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.

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.

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 nucleus tractus solitarii (NTS) is the primary central station that integrates visceral afferent information and regulates respiratory, gastrointestinal, cardiovascular, and other physiological functions. Leptin receptor b (LepRb)-expressing neurons of the NTS (NTSLepRb neurons) are implicated in central respiration regulation, respiratory facilitation, and respiratory drive enhancement. Furthermore, LepRb dysfunction is involved in obesity, insulin resistance, and sleep-disordered breathing. However, the monosynaptic inputs and outputs of NTSLepRb neurons in whole-brain mapping remain to be elucidated. Therefore, the exploration of its whole-brain connection system may provide strong support for comprehensively understanding the physiological and pathological functions of NTSLepRb neurons. In the present study, we used a cell type-specific, modified rabies virus and adeno-associated virus with the Cre-loxp system to map monosynaptic inputs and outputs of NTSLepRb neurons in LepRb-Cre mice. The results showed that NTSLepRb neurons received inputs from 48 nuclei in the whole brain from five brain regions, including especially the medulla. We found that NTSLepRb neurons received inputs from nuclei associated with respiration, such as the pre-Bötzinger complex, ambiguus nucleus, and parabrachial nucleus. Interestingly, some brain areas related to cardiovascular regulation-i.e., the ventrolateral periaqueductal gray and locus coeruleus-also sent a small number of inputs to NTSLepRb neurons. In addition, anterograde tracing results demonstrated that NTSLepRb neurons sent efferent projections to 15 nuclei, including the dorsomedial hypothalamic nucleus and arcuate hypothalamic nucleus, which are involved in regulation of energy metabolism and feeding behaviors. Quantitative statistical analysis revealed that the inputs of the whole brain to NTSLepRb neurons were significantly greater than the outputs. Our study comprehensively revealed neuronal connections of NTSLepRb neurons in the whole brain and provided a neuroanatomical basis for further research on physiological and pathological functions of NTSLepRb neurons.