Probes for INSULIN

Insulin acts on neurons and glial cells to regulate systemic glucose metabolism and feeding. However, the mechanisms of insulin access in discrete brain regions are incompletely defined. Here we show that insulin receptors in tanycytes, but not in brain endothelial cells, are required to regulate insulin access to the hypothalamic arcuate nucleus. Mice lacking insulin receptors in tanycytes (IR∆Tan mice) exhibit systemic insulin resistance, while displaying normal food intake and energy expenditure. Tanycytic insulin receptors are also necessary for the orexigenic effects of ghrelin, but not for the anorexic effects of leptin. IR∆Tan mice exhibit increased agouti-related peptide (AgRP) neuronal activity, while displaying blunted AgRP neuronal adaptations to feeding-related stimuli. Lastly, a highly palatable food decreases tanycytic and arcuate nucleus insulin signalling to levels comparable to those seen in IR∆Tan mice. These changes are rooted in modifications of cellular stress responses and of mitochondrial protein quality control in tanycytes. Conclusively, we reveal a critical role of tanycyte insulin receptors in gating feeding-state-dependent regulation of AgRP neurons and systemic insulin sensitivity, and show that insulin resistance in tanycytes contributes to the pleiotropic manifestations of obesity-associated insulin resistance.

26RFa (pyroglutamilated RFamide peptide [QRFP]) is a biologically active peptide that regulates glucose homeostasis by acting as an incretin and by increasing insulin sensitivity at the periphery. 26RFa is also produced by a neuronal population localised in the hypothalamus. In this study we investigated whether 26RFa neurons are involved in the hypothalamic regulation of glucose homeostasis.26Rfa+/+, 26Rfa-/- and insulin-deficient male C57Bl/6J mice were used in this study. Mice received an acute intracerebroventricular (i.c.v.) injection of 26RFa, insulin or the 26RFa receptor (GPR103) antagonist 25e and were subjected to IPGTTs, insulin tolerance tests, acute glucose-stimulated insulin secretion tests and pyruvate tolerance tests (PTTs). Secretion of 26RFa by hypothalamic explants after incubation with glucose, leptin or insulin was assessed. Expression and quantification of the genes encoding 26RFa, agouti-related protein, the insulin receptor and GPR103 were evaluated by quantitative reverse transcription PCR and RNAscope in situ hybridisation.Our data indicate that i.c.v.-injected 26RFa induces a robust antihyperglycaemic effect associated with an increase in insulin production by the pancreatic islets. In addition, we found that insulin strongly stimulates 26Rfa expression and secretion by the hypothalamus. RNAscope experiments revealed that neurons expressing 26Rfa are mainly localised in the lateral hypothalamic area, that they co-express the gene encoding the insulin receptor and that insulin induces the expression of 26Rfa in these neurons. Concurrently, the central antihyperglycaemic effect of insulin is abolished in the presence of a GPR103 antagonist and in 26RFa-deficient mice. Finally, our data indicate that the hypothalamic 26RFa neurons are not involved in the central inhibitory effect of insulin on hepatic glucose production, but mediate the central effects of the hormone on its own peripheral production.We have identified a novel mechanism in the hypothalamic regulation of glucose homeostasis, the 26RFa/GPR103 system, and we provide evidence that this neuronal peptidergic system is a key relay for the central regulation of glucose metabolism by insulin.

