Featured ArticlesVolume 32 | February 2020
|BMP4 gene therapy enhances insulin sensitivity but not adipose tissue browningOne of the early and central events in white adipocyte commitment is the action of bone morphogenetic protein 4 (BMP4). Overexpression or gene therapy with BMP4 can protect from obesity. Hoffmann and colleagues tested whether BMP4 gene therapy can also be used to treat already established obesity. Their results show that obesity is not reduced but that BMP4 improves whole-body insulin sensitivity, enhances insulin signaling in all key metabolic tissues, and reduces key gluconeogenic enzymes in the liver despite no weight loss.|
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Objective: Bone morphogenetic protein 4 (BMP4) adeno-associated viral vectors of serotype 8 (AAV8) gene therapy targeting the liver prevents the development of obesity in initially lean mice by browning the large subcutaneous white adipose tissue (WAT) and enhancing energy expenditure. Here, we examine whether this approach could also reduce established obesity.
Methods: Dietary-induced obese C57BL6/N mice received AAV8 BMP4 gene therapy at 17–18 weeks of age. They were kept on a high-fat diet and phenotypically characterized for an additional 10–12 weeks. Following termination, the mice underwent additional characterization in vitro.
Results: Surprisingly, we observed no effect on body weight, browning of WAT, or energy expenditure in these obese mice, but whole-body insulin sensitivity and glucose tolerance were robustly improved. Insulin signaling and insulin-stimulated glucose uptake were increased in both adipose cells and skeletal muscle. BMP4 also decreased hepatic glucose production and reduced gluconeogenic enzymes in the liver, but not in the kidney, in addition to enhancing insulin action in the liver.
Conclusions: Our findings show that BMP4 prevents, but does not reverse, established obesity in adult mice, while it improves insulin sensitivity independent of weight reduction. The BMP antagonist Noggin was increased in WAT in obesity, which may account for the lack of browning.[Hide abstract]
|The carbohydrate-insulin model does not explain the impact of macronutrients on body weightThe carbohydrate-insulin model (CIM) suggests that high levels of dietary carbohydrates elevate insulin secretion, which suppresses fatty acid oxidation and the release of fatty acids from adipose tissue while promoting lipogenesis. This creates a state of cellular “internal starvation” that drives both increased food intake and decreased energy expenditure, leading to obesity. Hu et al. directly tested specific predictions of the CIM in a mouse model. They found that only the changes in the post-prandial insulin and fasting glucose followed the CIM’s predictions in relation to the dietary carbohydrate. Fasting insulin, energy intake, energy expenditure, and body fat mass did not follow the trends predicted by the CIM.|
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Objective: The carbohydrate-insulin model (CIM) predicts that increases in fasting and post-prandial insulin in response to dietary carbohydrates stimulate energy intake and lower energy expenditures, leading to positive energy balance and weight gain. The objective of the present study was to directly test the CIM's predictions using C57BL/6 mice.
Methods: Diets were designed by altering dietary carbohydrates with either fixed protein or fat content and were fed to C57BL/6 mice acutely or chronically for 12 weeks. The body weight, body composition, food intake, and energy expenditures of the mice were measured. Their fasting and post-prandial glucose and insulin levels were also measured. RNA-seq was performed on RNA from the hypothalamus and subcutaneous white adipose tissue. Pathway analysis was conducted using IPA.
Results: Only the post-prandial insulin and fasting glucose levels followed the CIM's predictions. The lipolysis and leptin signaling pathways in the sWAT were inhibited in relation to the elevated fasting insulin, supporting the CIM's predicted impact of high insulin. However, because higher fasting insulin was unrelated to carbohydrate intake, the overall pattern did not support the model. Moreover, the hypothalamic hunger pathways were inhibited in relation to the increased fasting insulin, and the energy intake was not increased. The browning pathway in the sWAT was inhibited at higher insulin levels, but the daily energy expenditure was not altered.
