Modelling altered Glucocorticoid Sensitivity: From HPA axis to metabolic abnormalities in mice and humans

The primary determinants of tissue glucocorticoid action are glucocorticoid receptor (GR) density and intracellular levels of ligand, the latter determined both by activity of the hypothalamic-pituitary-adrenal (HPA) axis and cellular activity of 11beta- hydroxysteroid dehydrogenase (11beta-HSD) enzymes that interconvert active 11- hydroxy (corticosterone, cortisol) and inactive 11-keto (11-dehydrocorticosterone, cortisone) glucocorticoids. Here, the contribution of GR density and ligand levels in determining body composition and metabolic phenotype have been investigated in mice and in humans. Genetic evidence in humans implicates variations in the GR gene in the regulation of the HPA axis as well as the control of body fat distribution, metabolic parameters and blood pressure. Although GR deficient mouse models have been previously generated (with homozygous nulls dying at birth), the effects of altered GR density upon fat distribution and blood pressure have not been described. This study addresses the relationship between GR density and metabolic parameters, including body fat distribution, insulin resistance and hypertension. A novel line of mice harbouring a null mutation in the GR gene (GR+/-) was generated from an ES cell line in which a beta-galactosidase-neomycin phosphotransferase (beta geo) reporter cassette was fused with GR. The resulting fusion protein lacks part of the DNA binding domain and the entire ligand binding domain and is transcriptionally inactive. In addition, the beta-galactosidase enzyme activity “reports” activity of the GR gene promoter. GR-/- mice are present in a normal Mendelian ratio before birth. Intriguingly, 1 (of 36/146 expected if null allele not lethal) survived to adulthood suggesting this might be a hypomorphic rather than a null allele. Heterozygous 15 (GR+/-) mice showed 40-45% reductions in GR mRNA levels in the hippocampus, paraventricular nucleus of the hypothalamus, pituitary gland and adipose tissue, 30% in liver, 56% in muscle and 67% in adrenals. X-gal staining of GR+/- brain sections showed that GR-beta gal is present throughout, mirroring GR mRNA expression. Adult GR+/- mice had larger adrenals, higher evening plasma corticosterone levels and greater corticosterone responses following 10 minute restraint suggesting a hyperactive HPA axis. Compared to GR+/+ littermates, GR+/- mice had similar body weight gain on normal chow or high fat diet, with unaltered fat depot (inguinal, epididymal, mesenteric) weights and similar glucose and insulin tolerance. However, GR+/ - mice had higher (10%) systolic blood pressure, associated with activation of the renin-angiotensin system. Thus GR haploinsufficiency in mice causes increased blood pressure and accords with data associating GR polymorphisms with hypertension in humans. The role of altered GC sensitivity was also investigated in a mouse model of HPA axis hypoactivity pro-opiomelanocortin null (POMC) mice. POMC-null mice are obese due to central melanocortin deficiency. In contrast to most rodent models of obesity, POMC-null mice are also glucocorticoid deficient due to ACTH deficiency. Previous data have shown that glucocorticoid replacement in POMC-null mice exaggerated hyperphagia, obesity and insulin resistance and caused hypertension. Here, the contribution of peripheral glucocorticoid sensitivity was investigated. POMC-null mice have increased liver and retroperitoneal fat GR mRNA levels but, specifically in adipose tissue, decreased levels of mRNA encoding 11beta-HSD1, a reductase which regenerates active glucocorticoids, thus amplifying their action

