Single cell tracing of Pomc neurons reveals recruitment of 'Ghost' subtypes with atypical identity in a mouse model of obesity.

The hypothalamus contains a remarkable diversity of neurons that orchestrate behavioural and metabolic outputs in a highly plastic manner. Neuronal diversity is key to enabling hypothalamic functions and, according to the neuroscience dogma, it is predetermined during embryonic life. Here, by combining lineage tracing of hypothalamic pro-opiomelanocortin (Pomc) neurons with single-cell profiling approaches in adult male mice, we uncovered subpopulations of 'Ghost' neurons endowed with atypical molecular and functional identity. Compared to 'classical' Pomc neurons, Ghost neurons exhibit negligible Pomc expression and are 'invisible' to available neuroanatomical approaches and promoter-based reporter mice for studying Pomc biology. Ghost neuron numbers augment in diet-induced obese mice, independent of neurogenesis or cell death, but weight loss can reverse this shift. Our work challenges the notion of fixed, developmentally programmed neuronal identities in the mature hypothalamus and highlight the ability of specialised neurons to reversibly adapt their functional identity to adult-onset obesogenic stimuli.

mTORC1 in energy expenditure: consequences for obesity.

In eukaryotic cells, the mammalian target of rapamycin complex 1 (sometimes referred to as the mechanistic target of rapamycin complex 1; mTORC1) orchestrates cellular metabolism in response to environmental energy availability. As a result, at the organismal level, mTORC1 signalling regulates the intake, storage and use of energy by acting as a hub for the actions of nutrients and hormones, such as leptin and insulin, in different cell types. It is therefore unsurprising that deregulated mTORC1 signalling is associated with obesity. Strategies that increase energy expenditure offer therapeutic promise for the treatment of obesity. Here we review current evidence illustrating the critical role of mTORC1 signalling in the regulation of energy expenditure and adaptive thermogenesis through its various effects in neuronal circuits, adipose tissue and skeletal muscle. Understanding how mTORC1 signalling in one organ and cell type affects responses in other organs and cell types could be key to developing better, safer treatments targeting this pathway in obesity.

Poly-Agonist Pharmacotherapies for Metabolic Diseases: Hopes and New Challenges.

The use of glucagon-like peptide-1 (GLP-1) receptor-based multi-agonists in the treatment of type 2 diabetes and obesity holds great promise for improving glycaemic control and weight management. Unimolecular dual and triple agonists targeting multiple gut hormone-related pathways are currently in clinical trials, with recent evidence supporting their efficacy. However, significant knowledge gaps remain regarding the biological mechanisms and potential adverse effects associated with these multi-target agents. The mechanisms underlying the therapeutic efficacy of GLP-1 receptor-based multi-agonists remain somewhat mysterious, and hidden threats may be associated with the use of gut hormone-based polyagonists. In this review, we provide a critical analysis of the benefits and risks associated with the use of these new drugs in the management of obesity and diabetes, while also exploring new potential applications of GLP-1-based pharmacology beyond the field of metabolic disease.

Hypothalamic Glucose Hypersensitivity-Induced Insulin Secretion in the Obese Zücker Rat Is Reversed by Central Ghrelin Treatment.

Aims: Part of hypothalamic (mediobasal hypothalamus [MBH]) neurons detect changes in blood glucose levels that in turn coordinate the vagal control of insulin secretion. This control cascade requires the production of mitochondrial reactive oxygen species (mROS), which is altered in models of obesity and insulin resistance. Obese, insulin-resistant Zücker rats are characterized by hypothalamic hypersensitivity to glucose. This initiates an abnormal vagus-induced insulin secretion, associated with an overproduction of mROS in response to a low glucose dose. Here, we hypothesized that ghrelin, known to buffer reactive oxygen species (ROS) via mitochondrial function, may be a major component of the hypothalamic glucose hypersensitivity in the hypoghrelinemic obese Zücker rat. Results: Hypothalamic glucose hypersensitivity-induced insulin secretion of Zücker obese rats was reversed by ghrelin pretreatment. The overproduction of MBH mROS in response to a low glucose load no longer occurred in obese rats that had previously received the cerebral ghrelin infusion. This decrease in mROS production was accompanied by a normalization of oxidative phosphorylation (OXPHOS). Conversely, blocking the action of ghrelin with a growth hormone secretagogue receptor antagonist in a model of hyperghrelinemia (fasted rats) completely restored hypothalamic glucose sensing-induced insulin secretion that was almost absent in this physiological situation. Accordingly, ROS signaling and mitochondrial activity were increased by the ghrelin receptor antagonist. Innovation: These results demonstrate for the first time that ghrelin addressed only to the brain could have a protective effect on the defective control of insulin secretion in the insulin-resistant, hypoghrelinemic obese subject. Conclusions: Ghrelin, through its action on OXPHOS, modulates mROS signaling in response to cerebral hyperglycemia and the consequent vagal control of insulin secretion. In insulin-resistant obese states, brain hypoghrelinemia could be responsible for the nervous defect in insulin secretion.

