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• W2560644536 abstract "•Insulin promotes PI3 kinase signaling in MCH neurons and increases excitability•Obesity promotes insulin signaling in MCH neurons•Insulin action on MCH neurons controls locomotor activity and insulin sensitivity•Acute activation of MCH neurons impairs locomotor activity Melanin-concentrating-hormone (MCH)-expressing neurons (MCH neurons) in the lateral hypothalamus (LH) are critical regulators of energy and glucose homeostasis. Here, we demonstrate that insulin increases the excitability of these neurons in control mice. In vivo, insulin promotes phosphatidylinositol 3-kinase (PI3K) signaling in MCH neurons, and cell-type-specific deletion of the insulin receptor (IR) abrogates this response. While lean mice lacking the IR in MCH neurons (IRΔMCH) exhibit no detectable metabolic phenotype under normal diet feeding, they present with improved locomotor activity and insulin sensitivity under high-fat-diet-fed, obese conditions. Similarly, obesity promotes PI3 kinase signaling in these neurons, and this response is abrogated in IRΔMCH mice. In turn, acute chemogenetic activation of MCH neurons impairs locomotor activity but not insulin sensitivity. Collectively, our experiments reveal an insulin-dependent activation of MCH neurons in obesity, which contributes via distinct mechanisms to the manifestation of impaired locomotor activity and insulin resistance. Melanin-concentrating-hormone (MCH)-expressing neurons (MCH neurons) in the lateral hypothalamus (LH) are critical regulators of energy and glucose homeostasis. Here, we demonstrate that insulin increases the excitability of these neurons in control mice. In vivo, insulin promotes phosphatidylinositol 3-kinase (PI3K) signaling in MCH neurons, and cell-type-specific deletion of the insulin receptor (IR) abrogates this response. While lean mice lacking the IR in MCH neurons (IRΔMCH) exhibit no detectable metabolic phenotype under normal diet feeding, they present with improved locomotor activity and insulin sensitivity under high-fat-diet-fed, obese conditions. Similarly, obesity promotes PI3 kinase signaling in these neurons, and this response is abrogated in IRΔMCH mice. In turn, acute chemogenetic activation of MCH neurons impairs locomotor activity but not insulin sensitivity. Collectively, our experiments reveal an insulin-dependent activation of MCH neurons in obesity, which contributes via distinct mechanisms to the manifestation of impaired locomotor activity and insulin resistance. Obesity represents a major health burden currently affecting more than 30% of Western populations, and it results from a de-regulated balance between caloric intake and energy expenditure. Due to steadily rising rates of obesity, the incidence of type 2 diabetes mellitus (T2DM), and its associated comorbidities is also growing at an alarming pace (Ng et al., 2014Ng M. Fleming T. Robinson M. Thomson B. Graetz N. Margono C. Mullany E.C. Biryukov S. Abbafati C. Abera S.F. et al.Global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013: a systematic analysis for the Global Burden of Disease Study 2013.Lancet. 2014; 384: 766-781Abstract Full Text Full Text PDF PubMed Scopus (8094) Google Scholar). Regulation of blood glucose levels is a complex and highly integrated process involving peripheral tissues as well as the central nervous system (CNS) (Vogt and Brüning, 2013Vogt M.C. Brüning J.C. CNS insulin signaling in the control of energy homeostasis and glucose metabolism - from embryo to old age.Trends Endocrinol. Metab. 2013; 24: 76-84Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Resistance to the metabolic effects of the pancreas-derived hormone insulin in the periphery and CNS represents key features in the development of perturbed glucose homeostasis in T2DM (Belgardt and Brüning, 2010Belgardt B.F. Brüning J.C. CNS leptin and insulin action in the control of energy homeostasis.Ann. N Y Acad. Sci. 2010; 1212: 97-113Crossref PubMed Scopus (209) Google Scholar, Boucher et al., 2014Boucher J. Kleinridders A. Kahn C.R. Insulin receptor signaling in normal and insulin-resistant states.Cold Spring Harb. Perspect. Biol. 2014; 6: a009191Crossref Scopus (820) Google Scholar). However, recent studies also indicated that the simultaneous occurrence of insulin resistance in some tissues or cell types within the CNS with retained or even over-activated insulin action in other cell types, i.e., “selective hormone resistance,” might be more useful to explain the complex clinical outcome of this disease (Könner and Brüning, 2012Könner A.C. Brüning J.C. Selective insulin and leptin resistance in metabolic disorders.Cell Metab. 