Thermogenesis in brown adipose tissue (BAT) is well characterized as being under the control of the sympathetic nervous system. The energy-burning capacity of BAT makes it an attractive target for anti-obesity therapies. However, previous attempts to manipulate BAT’s sympathetic activation have lacked specificity. In this issue of the JCI, Bordicchia et al. provide new data indicating that cardiac natriuretic peptides (NPs) are also able to activate thermogenic machinery in adipose tissue. Their findings suggest a novel strategy to increase energy dissipation in adipose tissue, independent of adrenergic receptors.
Zfp521 is a novel antiadipogenic transcription factor that helps to determine the identity of a mesenchymal cell as bone or fat.
While there has been significant progress in determining the transcriptional cascade involved in terminal adipocyte differentiation, less is known about early events leading to lineage commitment and cell fate choice. It has been recently discovered that zinc finger protein 423 (Zfp423) is an early actor in adipose determination. Here, we show that a close paralog of Zfp423, Zfp521, acts as a key regulator of adipose commitment and differentiation in vitro and in vivo. Zfp521 exerts its actions by binding to early B cell factor 1 (Ebf1), a transcription factor required for the generation of adipocyte progenitors, and inhibiting the expression of Zfp423. Overexpression of Zfp521 in cells greatly inhibits adipogenic potential, whereas RNAi-mediated knock-down or genetic ablation of Zfp521 enhances differentiation. In addition, Zfp521−/− embryos exhibit increased mass of interscapular brown adipose tissue and subcutaneous white adipocytes, a cell autonomous effect. Finally, Ebf1 participates in a negative feedback loop to repress Zfp521 as differentiation proceeds. Because Zfp521 is known to promote bone development, our results suggest that it acts as a critical switch in the commitment decision between the adipogenic and osteogenic lineages.
Adipocytes or fat cells derive from the same stem cells that give rise to bone, cartilage, and muscle. Understanding the switch between bone and fat is of particular medical importance, with implications for diseases like osteoporosis and obesity. Here, we have identified a transcription factor called Zfp521 which is integral to adipogenesis. Prior work has shown that Zfp521 is important in bone formation. Using a variety of overexpression and knockout models, we show that Zfp521 blocks fat cell formation in cultured cells and in living animals. Furthermore, we show that Zfp521 performs this function by binding to another transcription factor, early B cell factor 1 (Ebf1), and repressing the expression of Zfp423, a transcriptional regulator of preadipocyte determination. Finally, we show that Zfp521 is itself repressed by Ebf1, providing a mechanism that turns the levels of this antiadipogenic protein off once the decision to undergo fat cell development has been made. We postulate that Zfp521 regulates one of the earliest events in the lineage switch between bone and fat.
Under-expression of the transcriptional coactivator PGC-1α is causally linked to certain neurodegenerative disorders, including Huntington’s Disease (HD). HD pathoprogression is also associated with aberrant NMDAR activity, in particular an imbalance between synaptic vs. extrasynaptic (NMDAREX) activity. Here we show that PGC-1α controls NMDAREX activity in neurons and that its suppression contributes to mutant Huntingtin (mHtt)-induced increases in NMDAREX activity and vulnerability to excitotoxic insults.
We found that knock-down of endogenous PGC-1α increased NMDAREX activity and vulnerability to excitotoxic insults in rat cortical neurons. In contrast exogenous expression of PGC-1α resulted in a neuroprotective reduction of NMDAREX currents without affecting synaptic NMDAR activity. Since HD models are associated with mHtt-mediated suppression of PGC-1α expression, as well as increased NMDAREX activity, we investigated whether these two events were linked. Expression of mHtt (148Q) resulted in a selective increase in NMDAREX activity, compared to wHtt (18Q), and increased vulnerability to NMDA excitotoxicity. Importantly, we observed that the effects of mHtt and PGC-1α knockdown on NMDAREX activity and vulnerability to excitotoxicity were non-additive and occluded each other, consistent with a common mechanism. Moreover, exogenous expression of PGC-1α reversed mtHtt-mediated increases in NMDAREX activity, and protected neurons against excitotoxic cell death. The link between mHtt, PGC-1α, and NMDAR activity was also confirmed in rat striatal neurons. Thus, targeting levels of PGC-1α expression may help reduce aberrant NMDAREX activity in disorders where PGC-1α is under-expressed.
In this study we investigated the relationship between activity and energy expenditure (EE) in mice. By determining the relationship between activity and EE over a 24 hr period in an individual mouse, activity was calculated to account for 26.6% ± 1.1% of total EE at 30°C. However, when comparing across multiple mice, only 9.53% ± 1.1% of EE from activity appeared to be independent of other components involved in the thermogenic response, suggesting other metabolic processes may mask the contribution of activity to EE. In line with this concept, below thermoneutrality mice still expended a substantial amount of energy on activity; however, at 24°C, 20°C, or 5°C, no independent effect of EE from activity on total daily EE could be detected. Overall these results suggest that when studying mice at temperatures below thermoneutrality, activity is unlikely to explain differences in EE between groups of animals.
