Reciprocal activity of AgRP and POMC neurons governs coordinated control of feeding and metabolism.

Agouti-related peptide (AgRP)-expressing and proopiomelanocortin (POMC)-expressing neurons reciprocally regulate food intake. Here, we combine non-interacting recombinases to simultaneously express functionally opposing chemogenetic receptors in AgRP and POMC neurons for comparing metabolic responses in male and female mice with simultaneous activation of AgRP and inhibition of POMC neurons with isolated activation of AgRP neurons or isolated inhibition of POMC neurons. We show that food intake is regulated by the additive effect of AgRP neuron activation and POMC neuron inhibition, while systemic insulin sensitivity and gluconeogenesis are differentially modulated by isolated-versus-simultaneous regulation of AgRP and POMC neurons. We identify a neurocircuit engaging Npy1R-expressing neurons in the paraventricular nucleus of the hypothalamus, where activated AgRP neurons and inhibited POMC neurons cooperate to promote food consumption and activate Th+ neurons in the nucleus tractus solitarii. Collectively, these results unveil how food intake is precisely regulated by the simultaneous bidirectional interplay between AgRP and POMC neurocircuits.

Dopamine-inhibited POMCDrd2+ neurons in the ARC acutely regulate feeding and body temperature.

Dopamine acts on neurons in the arcuate nucleus (ARC) of the hypothalamus, which controls homeostatic feeding responses. Here we demonstrate a differential enrichment of dopamine receptor 1 (Drd1) expression in food intake-promoting agouti related peptide (AgRP)/neuropeptide Y (NPY) neurons and a large proportion of Drd2-expressing anorexigenic proopiomelanocortin (POMC) neurons. Owing to the nature of these receptors, this translates into a predominant activation of AgRP/NPY neurons upon dopamine stimulation and a larger proportion of dopamine-inhibited POMC neurons. Employing intersectional targeting of Drd2-expressing POMC neurons, we reveal that dopamine-mediated POMC neuron inhibition is Drd2 dependent and that POMCDrd2+ neurons exhibit differential expression of neuropeptide signaling mediators compared with the global POMC neuron population, which manifests in enhanced somatostatin responsiveness of POMCDrd2+ neurons. Selective chemogenetic activation of POMCDrd2+ neurons uncovered their ability to acutely suppress feeding and to preserve body temperature in fasted mice. Collectively, the present study provides the molecular and functional characterization of POMCDrd2+ neurons and aids our understanding of dopamine-dependent control of homeostatic energy-regulatory neurocircuits.

HypoMap-a unified single-cell gene expression atlas of the murine hypothalamus.

The hypothalamus plays a key role in coordinating fundamental body functions. Despite recent progress in single-cell technologies, a unified catalog and molecular characterization of the heterogeneous cell types and, specifically, neuronal subtypes in this brain region are still lacking. Here, we present an integrated reference atlas, 'HypoMap,' of the murine hypothalamus, consisting of 384,925 cells, with the ability to incorporate new additional experiments. We validate HypoMap by comparing data collected from Smart-Seq+Fluidigm C1 and bulk RNA sequencing of selected neuronal cell types with different degrees of cellular heterogeneity. Finally, via HypoMap, we identify classes of neurons expressing glucagon-like peptide-1 receptor (Glp1r) and prepronociceptin (Pnoc), and validate them using single-molecule in situ hybridization. Collectively, HypoMap provides a unified framework for the systematic functional annotation of murine hypothalamic cell types, and it can serve as an important platform to unravel the functional organization of hypothalamic neurocircuits and to identify druggable targets for treating metabolic disorders.

NPY mediates the rapid feeding and glucose metabolism regulatory functions of AgRP neurons.

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.

New roles for dopamine D2 and D3 receptors in pancreatic beta cell insulin secretion.