2012; 16: 144-152Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). The insulin receptor (IR) is widely expressed in the CNS, and specific deletion of the IR in the brain of mice results in mild diet-sensitive obesity and hypothalamic hypogonadism (Brüning et al., 2000Brüning J.C. Gautam D. Burks D.J. Gillette J. Schubert M. Orban P.C. Klein R. Krone W. Müller-Wieland D. Kahn C.R. Role of brain insulin receptor in control of body weight and reproduction.Science. 2000; 289: 2122-2125Crossref PubMed Scopus (1780) Google Scholar). Moreover, insulin failed to efficiently suppress hepatic glucose production in hyperinsulinemic-euglycemic clamp studies in these mice, assigning insulin action in the CNS an important role in the regulation of peripheral glucose metabolism (Inoue et al., 2006Inoue H. Ogawa W. Asakawa A. Okamoto Y. Nishizawa A. Matsumoto M. Teshigawara K. Matsuki Y. Watanabe E. Hiramatsu R. et al.Role of hepatic STAT3 in brain-insulin action on hepatic glucose production.Cell Metab. 2006; 3: 267-275Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). We and others could subsequently demonstrate that functional insulin signaling, specifically in Agouti-related-peptide (AgRP)-expressing neurons of the hypothalamic arcuate nucleus (ARC), is a prerequisite for peripheral applied insulin to efficiently suppress hepatic glucose production (Könner et al., 2007Könner A.C. Janoschek R. Plum L. Jordan S.D. Rother E. Ma X. Xu C. Enriori P. Hampel B. Barsh G.S. et al.Insulin action in AgRP-expressing neurons is required for suppression of hepatic glucose production.Cell Metab. 2007; 5: 438-449Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar, Lin et al., 2010Lin H.V. Plum L. Ono H. Gutiérrez-Juárez R. Shanabrough M. Borok E. Horvath T.L. Rossetti L. Accili D. Divergent regulation of energy expenditure and hepatic glucose production by insulin receptor in agouti-related protein and POMC neurons.Diabetes. 2010; 59: 337-346Crossref PubMed Scopus (120) Google Scholar). The present study focuses on melanin-concentrating-hormone (MCH)-expressing neurons (MCH neurons), which are found exclusively in the lateral hypothalamus (LH) and the zona incerta of the brain (Bittencourt, 2011Bittencourt J.C. Anatomical organization of the melanin-concentrating hormone peptide family in the mammalian brain.Gen. Comp. Endocrinol. 2011; 172: 185-197Crossref PubMed Scopus (94) Google Scholar). The LH is a brain region historically implicated in regulation of feeding and satiety and is considered as the most extensively interconnected area of the hypothalamus, with projections to other hypothalamic regions, cortical/limbic areas, and the autonomic and motor systems of the brainstem (Brown et al., 2015Brown J.A. Woodworth H.L. Leinninger G.M. To ingest or rest? Specialized roles of lateral hypothalamic area neurons in coordinating energy balance.Front. Syst. Neurosci. 2015; 9: 9Crossref PubMed Scopus (74) Google Scholar). When administered via intracerebroventricular (i.c.v.) injection, MCH causes an acute robust increase in feeding (Qu et al., 1996Qu D. Ludwig D.S. Gammeltoft S. Piper M. Pelleymounter M.A. Cullen M.J. Mathes W.F. Przypek R. Kanarek R. Maratos-Flier E. A role for melanin-concentrating hormone in the central regulation of feeding behaviour.Nature. 1996; 380: 243-247Crossref PubMed Scopus (1192) Google Scholar), and chronic infusion leads to excess weight gain associated with decreased energy expenditure and increased energy storage (Ito et al., 2003Ito M. Gomori A. Ishihara A. Oda Z. Mashiko S. Matsushita H. Yumoto M. Ito M. Sano H. Tokita S. et al.Characterization of MCH-mediated obesity in mice.Am. J. Physiol. Endocrinol. Metab. 2003; 284: E940-E945Crossref PubMed Scopus (150) Google Scholar). Conversely, mice with targeted deletion of the Mch gene present with reduced body weight and leanness due to hypophagia and increased metabolic rate (Shimada et al., 1998Shimada M. Tritos N.A. Lowell B.B. Flier J.S. Maratos-Flier E. Mice lacking melanin-concentrating hormone are hypophagic and lean.Nature. 1998; 396: 670-674Crossref PubMed Scopus (990) Google Scholar). In contrast, MCH overexpression results in hyperphagia, obesity, and insulin resistance (Ludwig et al., 2001Ludwig D.S. Tritos N.A. Mastaitis J.W. Kulkarni R. Kokkotou E. Elmquist J. Lowell B. Flier J.S. Maratos-Flier E. Melanin-concentrating hormone overexpression in transgenic mice leads to obesity and insulin resistance.J. Clin. Invest. 