Phosphodiesterase‐3B‐cAMP Pathway of Leptin Signalling in the Hypothalamus is Impaired During the Development of Diet‐Induced Obesity in FVB/N Mice

The phosphodiesterase‐3B (PDE3B)‐cAMP pathway plays an important role in transducing the action of leptin in the hypothalamus. Obesity is usually associated with hyperleptinaemia and resistance to anorectic and body weight‐reducing effects of leptin. To determine whether the hypothalamic PDE3B‐cAMP pathway of leptin signalling is impaired during the development of diet‐induced obesity (DIO), we fed male FVB/N mice a high‐fat diet (HFD: 58% kcal as fat) or low‐fat diet (LFD: 6% kcal as fat) for 4 weeks. HFD fed mice developed DIO in association with hyperphagia, hyperleptinaemia and hyperinsulinaemia. Leptin (i.p.) significantly increased hypothalamic PDE3B activity and phosphorylated (p)‐Akt levels in LFD‐fed but not in HFD‐fed mice. However, basal p‐Akt levels in hypothalamus were increased in DIO mice. Additionally, amongst six‐microdissected brain nuclei examined, leptin selectively decreased cAMP levels in the arcuate nucleus (ARC) of LFD‐fed mice but failed to do so in HFD‐fed mice. We next tested whether both the PBE3B and Akt pathways of leptin signalling remained impaired in DIO mice on the HFD for 12 weeks (long‐term). DIO mice were hyperinsulinaemic and hyperleptinaemic in association with impaired glucose and insulin tolerance. Although, in LFD‐fed mice, leptin significantly increased PDE3B activity and p‐Akt levels in the hypothalamus, it failed to do so in HFD‐fed mice. Also, basal p‐Akt levels in the hypothalamus were increased in DIO mice and leptin had no further effect. Similarly, immunocytochemistry showed that leptin increased the number of p‐Akt‐positive cells in the ARC of LFD‐fed but not in HFD‐fed mice, and there was an increased basal number of p‐Akt positive cells in the ARC of DIO mice. These results suggest that the PDE3B‐cAMP‐ and Akt‐pathways of leptin signalling in the hypothalamus are impaired during the development of DIO. Thus, a defect in the regulation by leptin of the hypothalamic PDE3B‐cAMP pathway and Akt signalling may be one of the mechanisms of central leptin resistance and the development of DIO.     

Adaptation of intestinal secretomotor function and nutrient absorption in response to diet‐induced obesity

Background The gut plays a significant role in the development of obesity, notably through peptide signaling to the brain. However, few studies have investigated intestinal function per se in a rodent model of diet‐induced obesity (DIO). Our aim was to investigate intestinal secretomotor function and glucose transport in DIO and diet‐resistant (DR) rat jejunum. Methods Male outbred Sprague–Dawley rats were maintained on a medium high fat diet for 9–10 weeks and split into DIO and DR groups based on weight gain. Mucosal‐submucosal preparations of the proximal jejunum were mounted in Ussing chambers and voltage‐clamped at 0 mV. Glucose (10 mmol L^?1), 2‐deoxy‐D‐glucose (10 mmol L^?1), and leptin (10 nmol L^?1) were added to the luminal side of the tissue and veratridine (30 μmol L^?1), bethanechol (100 μmol L^?1), and forskolin (10 μmol L^?1) were added to the basolateral side of the tissue . Key results Secretomotor responses were significantly decreased in DIO jejunum compared to DR tissues. Glucose‐stimulated increases in I_sc in DR animals, that were sensitive to leptin inhibition, were significantly reduced in DIO rats. Decreased sodium glucose transporter‐1 mediated glucose transport was accompanied by a concomitant increase in the expression of jejunal glucose transporter‐2. Conclusions & Inferences These data suggest that submucosal nerve function is compromised in DIO rats and electrogenic glucose transport is significantly decreased. The latter may represent an adaptive response to limit nutrient absorption in the jejunum from DIO rats. However, the loss of secretomotor control may lead to an altered host defense with a resultant change in intestinal flora contributing to the maintenance of obesity.     

