Conjugated linoleic acid fails to worsen insulin resistance but induces hepatic steatosis in the presence of leptin in ob/ob mice

Conjugated linoleic acid (CLA) induces insulin resistance preceded by rapid depletion of the adipokines leptin and adiponectin, increased inflammation, and hepatic steatosis in mice. To determine the role of leptin in CLA-mediated insulin resistance and hepatic steatosis, recombinant leptin was coadministered with dietary CLA in ob/ob mice to control leptin levels and to, in effect, negate the leptin depletion effect of CLA. In a 2 x 2 factorial design, 6 week old male ob/ob mice were fed either a control diet or a diet supplemented with CLA and received daily intraperitoneal injections of either leptin or vehicle for 4 weeks. In the absence of leptin, CLA significantly depleted adiponectin and induced insulin resistance, but it did not increase hepatic triglyceride concentrations or adipose inflammation, marked by interleukin-6 and tumor necrosis factor-alpha mRNA expression. Insulin resistance, however, was accompanied by increased macrophage infiltration (F4/80 mRNA) in adipose tissue. In the presence of leptin, CLA depleted adiponectin but did not induce insulin resistance or macrophage infiltration. Despite this, CLA induced hepatic steatosis. In summary, CLA worsened insulin resistance without evidence of inflammation or hepatic steatosis in mice after 4 weeks. In the presence of leptin, CLA failed to worsen insulin resistance but induced hepatic steatosis in ob/ob mice.     

Changes in hepatic gene expression upon oral administration of taurine-conjugated ursodeoxycholic acid in ob/ob mice

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent and associated with considerable morbidities. Unfortunately, there is no currently available drug established to treat NAFLD. It was recently reported that intraperitoneal administration of taurine-conjugated ursodeoxycholic acid (TUDCA) improved hepatic steatosis in ob/ob mice. We hereby examined the effect of oral TUDCA treatment on hepatic steatosis and associated changes in hepatic gene expression in ob/ob mice. We administered TUDCA to ob/ob mice at a dose of 500 mg/kg twice a day by gastric gavage for 3 weeks. Body weight, glucose homeostasis, endoplasmic reticulum (ER) stress, and hepatic gene expression were examined in comparison with control ob/ob mice and normal littermate C57BL/6J mice. Compared to the control ob/ob mice, TUDCA treated ob/ob mice revealed markedly reduced liver fat stained by oil red O (44.2±5.8% vs. 21.1±10.4%, P<0.05), whereas there was no difference in body weight, oral glucose tolerance, insulin sensitivity, and ER stress. Microarray analysis of hepatic gene expression demonstrated that oral TUDCA treatment mainly decreased the expression of genes involved in de novo lipogenesis among the components of lipid homeostasis. At pathway levels, oral TUDCA altered the genes regulating amino acid, carbohydrate, and drug metabolism in addition to lipid metabolism. In summary, oral TUDCA treatment decreased hepatic steatosis in ob/ob mice by cooperative regulation of multiple metabolic pathways, particularly by reducing the expression of genes known to regulate de novo lipogenesis.     

Glucoregulatory effects of bombesin in lean and genetically obese hyperglycaemic (ob/ob) mice

1. Plasma glucose and insulin responses to bombesin were examined in 12-15-week-old 12 hr fasted lean and genetically obese hyperglycaemic (ob/ob) mice. 2. Bombesin (1 mg/kg ip) produced a prompt but transient increase of plasma insulin in lean mice (maximum increase of 50% at 5 min), and a more slowly generated but protracted insulin response in ob/ob mice (maximum increase of 80% at 30 min). Plasma glucose concentrations of both groups of mice were increased by bombesin (maximum increases of 40 and 48% respectively in lean and ob/ob mice at 15 min). 3. When administered with glucose (2 g/kg ip), bombesin (1 mg/kg ip) rapidly increased insulin concentrations of lean and ob/ob mice (maximum increases of 39 and 63% respectively at 5 min). Bombesin did not significantly alter the rise of plasma glucose after exogenous glucose administration to these mice. 4. The results indicate that bombesin exerts an insulin-releasing effect in lean and ob/ob mice. The greater insulin-releasing effect in ob/ob mice renders bombesin a possible component of the overactive entero-insular axis in the ob/ob mutant, especially if it acts within the islets as a neurotransmitter or paracrine agent.     