2001; 107: 379-386Crossref PubMed Scopus (545) Google Scholar). Furthermore, mice lacking MCH exhibit increased locomotor activity, and this increase becomes even more prominent when animals are placed on a high-fat diet (HFD) (Kokkotou et al., 2005Kokkotou E. Jeon J.Y. Wang X. Marino F.E. Carlson M. Trombly D.J. Maratos-Flier E. Mice with MCH ablation resist diet-induced obesity through strain-specific mechanisms.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2005; 289: R117-R124Crossref PubMed Scopus (127) Google Scholar). With respect to glucose homeostasis, aged Mch null mice exhibited better glucose tolerance and insulin sensitivity when compared to control animals, and aging-associated decreases in locomotor activity were also attenuated in these mice (Jeon et al., 2006Jeon J.Y. Bradley R.L. Kokkotou E.G. Marino F.E. Wang X. Pissios P. Maratos-Flier E. MCH-/- mice are resistant to aging-associated increases in body weight and insulin resistance.Diabetes. 2006; 55: 428-434Crossref PubMed Scopus (45) Google Scholar). In addition to these models of chronically altered MCH activity, acute ablation of neurons that express MCH in adult leptin-deficient ob/ob mice had no effect on food intake, body weight, or fertility but resulted in improved glucose tolerance (Wu et al., 2012Wu Q. Whiddon B.B. Palmiter R.D. Ablation of neurons expressing agouti-related protein, but not melanin concentrating hormone, in leptin-deficient mice restores metabolic functions and fertility.Proc. Natl. Acad. Sci. USA. 2012; 109: 3155-3160Crossref PubMed Scopus (88) Google Scholar). Collectively, all of these experiments point toward a critical regulatory function of MCH neurons in the integrated control of food intake, locomotor activity, and systemic glucose homeostasis (Burdakov et al., 2013Burdakov D. Karnani M.M. Gonzalez A. Lateral hypothalamus as a sensor-regulator in respiratory and metabolic control.Physiol. Behav. 2013; 121: 117-124Crossref PubMed Scopus (67) Google Scholar, Leinninger, 2011Leinninger G.M. Lateral thinking about leptin: a review of leptin action via the lateral hypothalamus.Physiol. Behav. 2011; 104: 572-581Crossref PubMed Scopus (38) Google Scholar). To investigate a role for insulin signaling in LH MCH-expressing cells in the regulation of energy and glucose homeostasis, we now have specifically altered insulin signaling in MCH-expressing cells of mice and reveal an important role for insulin action in this circuitry in control of locomotor activity and peripheral glucose metabolism under obese conditions. First, we performed perforated patch-clamp recordings in brain slices from reporter animals, which express enhanced GFP upon Cre-mediated recombination specifically in MCH neurons (Novak et al., 2000Novak A. Guo C. Yang W. Nagy A. Lobe C.G. Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision.Genesis. 2000; 28: 147-155Crossref PubMed Scopus (725) Google Scholar). These mice showed a pattern of GFP immunoreactivity in the LH, which is consistent with the previously described MCH expression pattern in the LH (Figures 1A and 1B ) (Kong et al., 2010Kong D. Vong L. Parton L.E. Ye C. Tong Q. Hu X. Choi B. Brüning J.C. Lowell B.B. Glucose stimulation of hypothalamic MCH neurons involves K(ATP) channels, is modulated by UCP2, and regulates peripheral glucose homeostasis.Cell Metab. 2010; 12: 545-552Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Furthermore, to demonstrate that this approach faithfully marks MCH neurons, we next performed immunohistochemistry for endogenous MCH in Mch-Cre reporter mice. This staining, indeed, revealed faithful genetic marking of MCH neurons in these animals (Figure S1). MCH-expressing neurons in the LH were initially identified by direct visualization of GFP fluorescence. Further, MCH neurons were electrophysiologically identified by the presence of a prominent outward rectification after hyperpolarization (Figure 1C) (Huang et al., 2007Huang H. Acuna-Goycolea C. Li Y. Cheng H.M. Obrietan K. van den Pol A.N. Cannabinoids excite hypothalamic melanin-concentrating hormone but inhibit hypocretin/orexin neurons: implications for cannabinoid actions on food intake and cognitive arousal.J. Neurosci. 2007; 27: 4870-4881Crossref PubMed Scopus (84) Google Scholar). For post hoc identification, cells were loaded with biocytin by converting the perforated-patch configuration to the whole-cell configuration at the end of the recording. The identity of MCH neurons was then confirmed by double-labeling confocal immunohistochemistry of biocytin and GFP (Figure 1D). When synaptic input was pharmacologically blocked, approximately 62% (13 of 21) of the MCH-GFP neurons were silent, while only 38% (8 of 21) of MCH-GFP neurons generated spontaneous action potentials (APs) (Figure 1E), which is consistent with previous findings (Kong et al., 2010Kong D. Vong L. Parton L.E. Ye C. Tong Q. Hu X. Choi B. Brüning J.C. Lowell B.B. Glucose stimulation of hypothalamic MCH neurons involves K(ATP) channels, is modulated by UCP2, and regulates peripheral glucose homeostasis.Cell Metab. 