► Under standard lab conditions, more-active mice do not expend more energy
Peroxisome proliferator-activated receptor-γ coactivators PGC1α and PGC1β modulate mitochondrial biogenesis and energy homeostasis. The function of these transcriptional coactivators is impaired in obesity, insulin resistance, and type 2 diabetes. We searched for transcriptomic, lipidomic, and electrophysiological alterations in PGC1β−/− hearts potentially associated with increased arrhythmic risk in metabolic diseases.
Methods and results
Microarray analysis in mouse PGC1β−/− hearts confirmed down-regulation of genes related to oxidative phosphorylation and the electron transport chain and up-regulation of hypertrophy- and hypoxia-related genes. Lipidomic analysis showed increased levels of the pro-arrhythmic and pro-inflammatory lipid, lysophosphatidylcholine. PGC1β−/− mouse electrocardiograms showed irregular heartbeats and an increased incidence of polymorphic ventricular tachycardia following isoprenaline infusion. Langendorff-perfused PGC1β−/− hearts showed action potential alternans, early after-depolarizations, and ventricular tachycardia. PGC1β−/− ventricular myocytes showed oscillatory resting potentials, action potentials with early and delayed after-depolarizations, and burst firing during sustained current injection. They showed abnormal diastolic Ca2+ transients, whose amplitude and frequency were increased by isoprenaline, and Ca2+ currents with negatively shifted inactivation characteristics, with increased window currents despite unaltered levels of CACNA1C RNA transcripts. Inwardly and outward rectifying K+ currents were all increased. Quantitiative RT-PCR demonstrated increased SCN5A, KCNA5, RYR2, and Ca2+-calmodulin dependent protein kinase II expression.
PGC1β−/− hearts showed a lysophospholipid-induced cardiac lipotoxicity and impaired bioenergetics accompanied by an ion channel remodelling and altered Ca2+ homeostasis, converging to produce a ventricular arrhythmic phenotype particularly during adrenergic stress. This could contribute to the increased cardiac mortality associated with both metabolic and cardiac disease attributable to lysophospholipid accumulation.
Mitochondria; Cardiac arrhythmia; Peroxisome proliferator-activated receptor-γ coactivator 1β; Metabolic disease; Lysophosphatidylcholine
Optimal lipid storage and mobilization are essential for efficient adipose tissue. Nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) regulates adipocyte differentiation and lipid deposition, but its role in lipolysis and dysregulation in obesity is not well defined. This investigation aimed to understand the molecular impact of dysfunctional PPARγ on the lipolytic axis and to explore whether these defects are also confirmed in common forms of human obesity. For this purpose, we used the P465L PPARγ mouse as a model of dysfunctional PPARγ that recapitulates the human pparγ mutation (P467L). We demonstrated that defective PPARγ impairs catecholamine-induced lipolysis. This abnormal lipolytic response is exacerbated by a state of positive energy balance in leptin-deficient ob/ob mice. We identified the protein kinase A (PKA) network as a PPARγ-dependent regulatory node of the lipolytic response. Specifically, defective PPARγ is associated with decreased basal expression of prkaca (PKAcatα) and d-akap1, the lipase genes Pnplaz (ATGL) and Lipe (HSL), and lipid droplet protein genes fsp27 and adrp in vivo and in vitro. Our data indicate that PPARγ is required for activation of the lipolytic regulatory network, dysregulation of which is an important feature of obesity-induced insulin resistance in humans.
Mitochondria dysfunction contributes to the pathophysiology of obesity, diabetes, neurodegeneration and ageing. The peroxisome proliferator-activated receptor-gamma coactivator-1β (PGC-1β) coordinates mitochondrial biogenesis and function as well as fatty acid metabolism. It has been suggested that endoplasmic reticulum (ER) stress may be one of the mechanisms linking mitochondrial dysfunction and these pathologies. Here we investigate whether PGC-1β ablation affects the ER stress response induced by specific nutritional and pharmacological challenges in the CNS. By using flow cytometry, western blot, real time PCR and several pharmacological and nutritional interventions in PGC-1β knock out and WT mice, we confirmed that PGC-1β coordinates mitochondria function in brain and reported for the first time that a) ablation of PGC-1β is associated with constitutive activation of mTORC1 pathway associated with increased basal GRP78 protein levels in hypothalamus and cortex of animals fed chow diet; and b) in animals fed chronically with high fat diet (HFD) or high protein diet (HPD), we observed a failure to appropriately induce ER stress response in the absence of PGC-1β, associated with an increase in mTOR pathway phosphorylation. This contrasted with the appropriate upregulation of ER stress response observed in wild type littermates. Additionally, inefficient in vitro induction of ER stress by thapsigargin seems result in apoptotic neuronal cell death in PGC-1β KO. Our data indicate that PGC-1β is required for a neuronal ER response to nutritional stress imposed by HFD and HPD diets and that genetic ablation of PGC-1β might increase the susceptibility to neuronal damage and cell death.
► The PGC-1β coordinates mitochondrial function and fatty acid metabolism. ► Ablation of PGC-1β associates with mTORC1 activation and basal increase of GRP78. ► Metabolic stress results in inefficient GRP78 increase in PGC-1β KO mice.