Insulin receptor tyrosine kinase activity is unaltered in ob/ob and db/db mouse skeletal muscle membranes

Insulin binding and insulin receptor tyrosine kinase activity were examined in two rodent models with genetic insulin resistance using partially-purified skeletal muscle membrane preparations. Insulin binding activity was decreased about 50% in both 12-week (219 +/- 184 vs 1255 +/- 158 fmoles/mg, p less than 0.01) and 24-week old (2120 +/- 60 vs 1081 +/- 60 fmoles/mg, p less than 0.01) ob/ob mice. In contrast, insulin binding to membrane derived from 24-week old db/db mice was not significantly different from lean controls (1371 +/- 212 vs 1253 +/- 247 fmoles/mg). Insulin-associated tyrosine kinase activity of membranes from ob/ob skeletal muscle was decreased, compared to its normal lean littermate, when compared on a per mg of protein basis in both 12-week (37 +/- 3 vs 21 +/- 3 pmoles/min/mg, p less than 0.05) and 24-week old (71 +/- 5 vs 37 +/- 6 pmoles/min/mg, p less than 0.01) mice. However, no significant differences in kinase activities were observed when the data were normalized and compared on a per fmole of insulin-binding activity basis for the 12-week (12 +/- 1 vs 11 +/- 2) and 24-week (27 +/- 2 vs 20 +/- 3) age groups. Insulin receptor tyrosine kinase activity of db/db skeletal muscle membranes was not different than its normal lean littermate whether expressed on a protein (34 +/- 7 vs 30 +/- 3) or fmole of insulin-binding activity (21 +/- 4 vs 18 +/- 4) basis. These data suggest that insulin receptor tyrosine kinase is not associated with the insulin resistance observed in ob/ob and db/db mice and demonstrate differences in receptor regulation between both animal models.     

The Amelioration of Hepatic Steatosis by Thyroid Hormone Receptor Agonists Is Insufficient to Restore Insulin Sensitivity in Ob/Ob Mice

Thyroid hormone receptor (TR) agonists have been proposed as therapeutic agents to treat non-alcoholic fatty liver disease (NAFLD) and insulin resistance. We investigated the ability of the TR agonists GC-1 and KB2115 to reduce hepatic steatosis in ob/ob mice. Both compounds markedly reduced hepatic triglyceride levels and ameliorated hepatic steatosis. However, the amelioration of fatty liver was not sufficient to improve insulin sensitivity in these mice and reductions in hepatic triglycerides did not correlate with improvements in insulin sensitivity or glycemic control. Instead, the effects of TR activation on glycemia varied widely and were found to depend upon the time of treatment as well as the compound and dosage used. Lower doses of GC-1 were found to further impair glycemic control, while a higher dose of the same compound resulted in substantially improved glucose tolerance and insulin sensitivity, despite all doses being equally effective at reducing hepatic triglyceride levels. Improvements in glycemic control and insulin sensitivity were observed only in treatments that also increased body temperature, suggesting that the induction of thermogenesis may play a role in mediating these beneficial effects. These data illustrate that the relationship between TR activation and insulin sensitivity is complex and suggests that although TR agonists may have value in treating NAFLD, their effect on insulin sensitivity must also be considered.     