Efficacy of glucagon-like peptide-1 and estrogen dual agonist in pancreatic islets protection and pre-clinical models of insulin-deficient diabetes.

We study the efficacy of a glucagon-like peptide-1 (GLP-1) and estrogen dual agonist (GLP1-E2) in pancreatic islet protection. GLP1-E2 provides superior protection from insulin-deficient diabetes induced by multiple low-dose streptozotocin (MLD-STZ-diabetes) and by the Akita mutation in mice than a GLP-1 monoagonist. GLP1-E2 does not protect from MLD-STZ-diabetes in estrogen receptor-α (ERα)-deficient mice and fails to prevent diabetes in Akita mice following GLP-1 receptor (GLP-1R) antagonism, demonstrating the requirement of GLP-1R and ERα for GLP1-E2 antidiabetic actions. In the MIN6 ß cell model, GLP1-E2 activates estrogen action following clathrin-dependent, GLP-1R-mediated internalization and lysosomal acidification. In cultured human islet, proteomic bioinformatic analysis reveals that GLP1-E2 amplifies the antiapoptotic pathways activated by monoagonists. However, in cultured mouse islets, GLP1-E2 provides antiapoptotic protection similar to monoagonists. Thus, GLP1-E2 promotes GLP-1 and E2 antiapoptotic signals in cultured islets, but in vivo, additional GLP1-E2 actions in non-islet cells expressing GLP-1R are instrumental to prevent diabetes.

Hypothalamic bile acid-TGR5 signaling protects from obesity.

Bile acids (BAs) improve metabolism and exert anti-obesity effects through the activation of the Takeda G protein-coupled receptor 5 (TGR5) in peripheral tissues. TGR5 is also found in the brain hypothalamus, but whether hypothalamic BA signaling is implicated in body weight control and obesity pathophysiology remains unknown. Here we show that hypothalamic BA content is reduced in diet-induced obese mice. Central administration of BAs or a specific TGR5 agonist in these animals decreases body weight and fat mass by activating the sympathetic nervous system, thereby promoting negative energy balance. Conversely, genetic downregulation of hypothalamic TGR5 expression in the mediobasal hypothalamus favors the development of obesity and worsens established obesity by blunting sympathetic activity. Lastly, hypothalamic TGR5 signaling is required for the anti-obesity action of dietary BA supplementation. Together, these findings identify hypothalamic TGR5 signaling as a key mediator of a top-down neural mechanism that counteracts diet-induced obesity.

CB1 and GLP-1 Receptors Cross Talk Provides New Therapies for Obesity.

Glucagon-like peptide 1 receptor (GLP-1R) agonists effectively improve glycemia and body weight in patients with type 2 diabetes and obesity but have limited weight-lowering efficacy and minimal insulin sensitizing action. In preclinical models, peripherally restricted cannabinoid receptor type 1 (CB1R) inhibitors, which are devoid of the neuropsychiatric adverse effects observed with brain-penetrant CB1R blockers, ameliorate obesity and its multiple metabolic complications. Using mouse models with genetic loss of CB1R or GLP-1R, we demonstrate that these two metabolic receptors modulate food intake and body weight via reciprocal functional interactions. In diet-induced obese mice, the coadministration of a peripheral CB1R inhibitor with long-acting GLP-1R agonists achieves greater reduction in body weight and fat mass than monotherapies by promoting negative energy balance. This cotreatment also results in larger improvements in systemic and hepatic insulin action, systemic dyslipidemia, and reduction of hepatic steatosis. Thus, peripheral CB1R blockade may allow safely potentiating the antiobesity and antidiabetic effects of currently available GLP-1R agonists.

