Melanocortin activation of nucleus of the solitary tract avoids anorectic tachyphylaxis and induces prolonged weight loss

To examine the role of the brain stem melanocortin system in long-term energy regulation, we assessed the effects of overproduction of proopiomelanocortin (POMC) in the caudal brain stem of F344xBN rats with adult-onset obesity. Recombinant adeno-associated viral vector encoding POMC gene was delivered to the nucleus of solitary tract (NTS) in the hindbrain, and food intake, body weight, glucose and fat metabolism, brown adipose tissue thermogenesis, and mRNA levels of neuropeptides and melanocortin receptors were assessed. POMC delivery resulted in sustained reduction in food intake and body weight over 42 days and improved insulin sensitivity. At death, in recombinant adeno-associated viral vector-POMC-treated rats vs. control rats, alpha-melanocyte-stimulating hormone in NTS increased nearly 21-fold, whereas hypothalamic alpha-melanocyte-stimulating hormone remained unchanged. Visceral adiposity decreased by 37%; tissue triglyceride content diminished by 26% and 47% in liver and muscle, respectively; serum triglyceride and nonesterified fatty acids were reduced by 35% and 34%, respectively; phosphorylation of acetyl-CoA carboxylase was elevated by 63% in soleus muscle; brown adipose tissue uncoupling protein 1 increased by 30%; and melanocortin 3 receptor expression declined by 60%, whereas neuropeptide Y, agouti-related protein, and MC4 receptor mRNA levels were unchanged in the NTS. In conclusion, POMC overexpression in the NTS produces a characteristic unabated hypophagia that is uniquely different from the anorexic tachyphylaxis following POMC overexpression in the hypothalamus. The sustained anorectic response may result from absence of compensatory elements in the NTS, such as increased agouti-related protein expression, suggesting melanocortin activation of the brain stem may be a viable strategy to alleviate obesity.     

Real-Time Continuous Glucose Monitoring Shows High Accuracy within 6 Hours after Sensor Calibration: A Prospective Study

Accurate and timely glucose monitoring is essential in intensive care units. Real-time continuous glucose monitoring system (CGMS) has been advocated for many years to improve glycemic management in critically ill patients. In order to determine the effect of calibration time on the accuracy of CGMS, real-time subcutaneous CGMS was used in 18 critically ill patients. CGMS sensor was calibrated with blood glucose measurements by blood gas/glucose analyzer every 12 hours. Venous blood was sampled every 2 to 4 hours, and glucose concentration was measured by standard central laboratory device (CLD) and by blood gas/glucose analyzer. With CLD measurement as reference, relative absolute difference (mean±SD) in CGMS and blood gas/glucose analyzer were 14.4%±12.2% and 6.5%±6.2%, respectively. The percentage of matched points in Clarke error grid zone A was 74.8% in CGMS, and 98.4% in blood gas/glucose analyzer. The relative absolute difference of CGMS obtained within 6 hours after sensor calibration (8.8%±7.2%) was significantly less than that between 6 to 12 hours after calibration (20.1%±13.5%, p<0.0001). The percentage of matched points in Clarke error grid zone A was also significantly higher in data sets within 6 hours after calibration (92.4% versus 57.1%, p<0.0001). In conclusion, real-time subcutaneous CGMS is accurate in glucose monitoring in critically ill patients. CGMS sensor should be calibrated less than 6 hours, no matter what time interval recommended by manufacturer.     

Tissue inhibitor of matrix metalloproteinase-3 expression in the mouse uterus during implantation and artificially induced decidualization