Fate of Adipose Progenitor Cells in Obesity-Related Chronic Inflammation

Adipose progenitor cells, or preadipocytes, constitute a small population of immature cells within the adipose tissue. They are a heterogeneous group of cells, in which different subtypes have a varying degree of commitment toward diverse cell fates, contributing to white and beige adipogenesis, fibrosis or maintenance of an immature cell phenotype with proliferation capacity. Mature adipocytes as well as cells of the immune system residing in the adipose tissue can modulate the function and differentiation potential of preadipocytes in a contact- and/or paracrine-dependent manner. In the course of obesity, the accumulation of immune cells within the adipose tissue contributes to the development of a pro-inflammatory microenvironment in the tissue. Under such circumstances, the crosstalk between preadipocytes and immune or parenchymal cells of the adipose tissue may critically regulate the differentiation of preadipocytes into white adipocytes, beige adipocytes, or myofibroblasts, thereby influencing adipose tissue expansion and adipose tissue dysfunction, including downregulation of beige adipogenesis and development of fibrosis. The present review will outline the current knowledge about factors shaping cell fate decisions of adipose progenitor cells in the context of obesity-related inflammation

Peripheral mechanisms contributing to the glucocorticoid hypersensitivity in proopiomelanocortin null mice treated with corticosterone.

Proopiomelanocortin (POMC) deficiency causes severe obesity through hyperphagia of hypothalamic origin. However, low glucocorticoid levels caused by adrenal insufficiency mitigate against insulin resistance, hyperphagia and fat accretion in Pomc-/- mice. Upon exogenous glucocorticoid replacement, corticosterone-supplemented (CORT) Pomc-/- mice show exaggerated responses, including excessive fat accumulation, hyperleptinaemia and insulin resistance. To investigate the peripheral mechanisms underlying this glucocorticoid hypersensitivity, we examined the expression levels of key determinants and targets of glucocorticoid action in adipose tissue and liver. Despite lower basal expression of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which generates active glucocorticoids within cells, CORT-mediated induction of 11beta-HSD1 mRNA levels was more pronounced in adipose tissues of Pomc-/- mice. Similarly, CORT treatment increased lipoprotein lipase mRNA levels in all fat depots in Pomc-/- mice, consistent with exaggerated fat accumulation. Glucocorticoid receptor (GR) mRNA levels were selectively elevated in liver and retroperitoneal fat of Pomc-/- mice but were corrected by CORT in the latter depot. In liver, CORT increased phosphoenolpyruvate carboxykinase mRNA levels specifically in Pomc-/- mice, consistent with their insulin-resistant phenotype. Furthermore, CORT induced hypertension in Pomc-/- mice, independently of adipose or liver renin-angiotensin system activation. These data suggest that CORT-inducible 11beta-HSD1 expression in fat contributes to the adverse cardiometabolic effects of CORT in POMC deficiency, whereas higher GR levels may be more important in liver

Hypoxia-driven metabolic reprogramming of adipocytes fuels cancer cell proliferation

OBJECTIVE: Obesity increases the risk of certain cancers, especially tumours that reside close to adipose tissue (breast and ovarian metastasis in the omentum). The obesogenic and tumour micro-environment share a common pathogenic feature, oxygen deprivation (hypoxia). Here we test how hypoxia changes the metabolome of adipocytes to assist cancer cell growth. METHODS: Human and mouse breast and ovarian cancer cell lines were co-cultured with human and mouse adipocytes respectively under normoxia or hypoxia. Proliferation and lipid uptake in cancer cells were measured by commercial assays. Metabolite changes under normoxia or hypoxia were measured in the media of human adipocytes by targeted LC/MS. RESULTS: Hypoxic cancer-conditioned media increased lipolysis in both human and mouse adipocytes. This led to increased transfer of lipids to cancer cells and consequent increased proliferation under hypoxia. These effects were dependent on HIF1α expression in adipocytes, as mouse adipocytes lacking HIF1α showed blunted responses under hypoxic conditions. Targeted metabolomics of the human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes media revealed that culture with hypoxic-conditioned media from non-malignant mammary epithelial cells (MCF10A) can alter the adipocyte metabolome and drive proliferation of the non-malignant cells. CONCLUSION: Here, we show that hypoxia in the adipose-tumour microenvironment is the driving force of the lipid uptake in both mammary and ovarian cancer cells. Hypoxia can modify the adipocyte metabolome towards accelerated lipolysis, glucose deprivation and reduced ketosis. These metabolic shifts in adipocytes could assist both mammary epithelial and cancer cells to bypass the inhibitory effects of hypoxia on proliferation and thrive

