The metabolic impact of ß-hydroxybutyrate on neurotransmission: Reduced glycolysis mediates changes in calcium responses and KATP channel receptor sensitivity.

Glucose is the main energy substrate for neurons, and ketone bodies are known to be alternative substrates. However, the capacity of ketone bodies to support different neuronal functions is still unknown. Thus, a change in energy substrate from glucose alone to a combination of glucose and ß-hydroxybutyrate might change neuronal function as there is a known coupling between metabolism and neurotransmission. The purpose of this study was to shed light on the effects of the ketone body ß-hydroxybutyrate on glycolysis and neurotransmission in cultured murine glutamatergic neurons. Previous studies have shown an effect of ß-hydroxybutyrate on glucose metabolism, and the present study further specified this by showing attenuation of glycolysis when ß-hydroxybutyrate was present in these neurons. In addition, the NMDA receptor-induced calcium responses in the neurons were diminished in the presence of ß-hydroxybutyrate, whereas a direct effect of the ketone body on transmitter release was absent. However, the presence of ß-hydroxybutyrate augmented transmitter release induced by the KATP channel blocker glibenclamide, thus giving an indirect indication of the involvement of KATP channels in the effects of ketone bodies on transmitter release. Energy metabolism and neurotransmission are linked and involve ATP-sensitive potassium (KATP ) channels. However, it is still unclear how and to what degree available energy substrate affects this link. We investigated the effect of changing energy substrate from only glucose to a combination of glucose and R-ß-hydroxybutyrate in cultured neurons. Using the latter combination, glycolysis was diminished, NMDA receptor-induced calcium responses were lower, and the KATP channel blocker glibenclamide caused a higher transmitter release.

mRNA distribution of CGRP and its receptor components in the trigeminovascular system and other pain related structures in rat brain, and effect of intracerebroventricular administration of CGRP on Fos expression in the TNC.

Calcitonin gene-related peptide (CGRP) infusion in humans provokes headache resembling spontaneous migraine, and CGRP receptor antagonists are effective against acute migraine. We hypothesized that CGRP infusion in the lateral ventricle (LV) will induce neuronal activation reflected by increase in Fos expression in the trigeminal nucleus caudalis (TNC). CGRP was infused intracerebroventricularly (i.c.v.) in freely moving rats to circumvent factors like anaesthesia, acute surgery and severe hypotension, three confounding factors for Fos expression. TNCs were isolated 2h after CGRP infusion. The level of Fos protein expression in TNC was analysed by immunohistochemistry (IHC). mRNA expression of CGRP and its receptor components in trigeminovascular and other pain processing structures in the brain was also studied. CGRP i.c.v. infusion did not induce Fos activation in the TNC. mRNA expression profile showed that CGRP and its receptor components were widely distributed in trigeminovascular and other pain processing structures. The widespread presence of CGRP receptor mRNA in the various central pain pathways suggests that CGRP might play a role in migraine pathogenesis.

mRNA expression of 5-hydroxytryptamine 1B, 1D, and 1F receptors and their role in controlling the release of calcitonin gene-related peptide in the rat trigeminovascular system.

Triptans, a family of 5-hydroxytryptamine (5-HT) 1B, 1D, and 1F receptor agonists, are used in the acute treatment of migraine attacks. The site of action and subtypes of the 5-HT(1) receptor that mediate the antimigraine effect have still to be identified. This study investigated the mRNA expression of these receptors and the role of 5-HT(1) receptor subtypes in controlling the release of calcitonin gene-related peptide (CGRP) in rat dura mater, trigeminal ganglion (TG), and trigeminal nucleus caudalis (TNC). The mRNA for each receptor subtype was quantified by quantitative real-time polymerase chain reaction. A high potassium concentration was used to release CGRP from dura mater, isolated TG, and TNC in vitro. The immunoreactive CGRP (iCGRP) release was measured by enzyme-linked immunoassay. The mRNA transcripts of the 3 5-HT(1) receptor subtypes were detected in the trigeminovascular system. Sumatriptan inhibited iCGRP release by 31% in dura mater, 44% in TG, and 56% in TNC. This effect was reversed by a 5-HT(1B/1D) antagonist (GR127395). The 5-HT(1F) agonist (LY-344864) was effective in the dura mater (26% iCGRP inhibition), and the 5-HT(1D) agonist (PNU-142633) had a significant effect in the TNC (48%), whereas the 5-HT(1B) agonist (CP-94253) was unable to reduce the iCGRP release in all tissues studied. We found that sumatriptan reduced the iCGRP release via activation of 5-HT(1D) and 5-HT(1F) receptor subtypes. The 5-HT(1F) receptor agonist was effective only in peripheral terminals in dura mater, whereas the 5-HT(1D) agonist had a preferential effect on central terminals in the TNC.

Activation of PAR-2 elicits NO-dependent and CGRP-independent dilation of the dural artery.