Conclusions: Two of the predictions were partially supported (and hence also partially not supported) and the other three predictions were not supported. We conclude that the CIM does not explain the impact of dietary macronutrients on adiposity in mice.[Hide abstract]
|Pharmacological antagonism of the incretin system protects against obesityThe incretins, glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 (GLP-1), are gut-derived peptides released in response to ingested nutrients that mediate important actions on nutrient metabolism. GLP-1 receptor agonists have been developed for the treatment of obesity independent of type 2 diabetes. Paradoxically, Glp1r-/- mice are protected against diet-induced obesity. Svendsen et al. hypothesized that developmental compensations might be present in animals with germ-line deletions of the incretin receptors. Therefore, they used acute pharmacologic antagonism in fully developed animals, but still found decreases in body weight under a high fat diet.
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Objective: Glucose-dependent insulinotropic polypeptide is an intestinally derived hormone that is essential for normal metabolic regulation. Loss of the GIP receptor (GIPR) through genetic elimination or pharmacological antagonism reduces body weight and adiposity in the context of nutrient excess. Interrupting GIPR signaling also enhances the sensitivity of the receptor for the other incretin peptide, glucagon-like peptide 1 (GLP-1). The role of GLP-1 compensation in loss of GIPR signaling to protect against obesity has not been directly tested.
Methods: We blocked the GIPR and GLP-1R with specific antibodies, alone and in combination, in healthy and diet-induced obese (DIO) mice. The primary outcome measure of these interventions was the effect on body weight and composition.
Results: Antagonism of either the GIPR or GLP-1R system reduced food intake and weight gain during high-fat feeding and enhanced sensitivity to the alternative incretin signaling system. Combined antagonism of both GIPR and GLP-1R produced additive effects to mitigate DIO. Acute pharmacological studies using GIPR and GLP-1R agonists demonstrated both peptides reduced food intake, which was prevented by co-administration of the respective antagonists.
Conclusions: Disruption of either axis of the incretin system protects against diet-induced obesity in mice. However, combined antagonism of both GIPR and GLP-1R produced additional protection against diet-induced obesity, suggesting additional factors beyond compensation by the complementary incretin axis. While antagonizing the GLP-1 system decreases weight gain, GLP-1R agonists are used clinically to target obesity. Hence, the phenotype arising from loss of function of GLP-1R does not implicate GLP-1 as an obesogenic hormone. By extension, caution is warranted in labeling GIP as an obesogenic hormone based on loss-of-function studies.[Hide abstract]
|Regulation of muscle and metabolic physiology by hypothalamic EPOAlthough erythropoietin (EPO) is a glycoprotein hormone that is essential for erythropoiesis, it has been shown to influence nonhematopoietic tissues and may impact body weight and glucose control. Apart from its peripheral production and actions, limited evidence exists showing that EPO or its receptor (EpoR) could be detected in the brain. Wang et al. observed that the hypothalamus is a key site for the production and action of neural EPO. They also found that brain EPO decreases with aging and dietary obesity. Hypothalamic EPO treatment in aging or obesity offered benefits in muscle protection as well as in metabolic protection against energy and glucose imbalance.
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Objective: The glycoprotein hormone erythropoietin (EPO) is required for erythropoiesis, and the kidney is the primary site of adult EPO synthesis. Limited evidence has suggested that EPO could be detectable in the brain under certain conditions, but it remains unknown if the brain might have its own EPO system for biological functions that are independent of peripheral EPO production and action. We performed this study to address this question using mice under normal conditions versus pathophysiological conditions including aging and dietary obesity.
Methods: EPO expression was measured in different brain regions as well as in the cerebrospinal fluid. Hypothalamic ventricular EPO was administered to physiologically examine possible therapeutic effects on the conditions of aging and dietary obesity. Body weight, body composition, insulin tolerance, and glucose tolerance were measured to assess the central effects of EPO on metabolic physiology, and muscle strength and histology were analyzed to assess the central effects of EPO on muscle function. In addition, β2-adrenergic receptor knockout bone marrow transplant was employed to determine the potential role of bone marrow in linking the brain to some of these peripheral functions.
Results: This study revealed that EPO is expressed in the ventromedial hypothalamus in addition to a few other brain regions and is present in the cerebrospinal fluid. Unlike blood EPO concentration, which increased with aging and dietary obesity, hypothalamic EPO decreased in these disease conditions. Therapeutically, aged mice were chronically treated with EPO in the hypothalamic ventricle, showing an increase in lean mass, while body weight and fat mass decreased as a result of a moderate reduction of food intake. Both muscle and metabolic functions were improved by this central treatment, and mechanistically, adrenergic signals to the bone marrow played a role in conveying hypothalamic EPO to these peripheral actions. Dietary obesity was also studied, showing that hypothalamic EPO treatment caused a reduction in food intake and obesity, leading to improved metabolic functions related to decreased fat as well as increased lean mass.
