Repression of latent NF-κB enhancers by PDX1 regulates ß cell functional heterogeneity.

Interactions between lineage-determining and activity-dependent transcription factors determine single-cell identity and function within multicellular tissues through incompletely known mechanisms. By assembling a single-cell atlas of chromatin state within human islets, we identified ß cell subtypes governed by either high or low activity of the lineage-determining factor pancreatic duodenal homeobox-1 (PDX1). ß cells with reduced PDX1 activity displayed increased chromatin accessibility at latent nuclear factor κB (NF-κB) enhancers. Pdx1 hypomorphic mice exhibited de-repression of NF-κB and impaired glucose tolerance at night. Three-dimensional analyses in tandem with chromatin immunoprecipitation (ChIP) sequencing revealed that PDX1 silences NF-κB at circadian and inflammatory enhancers through long-range chromatin contacts involving SIN3A. Conversely, Bmal1 ablation in ß cells disrupted genome-wide PDX1 and NF-κB DNA binding. Finally, antagonizing the interleukin (IL)-1ß receptor, an NF-κB target, improved insulin secretion in Pdx1 hypomorphic islets. Our studies reveal functional subtypes of single ß cells defined by a gradient in PDX1 activity and identify NF-κB as a target for insulinotropic therapy.

Time-restricted feeding mitigates obesity through adipocyte thermogenesis.

Misalignment of feeding rhythms with the light-dark cycle leads to disrupted peripheral circadian clocks and obesity. Conversely, restricting feeding to the active period mitigates metabolic syndrome through mechanisms that remain unknown. We found that genetic enhancement of adipocyte thermogenesis through ablation of the zinc finger protein 423 (ZFP423) attenuated obesity caused by consumption of a high-fat diet during the inactive (light) period by increasing futile creatine cycling in mice. Circadian control of adipocyte creatine metabolism underlies the timing of diet-induced thermogenesis, and enhancement of adipocyte circadian rhythms through overexpression of the clock activator brain and muscle Arnt-like protein-1 (BMAL1) ameliorated metabolic complications during diet-induced obesity. These findings uncover rhythmic creatine-mediated thermogenesis as an essential mechanism that drives metabolic benefits during time-restricted feeding.

P2Y1 purinergic receptor identified as a diabetes target in a small-molecule screen to reverse circadian ß-cell failure.

The mammalian circadian clock drives daily oscillations in physiology and behavior through an autoregulatory transcription feedback loop present in central and peripheral cells. Ablation of the core clock within the endocrine pancreas of adult animals impairs the transcription and splicing of genes involved in hormone exocytosis and causes hypoinsulinemic diabetes. Here, we developed a genetically sensitized small-molecule screen to identify druggable proteins and mechanistic pathways involved in circadian ß-cell failure. Our approach was to generate ß-cells expressing a nanoluciferase reporter within the proinsulin polypeptide to screen 2640 pharmacologically active compounds and identify insulinotropic molecules that bypass the secretory defect in CRISPR-Cas9-targeted clock mutant ß-cells. We validated hit compounds in primary mouse islets and identified known modulators of ligand-gated ion channels and G-protein-coupled receptors, including the antihelmintic ivermectin. Single-cell electrophysiology in circadian mutant mouse and human cadaveric islets revealed ivermectin as a glucose-dependent secretagogue. Genetic, genomic, and pharmacological analyses established the P2Y1 receptor as a clock-controlled mediator of the insulinotropic activity of ivermectin. These findings identify the P2Y1 purinergic receptor as a diabetes target based upon a genetically sensitized phenotypic screen.