2010; 12: 545-552Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). To analyze insulin-induced changes in electrophysiological properties, a series of hyperpolarizing and depolarizing current pulses were applied under current clamp from a membrane potential of −60 mV in synaptically isolated MCH neurons (Figure 1F). Insulin (200 nM) clearly increased evoked AP firing in nearly half of the tested MCH neurons (6 of 13; Figure 1G). In three MCH neurons, insulin decreased the evoked AP firing (Figure 1G, bottom). Insulin had no consistent effect on input resistance (control: 422.6 ± 54.9 MΩ; insulin: 411.4 ± 51.3 MΩ; p > 0.05, n = 13) or on membrane potential (control: −57 ± 1.7 mV; insulin: −57.2 ± 1.8 mV; p > 0.05, n = 13). Thus, insulin directly increases the excitability of a substantial subset of MCH neurons in the LH. Insulin-evoked phosphatidylinositol 3-kinase (PI3K) signaling controls many critical functions in diverse neuronal populations (Könner et al., 2007Könner A.C. Janoschek R. Plum L. Jordan S.D. Rother E. Ma X. Xu C. Enriori P. Hampel B. Barsh G.S. et al.Insulin action in AgRP-expressing neurons is required for suppression of hepatic glucose production.Cell Metab. 2007; 5: 438-449Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar, Könner et al., 2011Könner A.C. Hess S. Tovar S. Mesaros A. Sánchez-Lasheras C. Evers N. Verhagen L.A. Brönneke H.S. Kleinridders A. Hampel B. et al.Role for insulin signaling in catecholaminergic neurons in control of energy homeostasis.Cell Metab. 2011; 13: 720-728Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, Plum et al., 2006Plum L. Ma X. Hampel B. Balthasar N. Coppari R. Münzberg H. Shanabrough M. Burdakov D. Rother E. Janoschek R. et al.Enhanced PIP3 signaling in POMC neurons causes KATP channel activation and leads to diet-sensitive obesity.J. Clin. Invest. 2006; 116: 1886-1901Crossref PubMed Scopus (275) Google Scholar, Spanswick et al., 2000Spanswick D. Smith M.A. Mirshamsi S. Routh V.H. Ashford M.L. Insulin activates ATP-sensitive K+ channels in hypothalamic neurons of lean, but not obese rats.Nat. Neurosci. 2000; 3: 757-758Crossref PubMed Scopus (433) Google Scholar). Thus, we addressed whether insulin also activates this signaling branch in MCH-positive LH neurons. To genetically mark MCH-positive cells, we used a reporter mouse strain that expresses β-galactosidase (LacZ) after Cre-mediated recombination (MCH-LacZ) (Seibler et al., 2003Seibler J. Zevnik B. Küter-Luks B. Andreas S. Kern H. Hennek T. Rode A. Heimann C. Faust N. Kauselmann G. et al.Rapid generation of inducible mouse mutants.Nucleic Acids Res. 2003; 31: e12Crossref PubMed Scopus (239) Google Scholar). We performed double immunohistochemistry for immunoreactive phosphatidylinositol-3,4,5-trisphosphate (PIP3) and for LacZ in LH MCH neurons of MCH-LacZ mice that had been fasted and either injected with saline or stimulated with insulin for 10 min. Immunohistochemical assessment of PIP3 revealed that insulin strongly activated PIP3 formation in MCH-positive cells in the LH of control mice (Figure 2A). These results indicate that insulin activates PI3K signaling in the MCH neurons of control mice. To investigate the role of insulin action in MCH neurons in control of energy and glucose homeostasis, we generated mice with inactivation of the IR specifically in MCH-expressing neurons of the LH (IRΔMCH mice) by crossing IRlox/lox mice with animals expressing the Cre recombinase under control of the Mch gene (Kong et al., 2010Kong D. Vong L. Parton L.E. Ye C. Tong Q. Hu X. Choi B. Brüning J.C. Lowell B.B. Glucose stimulation of hypothalamic MCH neurons involves K(ATP) channels, is modulated by UCP2, and regulates peripheral glucose homeostasis.Cell Metab. 