Distinct mechanoreceptor pezo-1 isoforms modulate food intake in the nematode Caenorhabditis elegans.

Two PIEZO mechanosensitive cation channels, PIEZO1 and PIEZO2, have been identified in mammals, where they are involved in numerous sensory processes. While structurally similar, PIEZO channels are expressed in distinct tissues and exhibit unique properties. How different PIEZOs transduce force, how their transduction mechanism varies, and how their unique properties match the functional needs of the tissues they are expressed in remain all-important unanswered questions. The nematode Caenorhabditis elegans has a single PIEZO ortholog (pezo-1) predicted to have 12 isoforms. These isoforms share many transmembrane domains but differ in those that distinguish PIEZO1 and PIEZO2 in mammals. We used transcriptional and translational reporters to show that putative promoter sequences immediately upstream of the start codon of long pezo-1 isoforms predominantly drive green fluorescent protein (GFP) expression in mesodermally derived tissues (such as muscle and glands). In contrast, sequences upstream of shorter pezo-1 isoforms resulted in GFP expression primarily in neurons. Putative promoters upstream of different isoforms drove GFP expression in different cells of the same organs of the digestive system. The observed unique pattern of complementary expression suggests that different isoforms could possess distinct functions within these organs. We used mutant analysis to show that pharyngeal muscles and glands require long pezo-1 isoforms to respond appropriately to the presence of food. The number of pezo-1 isoforms in C. elegans, their putative differential pattern of expression, and roles in experimentally tractable processes make this an attractive system to investigate the molecular basis for functional differences between members of the PIEZO family of mechanoreceptors.

Decreased sensitivity to the anorectic effects of leptin in mice that lack a Pomc-specific neural enhancer.

Enhancer redundancy has been postulated to provide a buffer for gene expression against genetic and environmental perturbations. While work in Drosophila has identified functionally overlapping enhancers, work in mammalian models has been limited. Recently, we have identified two partially redundant enhancers, nPE1 and nPE2, that drive proopiomelanocortin gene expression in the hypothalamus. Here we demonstrate that deletion of nPE1 produces mild obesity while knockout of nPE2 has no discernible metabolic phenotypes. Additionally, we show that acute leptin administration has significant effects on nPE1 knockout mice, with food intake and body weight change significantly impacted by peripheral leptin treatment. nPE1 knockout mice became less responsive to leptin treatment over time as percent body weight change increased over 2 week exposure to peripheral leptin. Both Pomc and Agrp mRNA were not differentially affected by chronic leptin treatment however we did see a decrease in Pomc and Agrp mRNA in both nPE1 and nPE2 knockout calorie restricted mice as compared to calorie restricted PBS-treated WT mice. Collectively, these data suggest dynamic regulation of Pomc by nPE1 such that mice with nPE1 knockout become less responsive to the anorectic effects of leptin treatment over time. Our results also support our earlier findings in which nPE2 may only be critical in adult mice that lack nPE1, indicating that these neural enhancers work synergistically to influence metabolism.

Hypothalamic POMC or MC4R deficiency impairs counterregulatory responses to hypoglycemia in mice.

OBJECTIVE: Life-threatening hypoglycemia is a major limiting factor in the management of diabetes. While it is known that counterregulatory responses to hypoglycemia are impaired in diabetes, molecular mechanisms underlying the reduced responses remain unclear. Given the established roles of the hypothalamic proopiomelanocortin (POMC)/melanocortin 4 receptor (MC4R) circuit in regulating sympathetic nervous system (SNS) activity and the SNS in stimulating counterregulatory responses to hypoglycemia, we hypothesized that hypothalamic POMC as well as MC4R, a receptor for POMC derived melanocyte stimulating hormones, is required for normal hypoglycemia counterregulation. METHODS: To test the hypothesis, we induced hypoglycemia or glucopenia in separate cohorts of mice deficient in either POMC or MC4R in the arcuate nucleus (ARC) or the paraventricular nucleus of the hypothalamus (PVH), respectively, and measured their circulating counterregulatory hormones. In addition, we performed a hyperinsulinemic-hypoglycemic clamp study to further validate the function of MC4R in hypoglycemia counterregulation. We also measured Pomc and Mc4r mRNA levels in the ARC and PVH, respectively, in the streptozotocin-induced type 1 diabetes mouse model and non-obese diabetic (NOD) mice to delineate molecular mechanisms by which diabetes deteriorates the defense systems against hypoglycemia. Finally, we treated diabetic mice with the MC4R agonist MTII, administered stereotaxically into the PVH, to determine its potential for restoring the counterregulatory response to hypoglycemia in diabetes. RESULTS: Stimulation of epinephrine and glucagon release in response to hypoglycemia or glucopenia was diminished in both POMC- and MC4R-deficient mice, relative to their littermate controls. Similarly, the counterregulatory response was impaired in association with decreased hypothalamic Pomc and Mc4r expression in the diabetic mice, a phenotype that was not reversed by insulin treatment which normalized glycemia. In contrast, infusion of an MC4R agonist in the PVH restored the counterregulatory response in diabetic mice. CONCLUSION: In conclusion, hypothalamic Pomc as well as Mc4r, both of which are reduced in type 1 diabetic mice, are required for normal counterregulatory responses to hypoglycemia. Therefore, enhancing MC4R function may improve hypoglycemia counterregulation in diabetes.

