse model of the metabolic syndrome. RESULTS: Male NZO mice fed a semi-purified diet with sucrose exhibited an identical weight gain and diabetes incidence as controls without sucrose. In contrast, mice on a chow diet gained weight more slowly and developed diabetes approximately 10 weeks later than those on the semi-purified diet (energy density 3.05 vs. 3.85 kcal/g; fibre content 12.9 vs. 4.7%). In a second experimental series, neither the fat content (10 vs. 40% of the total energy) nor the quality of the fat (lard, safflower oil, or fish oil) of semi-purified diets modified weight gain. However, diabetes started approximately 2 weeks earlier and appeared more severe (blood glucose 30 vs. 20 mmol/l at week 13) in the high-fat diet group (energy density 4.58 kcal/g; fibre content 5.7%). CONCLUSIONS: Obesity in NZO mice develops independent of the dietary sucrose or fat content, and of the fat quality. However, the dietary fat content accelerates the onset of diabetes without enhancing adiposity. In contrast, chow diet exerts an anti-adipogenic/anti-diabetogenic effect that appears to be due to its lower caloric density and/or its higher fibre content.

Dissociation of lipotoxicity and glucotoxicity in a mouse model of obesity associated diabetes: role of forkhead box O1 (FOXO1) in glucose-induced beta cell failure.

AIMS/HYPOTHESIS: Carbohydrate-free diet prevents hyperglycaemia and beta cell destruction in the New Zealand Obese (NZO) mouse model. Here we have used a sequential dietary regimen to dissociate the effects of obesity and hyperglycaemia on beta cell function and integrity, and to study glucose-induced alterations of key transcription factors over 16 days. METHODS: Mice were rendered obese by feeding a carbohydrate-free diet for 18 weeks. Thereafter, a carbohydrate-containing diet was given. Plasma glucose, plasma insulin and total pancreatic insulin were determined, and forkhead box O1 protein (FOXO1) phosphorylation and the transcription factors pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 protein (NKX6.1) and v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A (avian) (MAFA) were monitored by immunohistochemistry for 16 days. RESULTS: Dietary carbohydrates produced a rapid and continuous increase in plasma glucose in NZO mice between day 2 and 16 after the dietary challenge. Hyperglycaemia caused a dramatic dephosphorylation of FOXO1 at day 2, followed by a progressive depletion of insulin stores. The loss of beta cells was triggered by apoptosis (detectable at day 8), associated with reduction of crucial transcription factors (PDX1, NKX6.1 and MAFA). Incubation of isolated islets from carbohydrate-restricted NZO mice or MIN6 cells with palmitate and glucose for 48 h resulted in a dephosphorylation of FOXO1 and thymoma viral proto-oncogene 1 (AKT) without changing the protein levels of both proteins. CONCLUSIONS/INTERPRETATION: The dietary regimen dissociates the effects of obesity (lipotoxicity) from those of hyperglycaemia (glucotoxicity) in NZO mice. Obese NZO mice are unable to compensate for the carbohydrate challenge by increasing insulin secretion or synthesising adequate amounts of insulin. In response to the hyperglycaemia, FOXO1 is dephosphorylated, leading to reduced levels of beta cell-specific transcription factors and to apoptosis of the cells.

Development of diabetes in obese, insulin-resistant mice: essential role of dietary carbohydrate in beta cell destruction.

AIMS/HYPOTHESIS: The role of dietary carbohydrate in the pathogenesis of type 2 diabetes is still a subject of controversial debate. Here we analysed the effects of diets with and without carbohydrate on obesity, insulin resistance and development of beta cell failure in the obese, diabetes-prone New Zealand Obese (NZO) mouse. MATERIALS AND METHODS: NZO mice were kept on a standard diet (4% [w/w] fat, 51% carbohydrate, 19% protein), a high-fat diet (15, 47 and 17%, respectively) and a carbohydrate-free diet in which carbohydrate was exchanged for fat (68 and 20%, respectively). Body composition and blood glucose were measured over a period of 22 weeks. Glucose tolerance tests and euglycaemic-hyperinsulinaemic clamps were performed to analyse insulin sensitivity. Islet morphology was assessed by immunohistochemistry. RESULTS: Mice on carbohydrate-containing standard or high-fat diets developed severe diabetes (blood glucose >16.6 mmol/l, glucosuria) due to selective destruction of pancreatic beta cells associated with severe loss of immunoreactivity of insulin, glucose transporter 2 (GLUT2) and musculoaponeurotic fibrosarcoma oncogene homologue A (MafA). In contrast, mice on the carbohydrate-free diet remained normoglycaemic and exhibited hyperplastic islets in spite of a morbid obesity associated with severe insulin resistance and a massive accumulation of macrophages in adipose tissue. CONCLUSIONS/INTERPRETATION: These data indicate that the combination of obesity, insulin resistance and the inflammatory response of adipose tissue are insufficient to cause beta cell destruction in the absence of dietary carbohydrate.