Development of glucose intolerance and impaired plasma insulin response to glucose in obese hyperglycaemic (ob/ob) mice

The development of glucose intolerance in Aston ob/ob mice showed a gross exaggeration of the age-related changes of glucose tolerance in lean (+/+) mice. Intraperitoneal glucose tolerance in ob/ob mice was poor at 5 weeks, improved by 10 weeks, but markedly worsened by 20 weeks. A 24 hour fast further exaggerated the glucose intolerance of ob/ob mice. Unlike lean mice, tolerance improved in ob/ob mice at 40 weeks. Alterations of insulin sensitivity and the plasma insulin response to glucose accounted in part for these observations. Insulin sensitivity deteriorated until 20 weeks, but improved at 40 weeks in both fed and 24 hour fasted ob/ob mice. A positive plasma insulin response to glucose was lost after 5 weeks in fed ob/ob mice. The severity of this abnormality corresponded with the extent of the basal hyperinsulinaemia. A 24 hour fast reduced plasma insulin concentrations and restored a positive plasma insulin response to glucose in ob/ob mice. The results suggest that the plasma insulin response to glucose in ob/ob mice is related to the secretory activity of the B-cells prior to stimulation. Furthermore, it is evident that factors in addition to insulin insensitivity and the impaired plasma insulin response to glucose contribute to the development of glucose intolerance in these mice.     

ApoB100 is required for increased VLDL-triglyceride secretion by microsomal triglyceride transfer protein in ob/ob mice

Microsomal triglyceride transfer protein (Mttp) is a key player in the assembly and secretion of hepatic very low density lipoproteins (VLDL). Here we determined the effects of Mttp overexpression on hepatic triglyceride (TG) and VLDL secretion in leptin-deficient (ob/ob) mice, specifically in relation to apolipoproteinB (apoB) isoforms. We crossed Apobec1(-/-) mice with congenic ob/ob mice to generate apoB100-only ob/ob mice (A-ob/ob). The obesity phenotype in both genotypes was similar, but A-ob/ob mice had greater hepatic TG content. Administration of recombinant adenovirus expressing murine Mttp cDNA (Ad-mMTP) increased hepatic Mttp content and activity and increased hepatic VLDL-TG secretion in A-ob/ob mice. However, despite equivalent overexpression of Mttp, there was no change in VLDL-TG secretion in ob/ob mice in a wild-type Apobec1 background. Metabolic labeling studies in primary hepatocytes from A-ob/ob mice demonstrated that Ad-mMTP increased triglyceride secretion without changing the synthesis and secretion of apoB100, suggesting greater incorporation of TG into existing VLDL particles rather than increased particle number. Ad-mMTP administration failed to increase hepatic VLDL secretion in lean Apobec1(-/-) mice or controls. By contrast, VLDL secretion increased and hepatic TG content decreased following Ad-mMTP administration to human APOB transgenic mice crossed into the Apobec1(-/-) line. These findings demonstrate that Ad-mMTP increases murine hepatic VLDL-TG secretion only in the apoB100 background, and even then only in situations with either increased hepatic TG accumulation or increased apoB100 expression.     

Sucrose diet induced enzymatic and hormonal responses affecting carbohydrate, lipid and energy metabolism in two species differing in insulin availability: spiny and ob/ob mice

The low-insulin responding spiny mice (Acomys cahirinus), maintained on a 50% sucrose diet vs isocaloric regular diet, responded with an impressive increase in the activity of hepatic enzymes of glycolysis and lipogenesis and in hyperlipidemia. There was no hyperinsulinemia or hyperglycemia and spiny mice did not gain weight on sucrose due to loss of adipose tissue. Serum T3 levels rose 1.8 fold and the activity of the hepatic mitochondrial FAD-glycerol-3-phosphate oxidase became induced 2.6 fold representing the enhancement of multiple, T3-dependent, energy-consuming metabolic cycles. An increased TG lipolysis in adipose tissue was also observed. C57BL/6J ob/ob mice were markedly hyperinsulinemic and gained weight on sucrose almost as much as those on regular diet, without changes in serum glucose or insulin. Serum triglyceride level decreased, whereas liver triglycerides accumulated markedly. The extent of the increase in hepatic enzyme activities related to lipogenesis was much lower both in the ob/ob mice and their lean siblings, than in spiny mice, but the basal enzyme activities in ob/ob mice were remarkably elevated. Serum T3 level was also elevated already on the regular diet and rose only slightly on sucrose. Basal glycerol phosphate oxidase activity in ob/ob mice exceeded that in spiny mice and rose only marginally on sucrose. Adipose tissue lipolysis was not increased. Thus, sucrose diet by enhancing the T3 production appeared to activate protective mechanism against weight gain in normoinsulinemic spiny mice, whereas the full expression of these mechanisms appeared to be precluded by the hyperinsulinemia of ob/ob mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased clearance of serum retinol-binding protein and elevated levels of transthyretin in insulin-resistant ob/ob mice