During implantation in mice, tissue inhibitor of matrix metalloproteinases-3 is believed to play a key role in inhibiting matrix metalloproteinase activity associated with embryo invasion and tissue remodeling. The first objective of this study was to quantitatively compare the steady-state mRNA levels of tissue inhibitors of matrix metalloproteinases between segments of the mouse uterus undergoing decidualization compared to those that are not during early pregnancy plus oil-induced decidualization. Steady-state tissue inhibitor of metalloproteinase-3 mRNA levels were significantly greater in implantation compared to interimplantation areas on days 6 and 7 of pregnancy and in stimulated compared to nonstimulated uterine horns at 48 and 72 hr after artificial induction of decidualization. Steady-state tissue inhibitor of metalloproteinase-1 mRNA levels were significantly greater in implantation compared to interimplantation areas on days 5-8 of pregnancy and in stimulated compared to nonstimulated uterine horns at 24, 48, and 72 hr after oil stimulation. Therefore, the steady-state mRNA levels of tissue inhibitors of metalloproteinase-1 and -3 increased in the uterus during decidualization. The second objective of this study was to determine if transforming growth factor-beta1 influences tissue inhibitors of metalloproteinase mRNA concentrations in mouse endometrial stromal cells. As determined by Northern blot analyses, transforming growth factor beta1 significantly increased tissue inhibitors of matrix metalloproteinases-1 and -3 mRNA levels in cultured mouse endometrial stromal cells isolated from uteri sensitized for decidualization. On the other hand, interleukin-1, epidermal growth factor, and leukemia inhibitory factor had no effect. The results of this study further characterize the tissue inhibitor of metalloproteinase expression in the uterus during implantation and artificially induced decidualization and the potential control of their expression in the stroma by transforming growth factor.     

Visfatin mRNA expression in human subcutaneous adipose tissue is regulated by exercise

Visfatin [pre-beta-cell colony-enhancing factor (PBEF)] is a novel adipokine that is produced by adipose tissue, skeletal muscle, and liver and has insulin-mimetic actions. Regular exercise enhances insulin sensitivity. In the present study, we therefore examined visfatin mRNA expression in abdominal subcutaneous adipose tissue and skeletal muscle biopsies obtained from healthy young men at time points 0, 3, 4.5, 6, 9, and 24 h in relation to either 3 h of ergometer cycle exercise at 60% of Vo(2 max) or rest. Adipose tissue visfatin mRNA expression increased threefold at the time points 3, 4.5, and 6 h in response to exercise (n = 8) compared with preexercise samples and compared with the resting control group (n = 7, P = 0.001). Visfatin mRNA expression in skeletal muscle was not influenced by exercise. The exercise-induced increase in adipose tissue visfatin was, however, not accompanied by elevated levels of plasma visfatin. Recombinant human IL-6 infusion to mimic the exercise-induced IL-6 response (n = 6) had no effect on visfatin mRNA expression in adipose tissue compared with the effect of placebo infusion (n = 6). The finding that exercise enhances subcutaneous adipose tissue visfatin mRNA expression suggests that visfatin has a local metabolic role in the recovery period following exercise.     

Continuous glucose monitoring in cystic fibrosis patients according to the glucose tolerance.

Cystic fibrosis (CF) is associated with a long preclinical state of abnormal glucose tolerance. The aim of this study was (i) to evaluate the profile of glucose tolerance in young adults with CF and (ii) to compare these results with those obtained by a continuous subcutaneous glucose monitoring (CGMS). CF subjects with fasting glycemia inferior to 126 mg/dl were included in the study. An oral glucose tolerance test (OGTT) identified the subjects either with a normal glucose tolerance (NGT), or impaired glucose tolerance (IGT), or diabetes. CGMS (Medtronic) was performed during 3 days to analyze mean glucose level, high glucose excursions, and glucose area under the curve (AUC). Forty-nine patients were included in the study. NGT (n=22), IGT (n=17), and diabetes groups (n=10) were comparable except with regard to age and BMI (p<0.001). HbA1c values in diabetes group were significantly higher (p<0.001) than in NGT and IGT groups. CGMS revealed peaks of glucose values superior to 200 mg/dl at least once after a meal in 8 patients (36%) with NGT, in 9 patients (52%) with IGT, and in all patients with diabetes (p<0.01). Mean CGMS glucose and glucose AUC values increased in patients with diabetes compared to patients with NGT and IGT (p<0.05). Peak of CGMS glucose reached 182+/-60 mg/dl in NGT group despite the normal glucose profile at OGTT. In conclusion, CGMS revealed pathological glucose excursions not only in patients with impaired glucose tolerance at OGTT but also in patients with a normal glycemic profile. CGMS could be a useful tool for the early detection of hyperglycemia in patients with CF.     