AAV-mediated IL-10 gene transfer counteracts inflammation in the hypothalamic arcuate nucleus and obesity induced by high-fat diet

Consumption of high-fat diet (HFD) induces energy imbalance and consequently obesity. In the pathogenesis of obesity, HFD triggers inflammation in the hypothalamus including arcuate nucleus (ARC). Interleukin-10 (IL-10) is a representative anti-inflammatory cytokine, known to ameliorate the adipose tissue inflammation and insulin resistance in obesity. However, the effect of IL-10 on the hypothalamic inflammation remains less defined. We here report the effect of over-expression of murine IL-10 using adeno-associated virus (AAV) vector on the inflammation in ARC and feeding behavior in HFD-induced obese (DIO) mice. DIO mice exhibited reduced POMC expression and elevated IKKs (IκB kinases) and SOCS3 expression in ARC. Overexpression of mIL-10 using AAV vector ameliorated obesity in parallel with restoration of ARC POMC expression in DIO mice. Moreover, IL-10 treatment suppressed IKKs activation and SOCS3 expression in ARC of DIO mice. These results suggest that IL-10 gene transfer provides an effective approach for counteracting HFD-induced inflammation and leptin resistance in ARC to prevent progression of obesity.     

Intrathecal administration of low‐dose nociceptin/orphanin FQ induces allodynia via c‐Jun N‐terminal kinase and monocyte chemoattractant protein‐1

Pathological chronic pain, which is frequently associated with prolonged tissue damage, inflammation, tumour invasion, and neurodegenerative diseases, gives rise to hyperalgesia and allodynia. We previously reported that intrathecal administration of nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for the orphan opioid receptor‐like receptor, in the femtomole range induces touch‐evoked allodynia. N/OFQ has been implicated in multiple signalling pathways, such as inhibition of cAMP production and Ca²⁺ channels, or activation of K⁺ channels and mitogen‐activated protein kinase, although the signalling pathways of N/OFQ‐induced allodynia remain unclear. To address these issues, we developed an ex vivo mitogen‐activated protein kinase assay by using intact slices of mouse spinal cord. N/OFQ markedly increased the phosphorylation of c‐Jun N‐terminal kinase (JNK) in the superficial dorsal horn of the spinal cord. The N/OFQ‐stimulated JNK phosphorylation was significantly inhibited by pertussis toxin, the phospholipase C inhibitor U73122, and the inositol trisphosphate receptor antagonist Xestospongin C. Intrathecal administration of the JNK inhibitor SP600125 inhibited N/OFQ‐evoked allodynia. The N/OFQ‐induced increase in JNK phosphorylation was observed in astrocytes that expressed glial fibrillary acidic protein. N/OFQ also induced monocyte chemoattractant protein‐1 (MCP‐1) release via the JNK pathway, and N/OFQ‐induced JNK phosphorylation was observed in MCP‐1‐immunoreactive astrocytes. Intrathecal administration of the MCP‐1 receptor antagonist RS504393 inhibited N/OFQ‐evoked allodynia. These results suggest that, in the spinal dorsal horn, N/OFQ induces allodynia through activation of JNK via the phospholipase C–inositol trisphosphate pathway, which is coupled to pertussis toxin‐sensitive G‐protein, and following the release of MCP‐1 from astrocytes.     

Both p110α and p110β Isoforms of Phosphatidylinositol 3‐OH‐Kinase are Required for Insulin Signalling in the Hypothalamus

