Central action of xenin affects the expression of lipid metabolism-related genes and proteins in mouse white adipose tissue

Xenin is a gastrointestinal hormone that reduces food intake when administered centrally and it has been hypothesized that central action of xenin participates in the regulation of whole-body metabolism. The present study was performed to address this hypothesis by investigating the central effect of xenin on the expression of genes and proteins that are involved in the regulation of lipid metabolism in white adipose tissue (WAT). Male obese ob/ob mice received intracerebroventricular (i.c.v.) injections of xenin (5 μg) twice 12 h apart. Food intake and body weight change during a 24-h period after the first injection were measured. Epididymal WAT was collected at the end of the 24-h treatment period and levels of lipid metabolism-related genes and proteins were measured. Xenin treatment caused significant reductions in food intake and body weight compared to control vehicle treatment. Levels of fatty acid synthase (FASN) protein were significantly reduced by xenin treatment, while levels of adipose triglyceride lipase (Atgl) and beta-3 adrenergic receptor (Adrb3) mRNA and phosphorylated hormone sensitive lipase (Ser⁶⁶⁰-pHSL and Ser⁵⁶³-pHSL) were significantly increased by xenin treatment. These findings suggest that central action of xenin causes alterations in lipid metabolism in adipose tissue toward reduced lipogenesis and increased lipolysis, possibly contributing to xenin-induced body weight reduction. Thus, enhancing central action of xenin and its downstream targets may be possible targets for the treatment of obesity by reducing the amount of stored fat in adipose tissue.     

The protective effects of the melanocortin receptor (MCR) agonist,melanotan-II (MTII), against binge-like ethanol drinking are facilitated by deletion of the MC3 receptor in mice

Recent data have implicated the melanocortin (MC) system in modulating voluntary ethanol consumption. Administration of melanotan-II (MTII), a nonselective melanocortin receptor (MCR) agonist, reduces voluntary ethanol consumption in C57BL/6J mice. Previous studies have demonstrated that central infusion of MTII effectively reduced voluntary ethanol drinking in mutant mice lacking normal expression of MC3R (MC3R^−/− mice) but failed to alter ethanol drinking in mice lacking expression of MC4R, demonstrating that central MTII administration reduces voluntary ethanol drinking by signaling through the MC4R. However, evidence shows that the neurocircuitry recruited during excessive binge-like ethanol drinking versus moderate ethanol drinking are not identical. Thus the present study sought to investigate the potential role of the MC3R in binge-like ethanol intake. To this end, the “drinking in the dark” (DID) procedure, a commonly used animal model of binge-like ethanol drinking, was employed. Wild-type MC3R^+/+ and MC3R^−/− mice were given intracerebroventricular (i.c.v.) infusion of MTII (0.0, 0.25, 0.50, or 1.0 μg) prior to the onset of a 4-h testing period in which mice were given access to 20% (v/v) ethanol. Immediately after the 4-h testing period, tail blood samples were collected from each animal in order to assess blood ethanol concentrations (BECs). Consistent with previous findings, central administration of MTII blunted binge-like ethanol drinking in both MC3R^+/+ and MC3R^−/− mice. Interestingly, all doses of MTII blunted binge-like ethanol drinking in MC3R^−/− mice during the first hour of testing, while only the 1.0 μg dose reduced binge-like drinking in MC3R^+/+ mice. Thus, MC3R^−/− mice were more sensitive to the protective effects of MTII. These data suggest that MC3Rs oppose the protective effects of MTII against binge-like ethanol drinking, and thus selective MC3R antagonists may have potential therapeutic roles in treating excessive ethanol drinking.     