PGC 1 beta; Endoplasmic reticulum stress; Mitochondria; mTOR; Amino acids; Brain
Pressure overload cardiac hypertrophy, a risk factor for heart failure, is associated with reduced mitochondrial fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) proteins that correlate in rodents with reduced PGC-1α expression.
To determine the role of PGC-1β in maintaining mitochondrial energy metabolism and contractile function in pressure overload hypertrophy.
Methods and Results
PGC-1β deficient (KO) mice and wildtype controls (WT) were subjected to transverse aortic constriction (TAC). Although LV function was modestly reduced in young KO hearts, there was no further decline with age so that LV function was similar between KO and WT when TAC was performed. WT – TAC mice developed relatively compensated LVH, despite reduced mitochondrial function and repression of OXPHOS and FAO genes. In non-stressed KO hearts, OXPHOS gene expression and palmitoyl-carnitine supported mitochondrial function were reduced to the same extent as banded WT, but FAO gene expression was normal. Following TAC, KO mice progressed more rapidly to heart failure and developed more severe mitochondrial dysfunction, despite a similar overall pattern of repression of OXPHOS and FAO genes as WT – TAC. However, relative to WT TAC, PGC-1β deficient mice exhibited greater degrees of oxidative stress, decreased cardiac efficiency, lower rates of glucose metabolism and repression of hexokinase II protein.
PGC-1β plays an important role in maintaining baseline mitochondrial function and cardiac contractile function following pressure overload hypertrophy by preserving glucose metabolism and preventing oxidative stress.
Mitochondria; Cardiac Hypertrophy; Heart Failure; Gene Expression
Individuals with metabolic syndrome are at high risk of developing chronic kidney disease (CKD) through unclear pathogenic mechanisms. Obesity and diabetes are known to induce glucolipotoxic effects in metabolically relevant organs. However, the pathogenic role of glucolipotoxicity in the aetiology of diabetic nephropathy is debated. We generated a murine model, the POKO mouse, obtained by crossing the peroxisome proliferator-activated receptor gamma 2 (PPARγ2) knockout (KO) mouse into a genetically obese ob/ob background. We have previously shown that the POKO mice showed: hyperphagia, insulin resistance, hyperglycaemia and dyslipidaemia as early as 4 weeks of age, and developed a complete loss of normal β-cell function by 16 weeks of age. Metabolic phenotyping of the POKO model has led to investigation of the structural and functional changes in the kidney and changes in blood pressure in these mice. Here we demonstrate that the POKO mouse is a model of renal disease that is accelerated by high levels of glucose and lipid accumulation. Similar to ob/ob mice, at 4 weeks of age these animals exhibited an increased urinary albumin:creatinine ratio and significantly increased blood pressure, but in contrast showed a significant increase in the renal hypertrophy index and an associated increase in p27Kip1 expression compared with their obese littermates. Moreover, at 4 weeks of age POKO mice showed insulin resistance, an alteration of lipid metabolism and glomeruli damage associated with increased transforming growth factor beta (TGFβ) and parathyroid hormone-related protein (PTHrP) expression. At this age, levels of proinflammatory molecules, such as monocyte chemoattractant protein-1 (MCP-1), and fibrotic factors were also increased at the glomerular level compared with levels in ob/ob mice. At 12 weeks of age, renal damage was fully established. These data suggest an accelerated lesion through glucolipotoxic effects in the renal pathogenesis in POKO mice.
Mice lacking Peroxisome Proliferator-Activated Receptor γ2 (PPARγ2) have unexpectedly normal glucose tolerance and mild insulin resistance. Mice lacking PPARγ2 were found to have elevated levels of Lipocalin prostaglandin D synthase (L-PGDS) expression in BAT and subcutaneous white adipose tissue (WAT). To determine if induction of L-PGDS was compensating for a lack of PPARγ2, we crossed L-PGDS KO mice to PPARγ2 KO mice to generate Double Knock Out mice (DKO). Using DKO mice we demonstrated a requirement of L-PGDS for maintenance of subcutaneous WAT (scWAT) function. In scWAT, DKO mice had reduced expression of thermogenic genes, the de novo lipogenic program and the lipases ATGL and HSL. Despite the reduction in markers of lipolysis in scWAT, DKO mice had a normal metabolic rate and elevated serum FFA levels compared to L-PGDS KO alone. Analysis of intra-abdominal white adipose tissue (epididymal WAT) showed elevated expression of mRNA and protein markers of lipolysis in DKO mice, suggesting that DKO mice may become more reliant on intra-abdominal WAT to supply lipid for oxidation. This switch in depot utilisation from subcutaneous to epididymal white adipose tissue was associated with a worsening of whole organism metabolic function, with DKO mice being glucose intolerant, and having elevated serum triglyceride levels compared to any other genotype. Overall, L-PGDS and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism.
Characterization of a peripheral hormonal system identifies the origin and mechanisms of regulation of glucocorticoid hormone oscillations in rats.