Conclusions: Hypothalamic EPO plays a role in the central regulation of muscle and metabolic physiology, while its decline contributes to aging and obesity physiology in a manner that is independent of peripheral EPO.[Hide abstract]
|Fasting induces remodeling of the orexigenic projections from the ARC to the PVHNeuronal populations of the hypothalamic arcuate nucleus (ARC) regulate food intake. ARC neurons strongly innervate the hypothalamic paraventricular nucleus (PVH), and these ARC-PVH projections are recognized as key regulators of food intake. Cabral, Fernandez, et al. provide neuroanatomical and functional evidence indicating that ARC-PVH projections undergo major morphological remodeling under fasting conditions, and growth hormone secretagogue receptor signaling is required for these effects.|
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Objective: Arcuate nucleus (ARC) neurons producing Agouti-related peptide (AgRP) and neuropeptide Y (NPY; ARCAgRP/NPY neurons) are activated under energy-deficit states. ARCAgRP/NPY neurons innervate the hypothalamic paraventricular nucleus (PVH), and ARC→PVH projections are recognized as key regulators of food intake. Plasma ghrelin levels increase under energy-deficit states and activate ARCAgRP/NPY neurons by acting on the growth hormone secretagogue receptor (GHSR). Here, we hypothesized that activation of ARCAgRP/NPY neurons in fasted mice would promote morphological remodeling of the ARCAgRP/NPY→PVH projections in a GHSR-dependent manner.
Methods: We performed 1) fluorescent immunohistochemistry, 2) imaging of green fluorescent protein (GFP) signal in NPY-GFP mice, and 3) DiI axonal labeling in brains of ad libitum fed or fasted mice with pharmacological or genetic blockage of the GHSR signaling and then estimated the density and strength of ARCAgRP/NPY→PVH fibers by assessing the mean fluorescence intensity, the absolute area with fluorescent signal, and the intensity of the fluorescent signal in the fluorescent area of the PVH.
Results: We found that 1) the density and strength of ARCAgRP/NPY fibers increase in the PVH of fasted mice, 2) the morphological remodeling of the ARCAgRP/NPY→PVH projections correlates with the activation of PVH neurons, and 3) PVH neurons are not activated in ARC-ablated mice. We also found that fasting-induced remodeling of ARCAgRP/NPY→PVH fibers and PVH activation are impaired in mice with pharmacological or genetic blockage of GHSR signaling.
Conclusions: This evidence shows that the connectivity between hypothalamic circuits controlling food intake can be remodeled in the adult brain, depending on the energy balance conditions, and that GHSR activity is a key regulator of this phenomenon.[Hide abstract]
|SWI/SNF complex subunit BAF60a represses hepatic ureagenesisHepatic ureagenesis plays a predominant role in maintaining nitrogen and ammonia homeostasis in mammals. Disruption of ureagenesis leads to hyperammonemia, accompanied by hepatic encephalopathy and liver fibrosis. BAF60a is a subunit of the SWItch/Sucrose NonFermentable (SWI/SNF) complexes that regulates a series of metabolic pathways as a transcription factor. Zhang, Dong, et al. have elucidated a BAF60a-orchestrated upstream regulatory mechanism in the urea cycle and provide a mechanism through which steatotic hepatocytes produce ammonia via formation of a BAF60a-YB-1 complex.|
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Objective: Ureagenesis predominantly occurs in the liver and functions to remove ammonia, and the dysregulation of ureagenesis leads to the development of hyperammonemia. Recent studies have shown that ureagenesis is under the control of nutrient signals, but the mechanism remains elusive. Therefore, intensive investigation of the molecular mechanism underlying ureagenesis will shed some light on the pathology of metabolic diseases related to ammonia imbalance.
Methods: Mice were fasted for 24 h or fed a high-fat diet (HFD) for 16 weeks. For human evaluation, we obtained a public data set including 41 obese patients with and without hepatic steatosis. We analyzed the expression levels of hepatic BAF60a under different nutrient status. The impact of BAF60a on ureagenesis and hyperammonemia was assessed by using gain- and loss-of-function strategies. The molecular chaperons mediating the effects of BAF60a on ureagenesis were validated by molecular biological strategies.
