Featured ArticlesVolume 17 | November 2018
|Sex dependent impact of gestational stress on predisposition to eating disorders and metabolic diseaseEating disorders (EDs) are damaging mental and metabolic illnesses. Recent evidence suggests that epigenetic mechanisms may be involved in initiating and maintaining EDs. Schroeder et al. explored the effects of chronic variable stress during the whole period of gestation, response to food restriction, and susceptibility to develop activity based-anorexia, binge eating, and metabolic syndrome in male and female offspring. They report that chronic prenatal stress induces sexually dimorphic effects on placental function, affecting fetal hypothalamic programming and subsequent basal metabolism and the response to a variety of metabolic challenges.|
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Objective: Vulnerability to eating disorders (EDs) is broadly assumed to be associated with early life stress. However, a careful examination of the literature shows that susceptibility to EDs may depend on the type, severity and timing of the stressor and the sex of the individual. We aimed at exploring the link between chronic prenatal stress and predisposition to EDs and metabolic disease.
Methods: We used a chronic variable stress protocol during gestation to explore the metabolic response of male and female offspring to food restriction (FR), activity-based anorexia (ABA), binge eating (BE) and exposure to high fat (HF) diet.
Results: Contrary to controls, prenatally stressed (PNS) female offspring showed resistance to ABA and BE and displayed a lower metabolic rate leading to hyperadiposity and obesity on HF diet. Male PNS offspring showed healthy responses to FR and ABA, increased propensity to binge and improved coping with HF compared to controls. We found that long-lasting abnormal responses to metabolic challenge are linked to fetal programming and adult hypothalamic dysregulation in PNS females, resulting from sexually dimorphic adaptations in placental methylation and gene expression.
Conclusions: Our results show that maternal stress may have variable and even opposing effects on ED risk, depending on the ED and the sex of the offspring.[Hide abstract]
|Reductions in glucokinase activity increase responses to hypoglycemiaMaintaining blood glucose within an appropriate range is crucial for survival. To defend against falling blood glucose, a series of robust counter-regulatory responses normally prevent hypoglycemia from occurring. The glucose-sensing apparatus in pancreatic β-cells includes the low affinity hexokinase glucokinase (GCK), which controls glycolytic flux into downstream metabolic sensing. Chakera, Hurst, Spyer, Ogunnowo-Bada, et al. examined the role of GCK in the hormonal protection against hypoglycemia. Their data identify a GCK-dependent glucose-sensing mechanism that boosts responses to falling glucose, augmenting reduction in insulin secretion and the release of glucagon and epinephrine.|
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Objective: Appropriate glucose levels are essential for survival; thus, the detection and correction of low blood glucose is of paramount importance. Hypoglycemia prompts an integrated response involving reduction in insulin release and secretion of key counter-regulatory hormones glucagon and epinephrine that together promote endogenous glucose production to restore normoglycemia. However, specifically how this response is orchestrated remains to be fully clarified. The low affinity hexokinase glucokinase is found in glucose-sensing cells involved in glucose homeostasis including pancreatic β-cells and in certain brain areas. Here, we aimed to examine the role of glucokinase in triggering counter-regulatory hormonal responses to hypoglycemia, hypothesizing that reduced glucokinase activity would lead to increased and/or earlier triggering of responses.
Methods: Hyperinsulinemic glucose clamps were performed to examine counter-regulatory responses to controlled hypoglycemic challenges created in humans with monogenic diabetes resulting from heterozygous glucokinase mutations (GCK-MODY). To examine the relative importance of glucokinase in different sensing areas, we then examined responses to clamped hypoglycemia in mice with molecularly defined disruption of whole body and/or brain glucokinase.