Serum retinol-binding protein (RBP4) is secreted by liver and adipocytes and is implicated in systemic insulin resistance in rodents and humans. RBP4 normally binds to the larger transthyretin (TTR) homotetramer, forming a protein complex that reduces renal clearance of RBP4. To determine whether alterations in RBP4-TTR binding contribute to elevated plasma RBP4 levels in insulin-resistant states, we investigated RBP4-TTR interactions in leptin-deficient ob/ob mice and high-fat-fed obese mice (HFD). Gel filtration chromatography of plasma showed that 88-94% of RBP4 is contained within the RBP4-TTR complex in ob/ob and lean mice. Coimmunoprecipitation with an RBP4 antibody brought down stoichiometrically equal amounts of TTR and RBP4, indicating that TTR was not more saturated with RBP4 in ob/ob mice than in controls. However, plasma TTR levels were elevated approximately fourfold in ob/ob mice vs. controls. RBP4 injected intravenously in lean mice cleared rapidly, whereas the t(1/2) for disappearance was approximately twofold longer in ob/ob plasma. Urinary fractional excretion of RBP4 was reduced in ob/ob mice, consistent with increased retention. In HFD mice, plasma TTR levels and clearance of injected RBP4 were similar to chow-fed controls. Hepatic TTR mRNA levels were elevated approximately twofold in ob/ob but not in HFD mice. Since elevated circulating RBP4 causes insulin resistance and glucose intolerance in mice, these findings suggest that increased TTR or alterations in RBP4-TTR binding may contribute to insulin resistance by stabilizing RBP4 at higher steady-state concentrations in circulation. Lowering TTR levels or interfering with RBP4-TTR binding may enhance insulin sensitivity in obesity and type 2 diabetes.     

Reduced inflammatory response and increased microcirculatory disturbances during hepatic ischemia-reperfusion injury in steatotic livers of ob/ob mice

Steatosis is a major risk factor for complications after liver surgery. Since neutrophil cytotoxicity is critical for ischemia-reperfusion injury in normal livers, the aim of the present study was to evaluate whether an exaggerated inflammatory response could cause the increased injury in steatotic livers. In C57Bl/6 mice, 60 min of warm hepatic ischemia triggered a gradual increase in hepatic neutrophil accumulation during reperfusion with peak levels of 100-fold over baseline at 12 h of reperfusion. Neutrophil extravasation and a specific neutrophil-induced oxidant stress (immunostaining for hypochlorous acid-modified epitopes) started at 6 h of reperfusion and peaked at 12-24 h. Ob/ob mice, which had a severe macrovesicular steatosis, suffered significantly higher injury (alanine transaminase activity: 18,000 +/- 2,100 U/l; 65% necrosis) compared with lean littermates (alanine transaminase activity: 4,900 +/- 720 U/l; 24% necrosis) at 6 h of reperfusion. However, 62% fewer neutrophils accumulated in steatotic livers. This correlated with an attenuated increase in mRNA levels of several proinflammatory genes in ob/ob mice during reperfusion. In contrast, sham-operated ob/ob mice had a 50% reduction in liver blood flow and 35% fewer functional sinusoids compared with lean littermates. These deficiencies in liver blood flow and the microcirculation were further aggravated only in ob/ob mice during reperfusion. The attenuated inflammatory response and reduced neutrophil-induced oxidant stress observed in steatotic livers during reperfusion cannot be responsible for the dramatically increased injury in ob/ob mice. In contrast, the aggravated injury appears to be mediated by ischemic necrosis due to massive impairment of blood and oxygen supply in the steatotic livers.     