Dietary manipulation reveals an unexpected inverse relationship between fat mass and adipose 11β-hydroxysteroid dehydrogenase type 1

Increased dietary fat intake is associated with obesity, insulin resistance, and metabolic disease. In transgenic mice, adipose tissue-specific overexpression of the glucocorticoid-amplifying enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) exacerbates high-fat (HF) diet-induced visceral obesity and diabetes, whereas 11β-HSD1 gene knockout ameliorates this, favoring accumulation of fat in nonvisceral depots. Paradoxically, in normal mice HF diet-induced obesity (DIO) is associated with marked downregulation of adipose tissue 11β-HSD1 levels. To identify the specific dietary fats that regulate adipose 11β-HSD1 and thereby impact upon metabolic disease, we either fed mice diets enriched (45% calories as fat) in saturated (stearate), monounsaturated (oleate), or polyunsaturated (safflower oil) fats ad libitum or we pair fed them a low-fat (11%) control diet for 4 wk. Adipose and liver mass and glucocorticoid receptor and 11β-HSD1 mRNA and activity levels were determined. Stearate caused weight loss and hypoinsulinemia, partly due to malabsorption, and this markedly increased plasma corticosterone levels and adipose 11β-HSD1 activity. Oleate induced pronounced weight gain and hyperinsulinemia in association with markedly low plasma corticosterone and adipose 11β-HSD1 activity. Weight gain and hyperinsulinemia was less pronounced with safflower compared with oleate despite comparable suppression of plasma corticosterone and adipose 11β-HSD1. However, with pair feeding, safflower caused a selective reduction in visceral fat mass and relative insulin sensitization without affecting plasma corticosterone or adipose 11β-HSD1. The dynamic depot-selective relationship between adipose 11β-HSD1 and fat mass strongly implicates a dominant physiological role for local tissue glucocorticoid reactivation in fat mobilization

Glucocorticoid receptor haploinsufficiency causes hypertension and attenuates hypothalamic-pituitary-adrenal axis and blood pressure adaptions to high-fat diet

Glucocorticoid hormones are critical to respond and adapt to stress. Genetic variations in the glucocorticoid receptor (GR) gene alter hypothalamic-pituitary-adrenal (HPA) axis activity and associate with hypertension and susceptibility to metabolic disease. Here we test the hypothesis that reduced GR density alters blood pressure and glucose and lipid homeostasis and limits adaption to obesogenic diet. Heterozygous GR βgeo/+ mice were generated from embryonic stem (ES) cells with a gene trap integration of a β-galactosidase-neomycin phosphotransferase (βgeo) cassette into the GR gene creating a transcriptionally inactive GR fusion protein. Although GRβgeo/+ mice have 50% less functional GR, they have normal lipid and glucose homeostasis due to compensatory HPA axis activation but are hypertensive due to activation of the renin-angiotensin- aldosterone system (RAAS). When challenged with a high-fat diet, weight gain, adiposity, and glucose intolerance were similarly increased in control and GRβgeo/+ mice, suggesting preserved control of intermediary metabolism and energy balance. However, whereas a high-fat diet caused HPA activation and increased blood pressure in control mice, these adaptions were attenuated or abolished in GRβgeo/+ mice. Thus, reduced GR density balanced by HPA activation leaves glucocorticoid functions unaffected but mineralocorticoid functions increased, causing hypertension. Importantly, reduced GR limits HPA and blood pressure adaptions to obesogenic diet

Adipocyte Pseudohypoxia Suppresses Lipolysis and Facilitates Benign Adipose Tissue Expansion

