Endothelial cell leptin receptor mutant mice have hyperleptinemia and reduced tissue uptake

Hyperleptinemia is usually associated with obesity and leptin resistance. Endothelial cell leptin receptor knockout (ELKO) mice without a signaling membrane‐bound leptin receptor in endothelia, however, have profound hyperleptinemia without signs of leptin resistance. Leptin mRNA in adipose tissue was unchanged. To test the hypothesis that the ELKO mutation results in delayed degradation and slowed excretion, we determined the kinetics of leptin transfer in groups of ELKO and wildtype mice after intravenous bolus injection of ¹²⁵I‐leptin and the reference substance ¹³¹I‐albumin. The degradation pattern of ¹²⁵I‐leptin in serum and brain homogenates at different time points between 10 and 60 min was measured by HPLC and acid precipitation. Although ELKO mice had reduced uptake of ¹²⁵I‐leptin uptake by the brain and several peripheral organs, leptin was more stable in blood and tissue. There was no change in the rate of renal excretion. ELISA showed that serum soluble leptin receptor, known to antagonize leptin transport, had a 400‐fold increase, probably contributing to the hyperleptinemia and reduced tissue uptake. Thus, the ELKO mutation unexpectedly increased the stability of leptin but suppressed its tissue uptake. These changes probably contribute to the known partial resistance of the ELKO mice to diet‐induced obesity. J. Cell. Physiol. 228: 1610–1616, 2013. © 2013 Wiley Periodicals, Inc.     

Hyperleptinemia Is Required for the Development of Leptin Resistance

Leptin regulates body weight by signaling to the brain the availability of energy stored as fat. This negative feedback loop becomes disrupted in most obese individuals, resulting in a state known as leptin resistance. The physiological causes of leptin resistance remain poorly understood. Here we test the hypothesis that hyperleptinemia is required for the development of leptin resistance in diet-induced obese mice. We show that mice whose plasma leptin has been clamped to lean levels develop obesity in response to a high-fat diet, and the magnitude of this obesity is indistinguishable from wild-type controls. Yet these obese animals with constant low levels of plasma leptin remain highly sensitive to exogenous leptin even after long-term exposure to a high fat diet. This shows that dietary fats alone are insufficient to block the response to leptin. The data also suggest that hyperleptinemia itself can contribute to leptin resistance by downregulating cellular response to leptin as has been shown for other hormones.     

Effects of cell-type specific leptin receptor mutation on leptin transport across the BBB

The functions of leptin receptors (LRs) are cell-type specific. At the blood-brain barrier, LRs mediate leptin transport that is essential for its CNS actions, and both endothelial and astrocytic LRs may be involved. To test this, we generated endothelia specific LR knockout (ELKO) and astrocyte specific LR knockout (ALKO) mice. ELKO mice were derived from a cross of Tie2-cre recombinase mice with LR-floxed mice, whereas ALKO mice were generated by a cross of GFAP-cre with LR-floxed mice, yielding mutant transmembrane LRs without signaling functions in endothelial cells and astrocytes, respectively. The ELKO mutation did not affect leptin half-life in blood or apparent influx rate to the brain and spinal cord, though there was an increase of brain parenchymal uptake of leptin after in situ brain perfusion. Similarly, the ALKO mutation did not affect blood-brain barrier permeation of leptin or its degradation in blood and brain. The results support our observation from cellular studies that membrane-bound truncated LRs are fully efficient in transporting leptin, and that basal levels of astrocytic LRs do not affect leptin transport across the endothelial monolayer. Nonetheless, the absence of leptin signaling at the BBB appears to enhance the availability of leptin to CNS parenchyma. The ELKO and ALKO mice provide new models to determine the dynamic regulation of leptin transport in metabolic and inflammatory disorders where cellular distribution of LRs is shifted.     

Socs3 deficiency in the brain elevates leptin sensitivity and confers resistance to diet-induced obesity

Leptin is an adipocyte-derived hormone that plays a key role in energy homeostasis, yet resistance to leptin is a feature of most cases of obesity in humans and rodents. In vitro analysis suggested that the suppressor of cytokine signaling-3 (Socs3) is a negative-feedback regulator of leptin signaling involved in leptin resistance. To determine the functional significance of Socs3 in vivo, we generated neural cell-specific SOCS3 conditional knockout mice using the Cre-loxP system. Compared to their wild-type littermates, Socs3-deficient mice showed enhanced leptin-induced hypothalamic Stat3 tyrosine phosphorylation as well as pro-opiomelanocortin (POMC) induction, and this resulted in a greater body weight loss and suppression of food intake. Moreover, the Socs3-deficient mice were resistant to high fat diet-induced weight gain and hyperleptinemia, and insulin-sensitivity was retained. These data indicate that Socs3 is a key regulator of diet-induced leptin as well as insulin resistance. Our study demonstrates the negative regulatory role of Socs3 in leptin signaling in vivo, and thus suppression of Socs3 in the brain is a potential therapy for leptin-resistance in obesity.     