Oscillating levels of adrenal glucocorticoid hormones are essential for optimal gene expression, and for maintaining physiological and behavioural responsiveness to stress. The biological basis for these oscillations is not known, but a neuronal “pulse generator” within the hypothalamus has remained a popular hypothesis. We demonstrate that pulsatile hypothalamic activity is not required for generating ultradian glucocorticoid oscillations. We show that a constant level of corticotrophin-releasing hormone (CRH) can activate a dynamic pituitary-adrenal peripheral network to produce ultradian adrenocorticotrophic hormone and glucocorticoid oscillations with a physiological frequency. This oscillatory response to CRH is dose dependent and becomes disrupted for higher levels of CRH. These data suggest that glucocorticoid oscillations result from a sub-hypothalamic pituitary-adrenal system, which functions as a deterministic peripheral hormone oscillator with a characteristic ultradian frequency. This constitutes a novel mechanism by which the level, rather than the pattern, of CRH determines the dynamics of glucocorticoid hormone secretion.
Glucocorticoid steroid hormones, such as cortisol and corticosterone, provide a rapid response to both physical and psychological stress, and act on areas of the brain that influence learning, memory, and behaviour. Glucocorticoids are released from the adrenal glands in near-hourly pulses, which results in oscillating glucocorticoid levels in the blood and in target organs. These hormone oscillations can become disrupted during ageing and in stress-related disease (e.g., major depression), so it is important to identify the underlying mechanisms that govern their dynamics. Although the origin of the oscillations is not known, it is assumed that they are generated by a neuronal “pulse generator” within the brain. In this study, we present data that challenge this hypothesis. We characterize a peripheral hormonal system and show that constant levels of corticotrophin-releasing hormone can induce and regulate hormone oscillations independent of the brain. We also describe mechanisms that can disrupt these oscillations. These findings have important implications for our understanding of glucocorticoid signalling in both health and disease, and will be important for the design of novel treatment strategies that take into account timing of hormone administration to patients undergoing steroid therapy for inflammatory or malignant disease.
Thermogenesis in brown adipose tissue (BAT) is fundamental to energy balance and is also relevant for humans. Bone morphogenetic proteins (BMPs) regulate adipogenesis, and, here, we describe a role for BMP8B in the direct regulation of thermogenesis. BMP8B is induced by nutritional and thermogenic factors in mature BAT, increasing the response to noradrenaline through enhanced p38MAPK/CREB signaling and increased lipase activity. Bmp8b−/− mice exhibit impaired thermogenesis and reduced metabolic rate, causing weight gain despite hypophagia. BMP8B is also expressed in the hypothalamus, and Bmp8b−/− mice display altered neuropeptide levels and reduced phosphorylation of AMP-activated protein kinase (AMPK), indicating an anorexigenic state. Central BMP8B treatment increased sympathetic activation of BAT, dependent on the status of AMPK in key hypothalamic nuclei. Our results indicate that BMP8B is a thermogenic protein that regulates energy balance in partnership with hypothalamic AMPK. BMP8B may offer a mechanism to specifically increase energy dissipation by BAT.
► BMP8B is expressed in BAT and is regulated in response to thermogenic stimuli ► BMP8B increases the thermogenic response of BAT to adrenergic stimulation ► Loss of BMP8B substantially increases susceptibility to diet-induced obesity ► BMP8B acts in the CNS to increase SNS activation of thermogenesis
Cold exposure and a high-fat diet trigger BMP8 signaling in brown adipose tissue and in the brain to boost energy expenditure.
Brown adipocytes can differentiate from white fat progenitor cells in mice exposed to cold or β3-adrenergic stimulation, and this process is regulated by a microRNA that regulates the expression of Hoxc8, a master regulator of brown adipogenesis.
The recent discovery of functional brown adipocytes in adult humans illuminates the potential of these cells in the treatment of obesity and its associated diseases. In rodents, brown adipocyte-like cells are known to be recruited in white adipose tissue (WAT) by cold exposure or β-adrenergic stimulation, but the molecular machinery underlying this phenomenon is not fully understood. Here, we show that inducible brown adipogenesis is mediated by the microRNA miR-196a. We found that miR-196a suppresses the expression of the white-fat gene Hoxc8 post-transcriptionally during the brown adipogenesis of white fat progenitor cells. In mice, miR-196a is induced in the WAT-progenitor cells after cold exposure or β-adrenergic stimulation. The fat-specific forced expression of miR-196a in mice induces the recruitment of brown adipocyte-like cells in WAT. The miR-196a transgenic mice exhibit enhanced energy expenditure and resistance to obesity, indicating the induced brown adipocyte-like cells are metabolically functional. Mechanistically, Hoxc8 targets and represses C/EBPβ, a master switch of brown-fat gene program, in cooperation with histone deacetylase 3 (HDAC3) through the C/EBPβ 3′ regulatory sequence. Thus, miR-196a induces functional brown adipocytes in WAT through the suppression of Hoxc8, which functions as a gatekeeper of the inducible brown adipogenesis. The miR-196a-Hoxc8-C/EBPβ signaling pathway may be a therapeutic target for inducing brown adipogenesis to combat obesity and type 2 diabetes.