Challenges of combining work and unpaid care, and solutions: A scoping review.

The number of people who combine work and unpaid care is increasing rapidly as more people need care, public and private care systems are progressively under pressure and more people are required to work for longer. Without adequate support, these working carers may experience detrimental effects on their well-being. To adequately support working carers, it is important to first understand the challenges they face. A scoping review was carried out, using Arksey and O'Malley's framework, to map the challenges of combining work and care and solutions described in the literature to address these challenges. The search included academic and grey literature between 2008 and 2018 and was conducted in April 2018, using electronic academic databases and reference list checks. Ninety-two publications were mapped, and the content analysed thematically. A conceptual framework was derived from the analysis which identified primary challenges (C1), directly resulting from combining work and care, primary solutions (S1) aiming to address these, secondary challenges (C2) resulting from solutions and secondary solutions (S2) aiming to address secondary challenges. Primary challenges were: (a) high and/or competing demands; (b) psychosocial/-emotional stressors; (c) distance; (d) carer's health; (e) returning to work; and (f) financial pressure. This framework serves to help those aiming to support working carers to better understand the challenges they face and those developing solutions for the challenges of combining work and care to consider potential consequences or barriers. Gaps in the literature have been identified and discussed.

Activation of hepatic estrogen receptor-α increases energy expenditure by stimulating the production of fibroblast growth factor 21 in female mice.

OBJECTIVE: The endogenous estrogen 17ß-estradiol (E2) promotes metabolic homeostasis in premenopausal women. In a mouse model of post-menopausal metabolic syndrome, we reported that estrogens increased energy expenditure, thus preventing estrogen deficiency-induced adiposity. Estrogens' prevention of fat accumulation was associated with increased serum concentrations of fibroblast growth factor 21 (FGF21), suggesting that FGF21 participates in estrogens' promotion of energy expenditure. METHODS: We studied the effect of E2 on FGF21 production and the role of FGF21 in E2 stimulation of energy expenditure and prevention of adiposity, using female estrogen receptor (ER)- and FGF21-deficient mice fed a normal chow and a cohort of ovariectomized women from the French E3N prospective cohort study. RESULTS: E2 acting on the hepatocyte ERα increases hepatic expression and production of FGF21 in female mice. In vivo activation of ERα increases the transcription of Fgf21 via an estrogen response element outside the promoter of Fgf21. Treatment with E2 increases oxygen consumption and energy expenditure and prevents whole body fat accumulation in ovariectomized female WT mice. The effect of E2 on energy expenditure is not observed in FGF21-deficient mice. While E2 treatment still prevents fat accumulation in FGF21-deficient mice, this effect is decreased compared to WT mice. In an observational cohort of ovariectomized women, E2 treatment was associated with lower serum FGF21 concentrations, which may reflect a healthier metabolic profile. CONCLUSIONS: In female mice, E2 action on the hepatocyte ERα increases Fgf21 transcription and FGF21 production, thus promoting energy expenditure and partially decreasing fat accumulation.

Loss of Nuclear and Membrane Estrogen Receptor-α Differentially Impairs Insulin Secretion and Action in Male and Female Mice.

Estrogens favor glucose homeostasis primarily through the estrogen receptor-α (ERα), but the respective importance of nuclear ERα (NOER) and membrane ERα (MOER) pools to glucose homeostasis are unknown. We studied glucose homeostasis, insulin secretion, and insulin sensitivity in male and female mice expressing either the NOER or the MOER. Male and female MOER mice exhibited fasting and fed hyperglycemia and glucose intolerance. Female MOER mice displayed impaired central insulin signaling associated with hyperinsulinemia and insulin resistance due to unrestrained hepatic gluconeogenesis, without alterations in glucose-stimulated insulin secretion (GSIS). In contrast, male MOER mice did not exhibit detectable insulin resistance, but showed impaired GSIS associated with reduced brain glucose sensing. Female NOER mice exhibited milder hepatic insulin resistance and glucose intolerance. In conclusion, nuclear ERα signaling is predominant in maintaining glucose homeostasis in mice of both sexes. Lack of nuclear ERα alters the central control of insulin sensitivity in females and predominantly impairs the central regulation of insulin secretion in males.