2010; 12: 545-552Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In contrast to control mice, insulin stimulation of IRΔMCH LacZ reporter mice, which express LacZ in IR-deficient cells, did not increase PIP3 in LacZ-positive cells compared to saline injection, indicating efficient deletion of the IR in MCH neurons of IRΔMCH mice (Figure 2B). To investigate the impact of IR inactivation on the regulation of energy homeostasis, we monitored body weight and other physiological parameters of control and IRΔMCH mice. On a normal chow diet, there was no difference in body weight, fat pad mass, brown adipose tissue (BAT) weight, circulating plasma leptin concentrations, or body fat content between control and IRΔMCH mice (Figures S2A–S2E), indicating that deletion of the IR in LH MCH neurons has no effect on energy homeostasis under normal chow diet conditions. Consistent with unaltered energy homeostasis in IRΔMCH mice, daily energy intake, O2 consumption as assessed by means of indirect calorimetry, and locomotor activity were not different from those of control mice (Figures S2F–S2H). Moreover, we assessed glucose metabolism in control and IRΔMCH mice. Mice lacking the IR specifically in MCH neurons do not show any differences in circulating plasma insulin concentrations, glucose tolerance, or insulin sensitivity when compared to control mice (Figures S2I–S2K). Taken together, IR inactivation in MCH cells does not affect energy homeostasis, locomotor activity, or glucose metabolism in lean mice. To address whether insulin action in MCH LH neurons affects energy and/or glucose homeostasis under conditions of diet-induced obesity, we analyzed control and IRΔMCH mice that were exposed to a HFD. This analysis revealed that HFD increased body weight, fat pad mass, BAT mass, body fat content, and circulating plasma leptin concentrations to a similar extent in control and IRΔMCH mice (Figures 2C–2G). Although the body weight of IRΔMCH mice shows a tendency to be decreased beginning from week 14 of age when compared to control mice on a HFD, this does not reach statistical significance (Figure 2C). Furthermore, daily food intakes and O2 consumption as assessed via indirect calorimetry were comparable between IRΔMCH and control mice on a HFD (Figures 2H and 2I). Interestingly, IRΔMCH mice on a HFD exhibited increased locomotor activity during both day and night phases when compared to control mice (Figure 2J). Moreover, HFD feeding increased circulating plasma insulin concentrations to the same extent in control and IRΔMCH mice (Figure 2K). However, IRΔMCH mice showed significantly improved insulin sensitivity as compared to control mice exposed to HFD, while glucose tolerance remained unaltered between the two groups (Figures 2L and 2M). To estimate gluconeogenesis in control and IRΔMCH mice, we next performed a pyruvate tolerance test in these mice. Interestingly, IRΔMCH mice showed a tendency to perform better in a pyruvate tolerance test than control mice on a HFD (Figure 2N), although this did not reach statistical significance. To further investigate the molecular basis of increased insulin sensitivity in IRΔMCH mice on a HFD, we analyzed insulin-stimulated signaling in liver and skeletal muscle. Following an overnight fast and peripheral injection of insulin, serine phosphorylation, and thus activation of the downstream serine/threonine kinase AKT, was significantly increased in the liver, but not skeletal muscle, of IRΔMCH mice, indicating that inactivation of the IR in MCH neurons improves insulin signaling at the level of AKT activation in the liver (Figure 2O). Collectively, these data indicated that impairing insulin action in MCH neurons of obese mice improved hepatic insulin action. To further specifically address the effect of impaired insulin action in MCH neurons on peripheral glucose homeostasis, we performed hyperinsulinemic-euglycemic clamp studies in HFD-fed control and IRΔMCH mice. Blood glucose levels were comparable between control and IRΔMCH mice throughout the clamp, and both groups reached a euglycemic state at the end of the experiment (Figure 3A). However, the glucose infusion rate required to maintain euglycemia during steady-state conditions was significantly increased in IRΔMCH mice (Figure 3B). Hepatic glucose production was not significantly different between control and IRΔMCH mice under basal-clamp conditions (Figure 3C). However, in response to insulin, IRΔMCH mice on a HFD suppressed hepatic glucose production more efficiently than control mice (Figure 3C). On the other hand, determination of tissue-specific glucose uptake into brain, white adipose tissue, and skeletal muscle showed similar rates of insulin-stimulated glucose uptake in control and IRΔMCH mice (Figure 3D). Taken together, IR inactivation in MCH cells resulted in increased insulin sensitivity and improved insulin’s ability to suppress hepatic glucose production under HFD conditions. As abrogation of IR signaling in MCH LH neurons improves insulin sensitivity, hepatic glucose metabolism and locomotor