Enhanced leptin sensitivity and attenuation of diet-induced obesity in mice with haploinsufficiency of Socs3

Leptin is an adipocyte-derived hormone that regulates energy balance and neuroendocrine function primarily by acting on specific hypothalamic pathways. Resistance to the weight reducing effects of leptin is a feature of most cases of human and rodent obesity, yet the molecular basis of leptin resistance is poorly understood. We have previously identified suppressor of cytokine signaling-3 (Socs3) as a leptin-induced negative regulator of leptin receptor signaling and potential mediator of leptin resistance. However, due to the non-viability of mice with targeted disruption of Socs3 (ref. 6), the importance of Socs3 in leptin action in vivo was unclear. To determine the functional significance of Socs3 in energy balance in vivo we undertook studies in mice with heterozygous Socs3 deficiency (Socs3(+/-)). We report here that Socs3(+/-) mice display greater leptin sensitivity than wild-type control mice: Socs3(+/-) mice show both enhanced weight loss and increased hypothalamic leptin receptor signaling in response to exogenous leptin administration. Furthermore, Socs3(+/-) mice are significantly protected against the development of diet-induced obesity and associated metabolic complications. The level of Socs3 expression is thus a critical determinant of leptin sensitivity and obesity susceptibility in vivo and this molecule is a potential target for therapeutic intervention.     

Low density lipoprotein (LDL) receptor-related protein 6 (LRP6) regulates body fat and glucose homeostasis by modulating nutrient sensing pathways and mitochondrial energy expenditure

Body fat, insulin resistance, and type 2 diabetes are often linked together, but the molecular mechanisms that unify their association are poorly understood. Wnt signaling regulates adipogenesis, and its altered activity has been implicated in the pathogenesis of type 2 diabetes and metabolic syndrome. LRP6(+/-) mice on a high fat diet were protected against diet-induced obesity and hepatic and adipose tissue insulin resistance compared with their wild-type (WT) littermates. Brown adipose tissue insulin sensitivity and reduced adiposity of LRP6(+/-) mice were accounted for by diminished Wnt-dependent mTORC1 activity and enhanced expression of brown adipose tissue PGC1-α and UCP1. LRP6(+/-) mice also exhibited reduced endogenous hepatic glucose output, which was due to diminished FoxO1-dependent expression of the key gluconeogenic enzyme glucose-6-phosphatase (G6pase). In addition, in vivo and in vitro studies showed that loss of LRP6 allele is associated with increased leptin receptor expression, which is a likely cause of hepatic insulin sensitivity in LRP6(+/-) mice. Our study identifies LRP6 as a nutrient-sensitive regulator of body weight and glucose metabolism and as a potential target for pharmacological interventions in obesity and diabetes.     

Altered food consumption in mice lacking lysophosphatidic acid receptor-1

The release of lysophosphatidic acid (LPA) by adipocytes has previously been proposed to play a role in obesity and associated pathologies such as insulin resistance and diabetes. In the present work, the sensitivity to diet-induced obesity was studied in mice lacking one of the LPA receptor subtype (LPA1R). Conversely to what was observed in wild type (WT) mice, LPA1R-KO-mice fed a high fat diet (HFD) showed no significant increase in body weight or fat mass when compared to low fat diet (LFD). In addition, in contrast to what was observed in WT mice, LPA1R-KO mice did not exhibit over-consumption of food associated with HFD. Surprisingly, when fed a LFD, LPA1R-KO mice exhibited significant higher plasma leptin concentration and higher level of adipocyte leptin mRNA than WT mice. In conclusion, LPA1R-KO mice were found to be resistant to diet-induced obesity consecutive to a resistance to fat-induced over-consumption of food that may result at least in part from alterations in leptin expression and production.     