Mitochondrial Dynamin-Related Protein 1 (DRP1) translocation in response to cerebral glucose is impaired in a rat model of early alteration in hypothalamic glucose sensing.

OBJECTIVE: Hypothalamic glucose sensing (HGS) initiates insulin secretion (IS) via a vagal control, participating in energy homeostasis. This requires mitochondrial reactive oxygen species (mROS) signaling, dependent on mitochondrial fission, as shown by invalidation of the hypothalamic DRP1 protein. Here, our objectives were to determine whether a model with a HGS defect induced by a short, high fat-high sucrose (HFHS) diet in rats affected the fission machinery and mROS signaling within the mediobasal hypothalamus (MBH). METHODS: Rats fed a HFHS diet for 3 weeks were compared with animals fed a normal chow. Both in vitro (calcium imaging) and in vivo (vagal nerve activity recordings) experiments to measure the electrical activity of isolated MBH gluco-sensitive neurons in response to increased glucose level were performed. In parallel, insulin secretion to a direct glucose stimulus in isolated islets vs. insulin secretion resulting from brain glucose stimulation was evaluated. Intra-carotid glucose load-induced hypothalamic DRP1 translocation to mitochondria and mROS (H2O2) production were assessed in both groups. Finally, compound C was intracerebroventricularly injected to block the proposed AMPK-inhibited DRP1 translocation in the MBH to reverse the phenotype of HFHS fed animals. RESULTS: Rats fed a HFHS diet displayed a decreased HGS-induced IS. Responses of MBH neurons to glucose exhibited an alteration of their electrical activity, whereas glucose-induced insulin secretion in isolated islets was not affected. These MBH defects correlated with a decreased ROS signaling and glucose-induced translocation of the fission protein DRP1, as the vagal activity was altered. AMPK-induced inhibition of DRP1 translocation increased in this model, but its reversal through the injection of the compound C, an AMPK inhibitor, failed to restore HGS-induced IS. CONCLUSIONS: A hypothalamic alteration of DRP1-induced fission and mROS signaling in response to glucose was observed in HGS-induced IS of rats exposed to a 3 week HFHS diet. Early hypothalamic modifications of the neuronal activity could participate in a primary defect of the control of IS and ultimately, the development of diabetes.

Estrogens Promote Misfolded Proinsulin Degradation to Protect Insulin Production and Delay Diabetes.

Conjugated estrogens (CE) delay the onset of type 2 diabetes (T2D) in postmenopausal women, but the mechanism is unclear. In T2D, the endoplasmic reticulum (ER) fails to promote proinsulin folding and, in failing to do so, promotes ER stress and ß cell dysfunction. We show that CE prevent insulin-deficient diabetes in male and in female Akita mice using a model of misfolded proinsulin. CE stabilize the ER-associated protein degradation (ERAD) system and promote misfolded proinsulin proteasomal degradation. This involves activation of nuclear and membrane estrogen receptor-α (ERα), promoting transcriptional repression and proteasomal degradation of the ubiquitin-conjugating enzyme and ERAD degrader, UBC6e. The selective ERα modulator bazedoxifene mimics CE protection of ß cells in females but not in males.

Androgen excess in pancreatic ß cells and neurons predisposes female mice to type 2 diabetes.