MTII-induced reduction of voluntary ethanol drinking is blocked by pretreatment with AgRP-(83-132)

Over the last 30 years, evidence has emerged indicating that the central melanocortin (MC) peptide system is involved with neurobiological responses to drugs of abuse. Recently, rats selectively bred for high ethanol preference were shown to have altered brain levels of MC receptor (MCR) and central infusion of the potent non-selective MCR agonist, melanotan-II (MTII), attenuates their high ethanol drinking. The goal of the present report was to further characterize the effects of MTII on voluntary ethanol consumption. In alcohol preferring C57BL/6 mice with an established history of ethanol drinking, intracerebroventricular (i.c.v.) infusion of a 5.0 microg dose of agouti-related protein (AgRP)-(83-132), a non-selective MCR antagonist, has no effect on 8-h ethanol drinking or food intake. However, pre-treatment with a 5.0 microg dose of (AgRP)-(83-132) significantly blocks MTII-induced (1.0 microg) reduction of 8-h ethanol drinking and food intake, consistent with a competitive antagonist action. I.c.v. infusion of MTII does not cause alteration of blood ethanol levels 2- or 4-h following intraperitoneal (i.p.) injection of a 4.0 g ethanol/kg dose. Finally, when given in an i.p. injection, a 150 microg dose of MTII reduces 8-h ethanol drinking. These data extend recent findings by showing that both central and peripheral administration of MTII reduces ethanol drinking by mice. Additionally, the ability of (AgRP)-(83-132) to block the effects of MTII implies that MTII-induced reduction of ethanol drinking is receptor mediated.     

Restorative effects of neurotrophin treatment on diabetes-induced cutaneous axon loss in mice

Chronic hyperglycemia in diabetes causes a variety of somatosensory deficits, including reduced cutaneous innervation of distal extremities. Deficient neurotrophin support has been proposed to contribute to the development of diabetic neuropathy. Here, studies were carried out in streptozotocin (STZ)-treated mice to determine whether (1) cutaneous innervation deficits develop in response to hyperglycemia, (2) neurotrophin production is altered in the skin, and (3) neurotrophin treatment improves cutaneous innervation deficits. Cutaneous innervation was quantified in the hindlimb skin using antibodies that label nerve growth factor- (NGF) responsive (CGRP), glial cell line-derived neurotrophic factor (GDNF)/neurturin (NTN) -responsive (P2X(3)), or all cutaneous axons (PGP 9.5). Diabetic mice displayed severely reduced cutaneous innervation for all three antibodies in both flank and footpad skin regions, similar to reports of cutaneous innervation loss in human diabetic patients. Qualitative assessment of mRNAs for NGF, GDNF, and NTN demonstrated that these mRNAs were expressed in hindlimb flank and footpad skin from diabetic mice. Next, diabetic mice were then treated intrathecally for 2 weeks with NGF, GDNF, or NTN. NGF treatment failed to improve cutaneous innervation, but stimulated axon branching. In comparison, GDNF and NTN treatment increased cutaneous innervation and axon branching. Our results reveal that similar to human diabetic patients, STZ-induced diabetes significantly reduces hindlimb cutaneous innervation in mice. Importantly, intrathecal treatment using GDNF or NTN strongly stimulated axon growth and branching, suggesting that administration of these trophic factors can improve cutaneous innervation deficits caused by diabetes.     

Effects of neuropeptide Y deficiency on hypothalamic agouti-related protein expression and responsiveness to melanocortin analogues

Central administration of neuropeptide Y (NPY) potently induces feeding and its abundance in the hypothalamus increases when energy stores fall. Consequently, NPY is considered to be a physiological effector of feeding behavior. Surprisingly, NPY-deficient (NPY-/-) mice feed and grow normally with ad libitum access to food and manifest a normal hyperphagic response after fasting, suggesting that other feeding effectors may compensate for the lack of NPY. Agouti-related protein (AgRP), a melanocortin receptor antagonist, can also stimulate feeding behavior when administered centrally and is coexpressed in a majority of hypothalmamic NPY-ergic neurons, making AgRP a candidate compensatory factor. To test this possibility, we evaluated AgRP mRNA and protein expression, as well as responsiveness to centrally administered AgRP in NPY-/- mice. These studies demonstrate that hypothalamic AgRP mRNA and immunoreactivity are upregulated with fasting and that these increases are not affected by NPY deficiency. Interestingly, NPY-/- mice are hypersensitive to central administration of AgRP(83-132), yet exhibit a normal response to centrally administered MTII, a melanocortin receptor agonist. These data suggest that if AgRP compensates for the lack of NPY in NPY-/- mice, it is not at the level of AgRP synthesis and may instead involve alterations in the postsynaptic signaling efficacy of AgRP. Moreover, the effects of AgRP are not limited to its actions at the melanocortin-4 receptor (MC4R), because MC4R-deficient (MC4R-/-) mice manifest a significant response to centrally administered AgRP. These data imply that AgRP has additional targets in the hypothalamus.     

