Featured ArticlesVolume 8 | February 2018
|Differential contribution of POMC and AgRP neurons to the regulation of autonomic nerve activity by leptinLeptin acts in the brain as part of a negative feedback mechanism to control adiposity through stimulation of sympathetic nerve activity (SNA). The arcuate nucleus (ARC) contains multiple unique leptin receptor (LepR) positive neuronal populations including those expressing proopiomelanocortin (POMC) and agouti-related peptide (AgRP). In the present study, Bell et al. show that POMC and AgRP neurons differentially contribute to leptin control of regional activity of the autonomic nervous system. These findings support the notion that leptin engages different neuronal populations in a selective manner to control the activity of the sympathetic and parasympathetic nervous systems.|
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Objectives: The autonomic nervous system is critically involved in mediating the control by leptin of many physiological processes. Here, we examined the role of the leptin receptor (LepR) in proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons in mediating the effects of leptin on regional sympathetic and parasympathetic nerve activity.
Methods: We analyzed how deletion of the LepR in POMC neurons (POMCCre/LepRfl/fl mice) or AgRP neurons (AgRPCre/LepRfl/fl mice) affects the ability of leptin to increase sympathetic and parasympathetic nerve activity. We also studied mice lacking the catalytic p110α or p110β subunits of phosphatidylinositol-3 kinase (PI3K) in POMC neurons.
Leptin-evoked increase in sympathetic nerve activity subserving thermogenic brown adipose tissue was partially blunted in mice lacking the LepR in either POMC or AgRP neurons. On the other hand, loss of the LepR in AgRP, but not POMC, neurons interfered with leptin-induced sympathetic nerve activation to the inguinal fat depot. The increase in hepatic sympathetic traffic induced by leptin was also reduced in mice lacking the LepR in AgRP, but not POMC, neurons whereas LepR deletion in either AgRP or POMC neurons attenuated the hepatic parasympathetic nerve activation evoked by leptin. Interestingly, the renal, lumbar and splanchnic sympathetic nerve activation caused by leptin were significantly blunted in POMCCre/LepRfl/fl mice, but not in AgRPCre/LepRfl/fl mice. However, loss of the LepR in POMC or AgRP neurons did not interfere with the ability of leptin to increase sympathetic traffic to the adrenal gland. Furthermore, ablation of the p110α, but not the p110β, isoform of PI3K from POMC neurons eliminated the leptin-elicited renal sympathetic nerve activation.
Conclusions: POMC and AgRP neurons are differentially involved in mediating the effects of leptin on autonomic nerve activity subserving various tissues and organs.[Hide abstract]
|Leptin transport across brain barriers controls food rewardLeptin is a key hormone controlling body weight by inhibiting food intake and stimulating energy expenditure. In addition, leptin receptor (LepR) regulates food reward. Until now, it was unclear whether leptin has to penetrate brain barriers to exert its central effects. To directly assess the role of LepR in leptin uptake by the brain and to elucidate LepR function in CNS barriers for body weight regulation, Di Spiezio and colleagues deleted all LepR isoforms selectively in brain endothelial cells and the choroid plexus. When deleting LepR in brain endothelial and epithelial cells, the uptake of leptin by the brain was reduced and body weight was increased on a high-fat diet but not on normal chow. Further experiments revealed that LepR in brain barriers inhibits food reward.|
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Objective: Leptin is a key hormone in the control of appetite and body weight. Predominantly produced by white adipose tissue, it acts on the brain to inhibit homeostatic feeding and food reward. Leptin has free access to circumventricular organs, such as the median eminence, but entry into other brain centers is restricted by the blood–brain and blood–CSF barriers. So far, it is unknown for which of its central effects leptin has to penetrate brain barriers. In addition, the mechanisms mediating the transport across barriers are unclear although high expression in brain barriers suggests an important role of the leptin receptor (LepR).
Methods: We selectively deleted LepR in brain endothelial and epithelial cells of mice (LepRbeKO). The expression of LepR in fenestrated vessels of the periphery and the median eminence as well as in tanycytes was not affected.
Results: Perfusion studies showed that leptin uptake by the brain depended on LepR in brain barriers. When being fed with a rewarding high-fat diet LepRbeKO mice gained more body weight than controls. The aggravated obesity of LepRbeKO mice was due to hyperphagia and a higher sensitivity to food reward.