Both insulin and leptin action in the brain are considered to involve activation of phosphoinositide 3‐kinase (PI3K), although the roles of different PI3K isoforms in insulin signalling in the hypothalamus are unknown. In the present study, we characterised the roles of these isoforms in hypothalamic insulin and leptin signalling and investigated the cross‐talk of both hormones. To evaluate PI3K levels in the hypothalamus, PI3K was immunoprecipitated using an antibody directed against the p85 subunit, and then total PI3K activity was measured in the presence of novel isoform‐selective pharmacological inhibitors of each isoform of PI3K. Subsequently, these inhibitors were administered into the lateral ventricle of male Sprague‐Dawley rats, followed by vehicle, insulin, leptin or both hormones 45 min later. PI3K activity was determined by immunohistochemical detection of phosphorylated AKT (S473). In a separate study, the effects of the inhibitors on the anorexigenic action of insulin and leptin were determined. Hypothalamic insulin signalling was specifically mediated by the combined actions of the class Ia isoforms p110α and p110β. Total hypothalamic PI3K activity was inhibited 65% by a p110α inhibitor, and 35% by a p110β inhibitor, with a combination of inhibitors being equally effective as the broad‐spectrum PI3K inhibitor wortmannin. Individual i.c.v. administration of p110α and p110β inhibitors partly prevented insulin‐induced phosphorylated AKT (S473) in the arcuate nucleus, whereas simultaneous application completely blocked insulin action. Unlike insulin, leptin did not induce phosphorylated AKT in the hypothalamus, as detected by immunohistochemistry, and the anorectic effects of leptin were not affected by pre‐treatment with a combination of p110α and p110β inhibitors. The enhanced anorectic effect of a combined i.c.v. application of both insulin and leptin could be prevented by pre‐treatment with the combination of p110α and p110β inhibitors. The data suggest that p110α and p110β isoforms of PI3K are necessary to mediate insulin action in the hypothalamus. The role of PI3K in leptin action is less clear, but it may be involved by means of an insulin‐dependent sensitisation of leptin action.     

Leptin Responsive and GABAergic Projections to the Rostral Preoptic Area in Mice

The adipocyte‐derived hormone leptin plays a critical role in the control of reproduction via signalling in hypothalamic neurones. The drivers of the hypothalamic‐pituitary‐gonadal axis, the gonadotrophin‐releasing hormone (GnRH) neurones, do not have the receptors for leptin. Therefore, intermediate leptin responsive neurones must provide leptin‐to‐GnRH signalling. We investigated the populations of leptin responsive neurones that provide input to the rostral preoptic area (rPOA) where GnRH cell bodies reside. Fluorescent retrograde tracer beads (RetroBeads; Lumafluor Inc., Naples, FL, USA) were injected into the rPOA of transgenic leptin receptor enhanced green fluorescent protein (Lepr‐eGFP) reporter mice. Uptake of the RetroBeads by Lepr‐eGFP neurones was assessed throughout the hypothalamus. RetroBead uptake was most evident in the medial arcuate nucleus (ARC), the dorsomedial nucleus (DMN) and the ventral premammillary nucleus (PMV) of the hypothalamus. The uptake of RetroBeads specifically by Lepr‐eGFP neurones was highest in the medial ARC (18% of tracer‐labelled neurones Lepr‐eGFP‐positive). Because neurones that are both leptin responsive and GABAergic play a critical role in the regulation of fertility by leptin, we next focussed on the location of these populations. To address whether GABAergic neurones in leptin‐responsive hypothalamic regions project to the rPOA, the experiment was repeated in GABA neurone reporter mice (Vgat‐tdTomato). Between 10% and 45% of RetroBead‐labelled neurones in the ARC were GABAergic, whereas uptake of tracer by GABAergic neurones in the DMN and PMV was very low (< 5%). These results show that both leptin responsive and GABAergic neurones from the ARC project to the region of the GnRH cell bodies. Our findings suggest that LEPR‐expressing GABA neurones from the ARC may be mediators of leptin‐to‐GnRH signalling.     

The neuroprotective action of SP600125, a new inhibitor of JNK, on transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 via nuclear and non-nuclear pathways

Increasing evidence suggests that c-Jun N-terminal kinase (JNK) is an important kinase mediating neuronal apoptosis in brain ischemia. To further study the roles of JNK activation in hippocampal CA1 neurons in a rat model of transient global ischemia, we assessed the effect of JNK inhibition by SP600125 on the degree of brain injury. Our results demonstrated that SP600125 significantly increased the number of surviving cells in hippocampal CA1 subfield and decreased the activation of p-JNK1/2 and p-JNK3 at 30 min and 3 days after brain ischemia. Moreover, SP600125 significantly diminished the increased levels of phosphorylated-c-Jun (Ser63/73) and phosphorylated-Bcl-2 (Ser87) at 3 h after brain ischemia. These results indicate that SP600125, a new inhibitor of JNK, protected transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 region at least via suppressing the activation of nuclear substrate (c-Jun) and inactivating non-nuclear substrate (Bcl-2) induced by ischemic insult. Thus, inhibiting JNK activity by SP600125 may represent a new and effective strategy to treat ischemic stoke.     