Mice Lacking C1q Are Protected from High Fat Diet-induced Hepatic Insulin Resistance and Impaired Glucose Homeostasis

Complement activation is implicated in the development of obesity and insulin resistance, and loss of signaling by the anaphylatoxin C3a prevents obesity-induced insulin resistance in mice. Here we have identified C1q in the classical pathway as required for activation of complement in response to high fat diets. After 8 weeks of high fat diet, wild-type mice became obese and developed glucose intolerance. This was associated with increased apoptotic cell death and accumulation of complement activation products (C3b/iC3b/C3c) in liver and adipose tissue. Previous studies have shown that high fat diet-induced apoptosis is dependent on Bid; here we report that Bid-mediated apoptosis was required for complement activation in adipose and liver. Although C1qa deficiency had no effect on high fat diet-induced apoptosis, accumulation of complement activation products and the metabolic complications of high fat diet-induced obesity were dependent on C1q. When wild-type mice were fed a high fat diet for only 3 days, hepatic insulin resistance was associated with the accumulation of C3b/iC3b/C3c in the liver. Mice deficient in C3a receptor were protected against this early high fat diet-induced hepatic insulin resistance, whereas mice deficient in the negative complement regulator CD55/DAF were more sensitive to the high fat diet. C1qa^−/− mice were also protected from high fat diet-induced hepatic insulin resistance and complement activation. Evidence of complement activation was also detected in adipose tissue of obese women compared with lean women. Together, these studies reveal an important role for C1q in the classical pathway of complement activation in the development of high fat diet-induced insulin resistance.     

Elevated hypothalamic TCPTP in obesity contributes to cellular leptin resistance

In obesity, anorectic responses to leptin are diminished, giving rise to the concept of "leptin resistance." Increased expression of protein tyrosine phosphatase 1B (PTP1B) has been associated with the attenuation of leptin signaling and development of cellular leptin resistance. Here we report that hypothalamic levels of the tyrosine phosphatase TCPTP are also elevated in obesity to attenuate the leptin response. We show that mice that lack TCPTP in neuronal cells have enhanced leptin sensitivity and are resistant to high-fat-diet-induced weight gain and the development of leptin resistance. Also, intracerebroventricular administration of a TCPTP inhibitor enhances leptin signaling and responses in mice. Moreover, the combined deletion of TCPTP and PTP1B in neuronal cells has additive effects in the prevention of diet-induced obesity. Our results identify TCPTP as a critical negative regulator of hypothalamic leptin signaling and causally link elevated TCPTP to the development of cellular leptin resistance in obesity.     

Comparison of the obesity phenotypes related to monosodium glutamate effect on arcuate nucleus and/or the high fat diet feeding in C57BL/6 and NMRI mice

In this study, susceptibility of inbred C57BL/6 and outbred NMRI mice to monosodium glutamate (MSG) obesity or diet-induced obesity (DIO) was compared in terms of food intake, body weight, adiposity as well as leptin, insulin and glucose levels. MSG obesity is an early-onset obesity resulting from MSG-induced lesions in arcuate nucleus to neonatal mice. Both male and female C57BL/6 and NMRI mice with MSG obesity did not differ in body weight from their lean controls, but had dramatically increased fat to body weight ratio. All MSG obese mice developed severe hyperleptinemia, more remarkable in females, but only NMRI male mice showed massive hyperinsulinemia and an extremely high HOMA index that pointed to development of insulin resistance. Diet-induced obesity is a late-onset obesity; it developed during 16-week-long feeding with high-fat diet containing 60 % calories as fat. Inbred C57BL/6 mice, which are frequently used in DIO studies, both male and female, had significantly increased fat to body weight ratio and leptin and glucose levels compared with their appropriate lean controls, but only female C57BL/6 mice had also significantly elevated body weight and insulin level. NMRI mice were less prone to DIO than C57BL/6 ones and did not show significant changes in metabolic parameters after feeding with high-fat diet.     

PTP1B regulates leptin signal transduction in vivo

Mice lacking the protein-tyrosine phosphatase PTP1B are hypersensitive to insulin and resistant to obesity. However, the molecular basis for resistance to obesity has been unclear. Here we show that PTP1B regulates leptin signaling. In transfection studies, PTP1B dephosphorylates the leptin receptor-associated kinase, Jak2. PTP1B is expressed in hypothalamic regions harboring leptin-responsive neurons. Compared to wild-type littermates, PTP1B(-/-) mice have decreased leptin/body fat ratios, leptin hypersensitivity, and enhanced leptin-induced hypothalamic Stat3 tyrosyl phosphorylation. Gold thioglucose treatment, which ablates leptin-responsive hypothalamic neurons, partially overcomes resistance to obesity in PTP1B(-/-) mice. Our data indicate that PTP1B regulates leptin signaling in vivo, likely by targeting Jak2. PTP1B may be a novel target to treat leptin resistance in obesity.     