Human insulin (INS) gene diverged from the ancestral genes of invertebrate and mammalian species millions of years ago. We previously found that mouse insulin gene (Ins2) isoforms are expressed in brain choroid plexus (ChP) epithelium cells where insulin secretion is regulated by serotonin and not by glucose. We further compared human INS isoform expression in postmortem ChP and islets of Langerhans. We uncovered novel INS upstream open reading frame (uORF) isoforms and their protein products. In addition, we found a novel alternatively spliced isoform that translates to a 74-amino acid (AA) proinsulin containing a shorter 19-AA C-peptide sequence, herein designated Cα-peptide. The middle portion of the conventional C-peptide contains β-sheet (GQVEL) and hairpin (GGGPG) motifs that are not present in Cα-peptide. Islet amyloid polypeptide (IAPP) is not expressed in ChP and its amyloid formation was inhibited in vitro by Cα-peptide more efficiently than by C-peptide. Of clinical relevance, the ratio of the 74-AA proinsulin to proconvertase processed Cα-peptide was significantly increased in islets from type 2 diabetes mellitus (T2DM) autopsy donors. Intriguingly, 100 years after the discovery of insulin we found that INS isoforms are present in ChP from insulin-deficient autopsy donors.

Activation of Agouti-Related Peptide (AgRP)-expressing neurons promotes feeding and insulin resistance. Here, we examine the contribution of neuropeptide Y (NPY)-dependent signaling to the diverse physiological consequences of activating AgRP neurons. NPY-deficient mice fail to rapidly increase food intake during the first hour of either chemo- or optogenetic activation of AgRP neurons, while the delayed increase in feeding is comparable between control and NPY-deficient mice. Acutely stimulating AgRP neurons fails to induce systemic insulin resistance in NPY-deficient mice, while increased locomotor activity upon AgRP neuron stimulation in the absence of food remains unaffected in these animals. Selective re-expression of NPY in AgRP neurons attenuates the reduced feeding response and reverses the protection from insulin resistance upon optogenetic activation of AgRP neurons in NPY-deficient mice. Collectively, these experiments reveal a pivotal role of NPY-dependent signaling in mediating the rapid feeding inducing effect and the acute glucose regulatory function governed by AgRP neurons

KEY POINTS: ICV insulin increased SNA and baroreflex control of SNA and HR dramatically more in obese male rats; in obese females, the responses were abolished. In obese males, the enhanced LSNA responses were associated with reduced tonic inhibition of LSNA by NPY in the PVN. Yet, PVN NPY injection decreased LSNA similarly in OP/OR/CON rats. Collectively, these results suggest that NPY inputs were decreased. In obese females, NPY inhibition in the PVN was maintained. Moreover, NPY neurons in the ArcN became resistant to the inhibitory effects of insulin. A HFD did not alter arcuate NPY neuronal InsR expression in males or females. Obesity-induced "selective sensitization" of the brain to the sympathoexcitatory effects of insulin and leptin may contribute to elevated basal SNA, and therefore hypertension development, in males with obesity. These data may explain in part why obesity increases SNA less in women compared to men. ABSTRACT: Obesity increases sympathetic nerve activity (SNA) in men, but not women; however, the mechanisms are unknown. We tested if intracerebroventricular insulin infusion increases SNA more in obese male than female rats and if sex differences are mediated by changes in tonic inhibition of SNA by Neuropeptide Y (NPY) in the paraventricular nucleus (PVN). When consuming a high fat diet, obesity prone (OP) rats accrued excess fat, whereas obesity resistant (OR) rats maintained adiposity as in rats eating a control (CON) diet. Insulin increased lumbar SNA (LSNA) similarly in CON/OR males and females under urethane-anesthesia. The LSNA response was magnified in OP males, but abolished in OP females. In males, blockade of PVN NPY Y1 receptors with BIBO3304 increased LSNA in CON/OR rats, but not OP rats. Yet, PVN nanoinjections of NPY decreased LSNA similarly between groups. Thus, tonic PVN NPY inhibition of LSNA may be lost in obese males, due to a decrease in NPY inputs. In contrast, in females, PVN BIBO3304 increased LSNA similarly in OP, OR and CON rats. After insulin, PVN BIBO3304 failed to increase LSNA in CON/OR females, but increased LSNA in OP females, suggesting that with obesity NPY neurons become resistant to the inhibitory effects of insulin. These sex differences were not associated with changes in arcuate NPY neuronal insulin receptor expression. Collectively, these data reveal a marked sex difference in the impact of obesity on insulin's sympathoexcitatory actions and implicate sexually dimorphic changes in NPY inhibition of SNA in the PVN as one mechanism.