Results: GCK-MODY patients displayed increased and earlier glucagon responses during hypoglycemia compared with a group of glycemia-matched patients with type 2 diabetes. Consistent with this, glucagon responses to hypoglycemia were also increased in I366F mice with mutated glucokinase and in streptozotocin-treated β-cell ablated diabetic I366F mice. Glucagon responses were normal in conditional brain glucokinase-knockout mice, suggesting that glucagon release during hypoglycemia is controlled by glucokinase-mediated glucose sensing outside the brain but not in β-cells. For epinephrine, we found increased responses in GCK-MODY patients, in β-cell ablated diabetic I366F mice and in conditional (nestin lineage) brain glucokinase-knockout mice, supporting a role for brain glucokinase in triggering epinephrine release.
Conclusions: Our data suggest that glucokinase in brain and other non β-cell peripheral hypoglycemia sensors is important in glucose homeostasis, allowing the body to detect and respond to a falling blood glucose.[Hide abstract]
|Deletion of the glucagon receptor gene before and after experimental diabetes reveals differential protection from hyperglycemiaGlucagon receptor (GCGR) signaling helps maintain glucose homeostasis by stimulating hepatic glucose production. Interestingly, in rodent models of type-1 diabetes, hyperglycemia is almost completely mitigated by deletion of Gcgr. Rivero-Gutierrez et al. created an inducible Gcgr knockout mouse model to compare acute and chronic loss of the GCGR during insulinopenic diabetes. Their results demonstrate that engagement of compensatory signals, specifically GLP-1 receptor signaling, rather than loss of GCGR activation per se, attenuates the development of hyperglycemia during insulinopenic conditions.|
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Objective: Mice with congenital loss of the glucagon receptor gene (Gcgr−/− mice) remain normoglycemic in insulinopenic conditions, suggesting that unopposed glucagon action is the driving force for hyperglycemia in Type-1 Diabetes Mellitus (T1DM). However, chronic loss of GCGR results in a neomorphic phenotype that includes hormonal signals with hypoglycemic activity. We combined temporally-controlled GCGR deletion with pharmacological treatments to dissect the direct contribution of GCGR signaling to glucose control in a common mouse model of T1DM.
Methods: We induced experimental T1DM by injecting the beta-cell cytotoxin streptozotocin (STZ) in mice with congenital or temporally-controlled Gcgr loss-of-function using tamoxifen (TMX).
Results: Disruption of Gcgr expression, using either an inducible approach in adult mice or animals with congenital knockout, abolished the response to a long-acting Gcgr agonist. Mice with either developmental Gcgr disruption or inducible deletion several weeks before STZ treatment maintained normoglycemia. However, mice with inducible knockout of the Gcgr one week after the onset of STZ diabetes had only partial correction of hyperglycemia, an effect that was reversed by GLP-1 receptor blockade. Mice with Gcgr deletion for either 2 or 6 weeks had similar patterns of gene expression, although the changes were generally larger with longer GCGR knockout.
Conclusions: These findings demonstrate that the effects of glucagon to mitigate diabetic hyperglycemia are not through acute signaling but require compensations that take weeks to develop.[Hide abstract]
|A PDX1-ATF transcriptional complex governs β cell survivalDiabetes results from the failure of insulin producing β cells to compensate for increased metabolic demand and stress. The homeodomain protein PDX1 is important for the function and survival of β cells. Juliana et al. identify previously undiscovered stress responsive transcriptional complexes containing PDX1, activating transcription factor 4 (ATF4), and ATF5 that regulate expression of stress and apoptosis genes to influence β cell fate decisions during stress.|
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Objective: Loss of insulin secretion due to failure or death of the insulin secreting β cells is the central cause of diabetes. The cellular response to stress (endoplasmic reticulum (ER), oxidative, inflammatory) is essential to sustain normal β cell function and survival. Pancreatic and duodenal homeobox 1 (PDX1), Activating transcription factor 4 (ATF4), and Activating transcription factor 5 (ATF5) are transcription factors implicated in β cell survival and susceptibility to stress. Our goal was to determine if a PDX1-ATF transcriptional complex or complexes regulate β cell survival in response to stress and to identify direct transcriptional targets.
