A brain leptin-renin angiotensin system interaction in the regulation of sympathetic nerve activity

The sympathetic nervous system, leptin, and renin-angiotensin system (RAS) have been implicated in obesity-associated hypertension. There is increasing evidence for the presence of both leptin and angiotensin II receptors in several key brain cardiovascular and metabolic control regions. We tested the hypothesis that the brain RAS plays a facilitatory role in the sympathetic nerve responses to leptin. In rats, intracerebroventricular (ICV) administration of losartan (5 μg) selectively inhibited increases in renal and brown adipose tissue (BAT) sympathetic nerve activity (SNA) produced by leptin (10 μg ICV) but did not reduce the SNA responses to corticotrophin-releasing factor (CRF) or the melanocortin receptor agonist MTII. In mice with deletion of angiotensin II type-1a receptors (AT(1a)R(-/-)), increases in renal and BAT SNA induced by leptin (2 μg ICV) were impaired whereas SNA responses to MTII were preserved. Decreases in food intake and body weight with ICV leptin did not differ in AT(1a)R(-/-) vs. AT(1a)R(+/+) mice. ICV leptin in rats increased AT(1a)R and angiotensin-converting enzyme (ACE) mRNA in the subfornical organ and AT(1a)R mRNA in the arcuate nucleus, suggesting leptin-induced upregulation of the brain RAS in specific brain regions. To evaluate the role of de novo production of brain angiotensin II in SNA responses to leptin, we treated rats with captopril (12.5 μg ICV). Captopril attenuated leptin effects on renal and BAT SNA. In conclusion, these studies provide evidence that the brain RAS selectively facilitates renal and BAT sympathetic nerve responses to leptin while sparing effects on food intake.     

Central leptin gene therapy fails to overcome leptin resistance associated with diet-induced obesity

The objective of this study was to determine if central overexpression of leptin could overcome the leptin resistance caused by 100 days of high-fat feeding. Three-month old-F344XBN male rats were fed either control low fat chow (Chow), which provides 15% of energy as fat, or a high-fat/high-sucrose diet (HF), which provides 59% of energy as fat. Over several weeks, the HF-fed animals spontaneously split into two groups of animals: those that became obese on the HF diet (DIO) and those that did not gain extra weight on the HF diet [diet resistant (DR)]. After 100 days of HF feeding, animals were given a single intracerebroventricular injection containing 5.75E10 particles of rAAV encoding leptin (rAAV-leptin) or control virus (rAAV-con). Chow animals responded robustly to rAAV-leptin, including significant anorexia, weight loss, and lipopenia. In contrast, DIO were completely unresponsive to rAAV-leptin. DR rats responded to rAAV-leptin, but in a more variable fashion than Chow. Unlike what was observed in Chow, the anorectic response to rAAV-leptin rapidly attenuated and was no longer significant by day 14 postvector delivery. Both DIO and DR animals were found to have reduced long-form leptin receptor expression and enhanced basal P-STAT-3 in the hypothalamus with respect to Chow. rAAV-leptin caused an increase in STAT3 phosphorylation and proopiomelanocortin expression in the hypothalamus and an increase in uncoupling protein-1 in brown adipose tissue in both Chow and DR animals, but failed to do so in DIO. This suggests that central overexpression of leptin is not a viable strategy to reverse diet-induced obesity.     

Reduced central leptin sensitivity in rats with diet-induced obesity

On low-fat chow diet, rats prone to diet-induced obesity (DIO) have increased arcuate nucleus neuropeptide Y (NPY) expression but similar leptin levels compared with diet-resistant (DR) rats (19). Here, body weight and leptin levels rose in DIO rats, and they defended their higher body weight after only 1 wk on a 31% fat high-energy (HE) diet. However, DIO NPY expression did not fall to DR levels until 4 wk when plasma leptin was 168% of DR levels. When switched to chow, DIO rats lost carcass fat (18). By 10 wk, leptin levels fell to 148% and NPY expression again rose to 150% of DR levels. During 4 wk of food restriction, DIO leptin fell by approximately 50% while NPY increased by 30%. While both returned to control levels by 8 wk, DIO rats still regained all lost weight when fed ad libitum. Finally, the anorexic effect of intracerebroventricular leptin (10 microg) was inversely correlated with subsequent 3-wk weight gain on HE diet. Thus NPY expression and food intake are less sensitive to the leptin's suppressive effects in DIO rats. While this may predispose them to develop DIO, it does not fully explain their defense of a higher body weight on HE diet.     