Obesity is caused by the accumulation of surplus energy in a fatty tissue called white adipose tissue (WAT) and can lead to important health problems such as diabetes. Mammals additionally possess brown adipose tissue (BAT), which serves to generate body heat to stabilize body temperature under exposure to cold, and is abundant in hibernating animals and human neonates. In performing its function BAT consumes energy, thereby reducing WAT fat accumulation. Recent studies have shown that exposure to a cold environment stimulates the partial conversion of WAT to BAT in mice, and given that human adults have a limited amount of BAT, such a conversion has the potential to afford a novel method of obesity control. Here, we analyze the molecular mechanism of this conversion using genetically manipulated mice and cells isolated from human adipose tissue. We find that the expression levels of a microRNA, miR-196a, positively correlate with the conversion of WAT to BAT under cold exposure conditions. We show that forced expression of miR-196a in mouse adipose tissue increases BAT content and energy expenditure, thereby rendering the animals resistant to obesity and diabetes. Mechanistically, we observe that miR-196a acts by inhibiting the expression of the homeotic gene Hoxc8, a repressor of brown adipogenesis. These findings introduce the therapeutic possibility of using microRNAs to control obesity and its associated diseases in humans.
Obesity-associated insulin resistance is characterized by a state of chronic, low-grade inflammation that is associated with the accumulation of M1 proinflammatory macrophages in adipose tissue. Although different evidence explains the mechanisms linking the expansion of adipose tissue and adipose tissue macrophage (ATM) polarization, in the current study we investigated the concept of lipid-induced toxicity as the pathogenic link that could explain the trigger of this response.
RESEARCH DESIGN AND METHODS
We addressed this question using isolated ATMs and adipocytes from genetic and diet-induced murine models of obesity. Through transcriptomic and lipidomic analysis, we created a model integrating transcript and lipid species networks simultaneously occurring in adipocytes and ATMs and their reversibility by thiazolidinedione treatment.
We show that polarization of ATMs is associated with lipid accumulation and the consequent formation of foam cell–like cells in adipose tissue. Our study reveals that early stages of adipose tissue expansion are characterized by M2-polarized ATMs and that progressive lipid accumulation within ATMs heralds the M1 polarization, a macrophage phenotype associated with severe obesity and insulin resistance. Furthermore, rosiglitazone treatment, which promotes redistribution of lipids toward adipocytes and extends the M2 ATM polarization state, prevents the lipid alterations associated with M1 ATM polarization.
Our data indicate that the M1 ATM polarization in obesity might be a macrophage-specific manifestation of a more general lipotoxic pathogenic mechanism. This indicates that strategies to optimize fat deposition and repartitioning toward adipocytes might improve insulin sensitivity by preventing ATM lipotoxicity and M1 polarization.
The progression towards type 2 diabetes depends on the allostatic response of pancreatic beta cells to synthesise and secrete enough insulin to compensate for insulin resistance. The endocrine pancreas is a plastic tissue able to expand or regress in response to the requirements imposed by physiological and pathophysiological states associated to insulin resistance such as pregnancy, obesity or ageing, but the mechanisms mediating beta cell mass expansion in these scenarios are not well defined. We have recently shown that ob/ob mice with genetic ablation of PPARγ2, a mouse model known as the POKO mouse failed to expand its beta cell mass. This phenotype contrasted with the appropriate expansion of the beta cell mass observed in their obese littermate ob/ob mice. Thus, comparison of these models islets particularly at early ages could provide some new insights on early PPARγ dependent transcriptional responses involved in the process of beta cell mass expansion
Here we have investigated PPARγ dependent transcriptional responses occurring during the early stages of beta cell adaptation to insulin resistance in wild type, ob/ob, PPARγ2 KO and POKO mice. We have identified genes known to regulate both the rate of proliferation and the survival signals of beta cells. Moreover we have also identified new pathways induced in ob/ob islets that remained unchanged in POKO islets, suggesting an important role for PPARγ in maintenance/activation of mechanisms essential for the continued function of the beta cell.
Our data suggest that the expansion of beta cell mass observed in ob/ob islets is associated with the activation of an immune response that fails to occur in POKO islets. We have also indentified other PPARγ dependent differentially regulated pathways including cholesterol biosynthesis, apoptosis through TGF-β signaling and decreased oxidative phosphorylation.
A genetic and pharmacological approach reveals novel insights into how changes in gut microbiota can subvert genetically predetermined phenotypes from lean to obese.
Environmental factors and host genetics interact to control the gut microbiota, which may have a role in the development of obesity and insulin resistance. TLR2-deficient mice, under germ-free conditions, are protected from diet-induced insulin resistance. It is possible that the presence of gut microbiota could reverse the phenotype of an animal, inducing insulin resistance in an animal genetically determined to have increased insulin sensitivity, such as the TLR2 KO mice. In the present study, we investigated the influence of gut microbiota on metabolic parameters, glucose tolerance, insulin sensitivity, and signaling of TLR2-deficient mice. We investigated the gut microbiota (by metagenomics), the metabolic characteristics, and insulin signaling in TLR2 knockout (KO) mice in a non-germ free facility. Results showed that the loss of TLR2 in conventionalized mice results in a phenotype reminiscent of metabolic syndrome, characterized by differences in the gut microbiota, with a 3-fold increase in Firmicutes and a slight increase in Bacteroidetes compared with controls. These changes in gut microbiota were accompanied by an increase in LPS absorption, subclinical inflammation, insulin resistance, glucose intolerance, and later, obesity. In addition, this sequence of events was reproduced in WT mice by microbiota transplantation and was also reversed by antibiotics. At the molecular level the mechanism was unique, with activation of TLR4 associated with ER stress and JNK activation, but no activation of the IKKβ-IκB-NFκB pathway. Our data also showed that in TLR2 KO mice there was a reduction in regulatory T cell in visceral fat, suggesting that this modulation may also contribute to the insulin resistance of these animals. Our results emphasize the role of microbiota in the complex network of molecular and cellular interactions that link genotype to phenotype and have potential implications for common human disorders involving obesity, diabetes, and even other immunological disorders.