Local insulin and the rapid regrowth of diabetic epidermal axons

Insulin deficiency may contribute toward the neurological deficits of diabetic polyneuropathy (DPN). In particular, the unique trophic properties of insulin, acting on sensory neuron and axon receptors offer an approach toward reversing loss of skin axons that develops during diabetes. Here we examined how local cutaneous insulin, acting on axon receptors, influences innervation of the epidermis. That cutaneous axons might be amenable to regrowth was suggested by confirming that a high proportion of epidermal axons expressed GAP43/B50, a growth associated protein. Also, IRβ (insulin receptor subunit β) mRNA was expressed and upregulated in the footpads of diabetic mice and protein expression was upregulated in their sensory dorsal root ganglia. Moreover, footpads expressed mRNAs of the downstream insulin transduction molecules, IRS-1 and IRS-2. IRβ protein was identified in dermal axons, some epidermal sensory axons, and in keratinocytes. In separate models of experimental diabetes, we identified a surprising and rapid local response of this axon population to insulin. C57BL/6J streptozotocin (STZ) injected mice, as a model of type 1 diabetes and dbdb mice, as a model of type 2 diabetes were both evaluated after 3 months of diabetes duration. Local hindpaw plantar injections of low dose subhypoglycemic insulin (that did not alter diabetic hyperglycemia) and carrier (into the opposite paw) were given over two days and innervation studied at 5 days. Insulin injections in both models were associated with an ipsilateral rise in the density of PGP 9.5 labeled diabetic epidermal axons at 5 days, compared to that of their contralateral carrier injected hindpaw. Nondiabetic controls did not have changes in innervation following insulin. In a separate cohort of STZ diabetic mice and controls evaluated for paw sensation, there was mild improvement in mechanical, but not thermal sensation at 2 weeks after insulin injection in diabetics but not controls. Fine unmyelinated epidermal axons have considerable plasticity. Here we identify a rapid improvement of skin innervation by doses of insulin insufficient to alter glycemia or innervation of the opposite paw. Local direct insulin signaling of receptors expressed on diabetic cutaneous axons may reverse retraction of their branches during experimental DPN.     

Melanocortin mediated inhibition of feeding behavior in rats

Melanocortinergic neurons are believed to play a role in the control of food intake. Melanocortin receptor agonists and antagonists modulate feeding in several mouse models of chemically and genetically induced hyperphagia. To date, little information is available describing the role of this neurological system in the control of the natural feeding cycle in genetically intact rats.To evaluate the involvement of melanocortins in spontaneous nocturnal feeding, the synthetic melanocortin receptor agonist, MTII and the antagonist, SHU9119 were administered ICV (third ventricle) alone and in combination. Dose-dependent inhibition or stimulation of food intake was observed with MTII or SHU9119, respectively. Co-injections containing equal concentrations of MTII and SHU9119 resulted in food intake that was indistinguishable from controls. Food intake patterns observed in studies in which various dose combinations of MTII and SHU9119 were co-injected are consistent with the concept that both affect feeding by acting on similar melanocortin receptors.The hypothesis that effects of melanocortins on feeding may be mediated via an NPY related pathway was tested by co-injecting MTII and NPY in a 2-h satiated food intake paradigm. MTII inhibited food intake induced by 5.0 μg hNPY in a dose dependent manner with the highest dose tested abolishing the NPY feeding response.The studies suggest that melanocortins act via specific receptors to control food intake in rats, possibly via an NPY related pathway. If similar neurochemical processes operate in humans, selectively modulating specific melanocortin receptor signaling may be an approach to the treatment of human obesity.     