Results: BAF60a was induced in the liver of both fasted and HFD-fed mice and was positively correlated with body mass index in obese patients. Liver-specific overexpression of BAF60a inhibited hepatic ureagenesis, leading to the increase of serum ammonia levels. Mechanistically, BAF60a repressed the transcription of Cps1, a rate-limiting enzyme, through interaction with Y-box protein 1 (YB-1) and by switching the chromatin structure of Cps1 promoter into an inhibitory state. More importantly, in response to different nutrient status, PGC-1α (as a transcriptional coactivator) and YB-1 competitively bound to BAF60a, thus selectively regulating hepatic fatty acid β-oxidation and ureagenesis.
Conclusions: The BAF60a-YB-1 axis represses hepatic ureagenesis, thereby contributing to hyperammonemia under overnutrient status. Therefore, hepatic BAF60a may be a novel therapeutic target for the treatment of overnutrient-induced urea cycle disorders and their associated diseases.[Hide abstract]
|Hepatic ERα accounts for sex differences in the ability to cope with excess dietary lipidsIn female mammals, liver estrogen receptor alpha (ERα) plays a central role in the adaptive response of hepatic metabolism to the energy requirements characterizing the different reproductive stages. Unlike in females, the expression of ERα in the adult male liver is very low. Although obesity is prevalent in females, fertile women are to some extent protected from obesity-associated fatty liver and cardio-metabolic disease, suggesting the involvement of estrogen signaling. Meda and colleagues highlight the essential role of hepatic ERα in the regulation of lipid metabolism in the liver of the two sexes when dietary lipids are in excess. In males, hepatic ERα action contributes to liver lipid accumulation; conversely, in females, ERα prevents the hepatic lipid deposition.
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Among obesity-associated metabolic diseases, non-alcoholic fatty liver disease (NAFLD) represents an increasing public health issue due to its emerging association with atherogenic dyslipidemia and cardiovascular diseases (CVDs). The lower prevalence of NAFLD in pre-menopausal women compared with men or post-menopausal women led us to hypothesize that the female-inherent ability to counteract this pathology might strongly rely on estrogen signaling.
Methods: Through liver metabolomics and transcriptomics we analyzed the relevance of hepatic ERα in the metabolic response of males and females to a diet highly enriched in fats (HFD) as a model of diet-induced obesity.
Results: The study shows that the hepatic ERα strongly contributes to the sex-specific response to an HFD and its action accounts for opposite consequences for hepatic health in males and females.
Conclusion: This study identified hepatic ERα as a novel target for the design of sex-specific therapies against fatty liver and its cardio-metabolic consequences.[Hide abstract]
|Single-cell ATAC-Seq in human pancreatic islets reveals T2D regulatory signaturesGenetic predisposition is one of the factors that can lead to type 2 diabetes (T2D). ATAC-seq is a high-throughput epigenomic profiling method to determine chromatin accessibility across samples in a tissue-wide manner. Rai, Quang, et al. use single-cell combinatorial indexing ATAC-seq that enables them to deconvolve cell populations and identify cell-type-specific regulatory signatures underlying T2D. They found T2D single nucleotide polymorphisms to be significantly enriched in beta cell-specific and across cell-type shared islet open chromatin, but not in alpha or delta cell-specific open chromatin. They also developed a novel deep learning-based strategy to improve signal recovery and feature reconstruction for low abundance cell populations and apply it successfully to delta cells (<5% of the total islet population) identified in the study.|
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Objective: Type 2 diabetes (T2D) is a complex disease characterized by pancreatic islet dysfunction, insulin resistance, and disruption of blood glucose levels. Genome-wide association studies (GWAS) have identified > 400 independent signals that encode genetic predisposition. More than 90% of associated single-nucleotide polymorphisms (SNPs) localize to non-coding regions and are enriched in chromatin-defined islet enhancer elements, indicating a strong transcriptional regulatory component to disease susceptibility. Pancreatic islets are a mixture of cell types that express distinct hormonal programs, so each cell type may contribute differentially to the underlying regulatory processes that modulate T2D-associated transcriptional circuits. Existing chromatin profiling methods such as ATAC-seq and DNase-seq, applied to islets in bulk, produce aggregate profiles that mask important cellular and regulatory heterogeneity.
