Leptin deficiency per se dictates body composition and insulin action in ob/ob mice.

Obese humans are often insulin- and leptin resistant. Since leptin can affect glucose metabolism, it is conceivable that a lack of leptin signal transduction contributes to insulin resistance. It remains unclear whether leptin affects glucose metabolism via peripheral and/or central mechanistic routes. In the present study, we aimed: (i) to determine the relative contributions of lack of leptin signal transduction and adiposity to insulin resistance and (ii) to establish the impact of central leptin action on glucose metabolism. To address the first point, ob/ob mice were subjected to severe calorie restriction, so that their body weight became similar to that of wild-type mice. Insulin sensitivity was measured in obese ob/ob, lean (food restricted) ob/ob and lean, weight-matched wild-type mice. To address the second point, leptin (or vehicle) was i.c.v. infused to the lateral cerebral ventricle of ob/ob mice and insulin sensitivity was determined. Hyperinsulinaemic euglyceamic clamps were used to quantify insulin sensitivity. Food restriction barely affected body composition, although it profoundly curtailed body weight. Insulin suppressed hepatic glucose production (HGP) to a greater extent in lean ob/ob than in obese ob/ob mice, but its impact remained considerably less than in wild-type mice (% suppression: 11.8 +/- 8.9 versus 1.3 +/- 1.1 versus 56.6 +/- 13.0%/nmol, for lean, obese ob/ob and wild-type mice, respectively; P < 0.05). The insulin-mediated glucose disposal (GD) of lean ob/ob mice was also in between that of obese ob/ob and wild-type mice (37.5 +/- 21.4 versus 25.1 +/- 14.6 versus 59.6 +/- 17.3 mumol/min/kg/nmol of insulin, respectively; P < 0.05 wild-type versus obese ob/ob mice). Leptin infusion acutely enhanced both hepatic insulin sensitivity (insulin-induced inhibition of HGP) and insulin-mediated GD (9.1 +/- 2.4 versus 5.0 +/- 2.7%/nmol of insulin, and 25.6 +/- 5.6 versus 13.6 +/- 4.8 mumol/min/kg/nmol of insulin, respectively; P < 0.05 for both comparisons) in ob/ob mice. Both a lack of leptin signals and adiposity may contribute to insulin resistance in obese individuals. Diminution of central leptin signalling can critically affect glucose metabolism in these individuals.

Dynamics of insulin signalling in liver during hyperinsulinemic euglycaemic clamp conditions in vivo and the effects of high-fat feeding in male mice.

Insulin is an important regulator of hepatic carbohydrate, lipid, and protein metabolism, and the regulation of these processes by insulin is disturbed under conditions of insulin resistance and type 2 diabetes. Despite these alterations, the impact of insulin resistance on insulin signalling in the liver is not well defined. Variations in time and dose of insulin stimulation as well as plasma glucose levels may underlie this. The present study aimed at determining the dynamics of activation of hepatic insulin signalling in vivo at insulin concentrations resembling those achieved after a meal, and addressing the effects of high-fat feeding. An unexpected finding of this study was the biphasic activation pattern of the IRS-PI3K-PKB/Akt pathway. Our findings indicate that the first burst of activation contributes to regulation of glucose metabolism. The physiological function of the second peak is still unknown, but may involve regulation of protein synthesis. Finally, high-fat feeding caused hepatic insulin resistance, as illustrated by a reduced suppression of hepatic glucose production. A sustained increased phosphorylation of the serine/threonine kinases p70S6kinase and Jun N-terminal kinase in the absence of insulin may underlie the abrogated phosphorylation of the IRS proteins and their downstream targets.

Sustained activation of the mammalian target of rapamycin nutrient sensing pathway is associated with hepatic insulin resistance, but not with steatosis, in mice.