Conclusions: The LepR-mediated transport of leptin across brain barriers in endothelial cells lining microvessels and in epithelial cells of the choroid plexus controls food reward but is apparently not involved in homeostatic control of feeding.[Hide abstract]
|Inhibition of ceramide synthesis restores insulin signaling and enhances β-cell function Obesity following a high fat diet (HFD) but also intra-hypothalamic injections of saturated fatty acids (FFAs) have been shown to induce central lipotoxicity. Central lipotoxicity has also been shown to be deleterious for the hypothalamic control of glucose homeostasis. Campana and colleagues deciphered the role of endogenous ceramide synthesis on the deregulation of hypothalamic insulin signaling and its consequences on glucose homeostasis. They found that de novo ceramide synthesis induced a hypothalamic insulin resistance. Interestingly, they also found that inhibition of de novo ceramide synthesis in the hypothalami of obese Zucker rats improved glucose tolerance.|
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Objectives: Hypothalamic lipotoxicity has been shown to induce central insulin resistance and dysregulation of glucose homeostasis; nevertheless, elucidation of the regulatory mechanisms remains incomplete. Here, we aimed to determine the role of de novo ceramide synthesis in hypothalamus on the onset of central insulin resistance and the dysregulation of glucose homeostasis induced by obesity.
Methods: Hypothalamic GT1-7 neuronal cells were treated with palmitate. De novo ceramide synthesis was inhibited either by pharmacological (myriocin) or molecular (si-Serine Palmitoyl Transferase 2, siSPT2) approaches. Obese Zucker rats (OZR) were intracerebroventricularly infused with myriocin to inhibit de novo ceramide synthesis. Insulin resistance was determined by quantification of Akt phosphorylation. Ceramide levels were quantified either by a radioactive kinase assay or by mass spectrometry analysis. Glucose homeostasis were evaluated in myriocin-treated OZR. Basal and glucose-stimulated parasympathetic tonus was recorded in OZR. Insulin secretion from islets and β-cell mass was also determined.
Results: We show that palmitate impaired insulin signaling and increased ceramide levels in hypothalamic neuronal GT1-7 cells. In addition, the use of deuterated palmitic acid demonstrated that palmitate activated several enzymes of the de novo ceramide synthesis pathway in hypothalamic cells. Importantly, myriocin and siSPT2 treatment restored insulin signaling in palmitate-treated GT1-7 cells. Protein kinase C (PKC) inhibitor or a dominant-negative PKCζ also counteracted palmitate-induced insulin resistance. Interestingly, attenuating the increase in levels of hypothalamic ceramides with intracerebroventricular infusion of myriocin in OZR improved their hypothalamic insulin-sensitivity. Importantly, central myriocin treatment partially restored glucose tolerance in OZR. This latter effect is related to the restoration of glucose-stimulated insulin secretion and an increase in β-cell mass of OZR. Electrophysiological recordings also showed an improvement of glucose-stimulated parasympathetic nerve activity in OZR centrally treated with myriocin.
Conclusion: Our results highlight a key role of hypothalamic de novo ceramide synthesis in central insulin resistance installation and glucose homeostasis dysregulation associated with obesity.[Hide abstract]
|A gut-brain axis regulating glucose metabolism mediated by bile acids and FGFBile acids modulate glucose homeostasis by direct actions on two receptors, G-protein coupled bile acid receptor 1 (GPBAR1) and farnesoid X receptor (FXR). One well known role for FXR is as a bile acid sensor within the enterocyte that increases the production of fibroblast growth factor 15 (FGF15; FGF19 is the human ortholog). Liu et al. provide evidence that bile acid signaling via FXR affects ileal FGF15/19 production, causing activation of FGFR1 on melanocortinergic neurons to significantly improve glucose tolerance. They identified a gut-brain axis wherein bile acid and FGF15/19 can improve glucose handling as well as short FGF-derived peptides that improve glucose homeostasis.|
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Objective: Bile acids have been implicated as important regulators of glucose metabolism via activation of FXR and GPBAR1. We have previously shown that FGF19 can modulate glucose handling by suppressing the activity of hypothalamic AGRP/NPY neurons. As bile acids stimulate the release of FGF19/FGF15 into the circulation, we pursued the potential of bile acids to improve glucose tolerance via a gut–brain axis involving FXR and FGF15/FGF19 within enterocytes and FGF receptors on hypothalamic AGRP/NPY neurons.
Methods: A 5-day gavage of taurocholic acid, mirroring our previous protocol of a 5-day FGF19 treatment, was performed. Oral glucose tolerance tests in mice with genetic manipulations of FGF signaling and melanocortin signaling were used to define a gut–brain axis responsive to bile acids.
Results: The taurocholic acid gavage led to increased serum concentrations of taurocholic acid as well as increases of FGF15 mRNA in the ileum and improved oral glucose tolerance in obese (ob/ob) mice. In contrast, lithocholic acid, an FXR antagonist but a potent agonist for GPBAR1, did not improve glucose tolerance. The positive response to taurocholic acid is dependent upon an intact melanocortinergic system as obese MC4R-null mice or ob/ob mice without AGRP did not show improvements in glucose tolerance after taurocholate gavage. We also tested the FGF receptor isoform necessary for the bile acid response, using AGRP:Fgfr1−/− and AGRP:Fgfr2−/− mice. While the absence of FGFR1 in AGRP/NPY neurons did not alter glucose tolerance after taurocholate gavage, manipulations of Fgfr2 caused bidirectional changes depending upon the experimental model. We hypothesized the existence of an endogenous hypothalamic FGF, most likely FGF17, that acted as a chronic activator of AGRP/NPY neurons. We developed two short peptides based on FGF8 and FGF17 that should antagonize FGF17 action. Both of these peptides improved glucose homeostasis after a 4-day course of central and peripheral injections. Significantly, daily average blood glucose from continuous glucose monitoring was reduced in all tested animals but glucose concentrations remained in the euglycemia range.