Circadian rhythms ­ 'inbuilt' 24-hour cycles ­ control many aspects of behaviour and physiology. In mammals, they operate in nearly all tissues, including those involved in glucose metabolism. Recent studies have shown that mice with faulty genes involved in circadian rhythms, the core clock genes, can develop diabetes. Diabetes arises when the body struggles to regulate blood sugar levels. In healthy individuals, the hormone insulin produced by beta cells in the pancreas regulates the amount of sugar in the blood. But when beta cells are faulty and do not generate sufficient insulin levels, or when insulin lacks the ability to stimulate cells to take up glucose, diabetes can develop. Marcheva, Weidemann, Taguchi et al. wanted to find out if diabetes caused by impaired clock genes could be treated by targeting pathways regulating the secretion of insulin. To do so, they tested over 2,500 potential drugs on genetically modified beta cells with faulty core clock genes. They further screened the drugs on mice with the same defect in their beta cells. Marcheva et al. identified one potential compound, the anti-parasite drug ivermectin, which was able to restore the secretion of insulin. When ivermectin was applied to both healthy mice and mice with faulty beta cells, the drug improved the control over glucose levels by activating a specific protein receptor that senses molecules important for storing and utilizing energy. The findings reveal new drug targets for treating forms of diabetes associated with deregulation of the pancreatic circadian clock. The drug screening strategy used in the study may also be applied to reveal mechanisms underlying other conditions associated with disrupted circadian clocks, including sleep loss and jetlag.

A window in time for ß-cell regeneration.

In vivo regeneration of ß cells provides hope for self-renewal of functional insulin-secreting cells following ß-cell failure, a historically fatal condition now sustainable only by administration of exogenous insulin. Despite advances in the treatment of diabetes mellitus, the path toward endogenous renewal of ß-cell populations has remained elusive. Intensive efforts have focused on elucidating pancreatic transcriptional programs that can drive the division and (trans-)differentiation of non-ß cells to produce insulin. A surprise has been the identification of an essential role of the molecular circadian clock in the regulation of competent insulin-producing ß cells. In this issue of Genes & Development, work by Petrenko and colleagues (pp. 1650-1665) now shows a requirement for the intrinsic clock in the regenerative capacity of insulin-producing cells following genetic ablation of ß cells. These studies raise the possibility that enhancing core clock activity may provide an adjuvant in cell replacement therapies.

A role for alternative splicing in circadian control of exocytosis and glucose homeostasis.

The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic ß cells that are perturbed in Clock-/- and Bmal1-/- ß-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant ß cells, including transcripts encoding Cask (calcium/calmodulin-dependent serine protein kinase) and Madd (MAP kinase-activating death domain). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd, and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in ß-cell function across the sleep/wake cycle.

NAD+ Controls Circadian Reprogramming through PER2 Nuclear Translocation to Counter Aging.

Disrupted sleep-wake and molecular circadian rhythms are a feature of aging associated with metabolic disease and reduced levels of NAD+, yet whether changes in nucleotide metabolism control circadian behavioral and genomic rhythms remains unknown. Here, we reveal that supplementation with the NAD+ precursor nicotinamide riboside (NR) markedly reprograms metabolic and stress-response pathways that decline with aging through inhibition of the clock repressor PER2. NR enhances BMAL1 chromatin binding genome-wide through PER2K680 deacetylation, which in turn primes PER2 phosphorylation within a domain that controls nuclear transport and stability and that is mutated in human advanced sleep phase syndrome. In old mice, dampened BMAL1 chromatin binding, transcriptional oscillations, mitochondrial respiration rhythms, and late evening activity are restored by NAD+ repletion to youthful levels with NR. These results reveal effects of NAD+ on metabolism and the circadian system with aging through the spatiotemporal control of the molecular clock.

A day in the life of chromatin: how enhancer-promoter loops shape daily behavior.

Each spring, we get out of bed 1 h ahead of our biological wake-up time due to the misalignment of internal clocks with the light-dark cycle. Genetic discoveries revealed that clock genes encode transcription factors that are expressed throughout many tissues, yet a gap has remained in understanding the temporal dynamics of transcription. Two groups now apply circular chromosome conformation capture and high-throughput sequencing to dissect how "time of day"-dependent changes in chromatin drive core clock oscillations. A surprise is the finding that disruption of enhancer-promoter contacts within chromatin leads to an advance in the "wake-up" time of mice. Furthermore, the assembly of transcriptionally active domains of chromatin requires the ordered recruitment of core clock transcription factors each day. These studies show that waking up involves highly dynamic changes in the three-dimensional positioning of genes within the cell.