Diet-induced obesity leads to the development of leptin resistance in vagal afferent neurons

Ingestion of high-fat, high-calorie diets is associated with hyperphagia, increased body fat, and obesity. The mechanisms responsible are currently unclear; however, altered leptin signaling may be an important factor. Vagal afferent neurons (VAN) integrate signals from the gut in response to ingestion of nutrients and express leptin receptors. Therefore, we tested the hypothesis that leptin resistance occurs in VAN in response to a high-fat diet. Sprague-Dawley rats, which exhibit a bimodal distribution of body weight gain, were used after ingestion of a high-fat diet for 8 wk. Body weight, food intake, and plasma leptin levels were measured. Leptin signaling was determined by immunohistochemical localization of phosphorylated STAT3 (pSTAT3) in cultured VAN and by quantifaction of pSTAT3 protein levels by Western blot analysis in nodose ganglia and arcuate nucleus in vivo. To determine the mechanism of leptin resistance in nodose ganglia, cultured VAN were stimulated with leptin alone or with lipopolysaccharide (LPS) and SOCS-3 expression measured. SOCS-3 protein levels in VAN were measured by Western blot following leptin administration in vivo. Leptin resulted in appearance of pSTAT3 in VAN of low-fat-fed rats and rats resistant to diet-induced obesity but not diet-induced obese (DIO) rats. However, leptin signaling was normal in arcuate neurons. SOCS-3 expression was increased in VAN of DIO rats. In cultured VAN, LPS increased SOCS-3 expression and inhibited leptin-induced pSTAT3 in vivo. We conclude that VAN of diet-induced obese rats become leptin resistant; LPS and SOCS-3 may play a role in the development of leptin resistance.     

Maternal obesity increases hypothalamic leptin receptor expression and sensitivity in juvenile obesity-prone rats

In rats selectively bred to develop diet-induced obesity (DIO) or to be diet-resistant (DR), DIO maternal obesity selectively enhances the development of obesity and insulin resistance in their adult offspring. We postulated that the interaction between genetic predisposition and factors in the maternal environment alter the development of hypothalamic peptide systems involved in energy homeostasis regulation. Maternal obesity in the current studies led to increased body and fat pad weights and higher leptin and insulin levels in postnatal day 16 offspring of both DIO and DR dams. However, by 6 wk of age, most of these intergroup differences disappeared and offspring of obese DIO dams had unexpected increases in arcuate nucleus leptin receptor mRNA, peripheral insulin sensitivity, diet- and leptin-induced brown adipose temperature increase and 24-h anorectic response compared with offspring of lean DIO, but not lean DR dams. On the other hand, while offspring of obese DIO dams did have the highest ventromedial nucleus melanocortin-4 receptor expression, their anorectic and brown adipose thermogenic responses to the melanocortin agonist, Melanotan II (MTII), did not differ from those of offspring of lean DR or DIO dams. Thus, during their rapid growth phase, juvenile offspring of obese DIO dams have alterations in their hypothalamic systems regulating energy homeostasis, which ameliorates their genetic and perinatally determined predisposition toward leptin resistance. Because they later go onto become more obese, it is possible that interventions during this time period might prevent the subsequent development of obesity.     

Diet-induced obesity alters AMP kinase activity in hypothalamus and skeletal muscle

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance and of the effects of leptin on food intake and fatty acid oxidation. Obesity is usually associated with resistance to the effects of leptin on food intake and body weight. To determine whether diet-induced obesity (DIO) impairs the AMPK response to leptin in muscle and/or hypothalamus, we fed FVB mice a high fat (55%) diet for 10-12 weeks. Leptin acutely decreased food intake by approximately 30% in chow-fed mice. DIO mice tended to eat less, and leptin had no effect on food intake. Leptin decreased respiratory exchange ratio in chow-fed mice indicating increased fatty acid oxidation. Respiratory exchange ratio was low basally in high fat-fed mice, and leptin had no further effect. Leptin (3 mg/kg intraperitoneally) increased alpha2-AMPK activity 2-fold in muscle in chow-fed mice but not in DIO mice. Leptin decreased acetyl-CoA carboxylase activity 40% in muscle from chow-fed mice. In muscle from DIO mice, acetyl-CoA carboxylase activity was basally low, and leptin had no further effect. In paraventricular, arcuate, and medial hypothalamus of chow-fed mice, leptin inhibited alpha2-AMPK activity but not in DIO mice. In addition, leptin increased STAT3 phosphorylation 2-fold in arcuate of chow-fed mice, but this effect was attenuated because of elevated basal STAT3 phosphorylation in DIO mice. Thus, DIO in FVB mice alters alpha2-AMPK in muscle and hypothalamus and STAT3 in hypothalamus and impairs further effects of leptin on these signaling pathways. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.     