Involvement of 3',5'-cyclic adenosine monophosphate-dependent protein kinase in regulation of Fos expression and tyrosine hydroxylase levels during morphine withdrawal in the hypothalamic paraventricular nucleus and medulla oblongata catecholaminergic cell groups

Morphine withdrawal stimulates the hypothalamic-pituitary-adrenocortical axis activity by activation of nucleus tractus solitarius (NTS)/ventrolateral medulla (VLM) noradrenergic pathways innervating the hypothalamic paraventricular nucleus (PVN). We investigated whether cAMP-dependent protein kinase (PKA) plays a role in this process by estimating changes in PKA immunoreactivity and the influence of inhibition of PKA on Fos protein expression and tyrosine hydroxylase (TH) immunoreactivity levels in the PVN and NTS/VLM during morphine withdrawal. Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (5 mg/kg s.c.). When opioid withdrawal was precipitated, an increase in PKA immunoreactivity levels was observed 90 min after naloxone administration in the PVN and NTS/VLM areas. Morphine withdrawal induced expression of Fos in the PVN and NTS/VLM, indicating an activation of neurones in those nuclei. TH immunoreactivity in NTS/VLM was increased 90 min after induction of morphine withdrawal, whereas there was a decrease in TH levels in the PVN at the same time point. When the selective PKA inhibitor HA-1004 was infused it greatly diminished the Fos expression observed in morphine-withdrawn rats. Furthermore, the changes in TH immunoreactivity were significantly modified by infusion of HA-1004. The present findings suggest that an up-regulated PKA-dependent transduction pathway might contribute to the activation of the hypothalamic-pituitary-adrenocortical axis in response to morphine withdrawal.     

First step toward near-infrared continuous glucose monitoring: in?vivo evaluation of antibody coupled biomaterials

Continuous glucose monitoring (CGM) is crucial in diabetic care. Long-term CGM systems however require an accurate sensor as well as a suitable measuring environment. Since large intravenous sensors are not feasible, measuring inside the interstitial fluid is considered the best alternative. This option, unfortunately, has the drawback of a lag time with blood glucose values. A good strategy to circumvent this is to enhance tissue integration and enrich the peri-implant vasculature. Implants of different optically transparent biomaterials (poly(methyl-methacrylate) [PMMA] and poly(dimethylsiloxane) [PDMS]) – enabling glucose monitoring in the near-infrared (NIR) spectrum – were surface-treated and subsequently implanted in goats at various implantation sites for up to 3 months. The overall in vivo biocompatibility, tissue integration, and vascularization at close proximity of the surfaces of these materials were assessed. Histological screening showed similar tissue reactions independent of the implantation site. No significant inflammation reaction was observed. Tissue integration and vascularization correlated, to some extent, with the biomaterial composition. A modification strategy, in which a vascular endothelial-cadherin antibody was coupled to the biomaterials surface through a dopamine layer, showed significantly enhanced vascularization 3 months after subcutaneous implantation. Our results suggest that the developed strategy enables the creation of tissue interactive NIR transparent packaging materials, opening the possibility of continuous glucose monitoring.     

Regulation of glucose transporter messenger RNA levels in rat adipose tissue by insulin