Direct thiazolidinedione action on isolated rat skeletal muscle fuel handling is independent of peroxisome proliferator-activated receptor-gamma-mediated changes in gene expression.

Thiazolidinediones (TZDs) are believed to induce insulin sensitization by modulating gene expression via agonistic stimulation of the nuclear peroxisome proliferator-activated receptor-gamma (PPAR-gamma). We have shown earlier that the TZD troglitazone inhibits mitochondrial fuel oxidation in isolated rat skeletal muscle. In the present study, rat soleus muscle strips were exposed to TZDs to examine whether the inhibition of fuel oxidation is mediated by PPAR-gamma activation. Our findings consistently indicated direct, acute, and PPAR-gamma-independent TZD action on skeletal muscle fuel metabolism. Rapid stimulation of lactate release by 20 micromol/l troglitazone within 30 min suggested that direct TZD action on skeletal muscle in vitro does not rely on changes in gene expression rates (12.6 +/- 0.6 [control] vs. 16.0 +/- 0.8 micromol. g(-1). h(-1) [troglitazone]; P < 0.01). This conclusion was supported by the failure of actinomycin D and cycloheximide to block the effects of troglitazone. Mitochondrial fuel oxidation was consistently inhibited by six different TZDs (percent inhibition of CO(2) production from palmitate after 25 h: troglitazone, -61 +/- 2%; pioglitazone, -43 +/- 7%; rosiglitazone, -22 +/- 6%; BM13.1258, -47 +/- 9%; BM15.2054, -51 +/- 4%; and T-174, -59 +/- 4% [P < 0.005 each]), but not by PPAR-gamma agonistic compounds not belonging to the TZD class (JTT-501, -5 +/- 7% [NS]; prostaglandin J(2), 17 +/- 7% [P < 0.05]), which further argues against dependence on PPAR-gamma activation. In summary, our findings provided good evidence that direct inhibition of mitochondrial fuel oxidation in isolated skeletal muscle is a group-specific effect of TZDs and is independent of PPAR-gamma-mediated gene expression.

UCP3 gene expression does not correlate with muscle oxidation rates in troglitazone-treated Zucker fatty rats.

Uncoupling protein-3 (UCP3), a mitochondrial carrier protein predominantly expressed in muscle, has been suggested to release stored energy as heat. The insulin-sensitizing thiazolidinediones enhance glucose disposal in skeletal muscle and have been reported to increase the expression of uncoupling proteins in various experimental systems. We therefore studied the effect of troglitazone treatment on UCP3 gene expression in muscles from lean and obese Zucker rats. In comparison with obese littermates, basal UCP3 mRNA levels in lean Zucker rats tended to be higher in white and red gastrocnemius muscles, but were lower in soleus (P<0.001) muscle and heart (P<0.01). In lean rats, troglitazone significantly increased UCP3 gene expression in white and red gastrocnemius and heart muscles (all P<0.01). In contrast, the drug reduced UCP3 mRNA expression in red gastrocnemius and soleus muscles of obese littermates (all P<0.001). The troglitazone-dependent decrease in UCP3 gene expression was accompanied by an increased weight gain in obese rats, while no such effect was observed in lean rats. In obese rats, improvement of insulin resistance by troglitazone was associated with increased rates of basal and insulin-stimulated CO(2) production from glucose measured in soleus muscle. These studies demonstrate that effects of troglitazone on UCP3 gene expression depend on the phenotype of Zucker rats and that troglitazone-induced metabolic improvements are not related to increased uncoupling resulting from upregulation of UCP3 mRNA expression in muscle.

Metabolic response to anisoosmolarity of rat skeletal muscle in vitro.

