Muscle-Liver Trafficking of BCAA-Derived Nitrogen Underlies Obesity-Related Glycine Depletion.

Glycine levels are inversely associated with branched-chain amino acids (BCAAs) and cardiometabolic disease phenotypes, but biochemical mechanisms that explain these relationships remain uncharted. Metabolites and genes related to BCAA metabolism and nitrogen handling were strongly associated with glycine in correlation analyses. Stable isotope labeling in Zucker fatty rats (ZFRs) shows that glycine acts as a carbon donor for the pyruvate-alanine cycle in a BCAA-regulated manner. Inhibition of the BCAA transaminase (BCAT) enzymes depletes plasma pools of alanine and raises glycine levels. In high-fat-fed ZFRs, dietary glycine supplementation raises urinary acyl-glycine content and lowers circulating triglycerides but also results in accumulation of long-chain acyl-coenzyme As (acyl-CoAs), lower 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in muscle, and no improvement in glucose tolerance. Collectively, these studies frame a mechanism for explaining obesity-related glycine depletion and also provide insight into the impact of glycine supplementation on systemic glucose, lipid, and amino acid metabolism.

1,25-Dihydroxyvitamin D3 enhances glucose-stimulated insulin secretion in mouse and human islets: a role for transcriptional regulation of voltage-gated calcium channels by the vitamin D receptor.

AIM: Vitamin D deficiency in rodents negatively affects glucose-stimulated insulin secretion (GSIS) and human epidemiological studies connect poor vitamin D status with type 2 diabetes. Previous studies performed primarily in rat islets have shown that vitamin D can enhance GSIS. However the molecular pathways linking vitamin D and insulin secretion are currently unknown. Therefore, experiments were undertaken to elucidate the transcriptional role(s) of the vitamin D receptor (VDR) in islet function. METHODS: Human and mouse islets were cultured with vehicle or 1,25-dihydroxyvitamin-D3 (1,25D3) and then subjected to GSIS assays. Insulin expression, insulin content, glucose uptake and glucose-stimulated calcium influx were tested. Microarray analysis was performed. In silico analysis was used to identify VDR response elements (VDRE) within target genes and their activity was tested using reporter assays. RESULTS: Vdr mRNA is abundant in islets and Vdr expression is glucose-responsive. Preincubation of mouse and human islets with 1,25D3 enhances GSIS and increases glucose-stimulated calcium influx. Microarray analysis identified the R-type voltage-gated calcium channel (VGCC) gene, Cacna1e, which is highly upregulated by 1,25D3 in human and mouse islets and contains a conserved VDRE in intron 7. Results from GSIS assays suggest that 1,25D3 might upregulate a variant of R-type VGCC that is resistant to chemical inhibition. CONCLUSION: These results suggest that the role of 1,25D3 in regulating calcium influx acts through the R-Type VGCC during GSIS, thereby modulating the capacity of beta cells to secrete insulin.

Bone remodeling effect of a chitosan and calcium phosphate-based composite.

Chitosan is a biocompatible polymer that has been widely studied for tissue engineering purposes. The aim of this research was to assess bone regenerative properties of an injectable chitosan and calcium phosphate-based composite and identify optimal degree of deacetylation (%DDA) of the chitosan polymer. Drill holes were generated on the left side of a mandible in Sprague-Dawley rats, and the hole was either left empty or filled with the implant. The animals were sacrificed at several time points after surgery (7-22 days) and bone was investigated using micro-CT and histology. No significant new bone formation was observed in the implants themselves at any time points. However, substantial new bone formation was observed in the rat mandible further away from the drill hole. Morphological changes indicating bone formation were found in specimens explanted on Day 7 in animals that received implant. Similar bone formation pattern was seen in control animals with an empty drill hole at later time points but not to the same extent. A second experiment was performed to examine if the %DDA of the chitosan polymer influenced the bone remodeling response. The results suggest that chitosan polymers with %DDA between 50 and 70% enhance the natural bone remodeling mechanism.

The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins.

Lysine acetylation (AcK), a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT), an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.

Role of calcium and EPAC in norepinephrine-induced ghrelin secretion.

Ghrelin is an orexigenic hormone secreted principally from a distinct population of gastric endocrine cells. Molecular mechanisms regulating ghrelin secretion are mostly unknown. Recently, norepinephrine (NE) was shown to enhance ghrelin release by binding to ß1-adrenergic receptors on ghrelin cells. Here, we use an immortalized stomach-derived ghrelin cell line to further characterize the intracellular signaling pathways involved in NE-induced ghrelin secretion, with a focus on the roles of Ca(2+) and cAMP. Several voltage-gated Ca(2+) channel (VGCC) family members were found by quantitative PCR to be expressed by ghrelin cells. Nifedipine, a selective L-type VGCC blocker, suppressed both basal and NE-stimulated ghrelin secretion. NE induced elevation of cytosolic Ca(2+) levels both in the presence and absence of extracellular Ca(2+). Ca(2+)-sensing synaptotagmins Syt7 and Syt9 were also highly expressed in ghrelin cell lines, suggesting that they too help mediate ghrelin secretion. Raising cAMP with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine also stimulated ghrelin secretion, although such a cAMP-mediated effect likely does not involve protein kinase A, given the absence of a modulatory response to a highly selective protein kinase A inhibitor. However, pharmacological inhibition of another target of cAMP, exchange protein-activated by cAMP (EPAC), did attenuate both basal and NE-induced ghrelin secretion, whereas an EPAC agonist enhanced basal ghrelin secretion. We conclude that constitutive ghrelin secretion is primarily regulated by Ca(2+) influx through L-type VGCCs and that NE stimulates ghrelin secretion predominantly through release of intracellular Ca(2+). Furthermore, cAMP and its downstream activation of EPAC are required for the normal ghrelin secretory response to NE.