Neuropeptide FF (NPFF) group peptides belong to the evolutionary conserved RF-amide peptide family. While they have been assigned a role as pain modulators, their roles in other aspects of physiology have received much less attention. NPFF peptides and their receptor NPFFR2 have strong and localized expression within the dorsal vagal complex that has emerged as the key centre for regulating glucose homeostasis. Therefore, we investigated the role of the NPFF system in the control of glucose metabolism and the histochemical and molecular identities of NPFF and NPFFR2 neurons.We examined glucose metabolism in Npff-/- and wild type (WT) mice using intraperitoneal (i.p.) glucose tolerance and insulin tolerance tests. Body composition and glucose tolerance was further examined in mice after 1-week and 3-week of high-fat diet (HFD). Using RNAScope double ISH, we investigated the neurochemical identity of NPFF and NPFFR2 neurons in the caudal brainstem, and the expression of receptors for peripheral factors in NPFF neurons.Lack of NPFF signalling in mice leads to improved glucose tolerance without significant impact on insulin excursion after the i.p. glucose challenge. In response to an i.p. bolus of insulin, Npff-/- mice have lower glucose excursions than WT mice, indicating an enhanced insulin action. Moreover, while HFD has rapid and potent detrimental effects on glucose tolerance, this diet-induced glucose intolerance is ameliorated in mice lacking NPFF signalling. This occurs in the absence of any significant impact of NPFF deletion on lean or fat masses, suggesting a direct effect of NPFF signalling on glucose metabolism. We further reveal that NPFF neurons in the subpostrema area (SubP) co-express receptors for peripheral factors involved in glucose homeostasis regulation such as insulin and GLP1. Furthermore, Npffr2 is expressed in the glutamatergic NPFF neurons in the SubP, and in cholinergic neurons of the dorsal motor nucleus of the vagus (DMV), indicating that central NPFF signalling is likely modulating vagal output to innervated peripheral tissues including those important for glucose metabolic control.NPFF signalling plays an important role in the regulation of glucose metabolism. NPFF neurons in the SubP are likely to receive peripheral signals and mediate the control of whole-body glucose homeostasis via centrally vagal pathways. Targeting NPFF and NPFFR2 signalling may provide a new avenue for treating type 2 diabetes and obesity.

Interleukin 1β (IL1β) is a pro-inflammatory cytokine that may play a crucial role in enteric neuroinflammation in type 1 diabetes. Therefore, our goal is to evaluate the effects of chronic hyperglycemia and insulin treatment on IL1β immunoreactivity in myenteric neurons and their different subpopulations along the duodenum-ileum-colon axis. Fluorescent immunohistochemistry was used to count IL1β expressing neurons as well as the neuronal nitric oxide synthase (nNOS)- and calcitonin gene-related peptide (CGRP)-immunoreactive myenteric neurons within this group. Tissue IL1β level was measured by ELISA in muscle/myenteric plexus-containing homogenates. IL1β mRNA was detected by RNAscope in different intestinal layers. The proportion of IL1β-immunoreactive myenteric neurons was significantly higher in the colon than in the small intestine of controls. In diabetics, this proportion significantly increased in all gut segments, which was prevented by insulin treatment. The proportion of IL1β-nNOS-immunoreactive neurons only increased in the diabetic colon, while the proportion of IL1β-CGRP-immunoreactive neurons only increased in the diabetic ileum. Elevated IL1β levels were also confirmed in tissue homogenates. IL1β mRNA induction was detected in the myenteric ganglia, smooth muscle and intestinal mucosa of diabetics. These findings support that diabetes-related IL1β induction is specific for the different myenteric neuronal subpopulations, which may contribute to diabetic motility disturbances.