Conclusion: We have defined a gut–brain axis that regulates glucose metabolism mediated by antagonistic fibroblast growth factors. From the intestine, bile acids stimulate FGF15 secretion, leading to activation of the FGF receptors in hypothalamic AGRP/NPY neurons. FGF receptor intracellular signaling subsequently silences AGRP/NPY neurons, leading to improvements of glucose tolerance that are likely mediated by the autonomic nervous system. Finally, short peptides that antagonize homodimeric FGF receptor signaling within the hypothalamus have beneficial effects on glucose homeostasis without inducing hypoglycemia. These peptides could provide a new mode of regulating glucose metabolism.[Hide abstract]
|Adropin: A link between the biological clock and cholesterol homeostasisAdropin is a product of the Energy Homeostasis Association (ENHO) gene. It is a secreted peptide implied in metabolic status signaling. Ghoshal et al. report that plasma adropin concentrations are inversely related to plasma levels of low-density lipoprotein cholesterol (LDL-C) in males, but not in females. They also report that the biological clock may be a plausible focal point linking Enho transcription with nutrient intake and cellular metabolic condition.|
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Objective: Identify determinants of plasma adropin concentrations, a secreted peptide translated from the Energy Homeostasis Associated (ENHO) gene linked to metabolic control and vascular function.
Methods: Associations between plasma adropin concentrations, demographics (sex, age, BMI) and circulating biomarkers of lipid and glucose metabolism were assessed in plasma obtained after an overnight fast in humans. The regulation of adropin expression was then assessed in silico, in cultured human cells, and in animal models.
Results: In humans, plasma adropin concentrations are inversely related to atherogenic LDL-cholesterol (LDL-C) levels in men (n = 349), but not in women (n = 401). Analysis of hepatic Enho expression in male mice suggests control by the biological clock. Expression is rhythmic, peaking during maximal food consumption in the dark correlating with transcriptional activation by RORα/γ. The nadir in the light phase coincides with the rest phase and repression by Rev-erb. Plasma adropin concentrations in nonhuman primates (rhesus monkeys) also exhibit peaks coinciding with feeding times (07:00 h, 15:00 h). The ROR inverse agonists SR1001 and the 7-oxygenated sterols 7-β-hydroxysterol and 7-ketocholesterol, or the Rev-erb agonist SR9009, suppress ENHO expression in cultured human HepG2 cells. Consumption of high-cholesterol diets suppress expression of the adropin transcript in mouse liver. However, adropin over expression does not prevent hypercholesterolemia resulting from a high cholesterol diet and/or LDL receptor mutations.
Conclusion: In humans, associations between plasma adropin concentrations and LDL-C suggest a link with hepatic lipid metabolism. Mouse studies suggest that the relationship between adropin and cholesterol metabolism is unidirectional, and predominantly involves suppression of adropin expression by cholesterol and 7-oxygenated sterols. Sensing of fatty acids, cholesterol and oxysterols by the RORα/γ ligand-binding domain suggests a plausible functional link between adropin expression and cellular lipid metabolism. Furthermore, the nuclear receptors RORα/γ and Rev-erb may couple adropin synthesis with circadian rhythms in carbohydrate and lipid metabolism.[Hide abstract]
|Cdkn2a deficiency promotes adipose tissue browningGenome-wide association studies have established the CDKN2A/B locus as a hotspot influencing genetic risk for different cardio-metabolic diseases including type 2 diabetes. While rodent and human studies have highlighted the potential role of CDKN2A/B on insulin secretion, the contribution of CDKN2A/B to the control of insulin sensitivity is still elusive. Rabhi, Hannou Gromada et al. demonstrate that the genetic deletion of Cdkn2a promotes energy expenditure and improves insulin sensitivity in diet-induced obesity. Their data suggest that Cdkn2a plays a key role in energy metabolism by regulating a white-to-brown fat transition.|
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Objective: Genome-wide association studies have reported that DNA polymorphisms at the CDKN2A locus modulate fasting glucose in human and contribute to type 2 diabetes (T2D) risk. Yet the causal relationship between this gene and defective energy homeostasis remains elusive. Here we sought to understand the contribution of Cdkn2a to metabolic homeostasis.
Methods: We first analyzed glucose and energy homeostasis from Cdkn2a-deficient mice subjected to normal or high fat diets. Subsequently Cdkn2a-deficient primary adipose cells and human-induced pluripotent stem differentiated into adipocytes were further characterized for their capacity to promote browning of adipose tissue. Finally CDKN2A levels were studied in adipocytes from lean and obese patients.