Selective Deletion of Renin-b in the Brain Alters Drinking and Metabolism.

The brain-specific isoform of renin (Ren-b) has been proposed as a negative regulator of the brain renin-angiotensin system (RAS). We analyzed mice with a selective deletion of Ren-b which preserved expression of the classical renin (Ren-a) isoform. We reported that Ren-bNull mice exhibited central RAS activation and hypertension through increased expression of Ren-a, but the dipsogenic and metabolic effects in Ren-bNull mice are unknown. Fluid intake was similar in control and Ren-bNull mice at baseline and both exhibited an equivalent dipsogenic response to deoxycorticosterone acetate-salt. Dehydration promoted increased water intake in Ren-bNull mice, particularly after deoxycorticosterone acetate-salt. Ren-bNull and control mice exhibited similar body weight when fed a chow diet. However, when fed a high-fat diet, male Ren-bNull mice gained significantly less weight than control mice, an effect blunted in females. This difference was not because of changes in food intake, energy absorption, or physical activity. Ren-bNull mice exhibited increased resting metabolic rate concomitant with increased uncoupled protein 1 expression and sympathetic nerve activity to the interscapular brown adipose tissue, suggesting increased thermogenesis. Ren-bNull mice were modestly intolerant to glucose and had normal insulin sensitivity. Another mouse model with markedly enhanced brain RAS activity (sRA mice) exhibited pronounced insulin sensitivity concomitant with increased brown adipose tissue glucose uptake. Altogether, these data support the hypothesis that the brain RAS regulates energy homeostasis by controlling resting metabolic rate, and that Ren-b deficiency increases brain RAS activity. Thus, the relative level of expression of Ren-b and Ren-a may control activity of the brain RAS.

Angiotensin AT1A receptors on leptin receptor-expressing cells control resting metabolism.

Leptin contributes to the control of resting metabolic rate (RMR) and blood pressure (BP) through its actions in the arcuate nucleus (ARC). The renin-angiotensin system (RAS) and angiotensin AT1 receptors within the brain are also involved in the control of RMR and BP, but whether this regulation overlaps with leptin's actions is unclear. Here, we have demonstrated the selective requirement of the AT1A receptor in leptin-mediated control of RMR. We observed that AT1A receptors colocalized with leptin receptors (LEPRs) in the ARC. Cellular coexpression of AT1A and LEPR was almost exclusive to the ARC and occurred primarily within neurons expressing agouti-related peptide (AgRP). Mice lacking the AT1A receptor specifically in LEPR-expressing cells failed to show an increase in RMR in response to a high-fat diet and deoxycorticosterone acetate-salt (DOCA-salt) treatments, but BP control remained intact. Accordingly, loss of RMR control was recapitulated in mice lacking AT1A in AgRP-expressing cells. We conclude that angiotensin activates divergent mechanisms to control BP and RMR and that the brain RAS functions as a major integrator for RMR control through its actions at leptin-sensitive AgRP cells of the ARC.

Nicotinamide riboside is uniquely and orally bioavailable in mice and humans.

Nicotinamide riboside (NR) is in wide use as an NAD+ precursor vitamin. Here we determine the time and dose-dependent effects of NR on blood NAD+ metabolism in humans. We report that human blood NAD+ can rise as much as 2.7-fold with a single oral dose of NR in a pilot study of one individual, and that oral NR elevates mouse hepatic NAD+ with distinct and superior pharmacokinetics to those of nicotinic acid and nicotinamide. We further show that single doses of 100, 300 and 1,000 mg of NR produce dose-dependent increases in the blood NAD+ metabolome in the first clinical trial of NR pharmacokinetics in humans. We also report that nicotinic acid adenine dinucleotide (NAAD), which was not thought to be en route for the conversion of NR to NAD+, is formed from NR and discover that the rise in NAAD is a highly sensitive biomarker of effective NAD+ repletion.

Suppression of Resting Metabolism by the Angiotensin AT2 Receptor.