The wake-active orexin system plays a central role in the dynamic regulation of glucose homeostasis. Here we show orexin receptor type 1 and 2 are predominantly expressed in dorsal raphe nucleus-dorsal and -ventral, respectively. Serotonergic neurons in ventral median raphe nucleus and raphe pallidus selectively express orexin receptor type 1. Inactivation of orexin receptor type 1 in serotonin transporter-expressing cells of mice reduced insulin sensitivity in diet-induced obesity, mainly by decreasing glucose utilization in brown adipose tissue and skeletal muscle. Selective inactivation of orexin receptor type 2 improved glucose tolerance and insulin sensitivity in obese mice, mainly through a decrease in hepatic gluconeogenesis. Optogenetic activation of orexin neurons in lateral hypothalamus or orexinergic fibers innervating raphe pallidus impaired or improved glucose tolerance, respectively. Collectively, the present study assigns orexin signaling in serotonergic neurons critical, yet differential orexin receptor type 1- and 2-dependent functions in the regulation of systemic glucose homeostasis.

Systemic insulin sensitivity shows a diurnal rhythm with a peak upon waking1,2. The molecular mechanism that underlies this temporal pattern is unclear. Here we show that the nuclear receptors REV-ERB-α and REV-ERB-β (referred to here as 'REV-ERB') in the GABAergic (γ-aminobutyric acid-producing) neurons in the suprachiasmatic nucleus (SCN) (SCNGABA neurons) control the diurnal rhythm of insulin-mediated suppression of hepatic glucose production in mice, without affecting diurnal eating or locomotor behaviours during regular light-dark cycles. REV-ERB regulates the rhythmic expression of genes that are involved in neurotransmission in the SCN, and modulates the oscillatory firing activity of SCNGABA neurons. Chemogenetic stimulation of SCNGABA neurons at waking leads to glucose intolerance, whereas restoration of the temporal pattern of either SCNGABA neuron firing or REV-ERB expression rescues the time-dependent glucose metabolic phenotype caused by REV-ERB depletion. In individuals with diabetes, an increased level of blood glucose after waking is a defining feature of the 'extended dawn phenomenon'3,4. Patients with type 2 diabetes with the extended dawn phenomenon exhibit a differential temporal pattern of expression of REV-ERB genes compared to patients with type 2 diabetes who do not have the extended dawn phenomenon. These findings provide mechanistic insights into how the central circadian clock regulates the diurnal rhythm of hepatic insulin sensitivity, with implications for our understanding of the extended dawn phenomenon in type 2 diabetes.

The growth hormone receptor (GHR) is expressed in brain regions that are known to participate in the regulation of energy homeostasis and glucose metabolism. We generated a novel transgenic mouse line (GHRcre) to characterize GHR-expressing neurons specifically in the arcuate nucleus of the hypothalamus (ARC). Here, we demonstrate that ARCGHR+ neurons are co-localized with agouti-related peptide (AgRP), growth hormone releasing hormone (GHRH), and somatostatin neurons, which are activated by GH stimulation. Using the designer receptors exclusively activated by designer drugs (DREADD) technique to control the ARCGHR+ neuronal activity, we demonstrate that the activation of ARCGHR+ neurons elevates a respiratory exchange ratio (RER) under both fed and fasted conditions. However, while the activation of ARCGHR+ promotes feeding, under fasting conditions, the activation of ARCGHR+ neurons promotes glucose over fat utilization in the body. This effect was accompanied by significant improvements in glucose tolerance, and was specific to GHR+ versus GHRH+ neurons. The activation of ARCGHR+ neurons increased glucose turnover and whole-body glycolysis, as revealed by hyperinsulinemic-euglycemic clamp studies. Remarkably, the increased insulin sensitivity upon the activation of ARCGHR+ neurons was tissue-specific, as the insulin-stimulated glucose uptake was specifically elevated in the skeletal muscle, in parallel with the increased expression of muscle glycolytic genes. Overall, our results identify the GHR-expressing neuronal population in the ARC as a major regulator of glycolysis and muscle insulin sensitivity in vivo.