Regulation of Insulin and Leptin Signaling by Muscle Suppressor of Cytokine Signaling 3 (SOCS3)

Skeletal muscle resistance to the key metabolic hormones, leptin and insulin, is an early defect in obesity. Suppressor of cytokine signaling 3 (SOCS3) is a major negative regulator of both leptin and insulin signaling, thereby implicating SOCS3 in the pathogenesis of obesity and associated metabolic abnormalities. Here, we demonstrate that SOCS3 mRNA expression is increased in murine skeletal muscle in the setting of diet-induced and genetic obesity, inflammation, and hyperlipidemia. To further evaluate the contribution of muscle SOCS3 to leptin and insulin resistance in obesity, we generated transgenic mice with muscle-specific overexpression of SOCS3 (MCK/SOCS3 mice). Despite similar body weight, MCK/SOCS3 mice develop impaired systemic and muscle-specific glucose homeostasis and insulin action based on glucose and insulin tolerance tests, hyperinsulinemic-euglycemic clamps, and insulin signaling studies. With regards to leptin action, MCK/SOCS3 mice exhibit suppressed basal and leptin-stimulated activity and phosphorylation of alpha2 AMP-activated protein kinase (α2AMPK) and its downstream target, acetyl-CoA carboxylase (ACC). Muscle SOCS3 overexpression also suppresses leptin-regulated genes involved in fatty acid oxidation and mitochondrial function. These studies demonstrate that SOC3 within skeletal muscle is a critical regulator of leptin and insulin action and that increased SOCS may mediate insulin and leptin resistance in obesity.     

Sub-chronic administration of the 11beta-HSD1 inhibitor, carbenoxolone, improves glucose tolerance and insulin sensitivity in mice with diet-induced obesity

We have examined the metabolic effects of daily administration of carbenoxolone (CBX), a naturally occurring 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) inhibitor, in mice with high fat diet-induced insulin resistance and obesity. Eight-week-old male Swiss TO mice placed on a synthetic high fat diet received daily intraperitoneal injections of either saline vehicle or CBX over a 16-day period. Daily administration of CBX had no effect on food intake, but significantly lowered body weight (1.1- to 1.2-fold) compared to saline-treated controls. Non-fasting plasma glucose levels were significantly decreased (1.6-fold) by CBX treatment on day 4 and remained lower throughout the treatment period. Circulating plasma corticosterone levels were not significantly altered by CBX treatment. Plasma glucose concentrations of CBX-treated mice were significantly reduced (1.4-fold) following an intraperitoneal glucose load compared with saline controls. Similarly, after 16-day treatment with CBX, exogenous insulin evoked a significantly greater reduction in glucose concentrations (1.4- to 1.8-fold). 11beta-HSD1 gene expression was significantly down-regulated in liver, whereas glucocorticoid receptor gene expression was increased in both liver and adipose tissue following CBX treatment. The reduced body weight and improved metabolic control in mice with high fat diet-induced obesity upon daily CBX administration highlights the potential value of selective 11beta-HSD1 inhibition as a new route for the treatment of type 2 diabetes and obesity.     

Catestatin (chromogranin A(352-372)) and novel effects on mobilization of fat from adipose tissue through regulation of adrenergic and leptin signaling

Chromogranin A knock-out (Chga-KO) mice display increased adiposity despite high levels of circulating catecholamines and leptin. Consistent with diet-induced obese mice, desensitization of leptin receptors caused by hyperleptinemia is believed to contribute to the obese phenotype of these KO mice. In contrast, obesity in ob/ob mice is caused by leptin deficiency. To characterize the metabolic phenotype, Chga-KO mice were treated with the CHGA-derived peptide catestatin (CST) that is deficient in these mice. CST treatment reduced fat depot size and increased lipolysis and fatty acid oxidation. In liver, CST enhanced oxidation of fatty acids as well as their assimilation into lipids, effects that are attributable to the up-regulation of genes promoting fatty acid oxidation (Cpt1α, Pparα, Acox, and Ucp2) and incorporation into lipids (Gpat and CD36). CST did not affect basal or isoproterenol-stimulated cAMP production in adipocytes but inhibited phospholipase C activation by the α-adrenergic receptor (AR) agonist phenylephrine, suggesting inhibition of α-AR signaling by CST. Indeed, CST mimicked the lipolytic effect of the α-AR blocker phentolamine on adipocytes. Moreover, CST reversed the hyperleptinemia of Chga-KO mice and improved leptin signaling as determined by phosphorylation of AMPK and Stat3. CST also improved peripheral leptin sensitivity in diet-induced obese mice. In ob/ob mice, CST enhanced leptin-induced signaling in adipose tissue. In conclusion, our results implicate CST in a novel pathway that promotes lipolysis and fatty acid oxidation by blocking α-AR signaling as well as by enhancing leptin receptor signaling.     