Simultaneous POMC gene transfer to hypothalamus and brainstem increases physical activity, lipolysis and reduces adult-onset obesity

Pro-opiomelanocortin (POMC) neurons are identified in two brain sites, the arcuate nucleus of the hypothalamus and nucleus of the solitary tract (NTS) in brainstem. Earlier pharmacological and POMC gene transfer studies demonstrate that melanocortin activation in either site alone improves insulin sensitivity and reduces obesity. The present study, for the first time, investigated the long-term efficacy of POMC gene transfer concurrently into both sites in the regulation of energy metabolism in aged F344xBN rats bearing adult-onset obesity. Pair feeding was included to reveal food-independent POMC impact on energy expenditure. We introduced adeno-associated virus encoding either POMC or green fluorescence protein to the two brain areas in 22-month-old rats, then recorded food intake and body weight, assessed oxygen consumption, serum leptin, insulin and glucose, tested voluntary wheel running, analysed POMC expression, and examined fat metabolism in brown and white adipose tissues. POMC mRNA was significantly increased in both the hypothalamus and NTS region at termination. Relative to pair feeding, POMC caused sustained weight reduction and additional fat loss, lowered fasting insulin and glucose, and augmented white fat hormone-sensitive lipase activity and brown fat uncoupling protein 1 level. By wheel running assessment, the POMC animals ran twice the distance as the Control or pair-fed rats. Thus, the dual-site POMC treatment ameliorated adult-onset obesity effectively, involving a moderate hypophagia lasting ～60 days, enhanced lipolysis and thermogenesis, and increased physical activity in the form of voluntary wheel running. The latter finding provides a clue for countering age-related decline in physical activity.     

Diabetes, leukoencephalopathy and rage

Longstanding diabetes mellitus damages kidney, retina, peripheral nerve and blood vessels, but brain is not usually considered a primary target. We describe direct involvement of the brain, particularly white matter, in long-term (9 months) experimental diabetes of mice, not previously modeled, correlating magnetic resonance (MR) imaging with quantitative histological assessment. Leukoencephalopathy and cerebral atrophy, resembling that encountered in diabetic humans, developed in diabetic mice and was accompanied by time-related development of cognitive changes in behavioural testing. Increased RAGE (receptor for advanced glycation end products) expression, a mediator of widespread diabetic complications, increased dramatically at sites of white matter damage in regions of myelination. RAGE expression was also elevated within neurons, astrocytes and microglia in grey matter and within oligodendrocytes in white matter. RAGE null diabetic mice had significantly less neurodegenerative changes when compared to wild-type diabetic mice. Our findings identify a robust and novel model of cerebral, particularly white matter, involvement with diabetes associated with abnormal RAGE signaling.     

Streptozotocin-induced diabetes causes metabolic changes and alterations in neurotrophin content and retrograde transport in the cervical vagus nerve.

Abnormal availability of neurotrophins, such as nerve growth factor (NGF), has been implicated in diabetic somatosensory polyneuropathy. However, the involvement of neurotrophins in diabetic neuropathy of autonomic nerves, particularly the vagus nerve which plays a critical role in visceral afferent and in autonomic motor functions, is unknown. To assess the effects of hyperglycemia on the neurotrophin content and transport in this system, cervical vagus nerves of streptozotocin (STZ)-induced diabetic rats were studied at 8, 16, and 24 weeks after the induction of diabetes. Elevations in vagus nerve hexose (glucose and fructose) and polyol levels (sorbitol), and their normalization with insulin treatment, verified that the STZ treatment resulted in hyperglycemia-induced metabolic abnormalities in the nerve. Neurotrophin (NGF and neurotrophin-3; NT-3) content and axonal transport were assessed in the cervical vagus nerves from nondiabetic control rats, STZ-induced diabetic rats, and diabetic rats treated with insulin. The NGF, but not the NT-3, content of intact vagus nerves from diabetic rats was increased at 8 and 16 weeks (but not at 24 weeks). Using a double-ligation model to assess the transport of endogenous neurotrophins, the retrograde transport of both NGF and NT-3 was found to be significantly reduced in the cervical vagus nerve at later stages of diabetes (16 and 24 weeks). Anterograde transport of NGF or NT-3 was not apparent in the vagus nerve of diabetic or control rats. These data suggest that an increase in vagus nerve NGF is an early, but transient, response to the diabetic hyperglycemia and that a subsequent reduction in neuronal access to NGF and NT-3 secondary to decreased retrograde axonal transport may play a role in diabetes-induced damage to the vagus nerve.     