Activation of the brain renin-angiotensin system (RAS) stimulates energy expenditure through increasing of the resting metabolic rate (RMR), and this effect requires simultaneous suppression of the circulating and/or adipose RAS. To identify the mechanism by which the peripheral RAS opposes RMR control by the brain RAS, we examined mice with transgenic activation of the brain RAS (sRA mice). sRA mice exhibit increased RMR through increased energy flux in the inguinal adipose tissue, and this effect is attenuated by angiotensin II type 2 receptor (AT2) activation. AT2 activation in inguinal adipocytes opposes norepinephrine-induced uncoupling protein-1 (UCP1) production and aspects of cellular respiration, but not lipolysis. AT2 activation also opposes inguinal adipocyte function and differentiation responses to epidermal growth factor (EGF). These results highlight a major, multifaceted role for AT2 within inguinal adipocytes in the control of RMR. The AT2 receptor may therefore contribute to body fat distribution and adipose depot-specific effects upon cardio-metabolic health.

Nicotinamide Riboside Opposes Type 2 Diabetes and Neuropathy in Mice.

Male C57BL/6J mice raised on high fat diet (HFD) become prediabetic and develop insulin resistance and sensory neuropathy. The same mice given low doses of streptozotocin are a model of type 2 diabetes (T2D), developing hyperglycemia, severe insulin resistance and diabetic peripheral neuropathy involving sensory and motor neurons. Because of suggestions that increased NAD(+) metabolism might address glycemic control and be neuroprotective, we treated prediabetic and T2D mice with nicotinamide riboside (NR) added to HFD. NR improved glucose tolerance, reduced weight gain, liver damage and the development of hepatic steatosis in prediabetic mice while protecting against sensory neuropathy. In T2D mice, NR greatly reduced non-fasting and fasting blood glucose, weight gain and hepatic steatosis while protecting against diabetic neuropathy. The neuroprotective effect of NR could not be explained by glycemic control alone. Corneal confocal microscopy was the most sensitive measure of neurodegeneration. This assay allowed detection of the protective effect of NR on small nerve structures in living mice. Quantitative metabolomics established that hepatic NADP(+) and NADPH levels were significantly degraded in prediabetes and T2D but were largely protected when mice were supplemented with NR. The data justify testing of NR in human models of obesity, T2D and associated neuropathies.

Dietary Sodium Suppresses Digestive Efficiency via the Renin-Angiotensin System.

Dietary fats and sodium are both palatable and are hypothesized to synergistically contribute to ingestive behavior and thereby obesity. Contrary to this hypothesis, C57BL/6J mice fed a 45% high fat diet exhibited weight gain that was inhibited by increased dietary sodium content. This suppressive effect of dietary sodium upon weight gain was mediated specifically through a reduction in digestive efficiency, with no effects on food intake behavior, physical activity, or resting metabolism. Replacement of circulating angiotensin II levels reversed the effects of high dietary sodium to suppress digestive efficiency. While the AT1 receptor antagonist losartan had no effect in mice fed low sodium, the AT2 receptor antagonist PD-123,319 suppressed digestive efficiency. Correspondingly, genetic deletion of the AT2 receptor in FVB/NCrl mice resulted in suppressed digestive efficiency even on a standard chow diet. Together these data underscore the importance of digestive efficiency in the pathogenesis of obesity, and implicate dietary sodium, the renin-angiotensin system, and the AT2 receptor in the control of digestive efficiency regardless of mouse strain or macronutrient composition of the diet. These findings highlight the need for greater understanding of nutrient absorption control physiology, and prompt more uniform assessment of digestive efficiency in animal studies of energy balance.

Risperidone-induced weight gain is mediated through shifts in the gut microbiome and suppression of energy expenditure.