Analysis of glucose transporter mRNA levels in adipose tissue from streptozotocin (STZ)-induced diabetic rats demonstrated a specific decrease (10-fold) in adipose tissue GLUT-4 mRNA with no significant effect on GLUT-1 mRNA levels. Treatment of STZ-diabetic rats with twice daily injections of insulin for 1-3 days resulted in a 16-fold increase in the relative amount of GLUT-4 mRNA to levels approximately 2-fold greater than those in control animals. However, after 7 days of insulin therapy the amount of GLUT-4 mRNA decreased approximately 2-fold back to the levels in the control animals. Normalization of the STZ-induced serum hyperglycemia by phlorizin treatment, which inhibits renal tubular reabsorption of glucose, had no effect on GLUT-4 mRNA in the absence of insulin. Similar to STZ-diabetes, fasting for 48 h also reduced adipose GLUT-4 mRNA levels. Parenteral administration of insulin with glucose over 7.5 h, but not glucose alone, increased the levels of the GLUT-4 mRNA 3- to 4-fold. These studies demonstrate that the relative glycemic state does not influence GLUT-4 glucose transporter mRNA expression in vivo and strongly suggests that insulin is a major factor regulating the levels of GLUT-4 mRNA in adipose tissue.     

Regulation of macrophage migration inhibitory factor (MIF) expression by glucose and insulin in adipocytes in vitro.

BACKGROUND: It has been reported that macrophage migration inhibitory factor (MIF) stimulated insulin secretion from pancreatic islet beta-cells in an autocrine manner, which suggests its pivotal role in the glucose metabolism. According to this finding, we evaluated MIF expression in cultured adipocytes and epididymal fat pads of obese and diabetic rats to investigate its role in adipose tissue. MATERIALS AND METHODS: The murine adipocyte cell line 3T3-L1 was used to examine MIF mRNA expression and production of MIF protein in response to various concentrations of glucose and insulin. Epididymal fat pads of Otsuka Long-Evans Tokushima fatty (OLETF) and Wistar fatty rats, animal models of obesity and diabetes, were subjected to Northern blot analysis to determine MIF mRNA levels. RESULTS: MIF mRNA of 3T3-L1 adipocytes was up-regulated by costimulation with glucose and insulin. Intracellular MIF content was significantly increased by stimulation, whereas its content in the culture medium was decreased. When the cells were treated with cytochalasin B, MIF secretion in the medium was increased. Pioglitazone significantly increased MIF content in the culture medium of 3T3-L1 cells. However, MIF mRNA expression of both epididymal fat pads of OLETF and Wistar fatty rats was down-regulated despite a high plasma glucose level. The plasma MIF level of Wistar fatty rats was significantly increased by treatment with pioglitazone. CONCLUSION: We show here that the intracellular glucose level is critical to determining the MIF mRNA level as well as its protein content in adipose tissue. MIF is known to play an important role in glucose metabolism as a positive regulator of insulin secretion. In this context, it is conceivable that MIF may affect the pathophysiology of obesity and diabetes.     

Control of IFN-alphaA by CD73: implications for mucosal inflammation

Inflammatory diseases influence tissue metabolism, altering regulation of extracellular adenine nucleotides, with a resultant protective influence of adenosine. Ecto-5'-nucleotidase (CD73) is a central surface enzyme generating extracellular adenosine. Thus, we hypothesized that CD73 is protective in mucosal inflammation as modeled by trinitrobenzene sulfonate (TNBS) colitis. Initial studies revealed a >3-fold induction of CD73 mRNA levels after TNBS colitis. Additionally, the severity of colitis was increased, as determined by weight loss and colonic shortening, in cd73(-/-) mice relative to cd73(+/+) controls. Likewise, enteral administration of the selective CD73 inhibitor alpha,beta-methylene ADP to cd73(+/+) mice resulted in a similar increase in severity of TNBS colitis. Gene array profiling of cytokine mRNA expression, verified by real-time PCR, revealed a >90% down-regulation of IFN-alphaA in cd73(-/-) mice and alpha,beta-methylene ADP-treated cd73(+/+) mice, compared with cd73(+/+) mice. Exogenous administration of recombinant IFN-alphaA partially protected TNBS-treated cd73(-/-) mice. Cytokine profiling revealed similar increases in both IFN-gamma and TNF-alpha mRNA in colitic animals, independent of genotype. However, IL-10 mRNA increased in wild-type mice on day 3 after TNBS administration, whereas cd73(-/-) mice mounted no IL-10 response. This IL-10 response was restored in the cd73(-/-) mice by exogenous IFN-alphaA. Further cytokine profiling revealed that this IL-10 induction is preceded by a transient IFN-alphaA induction on day 2 after TNBS exposure. Together, these studies indicate a critical regulatory role for CD73-modulated IFNalphaA in the acute inflammatory phase of TNBS colitis, thereby implicating IFN-alphaA as a protective element of adenosine signaling during mucosal inflammation.     