Results: We report that Cdkn2a deficiency protects mice against high fat diet-induced obesity, increases energy expenditure and modulates adaptive thermogenesis, in addition to improving insulin sensitivity. Disruption of Cdkn2a associates with increased expression of brown-like/beige fat markers in inguinal adipose tissue and enhances respiration in primary adipose cells. Kinase activity profiling and RNA-sequencing analysis of primary adipose cells further demonstrate that Cdkn2a modulates gene networks involved in energy production and lipid metabolism, through the activation of the Protein Kinase A (PKA), PKG, PPARGC1A and PRDM16 signaling pathways, key regulators of adipocyte beiging. Importantly, CDKN2A expression is increased in adipocytes from obese compared to lean subjects. Moreover silencing CDKN2A expression during human-induced pluripotent stem cells adipogenic differentiation promoted UCP1 expression.
Conclusion: Our results offer novel insight into brown/beige adipocyte functions, which has recently emerged as an attractive therapeutic strategy for obesity and T2D. Modulating Cdkn2a-regulated signaling cascades may be of interest for the treatment of metabolic disorders.[Hide abstract]
|Degradation of brown adipocyte purine nucleotides regulates UCP1 activityMitochondrial uncoupling protein 1 (UCP1) is essential for the thermogenic process in brown adipocytes and a target of the crucial regulatory signals. In the resting state, UCP1 is inhibited by the binding purine di- and triphosphate nucleotides (GDP, ADP, GTP, ATP). The concentration of purine nucleotides can be assumed to be well in a range in which their degradation may actively contribute to activation of UCP1. Fromme and colleagues found a decrease in purine nucleotide di- and triphosphate concentration upon adrenergic stimulation of primary brown adipocytes and present evidence that alterations in purine nucleotide metabolism are involved in the activation of UCP1-mediated thermogenesis.|
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Objective: Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in uncoupling protein 1 activation.
Methods: With mass spectrometry, purine nucleotide metabolites were quantified in cellular homogenates and supernatants of cultured primary brown adipocytes. We also determined oxygen consumption in response to a β-adrenergic agonist.
Results: Upon adrenergic activation, brown adipocytes decreased the intracellular concentration of inhibitory nucleotides (ATP, ADP, GTP and GDP) and released the respective degradation products. At the same time, an increase in cellular calcium occurred. None of these phenomena occurred in white adipocytes or myotubes. The brown adipocyte expression of enzymes implicated in purine metabolic remodeling is altered upon cold exposure. Pharmacological and genetic interference of purine metabolism altered uncoupling protein 1 mediated uncoupled respiration.
Conclusion: Adrenergic stimulation of brown adipocytes lowers the intracellular concentration of purine nucleotides, thereby contributing to uncoupling protein 1 activation.[Hide abstract]
|Elevating eosinophils in obese mice does not rescue metabolic impairmentsA recently identified role for eosinophils is their potential influence on whole-body metabolic fitness by regulating adipose tissue (AT) health. To date, interventional studies have targeted eosinophils via nonspecific means which increase eosinophils but also alter a cascade of upstream or off-target immune reactions, making it difficult to definitively conclude eosinophils are responsible for the observed improvements in metabolic fitness. Bolus et al. aimed to determine whether directly normalizing AT eosinophil numbers via rIL5 injections in obese mice would improve metabolic fitness. They found that AT eosinophils of obese mice were successfully restored to levels of lean mice, but none of the metabolic improvements seen in other hypereosinophilic models were observed.|
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Objective: Obesity is a metabolic disorder that has reached epidemic proportions worldwide and leads to increased risk for diabetes, cardiovascular disease, asthma, certain cancers, and various other diseases. Obesity and its comorbidities are associated with impaired adipose tissue (AT) function. In the last decade, eosinophils have been identified as regulators of proper AT function. Our study aimed to determine whether normalizing the number of AT eosinophils in obese mice, to those of lean healthy mice, would reduce obesity and/or improve metabolic fitness.
Methods: C57BL/6J mice fed a high fat diet (HFD) were simultaneously given recombinant interleukin-5 (rIL5) for 8 weeks to increase AT eosinophils. Metabolic fitness was tested by evaluating weight gain, AT inflammation, glucose, lipid, and mixed-meal tolerance, AT insulin signaling, energy substrate utilization, energy expenditure, and white AT beiging capacity.
Results: Eosinophils were increased ∼3-fold in AT of obese HFD-fed mice treated with rIL5, and thus were restored to levels observed in lean healthy mice. However, there were no significant differences in rIL5-treated mice among the above listed comprehensive set of metabolic assays, despite the increased AT eosinophils.