Methods: Pdx1, Atf4 and Atf5 were silenced by viral delivery of gRNAs or shRNAs to Min6 insulinoma cells or primary murine islets. Gene expression was assessed by qPCR, RNAseq analysis, and Western blot analysis. Chromatin enrichment was measured in the Min6 β cell line and primary isolated mouse islets by ChIPseq and ChIP PCR. Immunoprecipitation was used to assess interactions among transcription factors in Min6 cells and isolated mouse islets. Activation of caspase 3 by immunoblotting or by irreversible binding to a fluorescent inhibitor was taken as an indication of commitment to an apoptotic fate.
Results: RNASeq identified a set of PDX1, ATF4 and ATF5 co-regulated genes enriched in stress and apoptosis functions. We further identified stress induced interactions among PDX1, ATF4, and ATF5. PDX1 chromatin occupancy peaks were identified over composite C/EBP-ATF (CARE) motifs of 26 genes; assessment of a subset of these genes revealed co-enrichment for ATF4 and ATF5. PDX1 occupancy over CARE motifs was conserved in the human orthologs of 9 of these genes. Of these, Glutamate Pyruvate Transaminase 2 (Gpt2), Cation transport regulator 1 (Chac1), and Solute Carrier Family 7 Member 1 (Slc7a1) induction by stress was conserved in human islets and abrogated by deficiency of Pdx1, Atf4, and Atf5 in Min6 cells. Deficiency of Gpt2 reduced β cell susceptibility to stress induced apoptosis in both Min6 cells and primary islets.
Conclusions: Our results identify a novel PDX1 stress inducible complex (es) that regulates expression of stress and apoptosis genes to govern β cell survival.[Hide abstract]
|microRNA-205-5p is a modulator of insulin sensitivity that inhibits FOXO functionmiRNAs regulate gene expression in physiologic and disease conditions, including type 2 diabetes (T2D). Genome-wide association studies for T2D susceptibility loci indicate that most of the diabetes-associated variants localize to noncoding regions, raising the possibility that miRNAs transcribed from these regions contribute to disease development. Given the role of the transcription factor Forkhead Box Protein O (FOXO) in insulin action, Langlet et al. undertook a systematic search for FOXO-regulated hepatic miRNAs and identified miR-205-5p as an endogenous regulator of insulin sensitivity that coordinately targets components of the insulin signaling cascade, including FOXO itself.|
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Objective: Hepatic insulin resistance is a hallmark of type 2 diabetes and obesity. Insulin receptor signaling through AKT and FOXO has important metabolic effects that have traditionally been ascribed to regulation of gene expression. However, whether all the metabolic effects of FOXO arise from its regulation of protein-encoding mRNAs is unknown.
Methods: To address this question, we obtained expression profiles of FOXO-regulated murine hepatic microRNAs (miRNAs) during fasting and refeeding using mice lacking Foxo1, 3a, and 4 in liver (L-Foxo1,3a, 4).
Results: Out of 439 miRNA analyzed, 175 were differentially expressed in Foxo knockouts. Their functions were associated with insulin, Wnt, Mapk signaling, and aging. Among them, we report a striking increase of miR-205-5p expression in L-Foxo1,3a,4 knockouts, as well as in obese mice. We show that miR-205-5p gain-of-function increases AKT phosphorylation and decreases SHIP2 in primary hepatocytes, resulting in FOXO inhibition. This results in decreased hepatocyte glucose production. Consistent with these observations, miR-205-5p gain-of-function in mice lowered glucose levels and improved pyruvate tolerance.
Conclusions: These findings reveal a homeostatic miRNA loop regulating insulin signaling, with potential implications for in vivo glucose metabolism.[Hide abstract]
|l-Alanine activates hepatic AMPK and modulates systemic glucose metabolismPlasma amino acids are dysregulated in obesity. While increased branched chain amino acids may reflect inadequate insulin action, it has also been suggested that amino acids can directly modulate insulin action and may contribute to the pathophysiology of insulin resistance. However, the molecular mechanisms responsible for these effects remain uncertain. Adachi and colleagues report that l-alanine uniquely increases phosphorylation of the nutrient sensor AMP kinase (AMPK) and, in parallel, improves glucose tolerance in vivo in mice.|
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Objective: AMP activated protein kinase (AMPK) is recognized as an important nutrient sensor contributing to regulation of cellular, tissue, and systemic metabolism. We aimed to identify specific amino acids which could modulate AMPK and determine effects on cellular and systemic metabolism.