AIMS/HYPOTHESIS: Activation of nutrient sensing through mammalian target of rapamycin (mTOR) has been linked to the pathogenesis of insulin resistance. We examined activation of mTOR-signalling in relation to insulin resistance and hepatic steatosis in mice. MATERIALS AND METHODS: Chronic hepatic steatosis and hepatic insulin resistance were induced by high-fat feeding of male C57BL/6Jico mice for 6 weeks. In addition, acute hepatic steatosis in the absence of insulin resistance was induced by pharmacological blockade of beta-oxidation using tetradecylglycidic acid (TDGA). mTOR signalling was examined in liver homogenates. RESULTS: High-fat feeding caused obesity (p<0.001), hepatic steatosis (p<0.05) and hepatic insulin resistance (p<0.05). The phosphorylation of mTOR and its downstream targets p70S6 kinase and S6 ribosomal protein was two-fold higher in mice on a high-fat diet than in mice fed standard chow (all p<0.05) and associated with enhanced rates of protein synthesis. Acute induction of hepatic steatosis with TDGA had no effect on mTOR activity. The increased activity of the mTOR pathway in livers from mice on a high-fat diet could not be ascribed to diet-induced alterations in known modulators of mTOR activity such as circulating plasma leucine levels, phosphorylation of protein kinase B and AMP-activated protein kinase, and changes in mitochondrial function. CONCLUSIONS/INTERPRETATION: High-fat diet induces increase of the mTOR nutrient sensing pathway in association with hepatic insulin resistance, but not with hepatic lipid accumulation as such.

Intracerebroventricular administration of melanotan II increases insulin sensitivity of glucose disposal in mice.

AIMS/HYPOTHESIS: The present study was conducted to evaluate the effects of central administration of melanotan II (MTII), a melanocortin-3/4 receptor agonist, on hepatic and whole-body insulin sensitivity, independent of food intake and body weight. METHODS: Over a period of 24 h, 225 ng of MTII was injected in three aliquots into the left lateral ventricle of male C57Bl/6 mice. The animals had no access to food. The control group received three injections of distilled water. Whole-body and hepatic insulin sensitivity were measured by hyperinsulinaemic-euglycaemic clamp in combination with [(3)H]glucose infusion. Glut4 mRNA expression was measured in skeletal muscle. RESULTS: Plasma glucose and insulin concentrations under basal and hyperinsulinaemic conditions were similar in MTII- and placebo-treated mice. Endogenous glucose production (EGP) and glucose disposal in the basal state were significantly higher in MTII-treated mice than in the control group (71+/-22 vs 43+/-12 micromol.min(-1).kg(-1), p<0.01). During hyperinsulinaemia, glucose disposal was significantly higher in MTII-treated mice (151+/-20 vs 108+/-20 micromol.min(-1).kg(-1), p<0.01). In contrast, the inhibitory effect of insulin on EGP was not affected by MTII (relative decrease in EGP: 45+/-27 vs 50+/-20%). Glut4 mRNA expression in skeletal muscle was significantly increased in MTII-treated mice (307+/-94 vs 100+/-56%, p<0.01). CONCLUSIONS/INTERPRETATION: Intracerebroventricular administration of MTII acutely increases insulin-mediated glucose disposal but does not affect the capacity of insulin to suppress EGP in C57Bl/6 mice. These data indicate that central stimulation of melanocortin-3/4 receptors modulates insulin sensitivity in a tissue-specific manner, independent of its well-known impact on feeding and body weight.

High fat diet induced hepatic insulin resistance is not related to changes in hypothalamic mRNA expression of NPY, AgRP, POMC and CART in mice.

The hypothalamic circuitry, apart from its impact on food intake, modulates insulin sensitivity to adapt metabolic conditions in the face of environmental fluctuations in nutrient availability. The purpose of the present study was to investigate the effects of 2 weeks high fat feeding in wildtype mice on (1) insulin sensitivity and triglyceride accumulation in liver and muscle in relation to (2) mRNA expression levels of Neuropeptide Y (NPY), Agouti-related protein (AgRP), pro-opiomelanocortin (POMC), and cocaine- and amphetamine-regulated transcript (CART) in the hypothalamus. Two weeks of high fat feeding induced hepatic insulin resistance in the presence of increased hepatic triglyceride accumulation. In muscle, however, 2 weeks of high fat feeding did not result in changes in insulin sensitivity or in triglyceride content. mRNA expression levels of NPY, AgRP, POMC, and CART in the hypothalamus were not different between the groups. This study shows that 2 weeks of high fat feeding in mice does not affect mRNA expression levels of NPY, AgRP, POMC or CART, in the whole hypothalamus, despite induction of hepatic, but not peripheral, insulin resistance. Therefore, a major physiological role of these neuroendocrine factors in the induction of hepatic insulin resistance during a high fat diet seems less likely.

