Leptin-Dependent Control of Glucose Balance and Locomotor Activity by POMC Neurons

Leptin plays a pivotal role in regulation of energy balance. Via unknown central pathways, leptin also affects peripheral glucose homeostasis and locomotor activity. We hypothesized that, specifically, pro-opiomelanocortin (POMC) neurons mediate those actions. To examine this possibility, we applied Cre-Lox technology to express leptin receptors (ObRb) exclusively in POMC neurons of the morbidly obese, profoundly diabetic, and severely hypoactive leptin receptor-deficient Lepr^db/db mice. Here, we show that expression of ObRb only in POMC neurons leads to a marked decrease in energy intake and a modest reduction in body weight in Lepr^db/db mice. Remarkably, blood glucose levels are entirely normalized. This normalization occurs independently of changes in food intake and body weight. In addition, physical activity is greatly increased despite profound obesity. Our results suggest that leptin signaling exclusively in POMC neurons is sufficient to stimulate locomotion and prevent diabetes in the severely hypoactive and hyperglycemic obese Lepr^db/db mice.     

Loss of Atg12, but not Atg5, in pro-opiomelanocortin neurons exacerbates diet-induced obesity

The autophagy-related proteins ATG12 and ATG5 form a covalent complex essential for autophagy. Here, we demonstrate that ATG12 has distinct functions from ATG5 in pro-opiomelanocortin (POMC)-expressing neurons. Upon high-fat diet (HFD) consumption, mice lacking Atg12 in POMC-positive neurons exhibit accelerated weight gain, adiposity, and glucose intolerance, which is associated with increased food intake, reduced ambulation, and decreased LEP/leptin sensitivity. Importantly, although genetic deletion of either Atg12 or Atg5 renders POMC neurons autophagy-deficient, mice lacking Atg5 in POMC neurons do not exhibit these phenotypes. Hence, we propose nonautophagic functions for ATG12 in POMC neurons that counteract excessive weight gain in response to HFD consumption.     

Fish oil supplementation increases expression of mammary tumor apoptosis mediators and reduces inflammation in an obesity-associated HER-2 breast cancer model

Obesity is associated with inflammation and has been shown to increase breast cancer severity. The objective of this study was to examine the effect of fish oil (FO) supplementation in obesity-associated mammary tumorigenesis in the MMTV-neu(ndl)-YD5 mouse model of human epidermal growth factor receptor-2 positive BC. Female mice were fed one of three diets for 16 weeks: i) high fat diet [HF, % kacl: 41.2% lard, 18.7% corn oil (CO)], ii) an isocaloric HF plus menhaden FO diet (HF+FO, % kcal: 41.2 lard, 13.4% CO, 5.3% FO), iii) low fat diet (LF, % kcal: 4.7% lard, 6% CO). HF mice had increased body weight, visceral adipose weight and serum hormone concentrations (increased leptin and resistin; decreased adiponectin) versus LF, which was attenuated in the HF+FO group versus HF (P<.05). Compared to HF, tumor onset was delayed in HF+FO and LF mice (P<0.05). Compared to HF, HF+FO reduced mammary tumor multiplicity (-27%), tumor weight (-46%) and total tumor volume (-50%) (P<0.05). Additionally, HF+FO reduced mammary tumor multiplicity (-33%), tumor weight (-39%) and total tumor volume (-60%) versus LF. HF+FO improved mammary tumor apoptosis status with increased expression of pro-apoptotic Bad and decreased expression of anti-apoptotic Bcl-xLmediators versus HF (P<0.05). Additionally, HF+FO decreased tumor protein expression of activated Akt, NFκB p65 and STAT3, versus HF (P<0.05). Tumor mRNA expression of inflammatory mediators TNFα, IL-6 and leptin were reduced in HF+FO, whereas IL-10 expression was increased compared to HF (P<0.05). Collectively these results demonstrate the efficacy of FO supplementation for improving obesity-associated breast cancer outcomes.     