Prolyl hydroxylase enzymes (PHDs) sense cellular oxygen upstream of hypoxia-inducible factor (HIF) signaling, leading to HIF degradation in normoxic conditions. In this study, we demonstrate that adipose PHD2 inhibition plays a key role in the suppression of adipocyte lipolysis. Adipose Phd2 gene ablation in mice enhanced adiposity, with a parallel increase in adipose vascularization associated with reduced circulating nonesterified fatty acid levels and normal glucose homeostasis. Phd2 gene-depleted adipocytes exhibited lower basal lipolysis in normoxia and reduced β-adrenergic-stimulated lipolysis in both normoxia and hypoxia. A selective PHD inhibitor suppressed lipolysis in murine and human adipocytes in vitro and in vivo in mice. PHD2 genetic ablation and pharmacological inhibition attenuated protein levels of the key lipolytic effectors hormone-sensitive lipase and adipose triglyceride lipase (ATGL), suggesting a link between adipocyte oxygen sensing and fatty acid release. PHD2 mRNA levels correlated positively with mRNA levels of AB-hydrolase domain containing-5, an activator of ATGL, and negatively with mRNA levels of lipid droplet proteins, perilipin, and TIP47 in human subcutaneous adipose tissue. Therapeutic pseudohypoxia caused by PHD2 inhibition in adipocytes blunts lipolysis and promotes benign adipose tissue expansion and may have therapeutic applications in obesity or lipodystrophy

A Stratified Transcriptomics Analysis of Polygenic Fat and Lean Mouse Adipose Tissues Identifies Novel Candidate Obesity Genes

Obesity and metabolic syndrome results from a complex interaction between genetic and environmetal factors. In addition to brain-regulated processes, recent genome wide association studies have indicated that genes highly expressed in adipose tissue affect the distribution and function of fat and thus contribute to obesity. Using a stratified transcriptome gene enrichment approach we attempted to identify adipose tissue-specific obesity genes in the unique polygenic fat (F) mouse strain generated by selective breeding over 60 generations for divergent adiposity from a comparator lean (L) strain. To enrich for adipose tissue obesity genes a ˝snap-shot˝ pooled-sample transcriptome comparison of key fat depots and non adipose tissue (muscle, liver, kidney) was performed. Known obesity quantitative trait loci (QTL) information for the model allowed us to further filter genes for increased likelihood of being causal or secondary for obesity. This successfully identified several genes previously linked to obesity (C1qr1, and Np3r) as positional QTL candidate genes elevated specifically in F line adipose tissue.A number of novel obesity candidate genes were also identified (Thbs1, Ppp1rd, Tmepai, Trp53inp2, Ttc7b, Tuba1a, Fgf13, Fmr) that have inferred rolesin fat cell function. Quantitative microarray analysis was then applied to the most phenotypically divergent adipose depot after exaggerating F and L strain differences with chronic high fat feeding which revealed a dictinct gene expression profile of line, fat depot and diet-responsive inflammatory, angiogenic and metabolic pathaways. Selected candidate genes Npr3 and Thbs1, as well as Gys2, a non-QTL gene that otherwise passed our enrichment criteria were characterised, revealing novel functional effects consistent with a contribution to obesity. A focussed candidate gene enrichment strategy in the unique F and L model has identified novel adipose tissue-enriched genes contributing to obesity

Modelling altered glucocorticoid sensitivity : from HPA axis to metabolic abnormalities in mice and humans