An intricate interaction between genetic and environmental factors influences the development of obesity and diabetes. Previous studies have shown that mice lacking an important receptor of the innate immune system, Toll-like Receptor 2 (TLR2), are protected from insulin resistance. Given that the innate immune system has emerged as a key regulator of the gut microbiota, we undertook to investigate in this study whether the gut microbiota have a role in modulating the response to insulin. By rearing these TLR2 mutant mice in conventional facilities (as opposed to “germ-free” conditions) we figured that they would develop an altered gut microbiota. In contrast to previous studies, our results show that these TLR2 mutant mice now develop a diseased phenotype reminiscent of metabolic syndrome, including weight gain, and end up with gut microbiota similar to that found in obese mice and humans. These mice could be rescued by treatment with broad-spectrum antibiotics, which decimated the microbiota. Conversely, transplantation of the gut microbiota from these mice to wild-type mice induced weight gain and the metabolic syndrome phenotype. Our results indicate that the gut microbiota per se can subvert a genetically predetermined condition previously described as being protective towards obesity and insulin resistance into a phenotype associated with weight gain and its complications, such as glucose intolerance and diabetes.
The alveolar macrophage (AM) - first line of innate immune defence against pathogens and environmental irritants - constitutively expresses peroxisome-proliferator activated receptor γ (PPARγ). PPARγ ligand-induced activation keeps the AM quiescent, and thereby contributes to combat invaders and resolve inflammation by augmenting the phagocytosis of apoptotic neutrophils and inhibiting an excessive expression of inflammatory genes. Because of these presumed anti-inflammatory functions of PPARγ we tested the hypothesis, whether reduced functional receptor availability in mutant mice resulted in increased cellular and molecular inflammatory response during acute inflammation and/or in an impairment of its resolution.
To address this hypothesis we examined the effects of a carbon-nanoparticle (CNP) lung challenge, as surrogate for non-infectious environmental irritants, in a murine model carrying a dominant-negative point mutation in the ligand-binding domain of PPARγ (P465L/wt). Animals were instilled intratracheally with Printex 90 CNPs and bronchoalveolar lavage (BAL) was gained 24 h or 72 h after instillation to investigate its cellular and protein composition.
Higher BAL cell numbers - due to higher macrophage counts - were found in mutants irrespective of treatment. Neutrophil numbers in contrast were slightly lower in mutants. Intratracheal CNP instillation resulted in a profound recruitment of inflammatory neutrophils into the alveolus, but genotype related differences at acute inflammation (24 h) and resolution (72 h) were not observed. There were no signs for increased alveolar-capillary membrane damage or necrotic cell death in mutants as determined by BAL protein and lactate-dehydrogenase content. Pro-inflammatory macrophage-derived cytokine osteopontin was higher, but galectin-3 lower in female mutants. CXCL5 and lipocalin-2 markers, attributed to epithelial cell stimulation did not differ.
Despite general genotype-related differences, we had to reject our hypothesis of an increased CNP induced lung inflammation and an impairment of its resolution in PPARγ defective mice. Although earlier studies showed ligand-induced activation of nuclear receptor PPARγ to promote resolution of lung inflammation, its reduced activity did not provide signs of resolution impairment in the settings investigated here.
peroxisome-proliverator activated receptor γ; carbon-nano particle; pulmonary inflammation; chronic lung disease; challenge; immune cell; broncho-alveolar lavage (BAL); inflammatory marker
Genetic disruption of the pancreatic mesenchyme reveals that it is critical for the expansion of epithelial progenitors and for the proliferation of insulin-producing beta cells.
The developing pancreatic epithelium gives rise to all endocrine and exocrine cells of the mature organ. During organogenesis, the epithelial cells receive essential signals from the overlying mesenchyme. Previous studies, focusing on ex vivo tissue explants or complete knockout mice, have identified an important role for the mesenchyme in regulating the expansion of progenitor cells in the early pancreas epithelium. However, due to the lack of genetic tools directing expression specifically to the mesenchyme, the potential roles of this supporting tissue in vivo, especially in guiding later stages of pancreas organogenesis, have not been elucidated. We employed transgenic tools and fetal surgical techniques to ablate mesenchyme via Cre-mediated mesenchymal expression of Diphtheria Toxin (DT) at the onset of pancreas formation, and at later developmental stages via in utero injection of DT into transgenic mice expressing the Diphtheria Toxin receptor (DTR) in this tissue. Our results demonstrate that mesenchymal cells regulate pancreatic growth and branching at both early and late developmental stages by supporting proliferation of precursors and differentiated cells, respectively. Interestingly, while cell differentiation was not affected, the expansion of both the endocrine and exocrine compartments was equally impaired. To further elucidate signals required for mesenchymal cell function, we eliminated β-catenin signaling and determined that it is a critical pathway in regulating mesenchyme survival and growth. Our study presents the first in vivo evidence that the embryonic mesenchyme provides critical signals to the epithelium throughout pancreas organogenesis. The findings are novel and relevant as they indicate a critical role for the mesenchyme during late expansion of endocrine and exocrine compartments. In addition, our results provide a molecular mechanism for mesenchymal expansion and survival by identifying β-catenin signaling as an essential mediator of this process. These results have implications for developing strategies to expand pancreas progenitors and β-cells for clinical transplantation.