Androgen excess predisposes women to type 2 diabetes (T2D), but the mechanism of this is poorly understood. We report that female mice fed a Western diet and exposed to chronic androgen excess using dihydrotestosterone (DHT) exhibit hyperinsulinemia and insulin resistance associated with secondary pancreatic ß cell failure, leading to hyperglycemia. These abnormalities are not observed in mice lacking the androgen receptor (AR) in ß cells and partially in neurons of the mediobasal hypothalamus (MBH) as well as in mice lacking AR selectively in neurons. Accordingly, i.c.v. infusion of DHT produces hyperinsulinemia and insulin resistance in female WT mice. We observe that acute DHT produces insulin hypersecretion in response to glucose in cultured female mouse and human pancreatic islets in an AR-dependent manner via a cAMP- and mTOR-dependent pathway. Acute DHT exposure increases mitochondrial respiration and oxygen consumption in female cultured islets. As a result, chronic DHT exposure in vivo promotes islet oxidative damage and susceptibility to additional stress induced by streptozotocin via AR in ß cells. This study suggests that excess androgen predisposes female mice to T2D following AR activation in neurons, producing peripheral insulin resistance, and in pancreatic ß cells, promoting insulin hypersecretion, oxidative injury, and secondary ß cell failure.

Extranuclear Actions of the Androgen Receptor Enhance Glucose-Stimulated Insulin Secretion in the Male.

Although men with testosterone deficiency are at increased risk for type 2 diabetes (T2D), previous studies have ignored the role of testosterone and the androgen receptor (AR) in pancreatic ß cells. We show that male mice lacking AR in ß cells (ßARKO) exhibit decreased glucose-stimulated insulin secretion (GSIS), leading to glucose intolerance. The AR agonist dihydrotestosterone (DHT) enhances GSIS in cultured male islets, an effect that is abolished in ßARKO(-/y) islets and human islets treated with an AR antagonist. In ß cells, DHT-activated AR is predominantly extranuclear and enhances GSIS by increasing islet cAMP and activating the protein kinase A. In mouse and human islets, the insulinotropic effect of DHT depends on activation of the glucagon-like peptide-1 (GLP-1) receptor, and accordingly, DHT amplifies the incretin effect of GLP-1. This study identifies AR as a novel receptor that enhances ß cell function, a finding with implications for the prevention of T2D in aging men.

The role of androgens in metabolism, obesity, and diabetes in males and females.

OBJECTIVE: In men, androgen deprivation contributes to the development of metabolic syndrome and type 2 diabetes (T2D). In women, androgen excess predisposes to insulin resistance and T2D. There is a bidirectional modulation of glucose homeostasis by androgens in males and females that is analyzed in this review. METHODS: We reviewed the literature in both rodents and humans on the role of androgens and the androgen receptor (AR) in the control of glucose and energy metabolism in health, obesity, and T2D. RESULTS: Sex-specific activation of AR in the hypothalamus, skeletal muscle, liver, adipose tissue, and pancreatic islet ß-cells accounts for maintenance or disruption in energy metabolism and glucose homeostasis. CONCLUSIONS: We argue that AR is a target to prevent androgen-related metabolic disorders.

Central mechanisms of adiposity in adult female mice with androgen excess.

OBJECTIVE: Androgen excess in women is associated with visceral adiposity. However, little is known on the mechanism through which androgen promotes visceral fat accumulation. METHODS: To address this issue, female mice to chronic androgen excess using 5α-dihydrotestosterone (DHT) and studied the regulation of energy homeostasis was exposed. RESULTS: DHT induced a leptin failure to decrease body weight associated with visceral adiposity but without alterations in leptin anorectic action. This paralleled leptin's failure to upregulate brown adipose tissue expression of uncoupling protein-1, associated with decreased energy expenditure (EE). DHT decreased hypothalamic proopiomelanocortin (pomc) mRNA expression and increased POMC intensity in neuronal bodies of the arcuate nucleus while simultaneously decreasing the intensity of POMC projections to the dorsomedial hypothalamus (DMH). This was associated with a failure of the melanocortin 4 receptor agonist melanotan-II to suppress body weight. CONCLUSION: Taken together, these data indicate that androgen excess promotes visceral adiposity with reduced POMC neuronal innervation in the DMH, reduced EE but without hyperphagia.

Hypothalamic astroglial connexins are required for brain glucose sensing-induced insulin secretion.