Ischemia-reperfusion injury of peripheral nerve in experimental diabetic neuropathy

The pathogenesis of human diabetic neuropathy likely involves the interplay of hyperglycemia, ischemia, and oxidative stress. Mild-moderate ischemia-reperfusion to streptozotocin (STZ)-induced diabetes results in florid fiber degeneration in diabetic but not in normal nerves. Uncertainty exists as to the influence of duration of diabetes on this susceptibility. We therefore studied diabetic tibial and sciatic nerves using a rat ischemia-reperfusion (IR) model after 1 month and 4 months of diabetes utilizing electrophysiological, behavioral, and neuropathological methods. Electrophysiological abnormalities were present in 1-month diabetic rats (D) and persisted over 4 months. Behavioral scores were decreased markedly at 4 months (p<0.05). Endoneurial edema and ischemia fiber degeneration (IFD) were observed at both the 1-month (p<0.01 and p<0.001) and 4-month (p<0.001) durations in diabetic nerves, whereas only mild or no damage was observed in age-matched control nerves. These findings demonstrate that STZ-induced diabetes exacerbates the morphological and electrophysiological pathology in peripheral nerve to IR injury both in the early timepoint of 1 month and late timepoint of 4 months, although there was a gradation of injury, which is more severe at the later timepoint. Reperfusion exaggerated morphological pathology in 1-month STZ-induced diabetic peripheral nerve.     

Food deprivation induces adipose plasminogen activator inhibitor-1 (PAI-1) expression without accumulation of plasma PAI-1 in genetically obese and diabetic db/db mice

Relationships between energy intake and fibrinolytic functions have been documented in detail. We evaluated food deprivation (FD) as a means of modulating fibrinolytic activity in genetically obese and diabetic db/db mice and in their lean counterparts. Twelve hours of FD induced considerable gene expression of plasminogen activator inhibitor-1 (PAI-1) in both epididymal (3.8-fold, p < 0.05) and intestinal (2.4-fold, p < 0.05) adipose tissues without affecting plasma PAI-1 levels in db/db mice, whereas the FD did not affect these parameters in wild-type mice. Importantly, 24 hours of FD increased the plasma PAI-1 content in wild-type (1.9-fold, p < 0.01) but not in db/db mice, although adipose PAI-1 mRNA levels were significantly increased in db/db mice. The plasma PAI-1 content significantly correlated with hepatic PAI-1 mRNA levels in wild-type (r = 0.84, p < 0.01) and in db/db (r = 0.63, p < 0.01) mice. However, plasma PAI-1 did not correlate with adipose PAI-1 expression in db/db mice, although adipose tissue in general is thought to be the principal site of PAI-1 production in obesity. Hepatic PAI-1 expression was closely correlated with serum levels of free fatty acids in wild-type (r = 0.72, p < 0.01), but not in db/db mice. Adipose PAI-1 expression significantly correlated with serum corticosterone levels in both genotypes (wild-type, r = 0.52, p < 0.05; db/db, r = 0.51, p < 0.01), suggesting that adipose PAI-1 expression is up-regulated by fasting-induced glucocorticoids. The present findings suggested that fasting differentially affects fibrinolytic activity in obese and lean subjects and that PAI-1 expression in the liver as well as in adipose tissues comprises an important determinant of increased risk for cardiovascular disease in obesity.     