Methods: We performed an unbiased amino acid screen to identify activators of AMPK. Detailed analysis of cellular signaling and metabolism was performed in cultured hepatoma cells, and in vivo glucose metabolism and metabolomic patterns were assessed in both chow-fed mice and mice made obese by high-fat diet feeding.
Results: Alanine acutely activates AMP kinase in both cultured hepatic cells and in liver from mice treated in vivo with Ala. Oral alanine administration improves systemic glucose tolerance in both chow and high fat diet fed mice, with reduced efficacy of Ala in mice with reduced AMPK activity. Our data indicate that Ala activation of AMPK is mediated by intracellular Ala metabolism, which reduces TCA cycle metabolites, increases AMP/ATP ratio, and activates NH3 generation.
Conclusions: Ala may serve as a distinct amino acid energy sensor, providing a positive signal to activate the beneficial AMPK signaling pathway.[Hide abstract]
|Lipid droplet characteristics and distribution unmask the athlete’s paradoxIndividuals with type 2 diabetes present with increased storage of intramyocellular lipid (IMCL). Paradoxically, IMCL levels are also elevated in endurance trained athletes, who are very insulin sensitive. This phenomenon is known as the athlete’s paradox. Daemen, Gemmink, et al. examined the athlete’s paradox in individuals with similar levels of IMCL but over a wide range of insulin sensitivity. They found that insulin sensitive, trained individuals possess high levels of muscle fat that is dispersed in small lipid droplets in oxidative type I muscle fibers. On the other hand, in the insulin resistant type 2 diabetic state, most of the muscle fat is found in large lipid droplets in the subsarcolemmal space of type II muscle fibers. Therefore, the athlete’s paradox can be explained from a physiological perspective|
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Objective: Intramyocellular lipid (IMCL) storage negatively associates with insulin resistance, albeit not in endurance-trained athletes. We investigated the putative contribution of lipid droplet (LD) morphology and subcellular localization to the so-called athlete's paradox.
Methods: We performed quantitative immunofluorescent confocal imaging of muscle biopsy sections from endurance Trained, Lean sedentary, Obese, and Type 2 diabetes (T2DM) participants (n = 8/group). T2DM patients and Trained individuals were matched for IMCL content. Furthermore we performed this analysis in biopsies of T2DM patients before and after a 12-week exercise program (n = 8).
Results: We found marked differences in lipid storage morphology between trained subjects and T2DM: the latter group mainly store lipid in larger LDs in the subsarcolemmal (SS) region of type II fibers, whereas Trained store lipid in a higher number of LDs in the intramyofibrillar (IMF) region of type I fibers. In addition, a twelve-week combined endurance and strength exercise program resulted in a LD phenotype shift in T2DM patients partly towards an ‘athlete-like’ phenotype, accompanied by improved insulin sensitivity. Proteins involved in LD turnover were also more abundant in Trained than in T2DM and partly changed in an ‘athlete-like’ fashion in T2DM patients upon exercise training.
Conclusions: Our findings provide a physiological explanation for the athlete's paradox and reveal LD morphology and distribution as a major determinant of skeletal muscle insulin sensitivity.[Hide abstract]
|Quantitative mass spectrometry suggests prominent roles for β-MSH and desacetyl-α-MSH in energy homeostasisThe pro-opiomelanocortin (POMC) protein undergoes extensive proteolytic cleavage to produce neuropeptides that regulate food intake and energy expenditure. Since mice are unable to produce β-melanocyte stimulating hormone (β-MSH) from the POMC precursor, their utility as a model system for studying human-specific aspects of POMC processing is limited. Kirwan and colleagues analyzed POMC processing from hypothalamic neuron cell cultures as well as primary human brain samples via liquid chromatography tandem mass spectrometry (LC-MS/MS). They found that β-MSH and desacetyl-α-MSH were present at high concentrations, suggesting important roles for these peptides.