OBJECTIVES: The goal of this study was to determine the vascular effects of protease-activated receptor-2 (PAR-2) activation in the rat cranial vasculature. BACKGROUND: The role of PAR-2 in pain and inflammatory conditions has been established but the information available on its effects and receptor distribution in the trigeminal vascular axis is limited. We studied the dilatory function and expression of PAR-2 in the neuro-vascular circuit, critical in migraine pathogenesis. We also investigated the interaction of PAR-2 with calcitonin gene-related peptide (CGRP) and dural mast cells. METHODS: We used an improved model of intravital microscopy on the closed cranial window in rats to study the vascular effects of PAR-2 activating peptides (PAR-2 APs; SLIGRL-NH(2), 2-Furoyl-LIGRLO-NH(2)) in the dural vasculature. Measurement of immunoreactive CGRP in skull halves and in trigeminal nucleus caudalis was done by using an enzyme-linked immunosorbent assay. We also analyzed the presence of PAR-2 in different migraine relevant tissues by quantitative real-time PCR and Western blot analysis. RESULTS: PAR-2 APs and trypsin induced a dose-dependent increase in dural artery diameter. The topical application of a nonspecific nitric oxide synthase (NOS) inhibitor, L-N(G)-Nitroarginine methyl ester, attenuated SLIGRL-NH(2) responses. Olcegepant, a CGRP receptor antagonist, did not a have significant effect on the SLIGRL-NH(2) responses, though exogenous CGRP responses were completely blocked. There was no significant release of CGRP from skull halves incubated with SLIGRL-NH(2) as compared with those incubated with the corresponding negative peptide. Chronic mast cell degranulation did not change the vascular effects of PAR-2 APs. mRNA and protein expression of PAR-2 were found throughout trigeminovasuclar axis. CONCLUSION: PAR-2 activation leads to vasodilation of dural arteries and these responses are partially mediated by nitric oxide. As PAR-2 is present throughout trigeminovasuclar axis, it may have a role in migraine pathogenesis, independent of CGRP and mast cell mediated mechanism.

Meal-stimulated glucagon release is associated with postprandial blood glucose level and does not interfere with glycemic control in children and adolescents with new-onset type 1 diabetes.

CONTEXT: The role of glucagon in hyperglycemia in type 1 diabetes is unresolved, and in vitro studies suggest that increasing blood glucose might stimulate glucagon secretion. OBJECTIVE: Our objective was to investigate the relationship between postprandial glucose and glucagon level during the first 12 months after diagnosis of childhood type 1 diabetes. DESIGN: We conducted a prospective, noninterventional, 12-month follow-up study conducted in 22 centers in 18 countries. PATIENTS: Patients included 257 children and adolescents less than 16 yr old with newly diagnosed type 1 diabetes; 204 completed the 12-month follow-up. SETTING: The study was conducted at pediatric outpatient clinics. MAIN OUTCOME MEASURES: We assessed residual beta-cell function (C-peptide), glycosylated hemoglobin (HbA(1c)), blood glucose, glucagon, and glucagon-like peptide-1 (GLP-1) release in response to a 90-min meal stimulation (Boost) at 1, 6, and 12 months after diagnosis. RESULTS: Compound symmetric repeated-measurements models including all three visits showed that postprandial glucagon increased by 17% during follow-up (P = 0.001). Glucagon levels were highly associated with postprandial blood glucose levels because a 10 mmol/liter increase in blood glucose corresponded to a 20% increase in glucagon release (P = 0.0003). Glucagon levels were also associated with GLP-1 release because a 10% increase in GLP-1 corresponded to a 2% increase in glucagon release (P = 0.0003). Glucagon levels were not associated (coefficient -0.21, P = 0.07) with HbA(1c), adjusted for insulin dose. Immunohistochemical staining confirmed the presence of Kir6.2/SUR1 in human alpha-cells. CONCLUSION: Our study supports the recent in vitro data showing a stimulation of glucagon secretion by high glucose levels. Postprandial glucagon levels were not associated with HbA(1c), adjusted for insulin dose, during the first year after onset of childhood type 1 diabetes.

Co-localisation of the Kir6.2/SUR1 channel complex with glucagon-like peptide-1 and glucose-dependent insulinotrophic polypeptide expression in human ileal cells and implications for glycaemic control in new onset type 1 diabetes.

OBJECTIVE: The ATP-dependent K+-channel (K(ATP)) is critical for glucose sensing and normal glucagon and insulin secretion from pancreatic endocrine alpha- and beta-cells. Gastrointestinal endocrine L- and K-cells are also glucose-sensing cells secreting glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotrophic polypeptide (GIP) respectively. The aims of this study were to 1) investigate the expression and co-localisation of the K(ATP) channel subunits, Kir6.2 and SUR1, in human L- and K-cells and 2) investigate if a common hyperactive variant of the Kir6.2 subunit, Glu23Lys, exerts a functional impact on glucose-sensing tissues in vivo that may affect the overall glycaemic control in children with new-onset type 1 diabetes. DESIGN AND METHODS: Western blot and immunohistochemical analyses were performed for expression and co-localisation studies. Meal-stimulated C-peptide test was carried out in 257 children at 1, 6 and 12 months after diagnosis. Genotyping for the Glu23Lys variant was by PCR-restriction fragment length polymorphism. RESULTS: Kir6.2 and SUR1 co-localise with GLP-1 in L-cells and with GIP in K-cells in human ileum tissue. Children with type 1 diabetes carrying the hyperactive Glu23Lys variant had higher HbA1C at diagnosis (coefficient = 0.61%, P = 0.02) and 1 month after initial insulin therapy (coefficient = 0.30%, P = 0.05), but later disappeared. However, when adjusting HbA1C for the given dose of exogenous insulin, the dose-adjusted HbA1C remained higher throughout the 12 month study period (coefficient = 0.42%, P = 0.03). CONCLUSIONS: Kir6.2 and SUR1 co-localise in the gastrointestinal endocrine L- and K-cells. The hyperactive Glu23Lys variant of the K(ATP) channel subunit Kir6.2 may cause defective glucose sensing in several tissues and impaired glycaemic control in children with type 1 diabetes.