Effects of Gold Thioglucose Treatment on Central Corticotrophin‐Releasing Hormone Systems in Mice

Systemic administration of gold thioglucose (GTG) causes a hypothalamic lesion that extends from the ventral part of the ventromedial hypothalamus (VMH) to the dorsal part of the arcuate nucleus (ARC), resulting in hyperphagia and obesity in mice. In the present study, we used in situ hybridisation histochemistry to explore the effects of GTG on the central corticotrophin‐releasing hormone (CRH) system, which regulates feeding and energy homeostasis. Type 2 CRH receptor (CRHR‐2) mRNA expression decreased by 40% at 8 weeks in the VMH and by 40–60% at 2 and 8 weeks in the ARC after GTG injection. By contrast, CRHR‐2 mRNA expression in the hypothalamic paraventricular nucleus (PVN) and lateral septum was unchanged. Urocortin (Ucn) 3 mRNA expression in the perifornical area and medial amygdala decreased, whereas CRH mRNA expression in the PVN increased at 2 and 8 weeks after GTG injection. Ucn 1 mRNA expression in the Edingher–Westphal nucleus and Ucn 2 mRNA expression in the PVN were unchanged. Because Ucn 3 is an anorexigenic and a possible endogenous ligand for VMH CRHR‐2, our results suggest that decreased Ucn 3 expression and decreased VMH CRHR‐2 expression contribute, in part, to GTG‐induced hyperphagia and obesity. To determine whether VMH CRHR‐2 mediates the anorexigenic effects of Ucn 3, Ucn 3 was administered i.c.v. and food intake was measured 8 weeks after GTG treatment. Ucn 3 decreased cumulative food intake on days 4–7 after surgery compared to i.c.v. administration of vehicle in control mice. By contrast, the anorexigenic effects of i.c.v. Ucn 3 were abolished in GTG‐treated mice. Taken together, our results indicate that the Ucn 3 pathway, which innervates the VMH, is involved in appetite regulation via CRHR‐2. It remains to be determined whether CRHR‐2 in the ARC has additional roles in appetite regulation by Ucn 3.     

Comparison of the effect of an H(3)-inverse agonist on energy intake and hypothalamic histamine release in normal mice and leptin resistant mice with high fat diet-induced obesity

Leptin is a key signal linking peripheral adiposity levels to the regulation of energy homeostasis in the brain. The injection of leptin decreases body weight and food intake in lean rodents; however, in a rodent model of high fat diet-induced obesity (DIO), the exogenous leptin cannot improve adiposity. This ineffectiveness is known as leptin resistance, and the factors downstream of leptin signaling have received attention as viable targets in the treatment of obesity. We previously reported that the histaminergic system is one of the targets of leptin. In the present study, the effect of an H(3)-receptor inverse agonist on hypothalamic histamine release and energy intake was investigated in normal and DIO mice. Leptin (1.3 mg/kg, i.p.) significantly increased hypothalamic histamine release and reduced 12 h-energy intake in normal mice, but had no such effects in DIO mice. In contrast, clobenpropit (5 mg/kg, i.p.), an H(3)-inverse agonist, elicited a significant increase in histamine release in both types of mice. Clobenpropit did not reduce 12 h-energy intake; however, it decreased 3 h-energy intake in both types of mice. These results suggest that lack of the activation of the histaminergic system partly contributes to obesity in DIO mice and direct activation of the histaminergic system circumvents leptin resistance.     