Diet-induced obesity causes severe but reversible leptin resistance in arcuate melanocortin neurons

Despite high leptin levels, most obese humans and rodents lack responsiveness to its appetite-suppressing effects. We demonstrate that leptin modulates NPY/AgRP and alpha-MSH secretion from the ARH of lean mice. High-fat diet-induced obese (DIO) mice have normal ObRb levels and increased SOCS-3 levels, but leptin fails to modulate peptide secretion and any element of the leptin signaling cascade. Despite this leptin resistance, the melanocortin system downstream of the ARH in DIO mice is over-responsive to melanocortin agonists, probably due to upregulation of MC4R. Lastly, we show that by decreasing the fat content of the mouse's diet, leptin responsiveness of NPY/AgRP and POMC neurons recovered simultaneously, with mice regaining normal leptin sensitivity and glycemic control. These results highlight the physiological importance of leptin sensing in the melanocortin circuits and show that their loss of leptin sensing likely contributes to the pathology of leptin resistance.     

Maternal diet-induced obesity during suckling period programs offspring obese phenotype and hypothalamic leptin/insulin resistance

During the early post-natal period, offspring are vulnerable to environmental insults, such as nutritional and hormonal changes, which increase risk to develop metabolic diseases later in life. Our aim was to understand whether maternal obesity during lactation programs offspring to metabolic syndrome and obese phenotype, in addition we aimed to assess the peripheral glucose metabolism and hypothalamic leptin/insulin signaling pathways. At delivery, female Wistar rats were randomly divided in two groups: Control group (CO), mothers fed a standard rodent chow (Nuvilab); and Diet-induced obesity group (DIO), mothers who had free access to a diet performed with 33% ground standard rodent chow, 33% sweetened condensed milk (Nestlé), 7% sucrose and 27% water. Maternal treatment was performed throughout suckling period. All offspring received standard rodent chow from weaning until 91-day-old. DIO dams presented increased total body fat and insulin resistance. Consequently, the breast milk from obese dams had altered composition. At 91-day-old, DIO offspring had overweight, hyperphagia and higher adiposity. Furthermore, DIO animals had hyperinsulinemia and insulin resistance, they also showed pancreatic islet hypertrophy and increased pancreatic β-cell proliferation. Finally, DIO offspring showed low ObRb, JAK2, STAT-3, IRβ, PI3K and Akt levels, suggesting leptin and insulin hypothalamic resistance, associated with increased of hypothalamic NPY level and decreased of POMC. Maternal obesity during lactation malprograms rat offspring to develop obesity that is associated with impairment of melanocortin system. Indeed, rat offspring displayed glucose dyshomeostasis and both peripheral and central insulin resistance.     

Gamma-MSH/MC3R natriuretic signaling system: The possible explanation for contrasting blood pressure effects of agouti mutation and melanocortin receptor knockout

Overweight and obesity are the major risk factors of arterial hypertension. Recent studies indicate that adipose tissue hormone, leptin, is involved in the development of obesity-induced hypertension. Models of genetically determined obesity in rodents are commonly used to study the pathogenesis of obesity-associated hypertension. One of such models are agouti yellow obese (A^y/a) mice which ubiquitously overexpress agouti protein—an endogenous antagonist of melanocortin receptors normally synthesized only in the hair follicle. In A^y/a mice, agouti protein is synthesized also in the hypothalamus and blocks the anorectic effect of leptin mediated by alpha-melanocyte-stimulating hormone (α-MSH) which binds to melanocortin type 3 and 4 receptors (MC3R and MC4R). Consequently, A^y/a mice are hyperphagic, obese, hyperinsulinemic and hyperleptinemic. Blood pressure is increased in A^y/a mice due to increased serum leptin level. In contrast, blood pressure is reduced in MC4R-null mice despite obesity and hyperleptinemia, and is not increased by the administration of leptin in these animals, suggesting an essential role of the melanocortin pathway in the hypertensive effect of leptin. Herein, I propose the hypothesis which might explain why blood pressure is increased in A^y/a mice but reduced in MC4R−/− mice, although hypothalamic melanocortin signaling is impaired in both models. According to this proposal, in MC4R−/− mice the natriuretic effect of γ-MSH mediated by intrarenal MC3R is preserved and counteracts prohypertensive mechanisms triggered by leptin. In contrast, in A^y/a mice, ubiquitously expressed agouti protein blocks not only hypothalamic MC4R but also renal MC3R and thus impairs γ-MSH-induced natriuresis, leading to blood pressure elevation due to unopposed central and/or peripheral pressor effects of leptin.     