Although long-studied in the central nervous system, there is increasing evidence that dopamine (DA) has important roles in the periphery including in metabolic regulation. Insulin-secreting pancreatic ß-cells express the machinery for DA synthesis and catabolism, as well as all five DA receptors. In these cells, DA functions as a negative regulator of glucose-stimulated insulin secretion (GSIS), which is mediated by DA D2-like receptors including D2 (D2R) and D3 (D3R) receptors. However, the fundamental mechanisms of DA synthesis, storage, release, and signaling in pancreatic ß-cells and their functional relevance in vivo remain poorly understood. Here, we assessed the roles of the DA precursor L-DOPA in ß-cell DA synthesis and release in conjunction with the signaling mechanisms underlying DA's inhibition of GSIS. Our results show that the uptake of L-DOPA is essential for establishing intracellular DA stores in ß-cells. Glucose stimulation significantly enhances L-DOPA uptake, leading to increased DA release and GSIS reduction in an autocrine/paracrine manner. Furthermore, D2R and D3R act in combination to mediate dopaminergic inhibition of GSIS. Transgenic knockout mice in which ß-cell D2R or D3R expression is eliminated exhibit diminished DA secretion during glucose stimulation, suggesting a new mechanism where D2-like receptors modify DA release to modulate GSIS. Lastly, ß-cell-selective D2R knockout mice exhibit marked postprandial hyperinsulinemia in vivo. These results reveal that peripheral D2R and D3R receptors play important roles in metabolism through their inhibitory effects on GSIS. This opens the possibility that blockade of peripheral D2-like receptors by drugs including antipsychotic medications may significantly contribute to the metabolic disturbances observed clinically.

Effects of Moderate Chronic Food Restriction on the Development of Postprandial Dyslipidemia with Ageing.

Ageing is a major risk factor for the development of metabolic disorders linked to dyslipidemia, usually accompanied by increased adiposity. The goal of this work was to investigate whether avoiding an excessive increase in adiposity with ageing, via moderate chronic food restriction (FR), ameliorates postprandial dyslipidemia in a rat model of metabolic syndrome associated with ageing. Accordingly, we performed an oral lipid loading test (OLLT) in mature middle-aged (7 months) and middle-old-aged (24 months) Wistar rats fed ad libitum (AL) or under moderate FR for 3 months. Briefly, overnight fasted rats were orally administered a bolus of extra-virgin olive oil (1 mL/Kg of body weight) and blood samples were taken from the tail vein before fat load (t = 0) and 30, 60, 90, 120, 180, and 240 min after fat administration. Changes in serum lipids, glucose, insulin, and glucagon levels were measured at different time-points. Expression of liver and adipose tissue metabolic genes were also determined before (t = 0) and after the fat load (t = 240 min). Postprandial dyslipidemia progressively increased with ageing and this could be associated with hepatic ChREBP activity. Interestingly, moderate chronic FR reduced adiposity and avoided excessive postprandial hypertriglyceridemia in 7- and 24-month-old Wistar rats, strengthening the association between postprandial triglyceride levels and adiposity. The 24-month-old rats needed more insulin to maintain postprandial normoglycemia; nevertheless, hyperglycemia occurred at 240 min after fat administration. FR did not alter the fasted serum glucose levels but it markedly decreased glucagon excursion during the OLLT and the postprandial rise of glycemia in the 24-month-old rats, and FGF21 in the 7-month-old Wistar rats. Hence, our results pointed to an important role of FR in postprandial energy metabolism and insulin resistance in ageing. Lastly, our data support the idea that the vWAT might function as an ectopic site for fat deposition in 7-month-old and in 24-month-old Wistar rats that could increase their browning capacity in response to an acute fat load.

Ciliary gene RPGRIP1L is required for hypothalamic arcuate neuron development.

Intronic polymorphisms in the α-ketoglutarate-dependent dioxygenase gene (FTO) that are highly associated with increased body weight have been implicated in the transcriptional control of a nearby ciliary gene, retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L). Previous studies have shown that congenital Rpgrip1l hypomorphism in murine proopiomelanocortin (Pomc) neurons causes obesity by increasing food intake. Here, we show by congenital and adult-onset Rpgrip1l deletion in Pomc-expressing neurons that the hyperphagia and obesity are likely due to neurodevelopmental effects that are characterized by a reduction in the Pomc/Neuropeptide Y (Npy) neuronal number ratio and marked increases in arcuate hypothalamic-paraventricular hypothalamic (ARH-PVH) axonal projections. Biallelic RPGRIP1L mutations result in fewer cilia-positive human induced pluripotent stem cell-derived (iPSC-derived) neurons and blunted responses to Sonic Hedgehog (SHH). Isogenic human ARH-like embryonic stem cell-derived (ESc-derived) neurons homozygous for the obesity-risk alleles at rs8050136 or rs1421085 have decreased RPGRIP1L expression and have lower numbers of POMC neurons. RPGRIP1L overexpression increases POMC cell number. These findings suggest that apparently functional intronic polymorphisms affect hypothalamic RPGRIP1L expression and impact development of POMC neurons and their derivatives, leading to hyperphagia and increased adiposity.