Nilvadipine in mild to moderate Alzheimer disease: A randomised controlled trial.

BACKGROUND: This study reports the findings of the first large-scale Phase III investigator-driven clinical trial to slow the rate of cognitive decline in Alzheimer disease with a dihydropyridine (DHP) calcium channel blocker, nilvadipine. Nilvadipine, licensed to treat hypertension, reduces amyloid production, increases regional cerebral blood flow, and has demonstrated anti-inflammatory and anti-tau activity in preclinical studies, properties that could have disease-modifying effects for Alzheimer disease. We aimed to determine if nilvadipine was effective in slowing cognitive decline in subjects with mild to moderate Alzheimer disease. METHODS AND FINDINGS: NILVAD was an 18-month, randomised, placebo-controlled, double-blind trial that randomised participants between 15 May 2013 and 13 April 2015. The study was conducted at 23 academic centres in nine European countries. Of 577 participants screened, 511 were eligible and were randomised (258 to placebo, 253 to nilvadipine). Participants took a trial treatment capsule once a day after breakfast for 78 weeks. Participants were aged >50 years, meeting National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer's disease Criteria (NINCDS-ADRDA) for diagnosis of probable Alzheimer disease, with a Standardised Mini-Mental State Examination (SMMSE) score of ≥12 and <27. Participants were randomly assigned to 8 mg sustained-release nilvadipine or matched placebo. The a priori defined primary outcome was progression on the Alzheimer's Disease Assessment Scale Cognitive Subscale-12 (ADAS-Cog 12) in the modified intention-to-treat (mITT) population (n = 498), with the Clinical Dementia Rating Scale sum of boxes (CDR-sb) as a gated co-primary outcome, eligible to be promoted to primary end point conditional on a significant effect on the ADAS-Cog 12. The analysis set had a mean age of 73 years and was 62% female. Baseline demographic and Alzheimer disease-specific characteristics were similar between treatment groups, with reported mean of 1.7 years since diagnosis and mean SMMSE of 20.4. The prespecified primary analyses failed to show any treatment benefit for nilvadipine on the co-primary outcome (p = 0.465). Decline from baseline in ADAS-Cog 12 on placebo was 0.79 (95% CI, -0.07-1.64) at 13 weeks, 6.41 (5.33-7.49) at 52 weeks, and 9.63 (8.33-10.93) at 78 weeks and on nilvadipine was 0.88 (0.02-1.74) at 13 weeks, 5.75 (4.66-6.85) at 52 weeks, and 9.41 (8.09-10.73) at 78 weeks. Exploratory analyses of the planned secondary outcomes showed no substantial effects, including on the CDR-sb or the Disability Assessment for Dementia. Nilvadipine appeared to be safe and well tolerated. Mortality was similar between groups (3 on nilvadipine, 4 on placebo); higher counts of adverse events (AEs) on nilvadipine (1,129 versus 1,030), and serious adverse events (SAEs; 146 versus 101), were observed. There were 14 withdrawals because of AEs. Major limitations of this study were that subjects had established dementia and the likelihood that non-Alzheimer subjects were included because of the lack of biomarker confirmation of the presence of brain amyloid. CONCLUSIONS: The results do not suggest benefit of nilvadipine as a treatment in a population spanning mild to moderate Alzheimer disease. TRIAL REGISTRATION: Clinicaltrials.gov NCT02017340, EudraCT number 2012-002764-27.