Risperidone is a second-generation antipsychotic that causes weight gain. We hypothesized that risperidone-induced shifts in the gut microbiome are mechanistically involved in its metabolic consequences. Wild-type female C57BL/6J mice treated with risperidone (80 µg/day) exhibited significant excess weight gain, due to reduced energy expenditure, which correlated with an altered gut microbiome. Fecal transplant from risperidone-treated mice caused a 16% reduction in total resting metabolic rate in naïve recipients, attributable to suppression of non-aerobic metabolism. Risperidone inhibited growth of cultured fecal bacteria grown anaerobically more than those grown aerobically. Finally, transplant of the fecal phage fraction from risperidone-treated mice was sufficient to cause excess weight gain in naïve recipients, again through reduced energy expenditure. Collectively, these data highlight a major role for the gut microbiome in weight gain following chronic use of risperidone, and specifically implicates the modulation of non-aerobic resting metabolism in this mechanism.

A mitochondrial-targeted coenzyme q analog prevents weight gain and ameliorates hepatic dysfunction in high-fat-fed mice.

We hypothesized that the mitochondrial-targeted antioxidant, mitoquinone (mitoQ), known to have mitochondrial uncoupling properties, might prevent the development of obesity and mitigate liver dysfunction by increasing energy expenditure, as opposed to reducing energy intake. We administered mitoQ or vehicle (ethanol) to obesity-prone C57BL/6 mice fed high-fat (HF) or normal-fat (NF) diets. MitoQ (500 µM) or vehicle (ethanol) was added to the drinking water for 28 weeks. MitoQ significantly reduced total body mass and fat mass in the HF-fed mice but had no effect on these parameters in NF mice. Food intake was reduced by mitoQ in the HF-fed but not in the NF-fed mice. Average daily water intake was reduced by mitoQ in both the NF- and HF-fed mice. Hypothalamic expression of neuropeptide Y, agouti-related peptide, and the long form of the leptin receptor were reduced in the HF but not in the NF mice. Hepatic total fat and triglyceride content did not differ between the mitoQ-treated and control HF-fed mice. However, mitoQ markedly reduced hepatic lipid hydroperoxides and reduced circulating alanine aminotransferase, a marker of liver function. MitoQ did not alter whole-body oxygen consumption or liver mitochondrial oxygen utilization, membrane potential, ATP production, or production of reactive oxygen species. In summary, mitoQ added to drinking water mitigated the development of obesity. Contrary to our hypothesis, the mechanism involved decreased energy intake likely mediated at the hypothalamic level. MitoQ also ameliorated HF-induced liver dysfunction by virtue of its antioxidant properties without altering liver fat or mitochondrial bioenergetics.

Hypertension in mice with transgenic activation of the brain renin-angiotensin system is vasopressin dependent.

An indispensable role for the brain renin-angiotensin system (RAS) has been documented in most experimental animal models of hypertension. To identify the specific efferent pathway activated by the brain RAS that mediates hypertension, we examined the hypothesis that elevated arginine vasopressin (AVP) release is necessary for hypertension in a double-transgenic model of brain-specific RAS hyperactivity (the "sRA" mouse model). sRA mice experience elevated brain RAS activity due to human angiotensinogen expression plus neuron-specific human renin expression. Total daily loss of the 4-kDa AVP prosegment (copeptin) into urine was grossly elevated (≥8-fold). Immunohistochemical staining for AVP was increased in the supraoptic nucleus of sRA mice (~2-fold), but no quantitative difference in the paraventricular nucleus was observed. Chronic subcutaneous infusion of a nonselective AVP receptor antagonist conivaptan (YM-087, Vaprisol, 22 ng/h) or the V(2)-selective antagonist tolvaptan (OPC-41061, 22 ng/h) resulted in normalization of the baseline (~15 mmHg) hypertension in sRA mice. Abdominal aortas and second-order mesenteric arteries displayed AVP-specific desensitization, with minor or no changes in responses to phenylephrine and endothelin-1. Mesenteric arteries exhibited substantial reductions in V(1A) receptor mRNA, but no significant changes in V(2) receptor expression in kidney were observed. Chronic tolvaptan infusion also normalized the (5 mmol/l) hyponatremia of sRA mice. Together, these data support a major role for vasopressin in the hypertension of mice with brain-specific hyperactivity of the RAS and suggest a primary role of V(2) receptors.