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Objective: The lack of pro-opiomelanocortin (POMC)-derived melanocortin peptides results in hypoadrenalism and severe obesity in both humans and rodents that is treatable with synthetic melanocortins. However, there are significant differences in POMC processing between humans and rodents, and little is known about the relative physiological importance of POMC products in the human brain. The aim of this study was to determine which POMC-derived peptides are present in the human brain, to establish their relative concentrations, and to test if their production is dynamically regulated.
Methods: We analysed both fresh post-mortem human hypothalamic tissue and hypothalamic neurons derived from human pluripotent stem cells (hPSCs) using liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the sequence and quantify the production of hypothalamic neuropeptides, including those derived from POMC.
Results: In both in vitro and in vivo hypothalamic cells, LC-MS/MS revealed the sequence of hundreds of neuropeptides as a resource for the field. Although the existence of β-melanocyte stimulating hormone (MSH) is controversial, we found that both this peptide and desacetyl α-MSH (d-α-MSH) were produced in considerable excess of acetylated α-MSH. In hPSC-derived hypothalamic neurons, these POMC derivatives were appropriately trafficked, secreted, and their production was significantly (P < 0.0001) increased in response to the hormone leptin.
Conclusions: Our findings challenge the assumed pre-eminence of α-MSH and suggest that in humans, d-α-MSH and β-MSH are likely to be the predominant physiological products acting on melanocortin receptors.[Hide abstract]
|Glucose sensing in the upper intestine potentiates glucose absorptionOur understanding of how gastrointestinal tract tissues and pathways coordinate the regulation of glucose metabolism after consumption of glucose is still limited. Using a knockout model of the sweet taste receptor (STR) compound Taste receptor type 1 member 2 (T1R2), Smith, Azari, et al. demonstrate that STR-mediated glucose sensing in the upper intestine is required for the potentiation of glucose absorption. Their findings reveal a mechanism where regulation of STR signaling in L-cells controls glucose absorption in enterocytes to limit postprandial hyperglycemia in response to high consumption of dietary sugars.
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Objective: Beyond the taste buds, sweet taste receptors (STRs; T1R2/T1R3) are also expressed on enteroendocrine cells, where they regulate gut peptide secretion but their regulatory function within the intestine is largely unknown.
Methods: Using T1R2-knock out (KO) mice we evaluated the role of STRs in the regulation of glucose absorption in vivo and in intact intestinal preparations ex vivo.
Results: STR signaling enhances the rate of intestinal glucose absorption specifically in response to the ingestion of a glucose-rich meal. These effects were mediated specifically by the regulation of GLUT2 transporter trafficking to the apical membrane of enterocytes. GLUT2 translocation and glucose transport was dependent and specific to glucagon-like peptide 2 (GLP-2) secretion and subsequent intestinal neuronal activation. Finally, high-sucrose feeding in wild-type mice induced rapid downregulation of STRs in the gut, leading to reduced glucose absorption.
Conclusions: Our studies demonstrate that STRs have evolved to modulate glucose absorption via the regulation of its transport and to prevent the development of exacerbated hyperglycemia due to the ingestion of high levels of sugars.[Hide abstract]
|Periodized low protein-high carbohydrate diet confers potent, but transient, metabolic improvementsChronic caloric restriction (CR) extends health and lifespan in experimental models and likely also in humans. However, long-term adherence to this ascetic regimen is difficult, and chronic CR is not feasible to implement broadly as a lifestyle intervention in humans. Since recent studies suggest that protein restriction rather than calorie restriction per se might underlie the anti-aging effects of CR diets, Li and colleagues tested a periodized low protein high carbohydrate diet regimen. They found some potent metabolic improvements, but these reversed within 14 days of returning to the control diet.|
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Objective: Chronic ad libitum low protein-high carbohydrate diet (LPHC) increases health- and life-span in mice. A periodized (p) LPHC regimen would be a more practical long-term human lifestyle intervention, but the metabolic benefits of pLPHC are not known. Also, the interactions between LPHC diet and exercise training have not been investigated. Presently, we aimed to provide proof-of-concept data in mice of the efficacy of pLPHC and to explore the potential interactions with concurrent exercise training.