Liraglutide Modulates Appetite and Body Weight Through Glucagon-Like Peptide 1 Receptor-Expressing Glutamatergic Neurons.

Glucagon-like peptide 1 receptor (GLP-1R) agonists are U.S. Food and Drug Administration-approved weight loss drugs. Despite their widespread use, the sites of action through which GLP-1R agonists (GLP1RAs) affect appetite and body weight are still not fully understood. We determined whether GLP-1Rs in either GABAergic or glutamatergic neurons are necessary for the short- and long-term effects of the GLP1RA liraglutide on food intake, visceral illness, body weight, and neural network activation. We found that mice lacking GLP-1Rs in vGAT-expressing GABAergic neurons responded identically to controls in all parameters measured, whereas deletion of GLP-1Rs in vGlut2-expressing glutamatergic neurons eliminated liraglutide-induced weight loss and visceral illness and severely attenuated its effects on feeding. Concomitantly, deletion of GLP-1Rs from glutamatergic neurons completely abolished the neural network activation observed after liraglutide administration. We conclude that liraglutide activates a dispersed but discrete neural network to mediate its physiological effects and that these effects require GLP-1R expression on glutamatergic but not GABAergic neurons.

Essential Role for Hypothalamic Calcitonin Receptor‒Expressing Neurons in the Control of Food Intake by Leptin.

The adipocyte-derived hormone leptin acts via its receptor (LepRb) on central nervous system neurons to communicate the repletion of long-term energy stores, to decrease food intake, and to promote energy expenditure. We generated mice that express Cre recombinase from the calcitonin receptor (Calcr) locus (Calcrcre mice) to study Calcr-expressing LepRb (LepRbCalcr) neurons, which reside predominantly in the arcuate nucleus (ARC). Calcrcre-mediated ablation of LepRb in LepRbCalcrknockout (KO) mice caused hyperphagic obesity. Because LepRb-mediated transcriptional control plays a crucial role in leptin action, we used translating ribosome affinity purification followed by RNA sequencing to define the transcriptome of hypothalamic Calcr neurons, along with its alteration in LepRbCalcrKO mice. We found that ARC LepRbCalcr cells include neuropeptide Y (NPY)/agouti-related peptide (AgRP)/γ-aminobutyric acid (GABA) ("NAG") cells as well as non-NAG cells that are distinct from pro-opiomelanocortin cells. Furthermore, although LepRbCalcrKO mice exhibited dysregulated expression of several genes involved in energy balance, neither the expression of Agrp and Npy nor the activity of NAG cells was altered in vivo. Thus, although direct leptin action via LepRbCalcr cells plays an important role in leptin action, our data also suggest that leptin indirectly, as well as directly, regulates these cells.

Reduced renal sympathetic nerve activity contributes to elevated glycosuria and improved glucose tolerance in hypothalamus-specific Pomc knockout mice.

OBJECTIVE: Hypothalamic arcuate nucleus-specific pro-opiomelanocortin deficient (ArcPomc-/-) mice exhibit improved glucose tolerance despite massive obesity and insulin resistance. We demonstrated previously that their improved glucose tolerance is due to elevated glycosuria. However, the underlying mechanisms that link glucose reabsorption in the kidney with ArcPomc remain unclear. Given the function of the hypothalamic melanocortin system in controlling sympathetic outflow, we hypothesized that reduced renal sympathetic nerve activity (RSNA) in ArcPomc-/- mice could explain their elevated glycosuria and consequent enhanced glucose tolerance. METHODS: We measured RSNA by multifiber recording directly from the nerves innervating the kidneys in ArcPomc-/- mice. To further validate the function of RSNA in glucose reabsorption, we denervated the kidneys of WT and diabetic db/db mice before measuring their glucose tolerance and urine glucose levels. Moreover, we performed western blot and immunohistochemistry to determine kidney GLUT2 and SGLT2 levels in either ArcPomc-/- mice or the renal-denervated mice. RESULTS: Consistent with our hypothesis, we found that basal RSNA was decreased in ArcPomc-/- mice relative to their wild type (WT) littermates. Remarkably, both WT and db/db mice exhibited elevated glycosuria and improved glucose tolerance after renal denervation. The elevated glycosuria in obese ArcPomc-/-, WT and db/db mice was due to reduced renal GLUT2 levels in the proximal tubules. Overall, we show that renal-denervated WT and diabetic mice recapitulate the phenotype of improved glucose tolerance and elevated glycosuria associated with reduced renal GLUT2 levels observed in obese ArcPomc-/- mice. CONCLUSION: Hence, we conclude that ArcPomc is essential in maintaining basal RSNA and that elevated glycosuria is a possible mechanism to explain improved glucose tolerance after renal denervation in drug resistant hypertensive patients.