Developmental switch of leptin signaling in arcuate nucleus neurons.

Leptin is well known for its role in the regulation of energy homeostasis in adults, a mechanism that at least partially results from the inhibition of the activity of NPY/AgRP/GABA neurons (NAG) in the arcuate nucleus of the hypothalamus (ARH). During early postnatal development in the rodent, leptin promotes axonal outgrowth from ARH neurons, and preautonomic NAG neurons are particularly responsive to leptin's trophic effects. To begin to understand how leptin could simultaneously promote axonal outgrowth from and inhibit the activity of NAG neurons, we characterized the electrochemical effects of leptin on NAG neurons in mice during early development. Here, we show that NAG neurons do indeed express a functional leptin receptor throughout the early postnatal period in the mouse; however, at postnatal days 13-15, leptin causes membrane depolarization in NAG neurons, rather than the expected hyperpolarization. Leptin action on NAG neurons transitions from stimulatory to inhibitory in the periweaning period, in parallel with the acquisition of functional ATP-sensitive potassium channels. These findings are consistent with the idea that leptin provides an orexigenic drive through the NAG system to help rapidly growing pups meet their energy requirements.

Impairment of skeletal muscle insulin action with aging in Wistar rats: role of leptin and caloric restriction.

Insulin resistance develops with aging in rats in parallel to fat mass accretion, central leptin resistance and hyperleptinemia. Previous studies demonstrated that insulin resistance appears earlier in adipose tissue than in muscle during aging and pointed to a role of hyperleptinemia in the impairment of insulin action. Here we explored the evolution along aging of insulin sensitivity in soleus and EDL muscles by analyzing insulin signaling in vivo and insulin-dependent glucose transport ex vivo. A decrease in insulin action was observed in both muscles. Caloric restriction improves insulin sensitivity at early aging but not in older animals. We also tested the role of leptin on insulin action in skeletal muscle. Short-term pretreatment with leptin inhibits in vivo muscle insulin signaling and insulin-dependent glucose transport in isolated muscle strips. This effect is mediated by its action on early insulin signaling as well as by the inhibition of p38. In contrast, chronic central administration of leptin elicits an insulin sensitizing effect on soleus. These data suggest that leptin can act as muscle insulin sensitizer, when acting at central level, and as insulin antagonistic when interacting directly with soleus muscle. This effect may be relevant in situations of hyperleptinemia such as aging.

Age-associated decrease of SIRT1 expression in rat hippocampus: prevention by late onset caloric restriction.

We have studied the effect of aging and late onset caloric restriction (CR) on the expression of SIRT1 in hippocampus and cerebral cortex of the rat. Quantitative analysis showed that there is a significant reduction of SIRT1 protein levels in hippocampus with aging. Late onset, moderate CR prevented the deleterious effect of aging on SIRT1 content. Examination of SIRT1 immunoreactivity in coronal sections from hippocampus supported these results, and confirmed that old animals are able to respond to the beneficial effects of CR by regulating SIRT1 protein expression. Differences in the amounts of SIRT1 transcripts among animal groups were not found, which suggest that post-transcriptional mechanisms could be involved in the effects of aging and CR on SIRT1 expression.

Development of insulin resistance during aging: involvement of central processes and role of adipokines.

Aging in mammals associates with the development of peripheral insulin resistance. Additionally, adiposity usually increases with aging and this could play a relevant role in the gradual impairment of insulin action. In fact, fat accretion leads to changes in the expression and circulating concentrations of factors originated in adipose tissue like leptin, resistin and inflammatory cytokines which have been shown to modulate insulin signaling in insulin target tissues acting both, directly or through the central nervous system. Even insulin action on peripheral target tissues has been recently demonstrated to be partially mediated by its central action, suggesting that a decrease in central insulin action could be involved in the development of peripheral insulin resistance. In the present review we analyze the available research data on aging-associated insulin resistance making emphasis in the following aspects: 1) The time-course of development of overall insulin resistance and the evolution of changes in circulating adipokines; 2) The effect of caloric restriction and the decrease of adiposity in insulin action; 3) The influence of changes in the central action of factors like leptin or insulin in the development and maintenance of insulin resistance during aging.