Attenuation of leptin action and regulation of obesity by protein tyrosine phosphatase 1B

Common obesity is primarily characterized by resistance to the actions of the hormone leptin. Mice deficient in protein tyrosine phosphatase 1B (PTP1B) are resistant to diabetes and diet-induced obesity, prompting us to further define the relationship between PTP1B and leptin in modulating obesity. Leptin-deficient (Lep(ob/ob)) mice lacking PTP1B exhibit an attenuated weight gain, a decrease in adipose tissue, and an increase in resting metabolic rate. Furthermore, PTP1B-deficient mice show an enhanced response toward leptin-mediated weight loss and suppression of feeding. Hypothalami from these mice also display markedly increased leptin-induced Stat3 phosphorylation. Finally, substrate-trapping experiments demonstrate that leptin-activated Jak2, but not Stat3 or the leptin receptor, is a substrate of PTP1B. These results suggest that PTP1B negatively regulates leptin signaling, and provide one mechanism by which it may regulate obesity.     

Leptin

Leptin is an adipocyte hormone that signals nutritional status to the central nervous system (CNS) and peripheral organs. Leptin is also synthetized in the placenta and in gastrointestinal tract, although its role in these tissues is not yet clear. Circulating concentrations of leptin exhibit pulsatility and circadian rhythmicity. The levels of plasma leptin vary directly with body mass index and percentage body fat, and leptin contributes to the regulation of body weight. Leptin plasma concentrations are also influenced by metabolic hormones, sex, and body energy requirements. Defects in the leptin signaling pathway result in obesity in animal models. Only a few obese humans have been identified with mutations in the leptin gene or in the leptin receptor; however, most cases of obesity in humans are associated with high leptin levels. Thus, in humans obesity may represent a state of leptin resistance. Minute-to-minute fluctuations in peripheral leptin concentrations influence the activity of the hypothalamic-pituitary-ovarian and hypothalamic-pituitary-adrenal axes, indicating that leptin may be a modulator of reproduction, stress-related endocrine function, and behavior. This suggests potential roles for leptin or its antagonists in the diagnosis, pathophysiology and treatment of several human diseases.     

Postnatal growth after intrauterine growth restriction alters central leptin signal and energy homeostasis

Intrauterine growth restriction (IUGR) is closely linked with metabolic diseases, appetite disorders and obesity at adulthood. Leptin, a major adipokine secreted by adipose tissue, circulates in direct proportion to body fat stores, enters the brain and regulates food intake and energy expenditure. Deficient leptin neuronal signalling favours weight gain by affecting central homeostatic circuitry. The aim of this study was to determine if leptin resistance was programmed by perinatal nutritional environment and to decipher potential cellular mechanisms underneath.We clearly demonstrated that 5 months old IUGR rats develop a decrease of leptin sentivity, characterized by no significant reduction of food intake following an intraperitoneal injection of leptin. Apart from the resistance to leptin injection, results obtained from IUGR rats submitted to rapid catch-up growth differed from those of IUGR rats with no catch-up since we observed, for the first group only, fat accumulation, increased appetite for food rich in fat and increased leptin synthesis. Centrally, the leptin resistant state of both groups was associated with a complex and not always similar changes in leptin receptor signalling steps. Leptin resistance in IUGR rats submitted to rapid catch-up was associated with alteration in AKT and mTOR pathways. Alternatively, in IUGR rats with no catch-up, leptin resistance was associated with low hypothalamic expression of LepRa and LepRb. This study reveals leptin resistance as an early marker of metabolic disorders that appears before any evidence of body weight increase in IUGR rats but whose mechanisms could depend of nutritional environment of the perinatal period.     

Resistance to Diet-Induced Obesity in Mice with Synthetic Glyoxylate Shunt

Given the success in engineering synthetic phenotypes in microbes and mammalian cells, constructing non-native pathways in mammals has become increasingly attractive for understanding and identifying potential targets for treating metabolic disorders. Here, we introduced the glyoxylate shunt into mouse liver to investigate mammalian fatty acid metabolism. Mice expressing the shunt showed resistance to diet-induced obesity on a high-fat diet despite similar food consumption. This was accompanied by a decrease in total fat mass, circulating leptin levels, plasma triglyceride concentration, and a signaling metabolite in liver, malonyl-CoA, that inhibits fatty acid degradation. Contrary to plants and bacteria, in which the glyoxylate shunt prevents the complete oxidation of fatty acids, this pathway when introduced in mice increases fatty acid oxidation such that resistance to diet-induced obesity develops. This work suggests that using non-native pathways in higher organisms to explore and modulate metabolism may be a useful approach.