Embryonic development is a highly complex process that requires tight orchestration of cellular proliferation, differentiation, and migration as cells grow within loosely aggregated mesenchyme and more organized epithelial sheets to form organs and tissues. In addition to intrinsic cell-autonomous signals, these events are further regulated by environmental cues provided by neighboring cells. Prior work demonstrated a critical role for the surrounding mesenchyme in guiding epithelial growth during the early stages of pancreas development. However, it remained unclear whether the mesenchyme also guided the later stages of pancreas organogenesis when the functional exocrine and endocrine cells are formed. Here, we show that specific genetic ablation of the mesenchyme at distinct developmental stages in vivo results in the formation of a smaller, misshapen pancreas. Loss of the mesenchyme profoundly impairs the expansion of both endocrine and exocrine pancreatic progenitors, as well as the proliferative capacity of maturing cells, including insulin-producing beta-cells. Thus, our studies reveal unappreciated roles for the mesenchyme in guiding the formation of the epithelial pancreas throughout development. The results suggest that identifying the specific mesenchymal signals might help to optimize cell culture protocols that aim to achieve the differentiation of stem cells into insulin-producing beta cells.
Hypoxia-inducible factor (HIF) is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance.
The hypothalamus in the brain is a master regulator of feeding and body weight. The regulation of it is mediated by the ability of the hypothalamus to sense nutrients (most importantly glucose) and hormones (such as insulin and leptin). While hormone has been extensively studied, we know less about how the hypothalamus can sense nutrients. It is also unclear whether changes in hypothalamic nutrient sensing can influence the development of obesity and related disease, and could therefore be targeted for disease intervention. In this study, we show that a protein termed hypoxia-inducible factor (HIF) is normally present in the hypothalamus and able to respond to glucose. This glucose response leads to the up-regulation of a hypothalamic neuropeptide, POMC, a pivotal molecule that controls feeding and body weight balance. We then developed a mouse model in which HIF is disrupted in hypothalamic cells that express POMC. These mice displayed reduced hypothalamic sensitivity to glucose, resulting in overeating and susceptibility to obesity. Furthermore, we found that delivery of the HIF gene into the hypothalamus has strong anti-obesity effects in mice. We conclude that HIF is a molecular mediator of hypothalamic glucose sensing and can be potentially targeted for obesity therapeutics.
Two crucial biological processes are (1) the sensing and coordination of responses to low oxygen levels and (2) the control of food intake and energy expenditure. The hypoxia-inducible factor (HIF) family of proteins is known to regulate responses to low oxygen, whereas neuropeptides derived from proopiomelanocortin (POMC) are implicated in the control of food intake and energy expenditure. It is now becoming apparent that these two apparently disparate processes may be linked, with the exciting discovery that HIF proteins can act in the brain to regulate food intake and energy expenditure as reported in the current issue of PLoS Biology. This primer discusses the traditional role of HIF proteins in terms of responding to oxygen levels in the periphery and also their new role in coordinating responses to nutrients in the brain through regulation of POMC.
This study aims to investigate whether orexigenic antipsychotic drugs may induce dyslipidemia and glucose disturbances in female rats through direct perturbation of metabolically active peripheral tissues, independent of prior weight gain.
In the current study, we examined whether a single intraperitoneal injection of clozapine or olanzapine induced metabolic disturbances in adult female outbred Sprague–Dawley rats. Serum glucose and lipid parameters were measured during time-course experiments up to 48 h. Real-time quantitative PCR was used to measure specific transcriptional alterations in lipid and carbohydrate metabolism in adipose tissue depots or in the liver.
Our results demonstrated that acute administration of clozapine or olanzapine induced a rapid, robust elevation of free fatty acids and glucose in serum, followed by hepatic accumulation of lipids evident after 12–24 h. These metabolic disturbances were associated with biphasic patterns of gluconeogenic and lipid-related gene expression in the liver and in white adipose tissue depots.
Our results support that clozapine and olanzapine are associated with primary effects on carbohydrate and lipid metabolism associated with transcriptional changes in metabolically active peripheral tissues prior to the development of drug-induced weight gain.
Electronic supplementary material
The online version of this article (doi:10.1007/s00213-011-2397-y) contains supplementary material, which is available to authorized users.