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.     

Defense of body weight against chronic caloric restriction in obesity-prone and -resistant rats

Half of Sprague-Dawley rats develop and defend diet-induced obesity (DIO) or diet resistance (DR) when fed a high-energy (HE) diet. Here, adult male rats were made DIO or DR after 10 wk on HE diet. Then half of each group was food restricted for 8 wk on chow to maintain their body weights at 90% of their respective baselines. Rate and magnitude of weight loss were comparable, but maintenance energy intake and the degree of sympathetic activity (24-h urine norepinephrine) inhibition were 17 and 29% lower, respectively, in restricted DR than DIO rats. Restricted DIO rats reduced adipose depot weights, plasma leptin, and insulin levels by 35%. Restricted DR rats reduced none of these. When fed ad libitum, both DR and DIO rats returned to the body weights of their respective chow-fed phenotype controls within 2 wk. This was associated with increased adipose mass and leptin and insulin levels only in DIO rats. Thus DR rats appear to alter primarily their lean body mass, whereas DIO rats primarily alter their adipose mass during chronic caloric restriction and refeeding.     

Central effects of leptin on cardiovascular and neurohormonal responses in conscious rabbits

We determined the cardiovascular and neurohormonal responses to intracerebroventricular injection of leptin in conscious rabbits. Intracerebroventricular injection of leptin elicited dose-related increases in mean arterial pressure and renal sympathetic nerve activity while producing no consistent, significant increases in heart rate. Peak values of mean arterial pressure and renal sympathetic nerve activity induced by intracerebroventricular injection of 50 microgram of leptin (+17.3 +/- 1.2 mmHg and +47.9 +/- 12.0%) were obtained at 10 and 20 min after injection, respectively. Plasma catecholamine concentrations significantly increased at 60 min after intracerebroventricular injection of leptin (control vs. 60 min; epinephrine: 33 +/- 12 vs. 97 +/- 27 pg/ml, P < 0.05; norepinephrine: 298 +/- 39 vs. 503 +/- 86 pg/ml, P < 0.05). Intracerebroventricular injection of leptin also caused significant increases in plasma vasopressin and glucose levels. However, pretreatment with intravenous injection of pentolinium (5 mg/kg), a ganglion blocking agent, abolished these cardiovascular and neurohormonal responses. On the other hand, intravenous injection of the same dose of leptin (50 microgram) as used in the intracerebroventricular experiment failed to cause any cardiovascular and renal sympathetic nerve responses. These results suggest that intracerebroventricular leptin acts in the central nervous system and activates sympathoadrenal outflow, resulting in increases in arterial pressure and plasma glucose levels in conscious rabbits.     

High-fat diet is associated with blunted splanchnic sympathoinhibitory responses to gastric leptin and cholecystokinin: implications for circulatory control

Gastric leptin and cholecystokinin (CCK) act on vagal afferents to induce cardiovascular effects and reflex inhibition of splanchnic sympathetic nerve discharge (SSND) and may act cooperatively in these responses. We sought to determine whether these effects are altered in animals that developed obesity in response to a medium high-fat diet (MHFD). Male Sprague-Dawley rats were placed on a low-fat diet (LFD; n = 8) or a MHFD (n = 24) for 13 wk, after which the animals were anesthetized and artificially ventilated. Arterial pressure was monitored and blood was collected for the determination of plasma leptin and CCK. SSND responses to leptin (15 μg/kg) and CCK (2 μg/kg) administered close to the coeliac artery were evaluated. Collectively, MHFD animals had significantly higher plasma leptin but lower plasma CCK levels than LFD rats (P < 0.05), and this corresponded to attenuated or reversed SSND responses to CCK (LFD, -21 ± 2%; and MHFD, -12 ± 2%; P < 0.05) and leptin (LFD, -6 ± 2%; and MHFD, 4 ± 1%; P < 0.001). Alternatively, animals on the MHFD were stratified into obesity-prone (OP; n = 8) or obesity-resistant (OR; n = 8) groups according to their weight gain falling within the upper or lower tertile, respectively. OP rats had significantly higher resting arterial pressure, adiposity, and plasma leptin but lower plasma CCK compared with LFD rats (P < 0.05). The SSND responses to CCK or leptin were not significantly different between OP and OR animals. These results demonstrate that a high-fat diet is associated with blunted splanchnic sympathoinhibitory responses to gastric leptin and CCK and may impact on sympathetic vasomotor mechanisms involved in circulatory control.     