Hepatic steatosis: a mediator of the metabolic syndrome. Lessons from animal models.

Epidemiological studies in humans, as well as experimental studies in animal models, have shown an association between visceral obesity and dyslipidemia, insulin resistance, and type 2 diabetes mellitus. Recently, attention has been focused on the excessive accumulation of triglycerides (TG) in the liver as part of this syndrome. In this review, important principles of the pathophysiological involvement of the liver in the metabolic syndrome obtained in rodent models are summarized. We focus on non-alcoholic causes of steatosis, because the animal experiments we refer to did not include alcohol as an experimental condition. In general, there is continuous cycling and redistribution of non-oxidized fatty acids between different organs. The amount of TG in an intrinsically normal liver is not fixed but can readily be increased by nutritional, metabolic, and endocrine interactions involving TG/free fatty acid (FFA) partitioning and TG/FFA metabolism. Several lines of evidence indicate that hepatic TG accumulation is also a causative factor involved in hepatic insulin resistance. Complex interactions between endocrine, metabolic, and transcriptional pathways are involved in TG-induced hepatic insulin resistance. Therefore, the liver participates passively and actively in the metabolic derangements of the metabolic syndrome. We speculate that similar mechanisms may also be involved in human pathophysiology.

White adipose tissue: getting nervous.

Neuroendocrine research has altered the traditional perspective of white adipose tissue (WAT) as a passive store of triglycerides. In addition to fatty acids, WAT produces many hormones and can therefore be designated as a traditional endocrine gland actively participating in the integrative physiology of fuel and energy metabolism, eating behaviour and the regulation of hormone secretion and sensitivity. WAT is controlled by humoral factors, para- and intracrine factors and by neural regulation. Sympathetic nerve fibres innervate WAT and stimulate lipolysis, leading to the release of glycerol and free fatty acids. In addition, recent research in rats has clearly shown a functional parasympathetic innervation of WAT. There appears to be a distinct somatotopy within the parasympathetic nuclei: separate sets of autonomic neurones in the brain stem innervate either the visceral or the subcutaneous fat compartment. We therefore propose that the central nervous system (CNS) plays a major role in the hitherto unexplained regulation of body fat distribution. Parasympathectomy induces insulin resistance with respect to glucose and fatty acid uptake in the innervated fat depot and has selective effects on local hormone synthesis. Thus, the CNS is involved not only in the regulation of hormone production by WAT, but also in its hormone sensitivity. The developments in this research area are likely to increase our insights in the pathogenesis of metabolic disorders such as hypertriglyceridemia, diabetes mellitus type 2 and lipodystrophy syndromes.

Protection from obesity and insulin resistance in mice overexpressing human apolipoprotein C1.

Apolipoprotein (APO) C1 is a 6.6-kDa protein present in plasma and associated with lipoproteins. Using hyperinsulinemic-euglycemic clamp tests, we previously found that in APOC1 transgenic mice, the whole-body insulin-mediated glucose uptake is increased concomitant with a decreased fatty acid uptake. These latter results are confirmed in the present study, showing that APOC1 transgenic mice exhibit a 50% reduction in the uptake of the fatty acid analog 15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid in white adipose tissue stores. We next investigated whether APOC1 overexpression can modulate the initiation and/or development of obesity and insulin resistance. When crossbred on the genetically obese ob/ob background, APOC1 transgenic mice were fully protected from the development of obesity compared with ob/ob only mice, as reflected by a strong reduction in body weight (21 +/- 4 vs. 44 +/- 7 g), total adipose tissue stores (15 +/- 3 vs. 25 +/- 3% body wt), and average adipocyte size (7,689 +/- 624 vs. 15,295 +/- 1,289 microm(2)). Although less pronounced, APOC1 overexpression also reduced body weight on a wild-type background, solely due to a reduction in adipose tissue. Furthermore, despite elevated plasma free fatty acid and triglyceride levels, APOC1 overexpression significantly improved insulin sensitivity in ob/ob mice, as demonstrated by a strong reduction in plasma glucose and insulin levels, as well as a better performance in the glucose tolerance test. In conclusion, a marked reduction in the uptake of fatty acids into adipocytes may underlie the protection from obesity and insulin resistance in transgenic mice overexpressing human APOC1.