Lxralpha deficiency hampers the hepatic adaptive response to fasting in mice

Besides its well established role in control of cellular cholesterol homeostasis, the liver X receptor (LXR) has been implicated in the regulation of hepatic gluconeogenesis. We investigated the role of the major hepatic LXR isoform in hepatic glucose metabolism during the feeding-to-fasting transition in vivo. In addition, we explored hepatic glucose sensing by LXR during carbohydrate refeeding. Lxralpha(-/-) mice and their wild-type littermates were subjected to a fasting-refeeding protocol and hepatic carbohydrate fluxes as well as whole body insulin sensitivity were determined in vivo by stable isotope procedures. Lxralpha(-/-) mice showed an impaired response to fasting in terms of hepatic glycogen depletion and triglyceride accumulation. Hepatic glucose 6-phosphate turnover was reduced in 9-h fasted Lxralpha(-/-) mice as compared with controls. Although hepatic gluconeogenic gene expression was increased in 9-h fasted Lxralpha(-/-) mice compared with wild-type controls, the actual gluconeogenic flux was not affected by Lxralpha deficiency. Hepatic and peripheral insulin sensitivity were similar in Lxralpha(-/-) and wild-type mice. Compared with wild-type controls, the induction of hepatic lipogenic gene expression was blunted in carbohydrate-refed Lxralpha(-/-) mice, which was associated with lower plasma triglyceride concentrations. Yet, expression of "classic" LXR target genes Abca1, Abcg5, and Abcg8 was not affected by Lxralpha deficiency in carbohydrate-refed mice. In summary, these studies identify LXRalpha as a physiologically relevant mediator of the hepatic response to fasting. However, the data do not support a role for LXR in hepatic glucose sensing.     

Acylation-stimulating protein (ASP) deficiency induces obesity resistance and increased energy expenditure in ob/ob mice

Acylation-stimulating protein (ASP) acts as a paracrine signal to increase triglyceride synthesis in adipocytes. ASP administration results in more rapid postprandial lipid clearance. In mice, C3 (the precursor to ASP) knockout results in ASP deficiency and leads to reduced body fat and leptin levels. The protective potential of ASP deficiency against obesity and involvement of the leptin pathway were examined in ob/ob C3(-/-) double knockout mice (2KO). Compared with age-matched ob/ob mice, 2KO mice had delayed postprandial triglyceride and fatty acid clearance; associated with decreased body weight (4-17 weeks age: male: -13.7%, female: -20.6%, p < 0.0001) and HOMA (homeostasis model assessment) index (-37.7%), suggesting increased insulin sensitivity. By contrast, food intake in 2KO mice was +9.1% higher over ob/ob mice (p < 0.001, 2KO 5.1 +/- 0.2 g/day, ob/ob 4.5 +/- 0.2 g/day, wild type 2.6 +/- 0.1 g/day). The hyperphagia/leanness was balanced by a 28.5% increase in energy expenditure (oxygen consumption: 2KO, 131 +/- 8.9 ml/h; ob/ob, 102 +/- 4.5 ml/h; p < 0.01; wild type, 144 +/- 8.9 ml/h). These results suggest that the ASP regulation of energy storage may influence energy expenditure and dynamic metabolic balance.     

Acute hepatic steatosis in mice by blocking beta-oxidation does not reduce insulin sensitivity of very-low-density lipoprotein production