Hemodilutional anemia is associated with increased cerebral neuronal nitric oxide synthase gene expression.

Severe hemodilutional anemia may reduce cerebral oxygen delivery, resulting in cerebral tissue hypoxia. Increased nitric oxide synthase (NOS) expression has been identified following cerebral hypoxia and may contribute to the compensatory increase in cerebral blood flow (CBF) observed after hypoxia and anemia. However, changes in cerebral NOS gene expression have not been reported after acute anemia. This study tests the hypothesis that acute hemodilutional anemia causes cerebral tissue hypoxia, triggering changes in cerebral NOS gene expression. Anesthetized rats underwent hemodilution when 30 ml/kg of blood were exchanged with pentastarch, resulting in a final hemoglobin concentration of 51.0 +/- 1.2 g/l (n = 7 rats). Caudate tissue oxygen tension (Pbr(O(2))) decreased transiently from 17.3 +/- 4.1 to 14.4 +/- 4.1 Torr (P < 0.05), before returning to baseline after approximately 20 min. An increase in CBF may have contributed to restoring Pbr(O(2)) by improving cerebral tissue oxygen delivery. An increase in neuronal NOS (nNOS) mRNA was detected by RT-PCR in the cerebral cortex of anemic rats after 3 h (P < 0.05, n = 5). A similar response was observed after exposure to hypoxia. By contrast, no increases in mRNA for endothelial NOS or interleukin-1beta were observed after anemia or hypoxia. Hemodilutional anemia caused an acute reduction in Pbr(O(2)) and an increase in cerebral cortical nNOS mRNA, supporting a role for nNOS in the physiological response to acute anemia.     

Corticotrophin-releasing factor mediates hypophagia after adrenalectomy, increasing meal-related satiety responses

Adrenalectomy-induced hypophagia is associated with increased satiety-related responses, which involve neuronal activation of the nucleus of the solitary tract (NTS). Besides its effects on the pituitary–adrenal axis, corticotrophin-releasing factor (CRF) has been shown to play an important role in feeding behaviour, as it possesses anorexigenic effects. We evaluated feeding-induced CRF mRNA expression in the paraventricular nucleus (PVN) and the effects of pretreatment with CRF₂ receptor antagonist (Antisauvagine-30, AS30) on food intake and activation of NTS neurons in response to feeding in adrenalectomised (ADX) rats. Compared to the sham group, ADX increased CRF mRNA levels in the PVN of fasted animals, which was further augmented by refeeding. AS30 treatment did not affect food intake in the sham and ADX + corticosterone (B) groups; however, it reversed hypophagia in the ADX group. In vehicle-pretreated animals, refeeding increased the number of Fos and Fos/TH-immunoreactive neurons in the NTS in the sham, ADX and ADX + B groups, with the highest number of neurons in the ADX animals. Similarly to its effect on food intake, pretreatment with AS30 in the ADX group also reversed the increased activation of NTS neurons induced by refeeding while having no effect in the sham and ADX + B animals. The present results show that adrenalectomy induces an increase in CRF mRNA expression in the PVN potentiated by feeding and that CRF₂ receptor antagonist abolishes the anorexigenic effect and the increased activation of NTS induced by feeding in the ADX animals. These data indicate that increased activity of PVN CRF neurons modulates brainstem satiety-related responses, contributing to hypophagia after adrenalectomy.     