Methods: We present genome-wide single-cell chromatin accessibility profiles in >1,600 cells derived from a human pancreatic islet sample using single-cell combinatorial indexing ATAC-seq (sci-ATAC-seq). We also developed a deep learning model based on U-Net architecture to accurately predict open chromatin peak calls in rare cell populations.
Results: We show that sci-ATAC-seq profiles allow us to deconvolve alpha, beta, and delta cell populations and identify cell-type-specific regulatory signatures underlying T2D. Particularly, T2D GWAS SNPs are significantly enriched in beta cell-specific and across cell-type shared islet open chromatin, but not in alpha or delta cell-specific open chromatin. We also demonstrate, using less abundant delta cells, that deep learning models can improve signal recovery and feature reconstruction of rarer cell populations. Finally, we use co-accessibility measures to nominate the cell-specific target genes at 104 non-coding T2D GWAS signals.
Conclusions: Collectively, we identify the islet cell type of action across genetic signals of T2D predisposition and provide higher-resolution mechanistic insights into genetically encoded risk pathways.[Hide abstract]
|Itaconate modulates tricarboxylic acid and redox metabolism to mitigate reperfusion injuryReperfusion after cerebral ischemic insult is necessary for survival. However, it is associated with the induction of oxidative stress and inflammatory responses, leading to reperfusion injury. This can drive extensive tissue damage and increase the risk of sepsis and multiple organ failure. Cordes and colleagues tested itaconate as a possible protector from reperfusion injury. They found that in mouse models, itaconate reduced oxidative stress and improved cerebral hemodynamics, inflammation, neurological function, and survival.|
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Objective: Cerebral ischemia/reperfusion (IR) drives oxidative stress and injurious metabolic processes that lead to redox imbalance, inflammation, and tissue damage. However, the key mediators of reperfusion injury remain unclear, and therefore, there is considerable interest in therapeutically targeting metabolism and the cellular response to oxidative stress.
Methods: The objective of this study was to investigate the molecular, metabolic, and physiological impact of itaconate treatment to mitigate reperfusion injuries in in vitro and in vivo model systems. We conducted metabolic flux and bioenergetic studies in response to exogenous itaconate treatment in cultures of primary rat cortical neurons and astrocytes. In addition, we administered itaconate to mouse models of cerebral reperfusion injury with ischemia or traumatic brain injury followed by hemorrhagic shock resuscitation. We quantitatively characterized the metabolite levels, neurological behavior, markers of redox stress, leukocyte adhesion, arterial blood flow, and arteriolar diameter in the brains of the treated/untreated mice.
Results: We demonstrate that the “immunometabolite” itaconate slowed tricarboxylic acid (TCA) cycle metabolism and buffered redox imbalance via succinate dehydrogenase (SDH) inhibition and induction of anti-oxidative stress response in primary cultures of astrocytes and neurons. The addition of itaconate to reperfusion fluids after mouse cerebral IR injury increased glutathione levels and reduced reactive oxygen/nitrogen species (ROS/RNS) to improve neurological function. Plasma organic acids increased post-reperfusion injury, while administration of itaconate normalized these metabolites. In mouse cranial window models, itaconate significantly improved hemodynamics while reducing leukocyte adhesion. Further, itaconate supplementation increased survival in mice experiencing traumatic brain injury (TBI) and hemorrhagic shock.
Conclusions: We hypothesize that itaconate transiently inhibits SDH to gradually “awaken” mitochondrial function upon reperfusion that minimizes ROS and tissue damage. Collectively, our data indicate that itaconate acts as a mitochondrial regulator that controls redox metabolism to improve physiological outcomes associated with IR injury.[Hide abstract]
|mTORC1 restrains adipocyte lipolysisThe mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that nucleates two structurally and functionally distinct complexes, mTORC1 and mTORC2. The available data do not identify a clear or consistent role for adipocyte mTORC1 signaling in systemic lipid homeostasis. Paolella et al. demonstrate that mTORC1 signaling in adipose tissue is critical for the maintenance of plasma lipid homeostasis in the fed state. Lack of mTORC1 in adipocytes increases both lipolysis and circulating triglycerides in fed mice.|
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Objective: Pharmacological agents targeting the mTOR complexes are used clinically as immunosuppressants and anticancer agents and can extend the lifespan of model organisms. An undesirable side effect of these drugs is hyperlipidemia. Although multiple roles have been described for mTOR complex 1 (mTORC1) in lipid metabolism, the etiology of hyperlipidemia remains incompletely understood. The objective of this study was to determine the influence of adipocyte mTORC1 signaling in systemic lipid homeostasis in vivo.