Hypothalamic glucose detection participates in maintaining glycemic balance, food intake, and thermogenesis. Although hypothalamic neurons are the executive cells involved in these responses, there is increasing evidence that astrocytes participate in glucose sensing (GS); however, it is unknown whether astroglial networking is required for glucose sensitivity. Astroglial connexins 30 and 43 (Cx30 and Cx43) form hexameric channels, which are apposed in gap junctions, allowing for the intercellular transfer of small molecules such as glucose throughout the astroglial networks. Here, we hypothesized that hypothalamic glucose sensitivity requires these connexins. First, we showed that both Cxs are enriched in the rat hypothalamus, with highly concentrated Cx43 expression around blood vessels of the mediobasal hypothalamus (MBH). Both fasting and high glycemic levels rapidly altered the protein levels of MBH astroglial connexins, suggesting cross talk within the MBH between glycemic status and the connexins' ability to dispatch glucose. Finally, the inhibition of MBH Cx43 (by transient RNA interference) attenuated hypothalamic glucose sensitivity in rats, which was demonstrated by a pronounced decreased insulin secretion in response to a brain glucose challenge. These results illustrate that astroglial connexins contribute to hypothalamic GS.

Alteration of hypothalamic glucose and lactate sensing in 48h hyperglycemic rats.

Hypothalamic detection of nutrients is involved in the control of energy metabolism and is altered in metabolic disorders. Although hypothalamic detection of blood lactate lowers hepatic glucose production and food intake, it is unknown whether it also modulates insulin secretion. To address this, a lactate injection via the right carotid artery (cephalad) was performed in Wistar rats. This triggered a transient increase in insulin secretion. Rats made hyperglycemic for 48h exhibited prolonged insulin secretion in response to a glucose injection via the carotid artery, but lactate injection induced two types of responses: half of the HG rats showed no difference compared to controls and the other half had markedly decreased insulin secretion. Astroglial monocarboxylates transporters MCT1 and MCT4 isoforms transfer lactate from blood to astrocytes and release lactate to the extracellular space, whilst the neuronal MCT2 isoform permits neuronal lactate uptake. We found that astroglial MCT1 and MCT4, and neuronal MCT2 protein levels in the medio-basal hypothalamus (MBH) were not modified by 48h-hyperglycemia. Together, these results indicate that hypothalamic sensing of circulating lactate triggers insulin secretion. Both glucose and lactate sensing are altered in a model of hyperglycemia, without alteration of MBH MCTs protein levels.

Importance of mitochondrial dynamin-related protein 1 in hypothalamic glucose sensitivity in rats.

AIMS: Hypothalamic mitochondrial reactive oxygen species (mROS)-mediated signaling has been recently shown to be involved in the regulation of energy homeostasis. However, the upstream signals that control this mechanism have not yet been determined. Here, we hypothesize that glucose-induced mitochondrial fission plays a significant role in mROS-dependent hypothalamic glucose sensing. RESULTS: Glucose-triggered translocation of the fission protein dynamin-related protein 1 (DRP1) to mitochondria was first investigated in vivo in hypothalamus. Thus, we show that intracarotid glucose injection induces the recruitment of DRP1 to VMH mitochondria in vivo. Then, expression was transiently knocked down by intra-ventromedial hypothalamus (VMH) DRP1 siRNA (siDRP1) injection. 72 h post siRNA injection, brain intracarotid glucose induced insulin secretion, and VMH glucose infusion-induced refeeding decrease were measured, as well as mROS production. The SiDRP1 rats decreased mROS and impaired intracarotid glucose injection-induced insulin secretion. In addition, the VMH glucose infusion-induced refeeding decrease was lost in siDRP1 rats. Finally, mitochondrial function was evaluated by oxygen consumption measurements after DRP1 knock down. Although hypothalamic mitochondrial respiration was not modified in the resting state, substrate-driven respiration was impaired in siDRP1 rats and associated with an alteration of the coupling mechanism. INNOVATION AND CONCLUSION: Collectively, our results suggest that glucose-induced DRP1-dependent mitochondrial fission is an upstream regulator for mROS signaling, and consequently, a key mechanism in hypothalamic glucose sensing. Thus, for the first time, we demonstrate the involvement of DRP1 in physiological regulation of brain glucose-induced insulin secretion and food intake inhibition. Such involvement implies DRP1-dependent mROS production.