Expression of type-1 plasminogen activator inhibitor in the kidney of diabetic rat models

INTRODUCTION: Intrarenal coagulation and fibrinolysis are thought to be involved in the pathogenesis of diabetic nephropathy. However, gene expression of fibrinolytic factors in diabetic nephropathy has not been clearly defined. Therefore we determined the gene expression of fibrinolytic factors in the kidneys of diabetic rats. MATERIALS AND METHODS: As a model of type1 diabetes male Sprague-Dawley rats were used. They were divided into three groups: control, streptozotocin (STZ)-induced diabetic, and insulin-treated diabetic. Otsuka Long-Evans Tokushima Fatty (OLETF) rats were used as a model of type 2 diabetes; and Long-Evans Tokushima Otsuka (LETO) rats, as the control. Renal gene expressions of type-1 plasminogen activator inhibitor (PAI-1), tissue-type PA (tPA), and urokinase-type PA (uPA) were examined by real-time PCR. Localization of PAI-1 mRNA was investigated by in situ hybridization. RESULTS: Renal PAI-1 mRNA levels (versus control) were increased by 60-80% in STZ-induced diabetic rats (10 days or 3 weeks post STZ injection); and insulin treatment reduced this increased expression to the control level. In OLETF rats (38 weeks old), the renal PAI-1 mRNA level was 2.5-fold higher than that in age-matched LETO rats. Both tPA and uPA mRNA levels were significantly lower than those in LETO rats. PAI-1 mRNA was observed in intraglomerular cells and tubular epithelial cells of both models. CONCLUSIONS: Renal PAI-1 gene expression is up-regulated in both type 1 and type 2 diabetic rats, and changes in gene expressions of fibrinolytic factors may play important roles in the development and pathogenesis of diabetic nephropathy.     

Streptozotocin-induced diabetes selectively alters the potency of analgesia produced by mu-opioid agonists, but not by delta- and kappa-opioid agonists.

To investigate the possible mechanisms of the alterations in morphine-induced analgesia observed in diabetic mice, we examined the influence of streptozotocin-induced (STZ-induced) diabetes on analgesia mediated by the different opioid receptors. The antinociceptive potency of morphine (10 mg/kg), administered s.c., as determined by both the tail-pinch and the tail-flick test, was significantly reduced in diabetic mice as compared to that in controls. Mice with STZ-induced diabetes had significantly decreased sensitivity to intracerebroventricularly (i.c.v.) administered mu-opioid agonists, such as morphine (10 micrograms) and [D-Ala2,N-Me Phe4,Gly-ol5]enkephalin (DAMGO, 0.5 micrograms). However, i.c.v. administration of [D-Pen2,5]enkephalin (DPDPE, 5 micrograms), a delta-opioid agonist, and U-50,488H (50 micrograms), a kappa-opioid agonist, produced pronounced antinociception in both control and diabetic mice. Furthermore, there were no significant differences in antinociceptive potency between diabetic and control mice when morphine (1 microgram), DAMGO (10 micrograms), DPDPE (0.5 micrograms) or U-50,488H (50 micrograms) was administered intrathecally. In conclusion, mice with STZ-induced diabetes are selectively hyporesponsive to supraspinal mu-opioid receptor-mediated antinociception, but they are normally responsive to activation of delta- and kappa-opioid receptors.     

LPS-dependent suppression of social exploration is augmented in type 1 diabetic mice