Methods: A detailed phenotypic and molecular characterization of mice undergoing different durations of 14 d LPHC (5 E% protein)/14 d control diet cycles for up to 4 months with or without concurrent access to activity wheels allowing voluntary exercise training.
Results: pLPHC conferred metabolic benefits similar to chronic LPHC, including increased FGF21 and adaptive thermogenesis, obesity-protection despite increased total energy intake and improved insulin sensitivity. The improved insulin sensitivity showed large fluctuations between diet periods and was lost within 14 days of switching back to control diet. Parallel exercise training improved weight maintenance but impaired the FGF21 response to pLPHC whereas repeated pLPHC cycles progressively augmented this response. Both the FGF21 suppression by exercise and potentiation by repeated cycles correlated tightly with Nupr1 mRNA in liver, suggesting dependence on liver integrated stress response.
Conclusions: These results suggest that pLPHC may be a viable strategy to promote human health but also highlight the transient nature of the benefits and that the interaction with other lifestyle-interventions such as exercise training warrants consideration.[Hide abstract]
|Hepatic leptin receptors can compensate for IL-6Rα deficiency in hepatocellular carcinomaHepatocellular carcinoma (HCC) is a type of cancer that is inflammation-driven. Deficiency of interleukin 6 (IL-6) ameliorates HCC in mice. However, deficiency of the IL-6 receptor α (IL-6Rα) reduces tumor burden only in lean mice and not in obese mice. Mittenbühler et al. show that in obese mice, leptin receptor signaling compensates for the IL-6Rα deficiency and promotes HCC progression.
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Objective: The current obesity pandemic represents a major health burden, given that it predisposes to the development of numerous obesity-associated disorders. The obesity-derived adipokines not only impair systemic insulin action but also increase the incidence of hepatocellular carcinoma (HCC), a highly prevalent cancer with poor prognosis. Thus, worldwide incidences of HCC are expected to further increase, and defining the molecular as well as cellular mechanisms will allow for establishing new potential treatment options. The adipose tissue of obese individuals increases circulating leptin and interleukin-6 (IL-6) levels, which both share similar signaling capacities such as Signal Transducer and Activator of Transcription 3 (STAT3) and Phosphoinositide 3-kinase (PI3K)/Akt activation. While mouse models with deficient IL-6 signaling show an ameliorated but not absent Diethylnitrosamine (DEN)-induced HCC development, the morbid obesity in mice with mutant leptin signaling complicates the dissection of hepatic leptin receptor (LEPR) and IL-6 signaling in HCC development. Here we have investigated the function of compensating hepatic LEPR expression in HCC development of IL-6Rα-deficient mice.
Methods: We generated and characterized a mouse model of hepatic LEPR deficiency that was intercrossed with IL-6Rα-deficient mice. Cohorts of single and double knockout mice were subjected to the DEN-HCC model to ascertain liver cancer development and characterize metabolic alterations.
Results: We demonstrate that both high-fat diet (HFD)-induced obesity and IL-6Rα deficiency induce hepatic Lepr expression. Consistently, double knockout mice show a further reduction in tumor burden in DEN-induced HCC when compared to control and single LepRL−KO/IL-6Rα knock out mice, whereas metabolism remained largely unaltered between the genotypes.
Conclusions: Our findings reveal a compensatory role for hepatic LEPR in HCC development of IL-6Rα-deficient mice and suggest hepatocyte-specific leptin signaling as promoter of HCC under obese conditions.[Hide abstract]