In muscle-specific lipoprotein lipase-overexpressing mice, muscle triglyceride content is increased without inhibition of insulin-stimulated whole-body and muscle-specific glucose uptake.

In patients with type 2 diabetes, a strong correlation between accumulation of intramuscular triclycerides (TGs) and insulin resistance has been found. The aim of the present study was to determine whether there is a causal relation between intramuscular TG accumulation and insulin sensitivity. Therefore, in mice with muscle-specific overexpression of human lipoprotein lipase (LPL) and control mice, muscle TG content was measured in combination with glucose uptake in vivo, under hyperinsulinemic-euglycemic conditions. Overexpression of LPL in muscle resulted in accumulation of TGs in skeletal muscle (85.5 +/- 33.3 vs. 25.7 +/- 23.1 micromol/g tissue in LPL and control mice, respectively; P < 0.05). During the hyperinsulinemic clamp study, there were no differences in plasma glucose, insulin, and FFA concentrations between the two groups. Moreover, whole-body, as well as skeletal muscle, insulin-mediated glucose uptake did not differ between LPL-overexpressing and wild-type mice. Surprisingly, whole-body glucose oxidation was decreased by approximately 60% (P < 0.05), whereas nonoxidative glucose disposal was increased by approximately 50% (P < 0.05) in LPL-overexpressing versus control mice. In conclusion, overexpression of human LPL in muscle increases intramuscular TG accumulation, but does not affect whole-body or muscle-specific insulin-mediated uptake, findings that argue against a simple causal relation between intramuscular TG content and insulin resistance.

Down-regulation of intestinal scavenger receptor class B, type I (SR-BI) expression in rodents under conditions of deficient bile delivery to the intestine.

Scavenger receptor class B, type I (SR-BI) is expressed in the intestines of rodents and has been suggested to be involved in the absorption of dietary cholesterol. The aim of this study was to determine whether intestinal SR-BI expression is affected in animal models with altered bile delivery to the intestine and impaired cholesterol absorption. SR-BI protein and mRNA levels were determined in proximal and distal small intestine from control, bile-duct-ligated and bile-diverted rats and from control and bile-duct-ligated mice. Two genetically altered mouse models were studied: multidrug resistance-2 P-glycoprotein-deficient [Mdr2((-/-))] mice that produce phospholipid/cholesterol-free bile, and cholesterol 7alpha-hydroxylase-deficient [Cyp7a((-/-))] mice, which exhibit qualitative and quantitative changes in the bile-salt pool. Cholesterol-absorption efficiency was quantified using a dual-isotope ratio method. SR-BI was present at the apical membrane of enterocytes in control rats and mice and was more abundant in proximal than in distal segments of the intestine. In bile-duct-ligated animals, levels of SR-BI protein were virtually absent and mRNA levels were decreased by approximately 50%. Bile-diverted rats, Mdr2((-/-)) mice and Cyp7a((-/-)) mice showed decreased levels of intestinal SR-BI protein while mRNA levels were unaffected. Cholesterol absorption was reduced by >90% in bile-duct-ligated and bile-diverted animals and in Cyp7a((-/-)) mice, whereas Mdr2((-/-)) mice showed an approximately 50% reduction. This study shows that SR-BI is expressed at the apical membrane of enterocytes of rats and mice, mainly in the upper intestine where cholesterol absorption is greatest, and indicates that bile components play a role in post-transcriptional regulation of SR-BI expression. Factors associated with cholestasis appear to be involved in transcriptional control of intestinal SR-BI expression. The role of SR-BI in the cholesterol-absorption process remains to be defined.

Dietary cholesterol does not normalize low plasma cholesterol levels but induces hyperbilirubinemia and hypercholanemia in Mdr2 P-glycoprotein-deficient mice.