We have previously shown that type 2 diabetes (T2D) in the mouse is associated with increased responsivity to innate immune challenge. Here we demonstrate that in a mouse model of type 1 diabetes (T1D) LPS-dependent suppression of social exploration (SE) is augmented and dependent on hyperglycemia. T1D was induced in mice with intraperitoneal (i.p.) streptozotocin (STZ). After 4d, STZ treated mice had blood glucose levels of 417+/-34mg/dl compared to 160+/-11mg/dl in non-STZ treated mice. When these diabetic mice were challenged with i.p. lipopolysaccharide (LPS), LPS-induced depression of SE was nearly 2.7-fold greater in diabetic mice at 2h than in non-diabetic mice. Examination of peritoneal proinflammatory cytokine levels 2h after LPS administration showed that diabetic mice had 4-, 2.5- and 3.6-fold greater concentrations of IL-1beta, IL-6 and TNF-alpha, respectively, when compared to non-diabetic mice. Control of blood glucose levels with injected insulin in diabetic mice improved 2h post LPS-induced loss of SE by 3.9-fold. Interestingly, insulin given intracerebroventricularly to diabetic mice did not impact LPS-induced loss of SE but did increase basal SE 8, 12 and 24h later. Finally, administration of STZ to hyperglycemic/hyperinsulinemic db/db mice did not alter LPS-induced loss of SE. Taken together these findings indicate that mice with T1D have augmented loss of SE in response to LPS and this is due to hyperglycemia and not to insulin.     

Modulation of melanocortin-induced changes in spinal nociception by mu-opioid receptor agonist and antagonist in neuropathic rats

Co-localization of opioid and melanocortin receptor expression, especially at the spinal cord level in the dorsal horn and in the gray matter surrounding the central canal led to the suggestion that melanocortins might play a role in nociceptive processes. In the present studies, we aimed to determine the effects of melanocortins, administered intrathecally, on allodynia, and to ascertain whether there is an interaction between opioid and melanocortin systems at the spinal cord level. Neuropathic pain was induced by chronic constriction injury (CCI) of the right sciatic nerve in rats. Tactile allodynia was assessed using von Frey filaments, while thermal hyperlagesia was evaluated in cold water allodynia test. In the present experiments, melanocortin receptor antagonist, SHU9119 was much more potent than mu-opioid receptor agonist, morphine after their intrathecal (i.th.) administration in neuropathic rats. SHU9119 alleviated allodynia in a comparable manner to DAMGO, a selective and potent mu-opioid receptor agonist. Administration of melanocortin receptor agonist, melanotan-II (MTII) increased the sensitivity to tactile and cold stimulation. Moreover, we demonstrated that the selective blockade of mu-opioid receptor by cyprodime (CP) enhanced antiallodynic effect of SHU9119 as well as pronociceptive action of MTII, whereas the combined administration of mu receptor agonist (DAMGO) and SHU9119 significantly reduced the analgesic effect of those ligands. DAMGO also reversed the proallodynic effect of melanocortin receptor agonist, MTII. In conclusion, it seems that the endogenous opioidergic system acts as a functional antagonist of melanocortinergic system, and mu-opioid receptor activity appears to be involved in the modulation of melanocortin system function.     

Brain antioxidant capacity in rat models of betacytotoxic-induced experimental sporadic Alzheimer's disease and diabetes mellitus

It is believed that oxidative stress plays a central role in the pathogenesis of metabolic diseases like diabetes mellitus (DM) and its complications (like peripheral neuropathy) as well as in neurodegenerative disorders like sporadic Alzheimer's disease (sAD). Representative experimental models of these diseases are streptozotocin (STZ)-induced diabetic rats and STZ-intracerebroventricularly (STZ-icv) treated rats, in which antioxidant capacity against peroxyl (ORAC(-ROO)*) and hydroxyl (ORAC(-OH)*) free radical was measured in three different brain regions (hippocampus, cerebellum, and brain stem) by means of oxygen radical absorbance capacity (ORAC) assay. In the brain of both STZ-induced diabetic and STZ-icv treated rats decreased antioxidant capacity has been found demonstrating regionally specific distribution. In the diabetic rats these abnormalities were not associated with the development of peripheral diabetic neuropathy. Also, these abnormalities were not prevented by the icv pretreatment of glucose transport inhibitor 5-thio-D-glucose in the STZ-icv treated rats, suggesting different mechanism for STZ-induced central effects from those at the periphery. Similarities in the oxidative stress alterations in the brain of STZ-icv rats and humans with sAD could be useful in the search for new drugs in the treatment of sAD that have antioxidant activity.     