DBA/2J Mice Are Susceptible to Diabetic Nephropathy and Diabetic Exacerbation of IOP Elevation

Some pathological manifestations of diabetes in the eye include retinopathy, cataracts and elevated intraocular pressure (IOP). Loss of retinal ganglion cells (RGCs) in non-proliferative stages of diabetic retinopathy and small increases in IOP in diabetic patients has raised the possibility that diabetes affects the development and progression of ocular hypertension and glaucoma. The Ins2^Akita mutation is known to cause diabetes and retinopathy on a C57BL/6J (B6) background by as early as 3 months of age. Here, the impact of the Akita mutation on glaucoma was assessed using DBA/2J (D2) mice, a widely used mouse model of ocular hypertension induced glaucoma. In D2.Ins2^Akita/+ mice, the contribution of diabetes to vascular permeability, IOP elevation, RGC loss, and glaucoma development was assessed. D2.Ins2^Akita/+ mice developed a severe diabetic nephropathy and early mortality between 6–8 months of age. This agrees with previous reports showing that the D2 background is more susceptible to diabetes than the B6 background. In addition, D2.Ins2^Akita/+ mice had vascular leakage, astrocyte reactivity and a significant increase in IOP. However no RGC loss and no anterograde axonal transport dysfunction were found at 8.5 months of age. Therefore, our data show that despite severe diabetes and an increased IOP compared to controls, RGCs do not lose axon transport or degenerate. This may be due to a DBA/2J-specific genetic modifier(s) that could provide novel and important avenues for developing new therapies for diabetic retinopathy and possibly glaucoma.     

Hyperleptinemia elicited by the 5-HT precursor, 5-hydroxytryptophan in mice: involvement of insulin

Mechanisms for hyperleptinemia elicited by a serotonin (5-hydroxytryptamine, 5-HT) precursor, 5-hydroxytryptophan (5-HTP), were investigated. 5-HTP elicited apparent increases in serum leptin levels of mice. Administration of 5-HTP did not alter expression of leptin mRNA in white adipose tissues. Furthermore, neither 5-HTP nor 5-HT increased leptin secretion from isolated fat pads of mice. Since insulin is known to enhance leptin release, involvement of insulin in 5-HTP-induced hyperleptinemia was examined. 5-HTP significantly elevated serum insulin levels. In mice treated with streptozotocin, which depletes insulin, 5-HTP did not increase serum leptin levels. These results suggest that hyperinsulinemia participates the elevation of serum leptin levels elicited by 5-HTP.     

Somato-Dendritic Localization and Signaling by Leptin Receptors in Hypothalamic POMC and AgRP Neurons

Leptin acts via neuronal leptin receptors to control energy balance. Hypothalamic pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP)/Neuropeptide Y (NPY)/GABA neurons produce anorexigenic and orexigenic neuropeptides and neurotransmitters, and express the long signaling form of the leptin receptor (LepRb). Despite progress in the understanding of LepRb signaling and function, the sub-cellular localization of LepRb in target neurons has not been determined, primarily due to lack of sensitive anti-LepRb antibodies. Here we applied light microscopy (LM), confocal-laser scanning microscopy (CLSM), and electron microscopy (EM) to investigate LepRb localization and signaling in mice expressing a HA-tagged LepRb selectively in POMC or AgRP/NPY/GABA neurons. We report that LepRb receptors exhibit a somato-dendritic expression pattern. We further show that LepRb activates STAT3 phosphorylation in neuronal fibers within several hypothalamic and hindbrain nuclei of wild-type mice and rats, and specifically in dendrites of arcuate POMC and AgRP/NPY/GABA neurons of Leprb ^+/+ mice and in Leprb ^db/db mice expressing HA-LepRb in a neuron specific manner. We did not find evidence of LepRb localization or STAT3-signaling in axon-fibers or nerve-terminals of POMC and AgRP/NPY/GABA neurons. Three-dimensional serial EM-reconstruction of dendritic segments from POMC and AgRP/NPY/GABA neurons indicates a high density of shaft synapses. In addition, we found that the leptin activates STAT3 signaling in proximity to synapses on POMC and AgRP/NPY/GABA dendritic shafts. Taken together, these data suggest that the signaling-form of the leptin receptor exhibits a somato-dendritic expression pattern in POMC and AgRP/NPY/GABA neurons. Dendritic LepRb signaling may therefore play an important role in leptin’s central effects on energy balance, possibly through modulation of synaptic activity via post-synaptic mechanisms.     