Conclusion: We have shown that restoring obese AT eosinophils to lean healthy levels is not sufficient to allow for improvement in any of a range of metabolic features otherwise impaired in obesity. Thus, the mechanisms that identified eosinophils as positive regulators of AT function, and therefore systemic health, are more complex than initially understood and will require further study to fully elucidate.[Hide abstract]
|Stard13 controls insulin secretion through F-actin remodelingActin cytoskeleton remodeling is regulated by the Rho-family of small GTPases in different cell types, including the pancreatic β-cells. The actin cytoskeleton plays a complex role in regulating insulin release: it acts as a physical barrier, impeding the access of insulin granules to the cell periphery, but it also actively participates in it by providing a cytoskeletal track for insulin granule transport. To gain insight into how actin cytoskeleton regulates insulin secretion, Naumann et al. visualized F-actin in β-cells. Their results suggest a previously unappreciated role of the Rho GTPase activating protein Stard13 as a key component of the insulin secretion machinery through actin cytoskeletal remodeling.|
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Objective: Actin cytoskeleton remodeling is necessary for glucose-stimulated insulin secretion in pancreatic β-cells. A mechanistic understanding of actin dynamics in the islet is paramount to a better comprehension of β-cell dysfunction in diabetes. Here, we investigate the Rho GTPase regulator Stard13 and its role in F-actin cytoskeleton organization and islet function in adult mice.
Methods: We used Lifeact-EGFP transgenic animals to visualize actin cytoskeleton organization and dynamics in vivo in the mouse islets. Furthermore, we applied this model to study actin cytoskeleton and insulin secretion in mutant mice deleted for Stard13 selectively in pancreatic cells. We isolated transgenic islets for 3D-imaging and perifusion studies to measure insulin secretion dynamics. In parallel, we performed histological and morphometric analyses of the pancreas and used in vivo approaches to study glucose metabolism in the mouse.
Results: In this study, we provide the first genetic evidence that Stard13 regulates insulin secretion in response to glucose. Postnatally, Stard13 expression became restricted to the mouse pancreatic islets. We showed that Stard13 deletion results in a marked increase in actin polymerization in islet cells, which is accompanied by severe reduction of insulin secretion in perifusion experiments. Consistently, Stard13-deleted mice displayed impaired glucose tolerance and reduced glucose-stimulated insulin secretion.
Conclusion: Taken together, our results suggest a previously unappreciated role for the RhoGAP protein Stard13 in the interplay between actin cytoskeletal remodeling and insulin secretion.[Hide abstract]
|Hepatocyte estrogen receptor α promotes reverse cholesterol transportObese, premenopausal women are more insulin sensitive than body mass index-matched men and have lower risk of coronary heart disease (CHD) with a less atherogenic plasma lipid profile. After menopause, CHD risk in women approaches that of men of the same age, suggesting that sex differences in CHD risk are in part influenced by estrogen signaling pathways. Zhu et al. investigated whether hepatic estrogen signaling regulates reverse cholesterol transport (RCT) and is protective against early stages of atherosclerosis. They show that hepatocyte estrogen receptor α plays an important role in RCT to protect against lipid retention in the artery wall at early stages of atherosclerosis during Western diet feeding in female mice.|
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Objective: Hepatocyte deletion of estrogen receptor alpha (LKO-ERα) worsens fatty liver, dyslipidemia, and insulin resistance in high-fat diet fed female mice. However, whether or not hepatocyte ERα regulates reverse cholesterol transport (RCT) in mice has not yet been reported.
Methods and results: Using LKO-ERα mice and wild-type (WT) littermates fed a Western-type diet, we found that deletion of hepatocyte ERα impaired in vivo RCT measured by the removal of 3H-cholesterol from macrophages to the liver, and subsequently to feces, in female mice but not in male mice. Deletion of hepatocyte ERα decreased the capacity of isolated HDL to efflux cholesterol from macrophages and reduced the ability of isolated hepatocytes to accept cholesterol from HDL ex vivo in both sexes. However, only in female mice, LKO-ERα increased serum cholesterol levels and increased HDL particle sizes. Deletion of hepatocyte ERα increased adiposity and worsened insulin resistance to a greater degree in female than male mice. All of the changes lead to a 5.6-fold increase in the size of early atherosclerotic lesions in female LKO-ERα mice compared to WT controls.
Conclusions: Estrogen signaling through hepatocyte ERα plays an important role in RCT and is protective against lipid retention in the artery wall during early stages of atherosclerosis in female mice fed a Western-type diet.[Hide abstract]
|Hepatic regulation of VLDL receptor modulates NAFLDNon-alcoholic fatty liver disease (NAFLD) is currently the most common liver disorder. Recently, it has been reported that very low-density lipoprotein receptor (VLDLR) plays an important role in the development of hepatic steatosis. VLDLR is regulated by several transcription factors, including Peroxisome Proliferator-Activated Receptor (PPAR) γ. However, little is known about the effects of PPARβ/δ on VLDLR regulation in the liver. Zarei and colleagues show that Pparβ/δ deficiency regulates VLDLR levels, whereas fibroblast growth factor 21 (FGF21) deficiency exacerbates ER stress-induced VLDLR levels, contributing to the progression of hepatic steatosis.|
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Objective: The very low-density lipoprotein receptor (VLDLR) plays an important role in the development of hepatic steatosis. In this study, we investigated the role of Peroxisome Proliferator-Activated Receptor (PPAR)β/δ and fibroblast growth factor 21 (FGF21) in hepatic VLDLR regulation.