Feeding responses to a melanocortin agonist and antagonist in obesity induced by a palatable high-fat diet

Hypothalamic melanocortins are critical for the control of food intake, and alterations in POMC mRNA have been described in genetic models of obesity. However, the time course of changes in brain transmitters over the development of dietary obesity is less clear. Therefore, we examined the effect of diet-induced obesity on hypothalamic alpha-MSH content and feeding responsiveness to synthetic melanocortins. Male Sprague-Dawley rats fed a high-fat cafeteria diet (30% fat) or chow (5% fat) for 4 or 12 weeks were implanted with intracerebroventricular cannulae and feeding responses to the MC3/4R agonist MTII (0.5 nmol) and the selective MC4R antagonist HS014 (0.8 nmol) were determined. MTII had a long-lasting inhibitory effect on food intake. Chronically overfed animals had a significantly exaggerated inhibitory feeding response 15 and 24 h after MTII injection and lost more body weight (15 +/- 3 g) compared to control rats (4 +/- 4 g; P < 0.05). Daytime administration of HS014 significantly increased food intake in all rats to the same extent (P < 0.05). No change in hypothalamic alpha-MSH content was observed after 2 or 12 weeks of high-fat diet. The observation of increased responsiveness to the melanocortin agonist, in the face of a high-fat diet, suggests melanocortin analogues may have potential for the pharmacological treatment of obesity.     

Functional plasticity of central TRPV1 receptors in brainstem dorsal vagal complex circuits of streptozotocin-treated hyperglycemic mice

Emerging data indicate that central neurons participate in diabetic processes by modulating autonomic output from neurons in the dorsal motor nucleus of the vagus (DMV). We tested the hypothesis that synaptic modulation by transient receptor potential vanilloid type 1 (TRPV1) receptors is reduced in the DMV in slices from a murine model of type 1 diabetes. The TRPV1 agonist capsaicin robustly enhanced glutamate release onto DMV neurons by acting at preterminal receptors in slices from intact mice, but failed to do so in slices from diabetic mice. TRPV1 receptor protein expression in the vagal complex was unaltered. Brief insulin preapplication restored TRPV1-dependent modulation of glutamate release in a PKC- and PI3K-dependent manner. The restorative effect of insulin was prevented by brefeldin A, suggesting that insulin induced TRPV1 receptor trafficking to the terminal membrane. Central vagal circuits critical to the autonomic regulation of metabolism undergo insulin-dependent synaptic plasticity involving TRPV1 receptor modulation in diabetic mice after several days of chronic hyperglycemia.     

Cerebellar Glucose During Fasting and Acute Hyperglycemia in Nondiabetic Men and in Men with Type 1 Diabetes

In diabetic patients, proton magnetic resonance spectroscopy (¹H MRS) has revealed increased brain glucose concentration and metabolite alterations that indicate neuronal damage and glial cell activation. Cerebellum is known to be more resistant to hypoglycemia than cerebrum, but the effects of both chronic and acute hyperglycemia on the cerebellum are less well known. ¹H MRS was used to quantify brain glucose and metabolite levels in the cerebellum, cerebral cortex, cerebral white matter, and the thalamus of diabetic and nondiabetic men after an overnight fast and during a hyperglycemic normoinsulinemic clamp with blood glucose 12 mmol/l above baseline. Fasting glucose levels were twice as high in the cerebellum than in the cerebrum. During acute hyperglycemia, the cerebellar glucose concentration increased by 3.0 mmol/l, which equals that in the cortex, but is 35% more than in the thalamus and 173% more than in the white matter. Acute hyperglycemia also increased the cerebellar tissue water content by 10%. There were no differences between diabetic and nondiabetic participants. Notably, the patients with complication free type 1 diabetes showed brain metabolite alterations in the cerebral cortex and the white matter but not in the cerebellum. Our study suggests that diabetes does not alter glucose content or uptake in the cerebellum. The increase in tissue water during acute hyperglycemia may serve to protect the cerebellum from the potentially deleterious effects of the excess glucose.