Role of glucocorticoids in mediating effects of fasting and diabetes on hypothalamic gene expression

Background  

Fasting and diabetes are characterized by elevated glucocorticoids and reduced insulin, leptin, elevated hypothalamic AGRP and NPY mRNA, and reduced hypothalamic POMC mRNA. Although leptin replacement can reverse changes in hypothalamic gene expression associated with fasting and diabetes, leptin also normalizes corticosterone; therefore the extent to which the elevated corticosterone contributes to the regulation of hypothalamic gene expression in fasting and diabetes remains unclear. To address if elevated corticosterone is necessary for hypothalamic responses to fasting and diabetes, we assessed the effects of adrenalectomy on hypothalamic gene expression in 48-hour-fasted or diabetic mice. To assess if elevated corticosterone is sufficient for the hypothalamic responses to fasting and diabetes, we assessed the effect of corticosterone pellets implanted for 48 hours on hypothalamic gene expression.     

Obesity Induces Hypothalamic Endoplasmic Reticulum Stress and Impairs Proopiomelanocortin (POMC) Post-translational Processing

It was shown previously that abnormal prohormone processing or inactive proconverting enzymes that are responsible for this processing cause profound obesity. Our laboratory demonstrated earlier that in the diet-induced obesity (DIO) state, the appetite-suppressing neuropeptide α-melanocyte-stimulating hormone (α-MSH) is reduced, yet the mRNA of its precursor protein proopiomelanocortin (POMC) remained unaltered. It was also shown that the DIO condition promotes the development of endoplasmic reticulum (ER) stress and leptin resistance. In the current study, using an in vivo model combined with in vitro experiments, we demonstrate that obesity-induced ER stress obstructs the post-translational processing of POMC by decreasing proconverting enzyme 2, which catalyzes the conversion of adrenocorticotropin to α-MSH, thereby decreasing α-MSH peptide production. This novel mechanism of ER stress affecting POMC processing in DIO highlights the importance of ER stress in regulating central energy balance in obesity.     

Extracts of Rhizoma Polygonati Odorati Prevent High-Fat Diet-Induced Metabolic Disorders in C57BL/6 Mice

Polygonatum odoratum (Mill.) Druce belongs to the genus Polygonatum family of plants. In traditional Chinese medicine, the root of Polygonatum odoratum, Rhizoma Polygonati Odorati, is used both for food and medicine to prevent and treat metabolic disorders such as hyperlipidemia, hyperglycemia, obesity and cardiovascular disease. However, there is no solid experimental evidence to support these applications, and the underlying mechanism is also needed to be elucidated. Here, we examined the effect of the extract of Rhizoma Polygonati Odorati (ER) on metabolic disorders in diet-induced C57BL/6 obese mice. In the preventive experiment, the ER blocked body weight gain, and lowered serum total cholesterol (TC), triglyceride (TG) and fasting blood glucose, improved glucose tolerance test (GTT) and insulin tolerance test (ITT), reduced the levels of serum insulin and leptin, and increased serum adiponectin levels in mice fed with a high-fat diet significantly. In the therapeutic study, we induced obesity in the mice and treated the obese mice with ER for two weeks. We found that ER treatments reduced serum TG and fasting blood glucose, and improved glucose tolerance in the mice. Gene expression analysis showed that ER increased the mRNA levels of peroxisome proliferator-activated receptors (PPAR) γ and α and their downstream target genes in mice livers, adipose tissues and HepG2 cells. Our data suggest that ER ameliorates metabolic disorders and enhances the mRNA expression of PPARs in obese C57BL/6 mice induced by high-fat diet.     