The primary determinants of tissue glucocorticoid action are glucocorticoid receptor (GR) density and intracellular levels of ligand, the latter determined both by activity of the hypothalamic-pituitary-adrenal (HPA) axis and cellular activity of 11beta- hydroxysteroid dehydrogenase (11beta-HSD) enzymes that interconvert active 11- hydroxy (corticosterone, cortisol) and inactive 11-keto (11-dehydrocorticosterone, cortisone) glucocorticoids. Here, the contribution of GR density and ligand levels in determining body composition and metabolic phenotype have been investigated in mice and in humans. Genetic evidence in humans implicates variations in the GR gene in the regulation of the HPA axis as well as the control of body fat distribution, metabolic parameters and blood pressure. Although GR deficient mouse models have been previously generated (with homozygous nulls dying at birth), the effects of altered GR density upon fat distribution and blood pressure have not been described. This study addresses the relationship between GR density and metabolic parameters, including body fat distribution, insulin resistance and hypertension. A novel line of mice harbouring a null mutation in the GR gene (GR+/-) was generated from an ES cell line in which a beta-galactosidase-neomycin phosphotransferase (beta geo) reporter cassette was fused with GR. The resulting fusion protein lacks part of the DNA binding domain and the entire ligand binding domain and is transcriptionally inactive. In addition, the beta-galactosidase enzyme activity “reports” activity of the GR gene promoter. GR-/- mice are present in a normal Mendelian ratio before birth. Intriguingly, 1 (of 36/146 expected if null allele not lethal) survived to adulthood suggesting this might be a hypomorphic rather than a null allele. Heterozygous 15 (GR+/-) mice showed 40-45% reductions in GR mRNA levels in the hippocampus, paraventricular nucleus of the hypothalamus, pituitary gland and adipose tissue, 30% in liver, 56% in muscle and 67% in adrenals. X-gal staining of GR+/- brain sections showed that GR-beta gal is present throughout, mirroring GR mRNA expression. Adult GR+/- mice had larger adrenals, higher evening plasma corticosterone levels and greater corticosterone responses following 10 minute restraint suggesting a hyperactive HPA axis. Compared to GR+/+ littermates, GR+/- mice had similar body weight gain on normal chow or high fat diet, with unaltered fat depot (inguinal, epididymal, mesenteric) weights and similar glucose and insulin tolerance. However, GR+/ - mice had higher (10%) systolic blood pressure, associated with activation of the renin-angiotensin system. Thus GR haploinsufficiency in mice causes increased blood pressure and accords with data associating GR polymorphisms with hypertension in humans. The role of altered GC sensitivity was also investigated in a mouse model of HPA axis hypoactivity pro-opiomelanocortin null (POMC) mice. POMC-null mice are obese due to central melanocortin deficiency. In contrast to most rodent models of obesity, POMC-null mice are also glucocorticoid deficient due to ACTH deficiency. Previous data have shown that glucocorticoid replacement in POMC-null mice exaggerated hyperphagia, obesity and insulin resistance and caused hypertension. Here, the contribution of peripheral glucocorticoid sensitivity was investigated. POMC-null mice have increased liver and retroperitoneal fat GR mRNA levels but, specifically in adipose tissue, decreased levels of mRNA encoding 11beta-HSD1, a reductase which regenerates active glucocorticoids, thus amplifying their action.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Innate Immune Cells in the Adipose Tissue in Health and Metabolic Disease

Metabolic disorders, such as obesity, type 2 diabetes mellitus, and nonalcoholic fatty liver disease, are characterized by chronic low-grade tissue and systemic inflammation. During obesity, the adipose tissue undergoes immunometabolic and functional transformation. Adipose tissue inflammation is driven by innate and adaptive immune cells and instigates insulin resistance. Here, we discuss the role of innate immune cells, that is, macrophages, neutrophils, eosinophils, natural killer cells, innate lymphoid type 2 cells, dendritic cells, and mast cells, in the adipose tissue in the healthy (lean) and diseased (obese) state and describe how their function is shaped by the obesogenic microenvironment, and humoral, paracrine, and cellular interactions. Moreover, we particularly outline the role of hypoxia as a central regulator in adipose tissue inflammation. Finally, we discuss the long-lasting effects of adipose tissue inflammation and its potential reversibility through drugs, caloric restriction, or exercise training