Isolated rat hepatocytes exhibit an insulin-like anabolic response to hypoosmotic incubation and a glucagon-like catabolic response to hyperosmotic incubation. Recently, a distinct glycogenic response to hypoosmotic treatment was likewise reported for cultured rat myotubes. The present study examines the effects of anisoosmolar exposure on glucose metabolism in freshly isolated rat soleus muscle strips. Under the same experimental conditions as used for cultured myotubes, hypoosmolarity reduced net glycogen synthesis to 52%, while hyperosmolarity stimulated glycogen storage to 231% of isoosmolar control (nmol glucose incorporated into glycogen g(-1) x h(-1): hypoosmolar, 34+/-3; isoosmolar, 65+/-8; hyperosmolar, 150+/-11; p<0.01 each vs. isoosmolar). The responses of native skeletal muscle to anisoosmolarity are therefore in opposition to what has been described for hepatocytes and cultured myotubes. Further experiments on rat skeletal muscle revealed that the observed lack of a glycogenic response to hypoosmolarity persisted independent of medium composition, specifically with regard to prevailing glucose and K+ concentrations. In conclusion, hypoosmotic exposure inhibits glycogen synthesis in isolated rat soleus muscle, which clearly argues against the hypothesis that osmotic changes and cell swelling may be physiologically relevant stimulators of muscle glycogen synthesis.

Redistribution of substrates to adipose tissue promotes obesity in mice with selective insulin resistance in muscle.

Obesity and insulin resistance in skeletal muscle are two major factors in the pathogenesis of type 2 diabetes. Mice with muscle-specific inactivation of the insulin receptor gene (MIRKO) are normoglycemic but have increased fat mass. To identify the potential mechanism for this important association, we examined insulin action in specific tissues of MIRKO and control mice under hyperinsulinemic-euglycemic conditions. We found that insulin-stimulated muscle glucose transport and glycogen synthesis were decreased by about 80% in MIRKO mice, whereas insulin-stimulated fat glucose transport was increased threefold in MIRKO mice. These data demonstrate that selective insulin resistance in muscle promotes redistribution of substrates to adipose tissue thereby contributing to increased adiposity and development of the prediabetic syndrome.

Troglitazone directly inhibits CO(2) production from glucose and palmitate in isolated rat skeletal muscle.

Troglitazone is a nuclear peroxisome proliferator-activated receptor-gamma agonist with insulin-sensitizing properties that has been introduced for the treatment of type 2 diabetes. To further elucidate its mechanism of action, this study examined direct troglitazone effects on glucose and palmitate utilization in isolated rat soleus muscle. Exposure of muscle specimens for 25 h to 5 micromol/liter troglitazone resulted in the distinct inhibition of insulin-stimulated mitochondrial fuel oxidation as indicated by decreased rates of CO(2) produced from glucose (glucose converted to CO(2), nanomoles per gram per hour: control, 1461 +/- 192 versus troglitazone, 753 +/- 80, P <.0001) and palmitate (palmitate converted to CO(2), nanomoles per gram per hour: control, 75 +/- 5 versus troglitazone, 20 +/- 2, P <.0001). Blunted fuel oxidation was accompanied by increased rates of anaerobic glycolysis (lactate release, micromoles per gram per hour: control, 17.3 +/- 1.0 versus troglitazone, 49.2 +/- 2.7, P <.0001) and glucose transport ([(3)H]2-deoxyglucose transport, cpm per milligram per hour: control, 540 +/- 46 versus troglitazone, 791 +/- 61, P <.0001), as well as by decreased rates of glycogen synthesis (glucose incorporation into glycogen, micromoles per gram per hour: control, 2.00 +/- 0.26 versus troglitazone, 1.02 +/- 0.13, P <.001). Such shift toward anaerobic glucose utilization also was seen in the absence of insulin and with short-term troglitazone exposure for 90 min, indicating an underlying mechanism that is rapid and independent of concomitant insulin stimulation. The results demonstrate direct and acute inhibition of fuel oxidation to CO(2) by troglitazone in rat skeletal muscle in vitro.

Chronic and acute effects of thiazolidinediones BM13.1258 and BM15.2054 on rat skeletal muscle glucose metabolism.