Accumulation of triglycerides (TG) in the liver is generally associated with hepatic insulin resistance. We questioned whether acute hepatic steatosis induced by pharmacological blockade of beta-oxidation affects hepatic insulin sensitivity, i.e., insulin-mediated suppression of VLDL production and insulin-induced activation of phosphatidylinositol 3-kinase (PI3-kinase) and PKB. Tetradecylglycidic acid (TDGA), an inhibitor of carnitine palmitoyl transferase-1 (CPT1), was used for this purpose. Male C57BL/6J mice received 30 mg/kg TDGA or its solvent intraperitoneally and were subsequently fasted for 12 h. CPT1 inhibition resulted in severe microvesicular hepatic steatosis (19.9 +/- 8.3 vs. 112.4 +/- 25.2 nmol TG/mg liver, control vs. treated, P < 0.05) with elevated plasma nonesterified fatty acid (0.68 +/- 0.25 vs. 1.21 +/- 0.41 mM, P < 0.05) and plasma TG (0.39 +/- 0.16 vs. 0.60 +/- 0.10 mM, P < 0.05) concentrations. VLDL-TG production rate was not affected on CPT1 inhibition (74.9 +/- 15.2 vs. 79.1 +/- 12.8 mumol TG.kg(-1).min(-1), control vs. treated) although treated mice secreted larger VLDL particles (59.3 +/- 3.6 vs. 66.6 +/- 4.5 nm diameter, P < 0.05). Infusion of insulin under euglycemic conditions suppressed VLDL production rate in control and treated mice by 43 and 54%, respectively, with formation of smaller VLDL particles (51.2 +/- 2.5 and 53.2 +/- 2.8 nm diameter). Insulin-induced insulin receptor substrate (IRS)1- and IRS2-associated PI3-kinase activity and PKB-phosphorylation were not affected on TDGA treatment. In conclusion, acute hepatic steatosis caused by pharmacological inhibition of beta-oxidation is not associated with reduced hepatic insulin sensitivity, indicating that hepatocellular fat content per se is not causally related to insulin resistance.     

Effects of insulin therapy on liver fat content and hepatic insulin sensitivity in patients with type 2 diabetes

We determined whether insulin therapy changes liver fat content (LFAT) or hepatic insulin sensitivity in type 2 diabetes. Fourteen patients with type 2 diabetes (age 51+/-2 yr, body mass index 33.1+/-1.4 kg/m2) treated with metformin alone received additional basal insulin for 7 mo. Liver fat (proton magnetic resonance spectroscopy), fat distribution (MRI), fat-free and fat mass, and whole body and hepatic insulin sensitivity (6-h euglycemic hyperinsulinemic clamp combined with infusion of [3-(3)H]glucose) were measured. The insulin dose averaged 75+/-10 IU/day (0.69+/-0.08 IU/kg, range 24-132 IU/day). Glycosylated hemoglobin A1c (Hb A1c) decreased from 8.9+/-0.3 to 7.4+/-0.2% (P<0.001). Whole body insulin sensitivity increased from 2.21+/-0.38 to 3.08+/-0.40 mg/kg fat-free mass (FFM).min (P<0.05). This improvement could be attributed to enhanced suppression of hepatic glucose production (HGP) by insulin (HGP 1.04+/-0.28 vs. 0.21+/-0.19 mg/kg FFM.min, P<0.01). The percent suppression of HGP by insulin increased from 72+/-8 to 105+/-11% (P<0.01). LFAT decreased from 17+/-3 to 14+/-3% (P<0.05). The change in LFAT was significantly correlated with that in hepatic insulin sensitivity (r=0.56, P<0.05). Body weight increased by 3.0+/-1.1 kg (P<0.05). Of this, 83% was due to an increase in fat-free mass (P<0.01). Fat distribution and serum adiponectin concentrations remained unchanged while serum free fatty acids decreased significantly. Conclusions: insulin therapy improves hepatic insulin sensitivity and slightly but significantly reduces liver fat content, independent of serum adiponectin.     

Anti-diabetic effect of ginsenoside Re in ob/ob mice

We evaluated the anti-diabetic effects of ginsenoside Re in adult male C57BL/6J ob/ob mice. Diabetic ob/ob mice with fasting blood glucose levels of approximately 230 mg/dl received daily intraperitoneal injections of 7, 20 and 60 mg/kg ginsenoside Re for 12 consecutive days. Dose-related effects of ginsenoside Re on fasting blood glucose levels were observed. After the 20 mg/kg treatment, fasting blood glucose levels were reduced to 188+/-9.2 and 180+/-10.8 mg/dl on Day 5 and Day 12, respectively (both P<0.01 compared to vehicle group, 229+/-9.5 and 235+/-13.4 mg/dl, respectively). The EC(70) of ginsenoside Re was calculated to be 10.3 mg/kg and was used for subsequent studies. Consistent with the reduction in blood glucose, there were significant decreases in both fed and fasting serum insulin levels in mice treated with ginsenoside Re. With 12 days of ginsenoside treatment, glucose tolerance of ob/ob mice increased significantly, and the area under the curve for glucose decreased by 17.8% (P<0.05 compared to vehicle treatment). The hypoglycemic effect of the ginsenoside persisted even at 3 days of treatment cessation (blood glucose levels: 198+/-13.1 with ginsenoside treatment vs. 253+/-20.3 mg/dl with vehicle, P<0.01). There were no significant changes in body weight or body temperature. Preliminary microarray analysis revealed differential expression of skeletal muscle genes associated with lipid metabolism and muscle function. The results suggest that ginsenoside Re may prove to be useful in treating type 2 diabetes.     