Hypothalamic NPY,AGRP, and POMC mRNA responses to leptin and refeeding in mice

Food deprivation (FD) increases hypothalamic neuropeptide Y (NPY) and agouti-related protein (AGRP) mRNA levels and decreases proopiomelanocortin (POMC) mRNA levels; refeeding restores these levels. We determined the time course of changes in hypothalamic NPY, AGRP, and POMC mRNA levels on refeeding after 24 h FD in C57BL mice by in situ hybridization. After 24 h deprivation, mice were refed with either chow or a palatable mash containing no calories or were injected with murine leptin (100 microg) without food. Mice were perfused 2 or 6 h after treatment. Food deprivation increased hypothalamic NPY mRNA (108 +/- 6%) and AGRP mRNA (78 +/- 7%) and decreased hypothalamic POMC mRNA (-15 +/- 1%). Refeeding for 6 h, but not 2 h, was sufficient to reduce (but not restore) NPY mRNA, did not affect AGRP mRNA, and restored POMC mRNA levels to ad libitum control levels. Intake of the noncaloric mash had no effect on mRNA levels, and leptin administration after deprivation (at a dose sufficient to reduce refeeding in FD mice) was not sufficient to affect mRNA levels. These results suggest that gradual postabsorptive events subsequent to refeeding are required for the restoration of peptide mRNA to baseline levels after food deprivation in mice.     

Neotomodon alstoni mice present sex differences between lean and obese in daily hypothalamic leptin signaling

This article compared the effects of spontaneous obesity on the daily profile in the relative amount of the leptin receptor (LepRb), and its output. That is the precursor Pro-opiomelanocortin (POMC) over a 24-hour period and compared with differences in locomotion and food intake in periods of artificial light. Differences between lean and obese mice were examined, as were sex differences. Body weight, food intake and locomotor activity were monitored in freely moving lean and obese mice. Hypothalamic tissue was collected at 5 h, 10 h, 15 h, 19 h and 24 h. Samples were analyzed by western blotting to determine the relative presence of protein for LepRb, STAT3 phosphorylation (by pSTAT3/STAT3 ratio) and POMC. Obese mice were 60% less active in locomotion than lean mice during the night. While both locomotor activity and food intake were noticeably greater during the day in obese mice than in lean mice, the hypothalamus in obese mice showed a lower relative abundance of POMC and reduced pSTAT3/STAT3 ratio and leptin receptors. Behavioral and biochemical differences were more evident in obese females than in obese males. These results indicate that obesity in N. alstoni affects hypothalamic leptin signaling according to sex.     

Food intake reductions and increases in energetic responses by hindbrain leptin and melanotan II are enhanced in mice with POMC-specific PTP1B deficiency

Leptin regulates energy balance through central circuits that control food intake and energy expenditure, including proopiomelanocortin (POMC) neurons. POMC neuron-specific deletion of protein tyrosine phosphatase 1B (PTP1B) (Ptpn1(loxP/loxP) POMC-Cre), a negative regulator of CNS leptin signaling, results in resistance to diet-induced obesity and improved peripheral leptin sensitivity in mice, thus establishing PTP1B as an important component of POMC neuron regulation of energy balance. POMC neurons are expressed in the pituitary, the arcuate nucleus of the hypothalamus (ARH), and the nucleus of the solitary tract (NTS) in the hindbrain, and it is unknown how each population might contribute to the phenotype of POMC-Ptp1b(-/-) mice. It is also unknown whether improved leptin sensitivity in POMC-Ptp1b(-/-) mice involves altered melanocortin receptor signaling. Therefore, we examined the effects of hindbrain administration (4th ventricle) of leptin (1.5, 3, and 6 μg) or the melanocortin 3/4R agonist melanotan II (0.1 and 0.2 nmol) in POMC-Ptp1b(-/-) (KO) and control PTP1B(fl/fl) (WT) mice on food intake, body weight, spontaneous physical activity (SPA), and core temperature (T(C)). The results show that KO mice were hypersensitive to hindbrain leptin- and MTII-induced food intake and body weight suppression and SPA compared with WT mice. Greater increases in leptin- but not MTII-induced T(C) were also observed in KO vs. WT animals. In addition, KO mice displayed elevated hindbrain and hypothalamic MC4R mRNA expression. These studies are the first to show that hindbrain administration of leptin or a melanocortin receptor agonist alters energy balance in mice likely via participation of hindbrain POMC neurons.     