BACKGROUND/AIMS: Mdr2 P-glycoprotein deficiency in mice (Mdr2(-/-) leads to formation of cholesterol/cholesterol-depleted bile and reduced plasma HDL cholesterol. We addressed the questions: (1) does HDL in Mdr2(-/-) mice normalize upon phospholipid and/or cholesterol feeding, and (2): is the Mdr2(-/-) liver capable of handling excess dietary cholesterol. METHODS: Male and female Mdr2(-/-) and Mdr2(+/+) mice were fed diets with or without additional phosphatidylcholine and/or cholesterol. Plasma, hepatic and biliary lipids as well as liver function parameters and expression of transport proteins involved in bile formation were analyzed. RESULTS: Feeding excess phospholipids and/or cholesterol did not affect lipoprotein levels in Mdr2(+/+) or Mdr2(-/+) mice. Dietary cholesterol caused hyperbilirubinemia (male +100%; female +500%) and elevated plasma bile salts (male +200%; female +1250%) in Mdr2(-/-) mice only, independent of phospholipids. Bile flow nor biliary bile salt and bilirubin secretion were affected in cholesterol-fed Mdr2(-/-) mice. Elevated plasma bile salts may be related to cholesterol-induced reduction of hepatic Na+-taurocholate cotransporting protein expression in Mdr2(-/-) mice. CONCLUSION: Excess dietary phospholipids and cholesterol do not normalize low HDL associated with Mdr2 P-glycoprotein-deficiency. Induction of hyperbilirubinemia and hypercholanemia by dietary cholesterol in Mdr2(-/-) mice delineates the important role of biliary lipid secretion in normal hepatic functioning.

Postprandial chylomicron formation and fat absorption in multidrug resistance gene 2 P-glycoprotein-deficient mice.

BACKGROUND & AIMS: It has been proposed that biliary phospholipids fulfill specific functions in the absorption of dietary fat from the intestine, but the physiological significance has not been established. The aim of this study was to evaluate the role of biliary phospholipids in dietary fat absorption in vivo by using mice homozygous or heterozygous for disruption of the Mdr2 gene (Mdr2((-/-)), Mdr2((+/-))) and control (Mdr2((+/+))) mice. Mdr2((-/-)) mice do not secrete phospholipids and cholesterol into bile, and bile salt secretion is not impaired. Mdr2((+/-)) mice show only impaired (-40%) phospholipid secretion. METHODS: Methods included an analysis of time dependency of intestinal uptake and plasma appearance of intragastrically administered (radiolabeled) triglycerides and measurement of 3-day fecal fat balance with low- and high-fat diets. RESULTS: Intragastric administration of olive oil resulted in a rapid increase in plasma triglycerides in Mdr2((+/+)) and Mdr2((+/-)) but not in Mdr2((-/-)) mice. The "postprandial response" of plasma triglycerides could be partially restored in Mdr2((-/-)) mice by intraduodenal infusion of whole rat bile. After intragastric [(3)H]triolein administration in Triton WR1339-pretreated animals, the appearance of (3)H-triglycerides in plasma was reduced by 70% in Mdr2((-/-)) compared with Mdr2((+/+)) mice, excluding accelerated lipolysis as the cause of defective triglyceride response in Mdr2((-/-)) mice. (3)H-triglycerides accumulated in enterocytes in Mdr2((-/-)) mice. Surprisingly, the efficacy of fat absorption as derived from balance studies was not affected and was only minimally affected in Mdr2((-/-)) mice fed low (14 energy percent)- and high (35 energy percent)-fat diets, respectively (all >95%). CONCLUSIONS: The results show that biliary lipid secretion is necessary for postprandial appearance in plasma of chylomicrons in vivo but not for quantitative absorption of dietary lipids.

Apolipoprotein E participates in the regulation of very low density lipoprotein-triglyceride secretion by the liver.