Hypothalamic glycogen synthase kinase 3β has a central role in the regulation of food intake and glucose metabolism

GSK3β (glycogen synthase kinase 3β) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3β activity is implicated in disorders ranging from cancer to Alzheimer's disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3β in leptin-deficient Lepob/ob mice and show that intracerebroventricular injection of a GSK3β inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3β inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3β in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3β signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.     

Melanocortin receptors mediate the excitatory effects of blood-borne murine leptin on hypothalamic paraventricular neurons in rat

The central pathways and mediators involved in sympathoexcitatory responses to circulating leptin are not well understood, although the arcuate-paraventricular nucleus (ARC-PVN) pathway likely plays a critical role. In urethane-anesthetized rats, ipsilateral intracarotid artery (ICA) injection of murine leptin (100 microg/kg) activated most PVN neurons tested. These responses were reduced by intracerebroventricular injection of the melanocortin subtype 3 and 4 receptor (MC3/4-R) antagonist SHU-9119 (0.6 nmol). The MC3/4-R agonist MTII (0.6 nmol icv) activated PVN neurons. Some PVN neurons that were excited by ICA leptin were inhibited by local application of neuropeptide Y (NPY, 2.5 ng). ICA leptin (100 microg/kg) excited presympathetic rostral ventrolateral medulla neurons and renal sympathetic nerve activity without significant change in blood pressure or heart rate; these effects were mimicked by intracerebroventricular injection of MTII (0.6 nmol). These data provide in vivo electrophysiological evidence to support the hypothesis that circulating leptin activates the sympathetic nervous system by stimulating the release of alpha-melanocyte-stimulating hormone in the vicinity of PVN neurons that are inhibited by the orexogenic peptide NPY.     

Impaired activation of phosphatidylinositol 3-kinase by leptin is a novel mechanism of hepatic leptin resistance in diet-induced obesity

Obesity is associated with the development of leptin resistance. However, the effects of leptin resistance on leptin-regulated metabolic processes and the biochemical defects that cause leptin resistance are poorly understood. We have addressed in rats the effect of dietinduced obesity (DIO), a situation of elevated tissue lipid levels, on the well described lipid-lowering effect of leptin in liver, an action that is proposed to be important for the prevention of tissue lipotoxicity and insulin resistance. In addition, we have addressed the role of phosphatidylinositol 3-kinase (PI 3-kinase) in mediating the acute effects of leptin on hepatic lipid levels in lean and DIO animals. A 90-min leptin ( approximately 10 ng/ml) perfusion of isolated livers from lean animals decreased triglyceride levels by 42 +/- 5% (p = 0.006). However, leptin concentrations ranging from approximately 10 to approximately 90 ng/ml had no effect on triglyceride levels in livers from DIO animals. The acute lipid-lowering effect of leptin on livers from lean animals was mediated by a PI 3-kinase-dependent mechanism, because wortmannin and LY294002, the PI 3-kinase inhibitors, blocked the effects of leptin on hepatic triglyceride levels and leptin increased liver PI 3-kinase activity by 183 +/- 6% (p = 0.003) and insulin receptor substrate 1 tyrosine phosphorylation by 185 +/- 30% (p = 0.02) in the absence of PI 3-kinase inhibitors. Contrary to the effects of leptin in lean livers, leptin did not activate PI 3-kinase in livers from DIO rats. These data present evidence for a role for 1). leptin resistance in contributing to the excessive accumulation of tissue lipid in obesity, 2). PI 3-kinase in mediating the acute lipid-lowering effects of leptin in liver, and 3). defective leptin activation of PI 3-kinase as a novel mechanism of leptin resistance.