Serotonin systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors and induce anorexia via a leptin-independent pathway in mice

NEFA/nucleobindin2 (NUCB2), a novel satiety molecule, is associated with leptin-independent melanocortin signaling in the central nervous system. Here, we show that systemic administration of m-chlorophenylpiperazine (mCPP), a serotonin 5-HT1B/2C receptor agonist, significantly increased the expression of hypothalamic NUCB2 in wild-type mice. The increases in hypothalamic NUCB2 expression induced by mCPP were attenuated in 5-HT2C receptor mutant mice. Systemic administration of mCPP suppressed food intake in db/db mice with leptin receptor mutation as well as lean control mice. On the other hand, the expression of hypothalamic NUCB2 and proopiomelanocortin (POMC) was significantly decreased in hyperphagic and non-obese 5-HT2C receptor mutants compared with age-matched wild-type mice. Interestingly, despite increased expression of hypothalamic POMC, hypothalamic NUCB2 expression was decreased in 5-HT2C receptor mutant mice with heterozygous mutation of β-endorphin gene. These findings suggest that 5-HT systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors, and induce anorexia via a leptin-independent pathway in mice.     

Hypothalamic neuronal histamine in genetically obese animals: its implication of leptin action in the brain

Leptin regulates feeding behavior and energy metabolism by affecting hypothalamic neuromodulators. The present study was designed to examine hypothalamic neuronal histamine, a recently identified mediator of leptin signaling in the brain, in genetic obese animals. Concentrations of hypothalamic histamine and tele-methylhistamine (t-MH), a major histamine metabolite, were significantly lower in obese (ob/ob) and diabetic (db/db) mice, and Zucker fatty (fa/fa) rats, leptin-deficient and leptin-receptor defective animals, respectively, relative to lean littermates (P < 0.05 for each). A bolus infusion of leptin (1.0 microg) into the lateral ventricle (ilvt) significantly elevated the turnover rate of hypothalamic neuronal histamine, as assessed by pargyline-induced accumulation of t-MH, in ob/ob mice compared with phosphate-buffered saline (PBS) infusions (P < 0.05). However, this same treatment did not affect hypothalamic histamine turnover in db/db mice. In agouti yellow (A(y)/a) mice, animals defective in pro-opiomelanocortin (POMC) signaling, normal levels of histamine, and t-MH were seen in the hypothalamus at 4 weeks of age when obesity had not yet developed. These amine levels in A(y)/a mice showed no change until 16 weeks of age, although the mice were remarkably obese by this time. Infusions of corticotropin releasing hormone (CRH), one of neuropeptide related to leptin signaling, into the third ventricle (i3vt) increased histamine turnover in the hypothalamus of Wistar King A rats (P < 0.05 versus PBS infusion). Infusion of neuropeptide Y (NPY) or alpha-melanocyte stimulating hormone (MSH), a POMC-derived peptide failed to increase histamine turnover. These results indicate that lowered activity of hypothalamic neuronal histamine in ob/ob and db/db mice, and fa/fa rats may be due to insufficiency of leptin action in the brains of these animals. These results also suggest that disruption of POMC signaling in A(y)/a mice may not impact on neuronal histamine. Moreover, CRH but neither POMC-derived peptide nor NPY may act as a signal to neuronal histamine downstream of the leptin signaling pathway.     

Apelin alleviates diabetes-associated endoplasmic reticulum stress in the pancreas of Akita mice

Apelin, a newly identified bioactive adipokine, has been found to play important roles in multiple diseases, including diabetes, hypertension and cardiovascular diseases with unclear molecular mechanisms. The present study aimed to investigate the effects of apelin on endoplasmic reticulum (ER) stress in the pancreas of Akita mice, a well-established type 1 diabetic model. Apelin-13 (400 pmol/kg) was injected from tail vein for 10 weeks. The physiological characters of experimental animals were evaluated, pancreatic islet morphology and insulin content were assessed by immunohistochemistry, and ER stress markers in the pancreas were examined by Western blots. Our results indicate apelin treatment significantly ameliorates diabetes-induced reduction in pancreatic islet mass and insulin content. Further studies suggested apelin treatment alleviates ER stress by inhibiting the diabetes induced up-regulation of PERK and IRE1α and chaperones (GRP78, calnexin and Hsp70) levels in Akita mice. We also demonstrated that apelin treatment normalizes the diabetes induced alteration of AKT and ERK activations in the pancreas of Akita mice. Taken together, these results suggest a novel physiological role of apelin in alleviating ER stress in the pancreas of type 1 diabetes.