activity under HFD conditions, we asked whether insulin-dependent signaling might be overactivated in the LH under conditions of obesity. To this end, we analyzed PI3K signaling in the LH in control MCH-LacZ mice on a HFD. Notably, control mice exposed to a HFD showed a clearly higher immunoreactive PIP3, even after an overnight fast when compared to lean control mice (Figures 3E and 3F). Whereas, in lean mice, only ∼15% of MCH neurons showed high PIP3 immunoreactivity (Figure 2A), this proportion was increased to ∼34% in saline-treated control mice exposed to a HFD (Figures 3E and 3F). This increase in MCH cells showing high PIP3 accumulation was significantly attenuated in IRΔMCH mice (∼17%; Figures 3E and 3F), indicating that a HFD significantly increased PI3K activity in MCH LH neurons and that this activation to substantial proportion depends on IR signaling in these cells. To further investigate whether insulin-dependent activation of MCH neurons in obesity is causally linked to the obesity-associated alterations, we decided to investigate the effect of chemogenetically activating these neurons. To this end, we crossed mice that allow for Cre-dependent expression of stimulatory DREADD receptors (hM3DGq) from the ROSA26 locus (Steculorum et al., 2016Steculorum S.M. Ruud J. Karakasilioti I. Backes H. Engström Ruud L. Timper K. Hess M.E. Tsaousidou E. Mauer J. Vogt M.C. et al.AgRP neurons control systemic insulin sensitivity via myostatin expression in brown adipose tissue.Cell. 2016; 165: 125-138Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) with MCH-Cre mice (hM3DGqMCH-Cre). In hM3DGqMCH-Cre mice carrying fluorescently labeled MCH neurons, ex vivo electrophysiological recordings revealed that identified MCH neurons exhibited rapid membrane depolarization and a strong increase in AP firing frequency upon bath incubation with clozapine-N-oxide (CNO) (Figures 4A and 4B ), indicating successful expression of functional, activating hM3D receptors in MCH neurons. While CNO injection in the home cage environment had no effect on food intake in hM3DGqMCH-Cre mice and their littermate controls, CNO-dependent excitation of MCH neurons resulted in significantly reduced locomotor behavior during the light phase of the cycle, indicating that these neurons promote inactivity, but not feeding, when acutely chemogenetically activated (Figures 4C and 4D). Having established a suitable model for inducible stimulation of MCH neuron activity, we next assessed the potential effect of acute activation of MCH neurons on systemic glucose metabolism and insulin sensitivity during glucose tolerance or insulin tolerance tests. However, acutely activating MCH neurons had no effect on either glucose tolerance or insulin sensitivity (Figures 4E and 4F). Many studies over the past decades have established the LH as an important center in the regulation of feeding, energy homeostasis, sleep-wake cycle, and reward-related processes (Brown et al., 2015Brown J.A. Woodworth H.L. Leinninger G.M. To ingest or rest? Specialized roles of lateral hypothalamic area neurons in coordinating energy balance.Front. Syst. Neurosci. 2015; 9: 9Crossref PubMed Scopus (74) Google Scholar, Burdakov et al., 2013Burdakov D. Karnani M.M. Gonzalez A. Lateral hypothalamus as a sensor-regulator in respiratory and metabolic control.Physiol. Behav. 2013; 121: 117-124Crossref PubMed Scopus (67) Google Scholar). However, the signals promoting LH MCH neuron activity in obesity remained ill defined. From a mechanistic point of view, we demonstrate that insulin signaling in MCH-expressing cells activates PI3K signaling and that insulin’s ability to activate the PI3K pathway was blunted specifically in LH MCH neurons of IRΔMCH mice, indicating that insulin can stimulate PI3K in these cells in a cell-autonomous manner independent of synaptic transmission. Moreover, insulin regulates neuronal excitability of a subpopulation of MCH neurons. A potential mediator of insulin’s effects on firing frequency is, indeed, the PI3K pathway. Insulin in a PI3K-dependent activation of KATP channels can lead to cell-autonomous hyperpolarization of neurons (Könner et al., 2007Könner A.C. Janoschek R. Plum L. Jordan S.D. Rother E. Ma X. Xu C. Enriori P. Hampel B. Barsh G.S. et al.Insulin action in AgRP-expressing neurons is required for suppression of hepatic glucose production.Cell Metab. 2007; 5: 438-449Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar, Spanswick et al., 2000Spanswick D. Smith M.A. Mirshamsi S. Routh V.H. Ashford M.L. Insulin activates ATP-sensitive K+ channels in hypothalamic neurons of lean, but not obese rats.Nat. Neurosci. 