Antipsychotic; Animal model; Clozapine; Energy; Metabolism; Fatty acid; Lipid; Glucose; Diabetes; Obesity; Gene expression
In recent years, there has been accumulating evidence that microRNAs are key regulator molecules of gene expression. The cellular processes that are regulated by microRNAs include e.g. cell proliferation, programmed cell death and cell differentiation. Adipocyte differentiation is a highly regulated cellular process for which several important regulating factors have been discovered, but still not all are known to fully understand the underlying mechanisms. In the present study, we analyzed the expression of 597 microRNAs during the differentiation of mouse mesenchymal stem cells into terminally differentiated adipocytes by real-time RT-PCR. In total, 66 miRNAs were differentially expressed in mesenchymal stem cell-derived adipocytes compared to the undifferentiated progenitor cells. To further study the regulation of these 66 miRNAs in white adipose tissue in vivo and their dependence on PPARγ activity, mouse models of genetically or diet induced obesity as well as a mouse line expressing a dominant negative PPARγ mutant were employed.
The success of antipsychotic drug treatment in patients with schizophrenia is limited by the propensity of these drugs to induce hyperphagia, weight gain and other metabolic disturbances, particularly evident for olanzapine and clozapine. However, the molecular mechanisms involved in antipsychotic-induced hyperphagia remain unclear. Here, we investigate the effect of olanzapine administration on the regulation of hypothalamic mechanisms controlling food intake, namely neuropeptide expression and AMP-activated protein kinase (AMPK) phosphorylation in rats. Our results show that subchronic exposure to olanzapine upregulates neuropeptide Y (NPY) and agouti related protein (AgRP) and downregulates proopiomelanocortin (POMC) in the arcuate nucleus of the hypothalamus (ARC). This effect was evident both in rats fed ad libitum and in pair-fed rats. Of note, despite weight gain and increased expression of orexigenic neuropeptides, subchronic administration of olanzapine decreased AMPK phosphorylation levels. This reduction in AMPK was not observed after acute administration of either olanzapine or clozapine. Overall, our data suggest that olanzapine-induced hyperphagia is mediated through appropriate changes in hypothalamic neuropeptides, and that this effect does not require concomitant AMPK activation. Our data shed new light on the hypothalamic mechanism underlying antipsychotic-induced hyperphagia and weight gain, and provide the basis for alternative targets to control energy balance.
The authors describe a new approach to studying cellular lipid profiles and
propose a compensatory mechanism that may help maintain the normal membrane
function of adipocytes in the context of obesity.
Identification of early mechanisms that may lead from obesity towards
complications such as metabolic syndrome is of great interest. Here we performed
lipidomic analyses of adipose tissue in twin pairs discordant for obesity but
still metabolically compensated. In parallel we studied more evolved states of
obesity by investigating a separated set of individuals considered to be
morbidly obese. Despite lower dietary polyunsaturated fatty acid intake, the
obese twin individuals had increased proportions of palmitoleic and arachidonic
acids in their adipose tissue, including increased levels of ethanolamine
plasmalogens containing arachidonic acid. Information gathered from these
experimental groups was used for molecular dynamics simulations of lipid
bilayers combined with dependency network analysis of combined clinical,
lipidomics, and gene expression data. The simulations suggested that the
observed lipid remodeling maintains the biophysical properties of lipid
membranes, at the price, however, of increasing their vulnerability to
inflammation. Conversely, in morbidly obese subjects, the proportion of
plasmalogens containing arachidonic acid in the adipose tissue was markedly
decreased. We also show by in vitro Elovl6 knockdown that the lipid network
regulating the observed remodeling may be amenable to genetic modulation.
Together, our novel approach suggests a physiological mechanism by which
adaptation of adipocyte membranes to adipose tissue expansion associates with
positive energy balance, potentially leading to higher vulnerability to
inflammation in acquired obesity. Further studies will be needed to determine
the cause of this effect.
Obesity is characterized by excess body fat, which is predominantly stored in the
adipose tissue. When adipose tissue expands too much it stops storing lipid
appropriately. The excess lipid accumulates in organs such as muscle, liver, and
pancreas, causing metabolic disease. In this study, we aim to identify factors
that cause adipose tissue to malfunction when it reaches its limit of expansion.
We performed lipidomic analyses of human adipose tissue in twin pairs discordant
for obesity—that is, one of the twins was lean and one was obese—but
still metabolically healthy. We identified multiple changes in membrane
phospholipids. Using computer modeling, we show that “lean” and
“obese” membrane lipid compositions have the same physical
properties despite their different compositions. We hypothesize that this
represents allostasis—changes in lipid membrane composition in obesity
occur to protect the physical properties of the membranes. However, protective
changes cannot occur without a cost, and accordingly we demonstrate that
switching to the “obese” lipid composition is associated with higher
levels of adipose tissue inflammation. In a separate group of metabolically
unhealthy obese individuals we investigated how the processes that regulate the
“lean” and “obese” lipid profiles are changed. To
determine how these lipid membrane changes are regulated we constructed an
in silico network model that identified key control points
and potential molecular players. We validated this network by performing genetic
manipulations in cell models. Therapeutic targeting of this network may open new
opportunities for the prevention or treatment of obesity-related metabolic