Methods: We characterized systemic lipid metabolism in mice lacking the mTORC1 subunit Raptor (RaptoraKO), the key lipolytic enzyme ATGL (ATGLaKO), or both (ATGL-RaptoraKO) in their adipocytes.
Results: Mice lacking mTORC1 activity in their adipocytes failed to completely suppress lipolysis in the fed state and displayed prominent hypertriglyceridemia and hypercholesterolemia. Blocking lipolysis in their adipose tissue restored normal levels of triglycerides and cholesterol in the fed state as well as the ability to clear triglycerides in an oral fat tolerance test.
Conclusions: Unsuppressed adipose lipolysis in the fed state interferes with triglyceride clearance and contributes to hyperlipidemia. Adipose tissue mTORC1 activity is necessary for appropriate suppression of lipolysis and for the maintenance of systemic lipid homeostasis.[Hide abstract]
|Continuous glucose monitoring reveals glycemic variability and hypoglycemia after VSG in ratsWith the rising use of bariatric surgery, there is also greater awareness of associated complications, one of which is post-bariatric surgery hypoglycemia (PBH). Episodes of hypoglycemia impair cognition and increase the risk for syncope, cardiac arrhythmias, seizures, coma, and even death. While medication or lifestyle modifications can be used effectively to reduce recurrence in diabetic patients, this is not the case for bariatric surgery patients. Evers, Kim, and colleagues propose that rat models of VSG replicate many aspects of PBH in humans. They found that VSG increases glycemic variability, and in response to a mixed-meal liquid gavage, hypoglycemia is readily detectable. Blockade of GLP-1R signaling prevents falls in the glucose nadir but does not improve glycemic variability.|
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Objective: Post–bariatric surgery hypoglycemia (PBH) is defined as the presence of neuroglycopenic symptoms accompanied by postprandial hypoglycemia in bariatric surgery patients. Recent clinical studies using continuous glucose monitoring (CGM) technology revealed that PBH is more frequently observed in vertical sleeve gastrectomy (VSG) patients than previously recognized. PBH cannot be alleviated by current medication. Therefore, a model system to investigate the mechanism and treatment is required.
Methods: We used CGM in a rat model of VSG and monitored the occurrence of glycemic variability and hypoglycemia in various meal conditions for 4 weeks after surgery. Another cohort of VSG rats with CGM was used to investigate whether the blockade of glucagon-like peptide-1 receptor (GLP-1R) signaling alleviates these symptoms. A mouse VSG model was used to investigate whether the impaired glucose counterregulatory system causes postprandial hypoglycemia.
Results: Like in humans, rats have increased glycemic variability and hypoglycemia after VSG. Postprandial hypoglycemia was specifically detected after liquid versus solid meals. Further, the blockade of GLP-1R signaling raises the glucose nadir but does not affect glycemic variability.
Conclusions: Rat bariatric surgery duplicates many features of human post–bariatric surgery hypoglycemia including postprandial hypoglycemia and glycemic variability, while blockade of GLP-1R signaling prevents hypoglycemia but not the variability.[Hide abstract]
|Leptin receptor sequences crucial for the STAT3-Independent control of metabolismLeptin, a peptide hormone produced by white adipose tissue in proportion to energy stores, plays a central role in the control of feeding and energy balance. Leptin receptor (LepRb) mainly signals trough activation of signal transducer and activator of transcription 3 (STAT3); however, there is also a second, yet unidentified signal. Barnes et al. used CRISPR/Cas9-mediated mutagenesis to generate a panel of mouse lines containing truncations of LepRb. By studying these five novel mouse lines, they identified a region of the intracellular LepRb that is required to mediate the second signal and additionally found a region that mediates a previously undescribed LepRb inhibitory signal.|
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Objective: Leptin acts via its receptor, LepRb, on specialized neurons in the brain to modulate energy balance and glucose homeostasis. LepRb→STAT3 signaling plays a crucial role in leptin action, but LepRb also mediates an additional as-yet-unidentified signal (Signal 2) that is important for leptin action. Signal 2 requires LepRb regions in addition to those required for JAK2 activation but operates independently of STAT3 and LepRb phosphorylation sites.