Transforming growth factor-beta 1 stimulates glucose uptake and the expression of glucose transporter mRNA in quiescent Swiss mouse 3T3 cells

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional polypeptide that regulates the proliferation and differentiation of various types of animal cells. TGF-beta 1 stimulated glucose uptake and the expression of a brain-type glucose transporter (GLUT1) mRNA in quiescent mouse 3T3 cells. TGF-beta 1 also synergistically stimulated these activities when given together with calf serum, phorbol ester, fibroblast growth factor, or epidermal growth factor. The increases in glucose uptake and the GLUT1 mRNA level were induced by picomolar concentrations of TGF-beta 1 within 3 h of stimulation, reached a peak between 6 and 9 h, and then decreased gradually to basal levels before an increase in DNA synthesis. The stimulation of GLUT1 mRNA expression was completely abolished by actinomycin D, but was not affected by cycloheximide, suggesting that new protein synthesis was not required for the expression of GLUT1 mRNA. TGF-beta 1 had little mitogenic activity and did not affect serum-induced DNA synthesis in quiescent 3T3 cells. However, it stimulated DNA synthesis synergistically when given with fibroblast growth factor, epidermal growth factor, phorbol ester, or insulin. These results suggest that TGF-beta 1 mediates the stimulation of glucose uptake, GLUT1 mRNA expression, and DNA synthesis via a pathway(s) and cellular components distinct from those for other growth factors. The possible role of the TGF-beta 1-induced stimulation of glucose transport activity in the control of mouse fibroblast proliferation is also discussed.     

Reduction of AUF1-mediated follistatin mRNA decay during glucose starvation protects cells from apoptosis

Follistatin (FST) performs several vital functions in the cells, including protection from apoptosis during stress. The expression of FST is up-regulated in response to glucose deprivation by an unknown mechanism. We herein showed that the induction of FST by glucose deprivation was due to an increase in the half-life of its mRNA. We further identified an AU-rich element (ARE) in the 3′UTR of FST mRNA that mediated its decay. The expression of FST was elevated after knocking down AUF1 and reduced when AUF1 was further expressed. In vitro binding assays and RNA pull-down assays revealed that AUF1 interacted with FST mRNA directly via its ARE. During glucose deprivation, a majority of AUF1 shuttled from cytoplasm to nucleus, resulting in dissociation of AUF1 from FST mRNA and thus stabilization of FST mRNA. Finally, knockdown of AUF1 decreased whereas overexpression of AUF1 increased glucose deprivation-induced apoptosis. The apoptosis promoting effect of AUF1 was eliminated in FST expressing cells. Collectively, this study provided evidence that AUF1 is a negative regulator of FST expression and participates in the regulation of cell survival under glucose deprivation.     

Plasminogen Activator Inhibitor-1 Expression by Brain Microvessel Endothelial Cells Is Inhibited by Elevated Glucose

Abstract: Patients with diabetes are predisposed to microvascular disease. In the retina and brain, this is characterized by neovascularization and new capillary formation. Because of the potential importance of plasmin generation in these processes, we evaluated the effect of elevated glucose concentrations on expression of plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), and urokinase (uPA) in cultured bovine brain endothelial cells (BBEC) versus cultured bovine aortic endothelial cells (BAEC). We observed that BBEC PAI-1 mRNA levels were decreased fivefold in cells cultured in media containing 20 m M glucose compared with BBEC cultured in media with 5.5 m M glucose, whereas expression of PAI-1 mRNA in BAEC, bovine mesenteric endothelial cells, and human umbilical vein endothelial cells was not modulated under these conditions. Expression of PAI-1 protein was also inhibited by growth of BBEC in elevated glucose, but the effect was less marked than at the mRNA level. Elevated glucose did not decrease expression of PAI-1 protein by BAEC. Withdrawal of acidic fibroblast growth factor enhanced expression of PAI-1 mRNA and protein in BBEC. Expression of tPA mRNA was not affected by the glucose concentration of the medium, and uPA mRNA was not detected in our BBEC cultures. A decrease in the local tissue activity of PAI-1 by elevated glucose concentrations, with no effect on tPA or uPA expression, would lead to an increase in the plasmin activity and thereby predispose neural tissues, such as the cerebrum and retina, of diabetic patients to neovascularization.