Alkalinization potentiates vascular calcium deposition in an uremic milieu.

BACKGROUND: Vascular calcification is a serious complication of chronic kidney disease. Acid-base balance is a relevant, albeit somewhat forgotten factor in the regulation of calcium deposition. Hemodialysis patients undergo repeated episodes of alkaline loading from the dialysate, resulting in prolonged alkalinization. We have hypothesized that extracellular alkalinization may promote vascular calcification. METHODS: Primary cultures of vascular smooth muscle cells were induced to calcify by the phosphate donor beta-glycerophosphate, in the presence of normal or uremic sera from hemodialysis patients and at different pH conditions. The influence of sodium bicarbonate supplementation for 2 months on aorta calcification was studied in 5/6 nephrectomized uremic rats. RESULTS: Uremic serum increased vascular smooth muscle cell calcification (twofold over nonuremic human serum at day 12, p<0.001). Alkalinization of the extracellular medium also increased vascular smooth muscle cell calcification. Increasing the extracellular pH from 7.42 to 7.53 resulted in a 2.5-fold increase in calcium accumulation at day 12 (p<0.05). In vivo, arterial calcification was significantly higher in alkalinized uremic animals (aorta calcification index, uremic + sodium bicarbonate, 164 +/- 57 units, vs. uremic + vehicle, 56 +/- 14 units; p<0.01). CONCLUSIONS: Alkalinization increases vascular calcification in cultured cells and uremic rats. These data may be used to optimize dialysate composition and the degree of alkalinization in calcification-prone individuals with advanced renal disease.

Effects of chronic acarbose treatment on adipocyte insulin responsiveness, serum levels of leptin and adiponectin and hypothalamic NPY expression in obese diabetic Wistar rats.

1. Inhibitors of intestinal glucosidases have been shown to improve glycaemic control in diabetic and obese humans and animals. In the present study, we have investigated the effect of 3 months treatment with acarbose on adiposity, food intake and the modulation of hypothalamic neuropeptide Y (NPY) in obese diabetic Wistar (WDF) rats and the possible correlation between changes in overall insulin sensitivity and the level of circulating adipokines, leptin and adiponectin. In addition, we investigated the effect of acarbose on adipocyte insulin signalling. 2. Mature male WDF rats were randomly distributed to one of three treatment groups (no acarbose or 20 or 40 mg of acarbose/100 g diet). After 3 months, blood glucose, cholesterol, triglyceride, insulin, leptin and adiponectin were analysed. Insulin signalling was determined in isolated adipocytes as the stimulation of mitogen-activated protein kinase (MAPK) and Akt phosphorylation; the level of hypothalamic NPY was assessed by immunohistochemistry. 3. Acarbose-treated rats had lower levels of blood glucose, cholesterol, triglyceride, insulin and leptin and an increase in adiponectin compared with untreated animals. There were no changes in bodyweight and adiposity. Stimulation of adipocyte MAPK activity by insulin was higher in rats treated with both doses of acarbose, whereas higher stimulation of Akt phosphorylation was observed with the highest dose of acarbose. Although food intake was not significantly reduced in rats treated with acarbose, the acarbose-treated rats had lower NPY expression in the arcuate nucleus. 4. We conclude that the improvement in overall insulin sensitivity in WDF rats after prolonged acarbose treatment is paralleled by increases in circulating adiponectin and adipocyte insulin responsiveness. Acarbose neither decreases food intake nor reverts obesity, but decreases leptin levels and the expression of the orexigenic NPY in the hypothalamus.

Impaired central insulin response in aged Wistar rats: role of adiposity.