Methods: To identify LepRb sequences that mediate Signal 2, we used CRISPR/Cas9 to generate five novel mouse lines containing COOH-terminal truncation mutants of LepRb. We analyzed the metabolic phenotype and measures of hypothalamic function for these mouse lines.
Results: We found that deletion of LepRb sequences between residues 921 and 960 dramatically worsens metabolic control and alters hypothalamic function relative to smaller truncations. We also found that deletion of the regions including residues 1013–1053 and 960–1013 each decreased obesity compared to deletions that included additional COOH-terminal residues.
Conclusions: LepRb sequences between residues 921 and 960 mediate the STAT3 and LepRb phosphorylation-independent second signal that contributes to the control of energy balance and metabolism by leptin/LepRb. In addition to confirming the inhibitory role of the region (residues 961–1013) containing Tyr985, we also identified the region containing residues 1013–1053 (which contains no Tyr residues) as a second potential mediator of LepRb inhibition. Thus, the intracellular domain of LepRb mediates multiple Tyr-independent signals.[Hide abstract]
|Glucocorticoid induces β cell dysfunction by involving riborepressor GAS5 LincRNAGlucocorticoid (GC) therapy may have metabolic side effects, one of which is diabetes mellitus. In GC signaling, the non-coding RNA growth arrest-specific 5 (GAS5) acts as a riborepressor by directly interacting with the glucocorticoid receptor in a dexamethasone-dependent manner. However, the role of GAS5 in human pancreatic beta cell function has not been previously addressed. Esguerra et al. demonstrate the involvement of GAS5 in GC-mediated beta cell dysfunction. Modulation of GAS5 in the human beta cell alleviated the GC-induced insulin secretion defect, demonstrating the potential of this non-coding RNA as a novel therapeutic target in countering GC-mediated beta cell dysfunction.
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Objective: A widely recognized metabolic side effect of glucocorticoid (GC) therapy is steroid-induced diabetes mellitus (DM). However, studies on the molecular basis of GC-induced pancreatic beta cell dysfunction in human beta cells are lacking. The significance of non-coding RNAs in various cellular processes is emerging. In this study, we aimed to show the direct negative impact of GC on beta cell function and elucidate the role of riborepressor GAS5 lincRNA in the GC signaling pathway in human pancreatic beta cells.
Methods: Patients undergoing two weeks of high-dose prednisolone therapy were monitored for C-peptide levels. Human pancreatic islets and the human beta cell line EndoC-βH1 were incubated in pharmacological concentrations of dexamethasone. The GAS5 level was modulated using anti-sense LNA gapmeR or short oligonucleotides with GAS5 HREM (hormone response element motif). Immunoblotting and/or real-time PCR were used to assess changes in protein and RNA expression, respectively. Functional characterization included glucose-stimulated insulin secretion and apoptosis assays. Correlation analysis was performed on RNAseq data of human pancreatic islets.
Results: We found reduced C-peptide levels in patients undergoing high-dose GC therapy. Human islets and the human beta cell line EndoC-βH1 exposed to GC exhibited reduced insulin secretion and increased apoptosis. Concomitantly, reduced expression of important beta cell transcription factors, PDX1 and NKX6-1, as well as exocytotic protein SYT13 were observed. The expression of the glucocorticoid receptor was decreased, while that of serum and glucocorticoid-regulated kinase 1 (SGK1) was elevated. The expression of these genes was found to significantly correlate with GAS5 in human islet transcriptomics data. Increasing GAS5 levels using GAS5 HREM alleviated the inhibitory effects of dexamethasone on insulin secretion.
Conclusions: The direct adverse effect of glucocorticoid in human beta cell function is mediated via important beta cell proteins and components of the GC signaling pathway in an intricate interplay with GAS5 lincRNA, a potentially novel therapeutic target to counter GC-mediated beta cell dysfunction.[Hide abstract]