Episodic ß-cell death and dedifferentiation during diet-induced obesity and dysglycemia in male mice.

Loss of functional islet ß-cell mass through cellular death or dedifferentiation is thought to lead to dysglycemia during the progression from obesity to type 2 diabetes. To assess these processes in a mouse model of obesity, we performed measures of circulating cell-free differentially methylated insulin II ( Ins2) DNA as a biomarker of ß-cell death and aldehyde dehydrogenase 1 family member A3 (ALDH1A3) and forkhead box 01 (Foxo1) immunostaining as markers of ß-cell dedifferentiation. Eight-week-old, C57BL/6J mice were fed a low-fat diet (LFD; 10% kcal from fat) or a high-fat diet (HFD; 60% kcal from fat) and were followed longitudinally for up to 13 wk to measure glycemic control and ß-cell mass, death, and dedifferentiation. Compared with LFD controls, ß-cell mass increased during the feeding period in HFD animals, and statistically greater ß-cell death (unmethylated Ins2) was detectable at 2 and 6 wk after diet initiation. Those times correspond to periods when significant step increases in fasting glucose and glucose intolerance, respectively, were detected. ALDH1A3 and Foxo1 immunostaining of the pancreas revealed evidence of ß-cell dedifferentiation by 13 wk when fed an HFD, but not in LFD controls. In conclusion, early episodic ß-cell death may be a feature of cellular turnover correlated with changes in glycemia during ß-cell mass accrual in obesity, whereas ß-cell dedifferentiation may be a feature seen later in established disease.-Tersey, S. A., Levasseur, E. M., Syed, F., Farb, T. B., Orr, K. S., Nelson, J. B., Shaw, J. L., Bokvist, K., Mather, K. J., Mirmira, R. G. Episodic ß-cell death and dedifferentiation during diet-induced obesity and dysglycemia in male mice.

Identification of biomarkers for glycaemic deterioration in type 2 diabetes.

We identify biomarkers for disease progression in three type 2 diabetes cohorts encompassing 2,973 individuals across three molecular classes, metabolites, lipids and proteins. Homocitrulline, isoleucine and 2-aminoadipic acid, eight triacylglycerol species, and lowered sphingomyelin 42:2;2 levels are predictive of faster progression towards insulin requirement. Of ~1,300 proteins examined in two cohorts, levels of GDF15/MIC-1, IL-18Ra, CRELD1, NogoR, FAS, and ENPP7 are associated with faster progression, whilst SMAC/DIABLO, SPOCK1 and HEMK2 predict lower progression rates. In an external replication, proteins and lipids are associated with diabetes incidence and prevalence. NogoR/RTN4R injection improved glucose tolerance in high fat-fed male mice but impaired it in male db/db mice. High NogoR levels led to islet cell apoptosis, and IL-18R antagonised inflammatory IL-18 signalling towards nuclear factor kappa-B in vitro. This comprehensive, multi-disciplinary approach thus identifies biomarkers with potential prognostic utility, provides evidence for possible disease mechanisms, and identifies potential therapeutic avenues to slow diabetes progression.

Increased brain neuropeptide Y1 and Y2 receptor binding in NPY knock out mice does not result in increased receptor function.

The brain neuropeptide Neuropeptide Y (NPY) is an important modulator of a number of centrally mediated processes including feeding, anxiety-like behaviors, blood pressure and others. NPY produces its effects through at least four functional G-protein coupled receptors termed Y1, Y2, Y4 and Y5. In the brain, the Y1 and Y2 receptor subtypes are the predominant receptor population. To better understand the roles of NPY, genetically modified mice lacking NPY were produced but lacked the expected phenotypes. These mice have previously been reported to have a marked increase in Y2 receptor binding. In the present study, we found an upregulation of both Y1 and Y2 receptor binding and extended these findings to the female. These increases were as large as 10-fold or greater in many brain regions. To assess functional coupling of the receptors, we performed agonist-induced [(35)S]GTPgammaS autoradiography. In the mouse brain, the Y1/Y4/Y5 agonist Leu(31),Pro(34)-NPY increased [(35)S]GTPgammaS binding with a regional distribution consistent with that produced when labeling adjacent sections with [(125)I]-Leu(31),Pro(34)-PYY. In a few brain regions, minor increases were noted in the agonist-induced binding when comparing knock out mice to wild type. The Y2 agonist C2-NPY stimulated [(35)S]GTPgammaS binding in numerous brain areas with a regional distribution similar to the binding observed with [(125)I]-PYY3-36. Again, no major increases were noted in the functional activation of Y2 receptors between knock out and wild type mice. Therefore, the increased Y1 and Y2 binding observed in the NPY knock out mice does not represent an increase in NPY receptor mediated signaling and is likely due to an increase in spare (uncoupled) receptors.