Elevated hepatic mitochondrial and peroxisomal oxidative capacities in fed and starved adult obese (ob/ob) mice.

Hepatic mitochondrial and peroxisomal oxidative capacities were studied in young (4-5 weeks old) and adult (6-9 months old) lean and obese ob/ob mice that were fed or starved for 24 or 48 h. The adult obese mice showed elevated capacity for mitochondrial oxidation (ng-atoms of O consumed/min per mg of protein) of lipid and non-lipid substrates, with the exception of pyruvate + malate, and elevated activities of citrate synthase and total carnitine palmitoyltransferase. Oxidative rates and enzyme activities were not affected by starvation of lean or obese mice, and both males and females responded similarly. Peroxisomal palmitoyl-CoA oxidation (nmol/min per mg of peroxisomal protein) was also increased in livers of adult obese mice and did not change with starvation. In young mice, hepatic mitochondrial and peroxisomal oxidative capacities in lean and obese mice were comparable. The increased mitochondrial and peroxisomal oxidative capacities appear to develop with maturation in obese ob/ob mice.     

Sustained activation of PPARα by endogenous ligands increases hepatic fatty acid oxidation and prevents obesity in ob/ob mice

Obesity, a major health concern, results from an imbalance between energy intake and expenditure. Leptin-deficient ob/ob mice are paradigmatic of obesity, resulting from excess energy intake and storage. Mice lacking acyl-CoA oxidase 1 (Acox1), the first enzyme of the peroxisomal fatty acid β-oxidation system, are characterized by increased energy expenditure and a lean body phenotype caused by sustained activation of peroxisome proliferator-activated receptor α (PPARα) by endogenous ligands in liver that remain unmetabolized in the absence of Acox1. We generated ob/ob mice deficient in Acox1 (Acox1(-/-)) to determine how the activation of PPARα by endogenous ligands might affect the obesity of ob/ob mice. In contrast to Acox1(-/-) (14.3±1.2 g at 6 mo) and the Acox1-deficient (ob/ob) double-mutant mice (23.8±4.6 g at 6 mo), the ob/ob mice are severely obese (54.3±3.2 g at 6 mo) and had significantly more (P<0.01) epididymal fat content. The resistance of Acox1(-/-)/ob/ob mice to obesity is due to increased PPARα-mediated up-regulation of genes involved in fatty acid oxidation in liver. Activation of PPARα in Acox1-deficient ob/ob mice also reduces serum glucose and insulin (P<0.05) and improves glucose tolerance and insulin sensitivity. Further, PPARα activation reduces hepatic steatosis and increases hepatocellular regenerative response in Acox1(-/-)/ob/ob mice at a more accelerated pace than in mice lacking only Acox1. However, Acox1(-/-)/ob/ob mice manifest hepatic endoplasmic reticulum (ER) stress and also develop hepatocellular carcinomas (8 of 8 mice) similar to those observed in Acox1(-/-) mice (10 of 10 mice), but unlike in ob/ob (0 of 14 mice) and OB/OB (0 of 6 mice) mice, suggesting that superimposed ER stress and PPARα activation contribute to carcinogenesis in a fatty liver. Finally, absence of Acox1 in ob/ob mice can impart resistance to high-fat diet (60% fat)-induced obesity, and their liver had significantly (P<0.01) more cell proliferation. These studies with Acox1(-/-)/ob/ob mice indicate that sustained activation of lipid-sensing nuclear receptor PPARα attenuates obesity and restores glucose homeostasis by ameliorating insulin resistance but increases the risk for liver cancer development, in part related to excess energy combustion.     