ApoE-deficient mice on low fat diet show hepatic triglyceride accumulation and a reduced very low density lipoprotein (VLDL) triglyceride production rate. To establish the role of apoE in the regulation of hepatic VLDL production, the human APOE3 gene was introduced into apoE-deficient mice by cross-breeding with APOE3 transgenics (APOE3/apoe-/- mice) or by adenoviral transduction. APOE3 was expressed in the liver and, to a lesser extent, in brain, spleen, and lung of transgenic APOE3/apoe-/- mice similar to endogenous apoe. Plasma cholesterol levels in APOE/apoe-/- mice (3.4 +/- 0.5 mM) were reduced when compared with apoe-/- mice (12.6 +/- 1.4 mM) but still elevated when compared with wild type control values (1.9 +/- 0.1 mM). Hepatic triglyceride accumulation in apoE-deficient mice was completely reversed by introduction of the APOE3 transgene. The in vivo hepatic VLDL-triglyceride production rate was reduced to 36% of control values in apoE-deficient mice but normalized in APOE3/apoe-/- mice. Hepatic secretion of apoB was not affected in either of the strains. Secretion of (3)H-labeled triglycerides synthesized from [(3)H]glycerol by cultured hepatocytes from apoE-deficient mice was four times lower than by APOE3/apoe-/- or control hepatocytes. The average size of secreted VLDL particles produced by cultured apoE-deficient hepatocytes was significantly reduced when compared with those of APOE3/apoe-/- and wild type mice. Hepatic expression of human APOE3 cDNA via adenovirus-mediated gene transfer in apoE-deficient mice resulted in a reduction of plasma cholesterol depending on plasma apoE3 levels. The in vivo VLDL-triglyceride production rate in these mice was increased up to 500% compared with LacZ-injected controls and correlated with the amount of apoE3 per particle. These findings indicate a regulatory role of apoE in hepatic VLDL-triglyceride secretion, independent from its role in lipoprotein clearance.

Biliary phospholipid secretion is not required for intestinal absorption and plasma status of linoleic acid in mice.

Biliary phospholipids have been hypothesized to be important for essential fatty acid homeostasis. We tested this hypothesis by investigating the intestinal absorption and the status of linoleic acid in mdr2 Pgp-deficient mice which secrete phospholipid-free bile. In mice homozygous (-/-) for disruption of the mdr2 gene and wild-type (+/+) mice, dietary linoleic acid absorption was determined by 72 h balance techniques. After enteral administration, [(13)C]-linoleic acid absorption was determined by measuring [(13)C]-linoleic acid concentrations in feces and in plasma. The status of linoleic acid was determined in plasma and in liver by calculating the molar percentage of linoleic acid and the triene:tetraene ratio. Although plasma concentration of [(13)C]-linoleic acid at 2 h after enteral administration was significantly lower in (-/-) compared to (+/+) mice (P

Reduced plasma cholesterol and increased fecal sterol loss in multidrug resistance gene 2 P-glycoprotein-deficient mice.

BACKGROUND & AIMS: mdr2 P-glycoprotein (Pgp) deficiency in mice leads to the absence of biliary phospholipids and cholesterol in the presence of normal bile salt secretion. The aim of this study was to evaluate the importance of the biliary pathway in cholesterol homeostasis by determining the effects of mdr2 Pgp deficiency on hepatic and plasma lipid levels and cholesterol kinetics in chow-fed mice. METHODS: Hepatic lipid content, enzyme activities, plasma lipoprotein levels, and fecal sterol excretion were measured in wild-type (+/+) and mdr2 Pgp-deficient (-/-) mice. Cholesterol kinetics were determined using radiotracer techniques. RESULTS: No differences in hepatic lipid content were observed between (-/-) and (+/+) mice. Plasma high-density lipoprotein cholesterol and apolipoprotein A-I levels were strongly reduced in (-/-) mice compared with controls, whereas the apolipoprotein B contents of very-low-density lipoprotein and low-density lipoprotein were increased. Hepatic activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase was threefold greater in (-/-) mice than in controls; however, compartmental analysis of plasma cholesterol decay showed no differences in cholesterol synthesis between (-/-) and (+/+) mice. A dual isotope approach for estimating cholesterol absorption yielded approximately 50% lower values in (-/-) mice than in controls. Surprisingly, (-/-) mice showed a fourfold increase in fecal neutral sterol secretion. CONCLUSIONS: This study unequivocally establishes the important direct role of biliary lipids in the regulation of plasma lipid levels in mice.