Methods: Studies were conducted in wild-type and Pparβ/δ-null mice, primary mouse hepatocytes, human Huh-7 hepatocytes, and liver biopsies from control subjects and patients with moderate and severe hepatic steatosis.
Results: Increased VLDLR levels were observed in liver of Pparβ/δ-null mice and in Pparβ/δ-knocked down mouse primary hepatocytes through mechanisms involving the heme-regulated eukaryotic translation initiation factor 2α (eIF2α) kinase (HRI), activating transcription factor (ATF) 4 and the oxidative stress-induced nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathways. Moreover, by using a neutralizing antibody against FGF21, Fgf21-null mice and by treating mice with recombinant FGF21, we show that FGF21 may protect against hepatic steatosis by attenuating endoplasmic reticulum (ER) stress-induced VLDLR upregulation. Finally, in liver biopsies from patients with moderate and severe hepatic steatosis, we observed an increase in VLDLR levels that was accompanied by a reduction in PPARβ/δ mRNA abundance and DNA-binding activity compared with control subjects.
Conclusions: Overall, these findings provide new mechanisms by which PPARβ/δ and FGF21 regulate VLDLR levels and influence hepatic steatosis development.[Hide abstract]
|Pharmacological stimulation of p53 ameliorates NAFLD p53 is an intensively studied protein, primarily as a tumor suppressor. Although there is a large body of evidence showing that p53 promotes fatty acid catabolism while it inhibits anabolism, the possible contribution of p53 to the pathogenesis of nonalcoholic fatty liver disease (NAFLD) has only recently begun to be understood. Porteiro et al. demonstrate that chronic pharmacological stimulation of p53 with a low dose of doxorubicin improves liver injury in different models of diet-induced steatosis and steatohepatitis through stimulation of fatty acid oxidation and decrease of lipogenesis, inflammation, and ER stress.
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Objective: Recent reports have implicated the p53 tumor suppressor in the regulation of lipid metabolism. We hypothesized that the pharmacological activation of p53 with low-dose doxorubicin, which is widely used to treat several types of cancer, may have beneficial effects on nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH).
Methods: We used long-term pharmacological activation of p53 by i.p. or oral administration of low-dose doxorubicin in different animal models of NAFLD (high fat diet containing 45% and 60% kcal fat) and NASH (methionine- and choline-deficient diet and choline deficiency combined with high fat diet). We also administered doxorubicin in mice lacking p53 in the liver and in two human hepatic cells lines (HepG2 and THLE2).
Results: The attenuation of liver damage was accompanied by the stimulation of fatty acid oxidation and decrease of lipogenesis, inflammation, and ER stress. The effects of doxorubicin were abrogated in mice with liver-specific ablation of p53. Finally, the effects of doxorubicin on lipid metabolism found in animal models were also present in two human hepatic cells lines, in which the drug stimulated fatty acid oxidation and inhibited de novo lipogenesis at doses that did not cause changes in apoptosis or cell viability.
Conclusions: These data provide new evidence for targeting p53 as a strategy to treat liver disease.[Hide abstract]
|The EndoC-βH1 cell line is a valid model of human beta cellsThe insulin producing beta cell is central in the etiology of human diabetes as beta cell failure is the major determining factor for progression from impaired glucose tolerance to overt diabetes. New potent antidiabetic drugs are needed, and analyses using human beta cells are a necessity as the majority of in vitro research so far has been done on islets and cell lines from rodents with only sporadic follow-up using human islets. Tsonkova and colleagues performed a thorough phenotypic validation of the human beta cell line EndoC-βH1. They conclude that it is a valid cell line model of human beta cells that can be successfully used as a screening platform for early drug discovery.|
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Objective: To characterize the EndoC-βH1 cell line as a model for human beta cells and evaluate its beta cell functionality, focusing on insulin secretion, proliferation, apoptosis and ER stress, with the objective to assess its potential as a screening platform for identification of novel anti-diabetic drug candidates.
Methods: EndoC-βH1 was transplanted into mice for validation of in vivo functionality. Insulin secretion was evaluated in cells cultured as monolayer and as pseudoislets, as well as in diabetic mice. Cytokine induced apoptosis, glucolipotoxicity, and ER stress responses were assessed. Beta cell relevant mRNA and protein expression were investigated by qPCR and antibody staining. Hundreds of proteins or peptides were tested for their effect on insulin secretion and proliferation.
Transplantation of EndoC-βH1 cells restored normoglycemia in streptozotocin induced diabetic mice. Both in vitro and in vivo, we observed a clear insulin response to glucose, and, in vitro, we found a significant increase in insulin secretion from EndoC-βH1 pseudoislets compared to monolayer cultures for both glucose and incretins.