Acute inhibition of hepatic beta-oxidation in APOE*3Leiden mice does not affect hepatic VLDL secretion or insulin sensitivity

Hepatic VLDL and glucose production is enhanced in type 2 diabetes and associated with hepatic steatosis. Whether the derangements in hepatic metabolism are attributable to steatosis or to the increased availability of FA metabolites is not known. We used methyl palmoxirate (MP), an inhibitor of carnitine palmitoyl transferase I, to acutely inhibit hepatic FA oxidation and investigated whether the FAs were rerouted into VLDL secretion and whether this would affect hepatic glucose production. After an overnight fast, male APOE3*Leiden transgenic mice received an oral dose of 10 mg/kg MP. Administration of MP led to an 83% reduction in plasma beta-hydroxybutyrate (ketone body) levels compared with vehicle-treated mice (0.47 +/- 0.07 vs. 2.81 +/- 0.16 mmol/l, respectively; P < 0.01), indicative of impaired ketogenesis. Plasma FFA levels were increased by 32% and cholesterol and insulin levels were decreased by 17% and 50%, respectively, in MP-treated mice compared with controls. MP treatment led to a 30% increase in liver triglyceride (TG) content. Surprisingly, no effect on hepatic VLDL-TG production was observed between the groups at 8 h after MP administration. In addition, the capacity of insulin to suppress endogenous glucose production was unaffected in MP-treated mice compared with controls. In conclusion, acute inhibition of FA oxidation increases hepatic lipid content but does not stimulate hepatic VLDL secretion or reduce insulin sensitivity.     

Accretion of visceral fat and hepatic insulin resistance in pregnant rats

Insulin resistance (IR) is a hallmark of pregnancy. Because increased visceral fat (VF) is associated with IR in nonpregnant states, we reasoned that fat accretion might be important in the development of IR during pregnancy. To determine whether VF depots increase in pregnancy and whether VF contributes to IR, we studied three groups of 6-mo-old female Sprague-Dawley rats: 1) nonpregnant sham-operated rats (Nonpreg; n = 6), 2) pregnant sham-operated rats (Preg; n = 6), and 3) pregnant rats in which VF was surgically removed 1 mo before mating (PVF-; n = 6). VF doubled by day 19 of pregnancy (Nonpreg 5.1 +/- 0.3, Preg 10.0 +/- 1.0 g, P < 0.01), and PVF- had similar amounts of VF compared with Nonpreg (PVF- 4.6 +/- 0.8 g). Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp in late gestation in chronically catheterized unstressed rats. Glucose IR (mg.kg(-1).min(-1)) was highest in Nonpreg (19.4 +/- 2.0), lowest in Preg (11.1 +/- 1.4), and intermediate in PVF- (14.7 +/- 0.6; P < 0.001 between all groups). During the clamp, Nonpreg had greater hepatic insulin sensitivity than Preg [hepatic glucose production (HGP): Nonpreg 4.5 +/- 1.3, Preg 9.3 +/- 0.5 mg.kg(-1).min(-1); P < 0.001]. With decreased VF, hepatic insulin sensitivity was similar to nonpregnant levels in PVF- (HGP 4.9 +/- 0.8 mg.kg(-1).min(-1)). Both pregnant groups had lower peripheral glucose uptake compared with Nonpreg. In parallel with hepatic insulin sensitivity, hepatic triglyceride content was increased in pregnancy (Nonpreg 1.9 +/- 0.4 vs. Preg 3.2 +/- 0.3 mg/g) and decreased with removal of VF (PVF- 1.3 +/- 0.4 mg/g; P < 0.05). Accretion of visceral fat is an important component in the development of hepatic IR in pregnancy, and accumulation of hepatic triglycerides is a mechanism by which visceral fat may modulate insulin action in pregnancy.