Hormones and diet,but not body weight,control hypothalamic microglial activity

The arcuate nucleus (ARC) of the hypothalamus plays a key role in sensing metabolic feedback and regulating energy homeostasis. Recent studies revealed activation of microglia in mice with high‐fat diet (HFD)‐induced obesity (DIO), suggesting a potential pathophysiological role for inflammatory processes within the hypothalamus. To further investigate the metabolic causes and molecular underpinnings of such glial activation, we analyzed the microglial activity in wild‐type (WT), monogenic obese ob/ob (leptin deficient), db/db (leptin‐receptor mutation), and Type‐4 melanocortin receptor knockout (MC4R KO) mice on either a HFD or on standardized chow (SC) diet. Following HFD exposure, we observed a significant increase in the total number of ARC microglia, immunoreactivity of ionized calcium binding adaptor molecule 1 (iba1‐ir), cluster of differentiation 68 (CD68‐ir), and ramification of microglial processes. The ob/ob mice had significantly less iba1‐ir and ramifications. Leptin replacement rescued these phenomena. The db/db mice had similar iba1‐ir comparable with WT mice but had significantly lower CD68‐ir and more ramifications than WT mice. After 2 weeks of HFD, ob/ob mice showed an increase of iba1‐ir, and db/db mice showed increase of CD68‐ir. Obese MC4R KO mice fed a SC diet had comparable iba1‐ir and CD68‐ir with WT mice but had significantly more ramifications than WT mice. Intriguingly, treatment of DIO mice with glucagon‐like peptide‐1 receptor agonists reduced microglial activation independent of body weight. Our results show that diet type, adipokines, and gut signals, but not body weight, affect the presence and activity levels of hypothalamic microglia in obesity. GLIA 2013;62:17–25     

The Response of Glucose-Excited Neurones in the Ventromedial Hypothalamus to Decreased Glucose is Enhanced in a Murine Model of Type 2 Diabetes Mellitus

Obesity and type 2 diabetes mellitus (T2DM) are associated with dysfunctional insulin signalling and impaired central glucose sensing. Glucose sensing neurones reside in key areas of the brain involved in glucose and energy homeostasis (e.g. ventromedial hypothalamus; VMH). We have recently shown that insulin attenuates the ability of glucose-excited (GE) neurones to sense decreased glucose. We hypothesise that this effect of insulin on VMH GE neurones is impaired during T2DM when insulin signalling is dysfunctional. To test our hypotheses, we used whole cell patch clamp recording techniques to evaluate the effects of insulin on VMH GE neurones in brain slices from wild-type and diabetic ( db/db ) mice. The effects of decreasing glucose from 2.5 to 0.1 m m on VMH GE neurones were similar in wild-type and db/db mice. However, decreasing glucose from 2.5 to 0.5 m m decreased the action potential frequency, membrane potential and input resistance of VMH GE neurones to a significantly greater extent in db/db versus wild-type mice. Furthermore, insulin (5 n m ) blunted the effects of decreased glucose in wild-type, but not db/db mice. These differences in both glucose and insulin sensitivity between wild-type and db/db mice were completely ameliorated by the insulin sensitiser, Compound 2 (300 n m ). These data are consistent with our hypothesis that impaired insulin signalling in T2DM sensitises VMH GE neurones to decreased glucose.     

Morphometric analysis of medial basal hypothalamic neuronal degeneration in diabetes (db/db) mutant C57BL/KsJ mice: relation to age and hyperglycemia

Age- and diabetes-related neuronal degenerative changes were morphometrically evaluated in the arcuate (ARC) and ventromedial (VMH) hypothalamic nuclei of control (+/?) and diabetic (db/db) C57BL/KsJ mice between 4 and 16 weeks of age. By 4 weeks of age, (db/db) mice exhibited marked obesity and hyperglycemia relative to controls. An increase in the population of degenerated ARC neurons was detected at 8 weeks of age in (db/db) mice relative to (+/?) animals. By 16 weeks of age, a significant increase in the number of degenerated VMH neurons in (db/db) mice was found, relative to controls. In addition, the neuronal density (neurons/mm2 area of nucleus) of both the ARC and VMH nuclei was found to be depressed in (db/db) mice, relative to controls, by 16 weeks of age. These data suggest that the normal degenerative loss of ARC and VMH neurons that occurs with age in normal mice is enhanced in the (db/db) mouse. These findings suggest that a functional alteration in hypothalamic nuclei which are recognized to modulate autonomic, pancreatic and pituitary activity may be associated with the onset or expression of the diabetic condition in the C57BL/KsJ (db/db) mouse.