2000; 3: 757-758Crossref PubMed Scopus (433) Google Scholar). Since insulin leads to an increased excitability in MCH-expressing neurons, it is unlikely that KATP channels are the downstream target of PI3K signaling in MCH neurons. However, a previous report suggests that insulin can excite POMC neurons via canonical transient receptor potential channels (TRPCs; e.g., TRPC5) in a PI3K-dependent manner (Qiu et al., 2014Qiu J. Zhang C. Borgquist A. Nestor C.C. Smith A.W. Bosch M.A. Ku S. Wagner E.J. Rønnekleiv O.K. Kelly M.J. Insulin excites anorexigenic proopiomelanocortin neurons via activation of canonical transient receptor potential channels.Cell Metab. 2014; 19: 682-693Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Clearly, future studies will have to further address the detailed intracellular mechanism(s) through which insulin controls MCH neuron excitability. Kong and colleagues could elegantly demonstrate that glucose excitation of MCH-expressing neurons in the LH is mediated by KATP channels and is negatively regulated by the mitochondrial protein UCP2 and that glucose sensing by MCH neurons plays an important role in regulating glucose homeostasis (Kong et al., 2010Kong D. Vong L. Parton L.E. Ye C. Tong Q. Hu X. Choi B. Brüning J.C. Lowell B.B. Glucose stimulation of hypothalamic MCH neurons involves K(ATP) channels, is modulated by UCP2, and regulates peripheral glucose homeostasis.Cell Metab. 2010; 12: 545-552Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). However, this study left open the question of how MCH neurons affect blood glucose levels. The results of the present study clearly demonstrate that insulin signaling in MCH neurons affects blood glucose levels by controlling hepatic glucose production and insulin sensitivity. On the other hand, insulin-stimulated glucose uptake rates into muscle and/or fat were unaltered in these mice. However, while chronically altering insulin action in MCH neurons affects glucose homeostasis, acute chemogenetic activation of these cells failed to affect either insulin or glucose tolerance. These findings indicate that MCH-neuron-dependent regulation of locomotor activity and systemic insulin sensitivity clearly engage alternative mechanisms that remain to be specified. On the other hand, chronic chemogenetic activation of MCH neurons might influence insulin sensitivity. Alternatively, the subpopulation of insulin-responsive neurons may have opposing functions to the non-insulin-responsive MCH neurons in control of glucose homeostasis; thus, global chemogenetic activation of all MCH neurons may override the acute glucose regulatory function of insulin-responsive MCH cells. Thus, the nature of this potential MCH-neuron heterogeneity will have to be investigated in future studies. Under normal chow diet conditions, insulin-dependent effects on MCH-expressing cells in the LH seem not to occur, because IRΔMCH mice are phenotypically indistinguishable from control mice under these conditions. Only when insulin levels rise, as upon high-fat feeding, does insulin action seem to reach a threshold for strong PI3K activation and subsequent activation of the insulin signaling pathways. This is directly supported by the finding of the present study that HFD feeding results in increased PIP3 formation in MCH-expressing cells in the LH and that this response was abrogated in IRΔMCH mice. These findings are in line for what we previously reported in VMH neurons upon HFD feeding (Klöckener et al., 2011Klöckener T. Hess S. Belgardt B.F. Paeger L. Verhagen L.A. Husch A. Sohn J.W. Hampel B. Dhillon H. Zigman J.M. et al.High-fat feeding promotes obesity via insulin receptor/PI3K-dependent inhibition of SF-1 VMH neurons.Nat. Neurosci. 2011; 14: 911-918Crossref PubMed Scopus (183) Google Scholar). Indeed, studies have indicated that selective hormone resistance represents the rule rather than exception in the manifestation of insulin resistance associated with obesity (Könner and Brüning, 2012Könner A.C. Brüning J.C. Selective insulin and leptin resistance in metabolic disorders.Cell Metab. 