Conclusions: Overall, the EndoC-βH1 cells strongly resemble human islet beta cells in terms of glucose and incretin stimulated insulin secretion capabilities. The cell line has an active cytokine induced caspase 3/7 apoptotic pathway and is responsive to ER stress initiation factors. The cells' ability to proliferate can be further increased by already known compounds as well as by novel peptides and proteins. Based on its robust performance during the functionality assessment assays, the EndoC-βH1 cell line was successfully used as a screening platform for identification of novel anti-diabetic drug candidates.[Hide abstract]
|Androgen receptor overexpression in prostate cancer in type 2 diabetes In contrast to numerous other malignancies, the incidence of prostate cancer is not increased in case of concurrent type 2 diabetes mellitus. Nevertheless, prostate cancer survival is clearly reduced when type 2 diabetes is present. One of the crucial drivers for prostate cell growth is androgen signaling. To better understand why prostate cancer survival is reduced in type 2 diabetes, Lutz et al. performed gene expression analysis of key proteins involved in androgen signaling and steroid modulators thereof using prostate tissue samples of men with and without diabetes. They report enhanced expression of androgen receptor in prostate cancer and stronger activation of androgen signaling in men with type 2 diabetes.|
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Objective: While prostate cancer does not occur more often in men with diabetes, survival is markedly reduced in this patient group. Androgen signaling is a known and major driver for prostate cancer progression. Therefore, we analyzed major components of the androgen signaling chain and cell proliferation in relation to type 2 diabetes.
Methods: Tumor content of 70 prostate tissue samples of men with type 2 diabetes and 59 samples of patients without diabetes was quantified by an experienced pathologist, and a subset of 51 samples was immunohistochemically stained for androgen receptor (AR). mRNA expression of AR, insulin receptor isoform A (IR-A) and B (IR-B), IGF-1 receptor (IGF1R), Cyp27A1 and Cyp7B1, PSA gene KLK3, PSMA gene FOLH1, Ki-67 gene MKI67, and estrogen receptor beta (ESR2) were analyzed by RT-qPCR.
Results: AR mRNA and protein expression were associated with the tumor content only in men with diabetes. AR expression also correlated with downstream targets PSA (KLK3) and PSMA (FOLH1) and increased cell proliferation. Only in diabetes, AR expression was correlated to higher IR-A/IR-B ratio and lower IR-B/IGF1R ratio, thus, in favor of the mitogenic isoforms. Reduced Cyp27A1 and increased Cyp7B1 expressions in tumor suggest lower levels of protective estrogen receptor ligands in diabetes.
Conclusions: We report elevated androgen receptor signaling and activity presumably due to altered insulin/IGF-1 receptors and decreased levels of protective estrogen receptor ligands in prostate cancer in men with diabetes. Our results reveal new insights why these patients have a worse prognosis. These findings provide the basis for future clinical trials to investigate treatment response in patients with prostate cancer and diabetes.[Hide abstract]
|Adiponectin release and insulin receptor targeting share endosomal trafficking routes The adipose tissue releases a variety of bioactive molecules into the circulation, such as adipokines, cytokines, and lipid metabolites. The precise intracellular trafficking pathways and particular molecular components mediating the targeting and release of bioactive molecules are still largely elusive. To explore the impact of ADP-ribosylation factor related protein 1 (ARFRP1) as part of the vesicle trafficking machinery on the secretory capacity of mature adipocytes, Rödiger and colleagues generated a mouse model with an inducible fat-specific disruption of Arfrp1. They demonstrate that the loss of Arfrp1 from differentiated adipocytes diminishes adiponectin secretion and plasma membrane localization of the insulin receptor, which is associated with detrimental effects on adipocyte metabolism and glucose homeostasis.|
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Objective: Intracellular vesicle trafficking maintains cellular structures and functions. The assembly of cargo-laden vesicles at the trans-Golgi network is initiated by the ARF family of small GTPases. Here, we demonstrate the role of the trans-Golgi localized monomeric GTPase ARFRP1 in endosomal-mediated vesicle trafficking of mature adipocytes.
Methods: Control (Arfrp1flox/flox) and inducible fat-specific Arfrp1 knockout (Arfrp1iAT−/−) mice were metabolically characterized. In vitro experiments on mature 3T3-L1 cells and primary mouse adipocytes were conducted to validate the impact of ARFRP1 on localization of adiponectin and the insulin receptor. Finally, secretion and transferrin-based uptake and recycling assays were performed with HeLa and HeLa M-C1 cells.