Distribution of nociceptin and Ro64-6198 activated [35S]-GTPgammaS binding in the rat brain.

The peptide, nociceptin, was discovered as the endogenous ligand for the opioid-like receptor, ORL1. Since its discovery, this peptide has been shown to modulate the perception of pain, modulate feeding and produce behavioral effects in rodent models of mood disorders. Recently, the non-peptide agonist {(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one} (Ro64-6198) of the ORL1 receptor has been reported in the literature. In the present study, we compared the distribution and potency of Ro64-6198 with nociceptin for their ability to stimulate [(35)S]-GTPgammaS binding to sections of rat brain. In initial studies, Ro64-6198 inhibited (125)I-nociceptin binding to the hORL1 receptors with a K(i) of 1.75 nM compared with 0.20 nM for nociceptin. To assess agonist potency in a whole cell assay, a cell line expressing the hORL1 receptor and G(alpha15) was created and used for calcium mobilization studies. In this assay system, Ro64-6198 increased intracellular calcium with an EC(50) of 52nM compared with 24 nM for nociceptin. Having verified the agonist properties of Ro64-6198, we then assessed the potency and distribution of ORL1 receptor activation in rat brain sections. In dose-response studies, Ro64-6198 increased [(35)S]-GTPgammaS binding to a variety of brain regions with EC(50) values ranging from 84.9 to 2,143 nM depending on the brain regions evaluated. These potencies were similar to that seen for nociceptin, but substantially lower than values established using [(125)I] nociceptin binding to the cloned human ORL1 receptor. In general, the brain distribution of agonist stimulated [(35)S]-GTPgammaS binding was similar when either Ro64-6198 or nociceptin were used. Using these techniques, we have demonstrated, for the first time that Ro64-6198 activates [(35)S]-GTPgammaS binding to rat brain sections and this compound stimulates a similar population of receptors as nociceptin.

Functional autoradiography of neuropeptide Y Y1 and Y2 receptor subtypes in rat brain using agonist stimulated [35S]GTPgammaS binding.

Neuropeptide Y, one of the most abundant brain peptides, has been found to modulate several important biological functions via a family of G-protein coupled receptors. To investigate the localization of functional NPY receptor subtypes in the rat brain, we performed agonist-induced [35S]GTPgammaS autoradiography. The Y1/Y4/Y5 agonist Leu(31), Pro(34)-NPY increased [35S]GTPgammaS binding in several brain areas with a regional distribution consistent with that produced when labeling adjacent sections with [125I]-Leu(31), Pro(34)-PYY. The Y1 selective antagonist BIBP3226 antagonized the Leu(31), Pro(34)-NPY stimulated increase in [35S]GTPgammaS binding in all areas examined. The Y2 agonist C2-NPY stimulated [35S]GTPgamma binding in numerous brain areas with a regional distribution similar to the binding observed with [125I]-PYY 3-36. No increase in [35S]GTPgammaS binding above basal was observed in any brain area evaluated using Y4 and Y5 selective agonists. This study demonstrates abundant Y1 and Y2 receptor activation in the rat brain, while evidence for functional Y4 and Y5 receptors was not observed.

Suppression of alcohol self-administration and reinstatement of alcohol seeking by melanin-concentrating hormone receptor 1 (MCH1-R) antagonism in Wistar rats.

RATIONALE: Melanin-concentrating hormone (MCH) is involved in regulation of appetitive behaviors as well as emotional reactivity and reward, behavioral domains relevant to alcohol addiction. MATERIALS AND METHODS: We evaluated the effects of the non-peptide MCH1 receptor antagonist, GW803430 [6-(4-chloro-phenyl)-3-[3-methoxy-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-3H-thieno[3,2-d]pyrimidin-4-one; 3-30 mg/kg, i.p.] on alcohol-related behaviors in Wistar rats. RESULTS: Ex vivo binding experiments demonstrated that the GW803430 dose range used resulted in high central MCH1 receptor occupancy. Alcohol self-administration was dose-dependently and potently suppressed, by approximately 80% at the highest dose. Reinstatement of alcohol-seeking induced by alcohol-associated cues was essentially eliminated. In contrast, reinstatement induced by footshock stress was not significantly altered. Taste preference for a quinine/saccharin solution, locomotor activity, and alcohol elimination were unaffected. CONCLUSION: Together, these observations support a specific involvement of the MCH system in mediating alcohol reward and cue-induced relapse to alcohol seeking. MCH1-R antagonism may constitute an attractive treatment target for alcohol use disorders.