Insulin, like leptin, is considered as a lipostatic signal acting at a central level. Aging and age-associated adiposity have been related to the development of leptin resistance in Wistar rats. In the present article, hypothalamic insulin response during aging has been studied in Wistar rats. Thus, the effects of intracerebroventricular infusion of insulin during a week on food intake and body weight as well as insulin signal transduction after acute intracerebroventricular insulin administration have been studied in 3-, 8-, and 24-month-old rats. To explore the possible role of age-associated adiposity, these experiments were also performed in 8- and 24-month-old rats after 3 months of food restriction to reduce visceral adiposity index to values below those of young animals. Intracerebroventricular administration of insulin during a week was more efficient at reducing food intake and body weight in 3-month-old rats than in 8- and 24-month-old rats. Hypothalamic insulin-stimulated insulin receptor, GSK3, AKT, and p70S6K phosphorylation decreased with aging. Insulin receptor and IRS-2 phosphoserine was increased in 24-month-old rats. Food restriction improved both insulin responsiveness and insulin signaling. These data suggest that Wistar rats develop hypothalamic insulin resistance with aging. This can be explained by alterations of the signal transduction pathway. The fact that food restriction improves central insulin response and signal transduction points to the age-associated adiposity as a key player in the development of central insulin resistance.

Mechanisms of endothelial cell protection by blockade of the JAK2 pathway.

Inhibition of the JAK2/STAT pathway has been implicated recently in cytoprotective mechanisms in both vascular smooth muscle cells and astrocytes. The advent of JAK2-specific inhibitors provides a practical tool for the study of this pathway in different cellular types. An interest in finding methods to improve endothelial cell (EC) resistance to injury led us to examine the effect of JAK2/STAT inhibition on EC protection. Furthermore, the signaling pathways involved in JAK2/STAT inhibition-related actions were examined. Our results reveal, for the first time, that blockade of JAK2 with the tyrosine kinase inhibitor AG490 strongly protects cultured EC against cell detachment-dependent death and serum deprivation and increases reseeding efficiency. Confirmation of the specificity of the effects of JAK2 inhibition was attained by finding protective effects on transfection with a dominant negative JAK2. Furthermore, AG490 blocked serum deprivation-induced phosphorylation of JAK2. In terms of mechanism, treatment with AG490 induces several relevant responses, both in monolayer and detached cells. These mechanisms include the following: 1) Increase and nuclear translocation of the active, dephosphorylated form of beta-catenin. In functional terms, this translocation is transcriptionally active, and its protective effect is further supported by the stimulation of EC cytoprotection by transfectionally induced excess of beta-catenin. 2) Increase of platelet endothelial cell adhesion molecule (PECAM)/CD31 levels. 3) Increase in total and phosphorylated AKT. 4) Increase in phosphorylated glycogen synthase kinase (GSK)3alpha/beta. The present findings imply potential practical applications of JAK2 inhibition on EC. These applications affect not only EC in the monolayer but also circulating detached cells and involve mechanistic interactions not previously described.

[Response to hypoxia. A systemic mechanism based on the control of gene expression]. / Respuesta a la hipoxia. Un mecanismo sistemico basado en el control de la expresion genica.

New, critically important data have been recently generated about the response to hypoxia. This response can be schematized in three main systems or functions, ie, detectional or oxygen sensing, regulatory, which controls gene expression and effector. The principal organizer of the regulatory branch is a specific transcription factor, the hypoxia-inducible factor 1 (HIF-1). In the presence of oxygen, the alpha subunit of HIF-1 (HIF-1alpha) is modified by hydroxylases, that represent the central point of the oxygen sensing mechanism. This type of hydroxylation induces HIF-1alpha catabolism by the proteosome. On the contrary, in hypoxia, or in the presence of certain growth factors that increase HIF-1alpha synthesis, HIF-1alpha translocates to the nucleus, where it binds HIF-1beta, and thence acts on transcription of genes carrying hypoxia responsive elements (HRE) on their promoters. These genes regulate the synthesis of an ample series of proteins, which span from respiratory enzymes and transporters to hormones regulating circulation and erythropoiesis. The role of HIF-1alpha is not restricted to the mere induction of adaptation to decreased oxygen: instead, it significantly participates in cell repairing mechanisms. A simple list of some of the stimulatory or inhibitory alterations of pathophysiological importance involving the HIF-1 system, would include: chronic lung disease, smoking adaptation, anemia/hemorrhage, ischemia/reperfusion, growth, vascularization and cell resistance of tumors, preeclampsia and intrauterine growth retardation, retinal hyper o hypovascularization, drug intoxications, bowel inflammatory disease and wound repair. This list illustrates by itself the importance of the mechanism herein reviewed.