Hypothalamic POMC Deficiency Improves Glucose Tolerance Despite Insulin Resistance by Increasing Glycosuria.

Hypothalamic proopiomelanocortin (POMC) is essential for the physiological regulation of energy balance; however, its role in glucose homeostasis remains less clear. We show that hypothalamic arcuate nucleus (Arc)POMC-deficient mice, which develop severe obesity and insulin resistance, unexpectedly exhibit improved glucose tolerance and remain protected from hyperglycemia. To explain these paradoxical phenotypes, we hypothesized that an insulin-independent pathway is responsible for the enhanced glucose tolerance. Indeed, the mutant mice demonstrated increased glucose effectiveness and exaggerated glycosuria relative to wild-type littermate controls at comparable blood glucose concentrations. Central administration of the melanocortin receptor agonist melanotan II in mutant mice reversed alterations in glucose tolerance and glycosuria, whereas, conversely, administration of the antagonist Agouti-related peptide (Agrp) to wild-type mice enhanced glucose tolerance. The glycosuria of ArcPOMC-deficient mice was due to decreased levels of renal GLUT 2 (rGLUT2) but not sodium-glucose cotransporter 2 and was associated with reduced renal catecholamine content. Epinephrine treatment abolished the genotype differences in glucose tolerance and rGLUT2 levels, suggesting that reduced renal sympathetic nervous system (SNS) activity is the underlying mechanism for the observed glycosuria and improved glucose tolerance in ArcPOMC-deficient mice. Therefore, the ArcPOMC-SNS-rGLUT2 axis is potentially an insulin-independent therapeutic target to control diabetes.

NILVAD protocol: a European multicentre double-blind placebo-controlled trial of nilvadipine in mild-to-moderate Alzheimer's disease.

INTRODUCTION: This study is a European multicentre, randomised, double-blind, placebo-controlled trial investigating the efficacy and safety of nilvadipine as a disease course modifying treatment for mild-to-moderate Alzheimer's disease (AD) in a phase III study that will run for a period of 82 weeks with a treatment period of 78 weeks. METHODS AND ANALYSIS: Adult patients, males and females over 50 years with mild-to-moderate AD as defined by the National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer's disease and Related Disorders Association (NINCDS-ADRDA) criteria, will be included in the study. It aims to recruit a total of 500 patients with AD; 250 in the nilvadipine group and 250 in the placebo group. Participants will be randomised to receive nilvadipine, an 8 mg overencapsulated, sustained release capsule, or a matching overencapsulated placebo (sugar pill) for a period of 78 weeks of treatment. The primary efficacy outcome measure in this study is the change in cognitive function as assessed by the Alzheimer's disease Assessment Scale (ADAS-Cog 12) from baseline to the end of treatment duration (78 weeks). There are two key secondary outcome measures, the Clinical Dementia Rating Scale Sum of Boxes (CDR-sb) and the Disability Assessment for Dementia (DAD). If a statistically significant effect is seen in the primary outcome, CDR-sb will be considered to be a coprimary end point and only the DAD will contribute to the secondary outcome analysis. ETHICS AND DISSEMINATION: The study and all subsequent amendments have received ethical approval within each participating country according to national regulations. Each participant will provide written consent to participate in the study. All participants will remain anonymised throughout and the results of the study will be published in an international peer-reviewed journal. TRIAL REGISTRATION NUMBER: EUDRACT Reference Number: 2012-002764-27.

The discovery and structure-activity relationships of indole-based inhibitors of glutamate carboxypeptidase II.

A series of N-substituted 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acids were synthesized as inhibitors of glutamate carboxypeptidase II (GCPII). Those containing carboxybenzyl or carboxyphenyl groups at the N-position exhibited potent inhibitory activity against GCPII. These indole-based compounds represent the first example of achiral GCPII inhibitors and demonstrate greater tolerance of the GCPII active site for ligands with significant structural difference from the endogenous substrate, N-acetyl-aspartylglutamate.