Results: We identified the ARFRP1-based sorting machinery to be involved in vesicle trafficking relying on the endosomal compartment for cell surface delivery. Secretion of adiponectin from fat depots was selectively reduced in Arfrp1iAT−/− mice, and Arfrp1-depleted 3T3-L1 adipocytes revealed an accumulation of adiponectin in Rab11-positive endosomes. Plasma adiponectin deficiency of Arfrp1iAT−/− mice resulted in deteriorated hepatic insulin sensitivity, increased gluconeogenesis and elevated fasting blood glucose levels. Additionally, the insulin receptor, undergoing endocytic recycling after ligand binding, was less abundant at the plasma membrane of adipocytes lacking Arfrp1. This had detrimental effects on adipose insulin signaling, followed by insufficient suppression of basal lipolytic activity and impaired adipose tissue expansion.
Conclusions: Our findings suggest that adiponectin secretion and insulin receptor surface targeting utilize the same post-Golgi trafficking pathways that are essential for an appropriate systemic insulin sensitivity and glucose homeostasis.[Hide abstract]
|Cadm2 regulates body weight and energy homeostasisCadm2 is an immunoglobulin domain-containing adhesion protein that mediates the assembly of pre-synaptic specializations in neurons in the brain to direct interactions across the nascent and mature synaptic cleft. Recently, it has been shown that the closely related Cadm1 regulates body weight and energy homeostasis via its expression within the hippocampus and hypothalamus. Here, Yan and colleagues illustrate that loss of Cadm2 protects mice from obesity and hyperglycemia by regulating locomotor activity and thermogenesis, further underlining the functional role of this gene family in systemic energy homeostasis via the brain.|
Abstract | PDF
Objective: Obesity is strongly linked to genes regulating neuronal signaling and function, implicating the central nervous system in the maintenance of body weight and energy metabolism. Genome-wide association studies identified significant associations between body mass index (BMI) and multiple loci near Cell adhesion molecule2 (CADM2), which encodes a mediator of synaptic signaling enriched in the brain. Here we sought to further understand the role of Cadm2 in the pathogenesis of hyperglycemia and weight gain.
Methods: We first analyzed Cadm2 expression in the brain of both human subjects and mouse models and subsequently characterized a loss-of-function mouse model of Cadm2 for alterations in glucose and energy homeostasis.
Results: We show that the risk variant rs13078960 associates with increased CADM2 expression in the hypothalamus of human subjects. Increased Cadm2 expression in several brain regions of Lepob/ob mice was ameliorated after leptin treatment. Deletion of Cadm2 in obese mice (Cadm2/ob) resulted in reduced adiposity, systemic glucose levels, and improved insulin sensitivity. Cadm2-deficient mice exhibited increased locomotor activity, energy expenditure rate, and core body temperature identifying Cadm2 as a potent regulator of systemic energy homeostasis.
Conclusions: Together these data illustrate that reducing Cadm2 expression can reverse several traits associated with the metabolic syndrome including obesity, insulin resistance, and impaired glucose homeostasis.[Hide abstract]
|Targeting erythropoietin protects against proteinuria Erythropoietin (EPO) is a glycoprotein hormone traditionally considered essential for erythropoiesis. Recent studies revealed a relationship between EPO and the progression of diabetic complications, such as diabetic nephropathy (DN), a common diabetes microvascular complication. DN ranks first as the cause of end-stage renal disease (ESRD). She et al. provide evidence that EPO is a clinically-protective factor in the progression of diabetic complications. They identify EPO as an active renal antiapoptotic factor, protecting kidney from hyperglycemia-induced damage and proteinuria in an EPO receptor-dependent manner both in zebrafish and in type 2 diabetic patients.|
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Objective: Adult human kidneys produce erythropoietin (EPO), which regulates red blood cell formation; however, whether EPO also functions directly on kidney development and controls diabetic kidney disease remains unknown. Here we analyzed the role of EPO in kidney development and under hyperglycemic conditions in zebrafish and in humans.
Methods: Diabetic patients and respective controls were enrolled in two cohorts. Serum EPO level and urine protein change upon human EPO administration were then analyzed. Transient knockdown and permanent knockout of EPO and EPOR in renal TG(WT1B:EGFP) zebrafish were established using the morpholino technology and CRISPR/Cas9 technology. Zebrafish embryos were phenotypically analyzed using fluorescence microscopy, and functional assays were carried out with the help of TexasRed labeled 70 kDa Dextran. Apoptosis was determined using the TUNEL assay and Annexin V staining, and caspase inhibitor zVADfmk was used for rescue experiments.
Results: In type 2 diabetic patients, serum EPO level decreased with the duration of diabetes, which was linked to reduced kidney function. Human recombinant EPO supplementation ameliorated proteinuria in diabetic nephropathy patients. In zebrafish, loss-of-function studies for EPO and EPOR, showed morphological and functional alterations within the pronephros, adversely affecting pronephric structure, leading to slit diaphragm dysfunction by increasing apoptosis within the pronephros. Induction of hyperglycemia in zebrafish embryos induced pronephros alterations which were further worsened upon silencing of EPO expression.
Conclusions: EPO was identified as a direct renal protective factor, promoting renal embryonic development and protecting kidneys from hyperglycemia induced nephropathy.[Hide abstract]