2012; 16: 144-152Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Collectively, the present study establishes that deletion of the IR and subsequent inactivation of insulin-stimulated signaling partially improve diet-induced impairment of locomotor activity and peripheral glucose metabolism and insulin sensitivity. IRΔMCH mice show enhanced locomotor activity in the absence of detectable changes in food intake or energy expenditure. However, the sensitivity to detect changes in energy expenditure via indirect calorimetry may be too limited to observe the expected increase resulting from the increased locomotor activity detected in our model. Mice lacking the IR in MCH-expressing cells suppress hepatic glucose production more efficiently than control animals on a HFD. Moreover, IRΔMCH mice on a HFD exhibit an increase in locomotor activity during both the day and night phases, and chemogenetic activation of MCH-expressing neurons decreases the activity in lean mice, which is in line with previous findings indicating that MCH receptor signaling is involved in the regulation of locomotor behavior (Brown et al., 2015Brown J.A. Woodworth H.L. Leinninger G.M. To ingest or rest? Specialized roles of lateral hypothalamic area neurons in coordinating energy balance.Front. Syst. Neurosci. 2015; 9: 9Crossref PubMed Scopus (74) Google Scholar, Burdakov et al., 2013Burdakov D. Karnani M.M. Gonzalez A. Lateral hypothalamus as a sensor-regulator in respiratory and metabolic control.Physiol. Behav. 2013; 121: 117-124Crossref PubMed Scopus (67) Google Scholar). Further detailed studies elucidating a downstream neuronal subpopulation (or subpopulations) and cellular mechanisms responsible for the observed effects may thus define potential targets for the treatment of obesity and T2DM. All animal procedures were conducted in compliance with protocols approved by local government authorities (Bezirksregierung Köln) and were in accordance with NIH guidelines. Mice were housed at 22°C–24°C using a 12-hr/12-hr light/dark cycle. Animals had ad libitum access to water at all times, and food was only withdrawn if required for an experiment. All experiments have been performed in adult mice. Food intake and indirect calorimetry measurements were made in a PhenoMaster System (TSE Systems), as previously described (Jordan et al., 2011Jordan S.D. Krüger M. Willmes D.M. Redemann N. Wunderlich F.T. Brönneke H.S. Merkwirth C. Kashkar H. Olkkonen V.M. Böttger T. et al.Obesity-induced overexpression of miRNA-143 inhibits insulin-stimulated AKT activation and impairs glucose metabolism.Nat. Cell Biol. 2011; 13: 434-446Crossref PubMed Scopus (407) Google Scholar). For insulin signaling, overnight-fasted mice were injected with 0.75 U/kg (of body weight) of human regular insulin or insulin diluent, diluted in 0.9% saline, into the peritoneal cavity. Mice were sacrificed 15 min after injection, samples of liver and skeletal muscle were collected, and proteins were extracted from tissues for western blot analysis. All values are expressed as the mean ± SEM, unless indicated otherwise in the figure legends. Statistical analyses were conducted using GraphPad PRISM (version 6.0h). Datasets with only two independent groups were analyzed for statistical significance using an unpaired two-tailed Student’s t test, unless indicated otherwise in the figure legends. Datasets with more than two groups were analyzed using a one-way ANOVA followed by Tukey’s post hoc test. Datasets subjected to two independent factors were analyzed using a two-way ANOVA followed by Holm-Sidak’s post hoc test. All p values ≤ 0.05 were considered significant (∗p ≤ 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001). A.C.H. and J.C.B. conceived the project, designed the experiments, analyzed the data, and wrote the manuscript with input from the other authors. A.C.H. performed all the experiments apart from experiments with hM3DGqMCH-Cre mice (J.R. and H.J.) and electrophysiological recordings (H.V., S.H., and P.K.). We acknowledge Nadine Evers, Jens Alber, Brigitte Hampel, Pia Scholl, Sigrid Irlenbusch, and Kerstin Marohl for outstanding technical assistance. We thank Brad B. Lowell for providing the MCH-Cre mice used in this study. This work was supported by a grant from the DFG (BR 1492/7-1) to J.C.B., and we received funding by the DFG within the framework of the TRR 134 and within the Excellence Initiative by German Federal and State Governments (CECAD). This work was funded (in part) by the Helmholtz Alliance (Imaging and Curing Environmental Metabolic Diseases [ICEMED, HA-314]) through the Initiative and Networking Fund of the Helmholtz Association. Moreover, the research leading to these results has received funding from the European Union Seventh Framework Program (FP7/2007-2013) under grant agreement 266408. J.R. was supported by the Swedish Medical Research Council (2013-530). Download .pdf (4.55 MB) Help with pdf files Document S1. Supplemental Experimental Procedures and Figures S1 and S2" @default.
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