1 New thiazolidinediones BM13.1258 and BM15.2054 were studied with regard to their PPARgamma-agonistic activities and to their acute and chronic effects on glucose metabolism in soleus muscle strips from lean and genetically obese rats. 2 Both BM13.1258 and BM15.2054 revealed to be potent PPARgamma-activators in transient transfection assays in vitro. 3 In insulin-resistant obese rats, but not in lean rats, 10 days of oral treatment with either compound increased the stimulatory effect of insulin on muscle glycogen synthesis to a similar extent (insulin-induced increment in micromol glucose incorporated into glycogen g-1 h-1: control, +1.19+/-0.28; BM13.1258, +2.50+/-0.20; BM15.2054, +2.55+/-0.46; P<0.05 vs control each). 4 In parallel to insulin sensitization, mean glucose oxidation increased insulin-independently in response to BM13.1258 (to 191 and 183% of control in the absence and presence of insulin, respectively; P<0.01 each), which was hardly seen in response to BM15.2054 (to 137 and 124% of control, respectively; ns). 5 Comparable effects on PPARgamma activation and on amelioration of insulin resistance by BM13.1258 and BM15.2054 were therefore opposed by different effects on glucose oxidation. 6 In contrast to chronic oral treatment, acute exposure of muscles to BM13.1258 or BM15.2054 in vitro elicited a distinct catabolic response of glucose metabolism in specimens from both lean and obese rats. 7 The results provide evidence that BM13.1258 and BM15.2054 can affect muscle glucose metabolism via more than one mechanism of action. 8 Further efforts are required to clarify, to what extent other mechanisms besides insulin sensitization via the activation of PPARgamma are involved in the antidiabetic actions of thiazolidinediones.

Acute and chronic exposure to tumor necrosis factor-alpha fails to affect insulin-stimulated glucose metabolism of isolated rat soleus muscle.

To better understand the effects of tumor necrosis factor-alpha (TNF alpha) on insulin sensitivity, direct interaction of the peptide with freshly isolated rat soleus muscle strips was investigated. Muscles were exposed to TNF alpha at concentrations ranging from 0.01-5 nmol/liter. Rates of insulin-stimulated (5 or 100 nmol/liter) glucose metabolism were determined after periods of TNF alpha preexposure of 30 min, 6 h, and 24 h. Independent of exposure time, TNF alpha failed to exert any significant effect on rates of 3H-2-deoxy-glucose transport (stimulation by 100 nmol/liter insulin after preincubation without vs. with 5 nmol/liter TNF alpha, cpm/mg x h: 30 min, 779 +/- 29 vs. 725 +/- 29; 6 h, 652 +/- 56 vs. 617 +/- 60; 24 h, 911 +/- 47 vs. 936 +/- 31) or glucose incorporation into glycogen (micromol/g x h: 30 min, 5.19 +/- 0.22 vs. 5.25 +/- 0.41; 6 h, 2.08 +/- 0.10 vs. 2.09 +/- 0.17; 24 h, 2.51 +/- 0.21 vs. 2.41 +/- 0.26). In parallel, TNF alpha neither affected insulin-stimulated rates of glucose oxidation (CO2 production) and anaerobic glycolysis (lactate release), nor muscle glycogen content. In conclusion, these findings do not support the hypothesis of muscle insulin desensitization by TNF alpha via autocrine or paracrine mechanisms. The obtained data favor the concept that TNF alpha-dependent muscle insulin resistance in vivo depends on indirect effects rather than direct interaction of the peptide with skeletal muscle.

Acute non-insulin-like stimulation of rat muscle glucose metabolism by troglitazone in vitro.

1. The direct short-term effects of troglitazone on parameters of glucose metabolism were investigated in rat soleus muscle strips. 2. In muscle strips from Sprague-Dawley rats, troglitazone (3.25 micromol l(-1)) increased basal and insulin-stimulated glucose transport by 24% and 41%, respectively (P<0.01 each). 3. In the presence of 5 nmol l(-1) insulin, stimulation of glucose transport by 3.25 micromol l(-1) troglitazone was accompanied by a 36% decrease in glycogen synthesis, while glycolysis was increased (112% increase in lactate production) suggesting a catabolic response of intracellular glucose handling. 4. Whereas insulin retained its stimulant effect on [3H]-2-deoxy-glucose transport in hypoxia-stimulated muscle (by 44%; c.p.m. mg(-1) h(-1): 852+/-77 vs 1229+/-75, P<0.01), 3.25 micromol l(-1) troglitazone failed to increase glucose transport under hypoxic conditions (789+/-40 vs 815+/-28, NS) suggesting that hypoxia and troglitazone address a similar, non-insulin-like mechanism. 5. No differences between troglitazone and hypoxia were identified in respective interactions with insulin. 6. Troglitazone acutely stimulated muscle glucose metabolism in a hypoxia/contraction-like manner, but it remains to be elucidated whether this contributes to the long-term antidiabetic and insulin enhancing potential in vivo or is to be regarded as an independent pharmacological effect.