Objective: Central melanocortin pathways are well-established regulators of energy balance. However, scant data exist about the role of systemic melanocortin peptides. We set out to determine if peripheral a-melanocyte stimulating hormone (α-MSH) plays a role in glucose homeostasis and tested the hypothesis that the pituitary is able to sense a physiological increase in circulating glucose and responds by secreting α-MSH.
Methods: We established glucose-stimulated α-MSH secretion using humans, non-human primates, and mouse models. Continuous α-MSH infusions were performed during glucose tolerance tests and hyperinsulinemic-euglycemic clamps to evaluate the systemic effect of α-MSH in glucose regulation. Complementary ex vivo and in vitro techniques were employed to delineate the direct action of α-MSH via the melanocortin 5 receptor (MC5R)–PKA axis in skeletal muscles. Combined treatment of non-selective/selective phosphodiesterase inhibitor and α-MSH was adopted to restore glucose tolerance in obese mice.
Results: Here we demonstrate that pituitary secretion of α-MSH is increased by glucose. Peripheral α-MSH increases temperature in skeletal muscles, acts directly on soleus and gastrocnemius muscles to significantly increase glucose uptake, and enhances whole-body glucose clearance via the activation of muscle MC5R and protein kinase A. These actions are absent in obese mice, accompanied by a blunting of α-MSH-induced cAMP levels in skeletal muscles of obese mice. Both selective and non-selective phosphodiesterase inhibition restores α-MSH induced skeletal muscle glucose uptake and improves glucose disposal in obese mice.
Conclusions: These data describe a novel endocrine circuit that modulates glucose homeostasis by pituitary α-MSH, which increases muscle glucose uptake and thermogenesis through the activation of a MC5R-PKA-pathway, which is disrupted in obesity.[Hide abstract]
Objective: Brain regulation of glucose homeostasis is sexually dimorphic; however, the impact sex hormones have on specific neuronal populations within the ventromedial hypothalamic nucleus (VMN), a metabolically sensitive brain region, has yet to be fully characterized. Glucose-excited (GE) and -inhibited (GI) neurons are located throughout the VMN and may play a critical role in glucose and energy homeostasis. Within the ventrolateral portion of the VMN (VL-VMN), glucose sensing neurons and estrogen receptor (ER) distributions overlap. We therefore tested the hypothesis that VL-VMN glucose sensing neurons were sexually dimorphic and regulated by 17β-estradiol (17βE).
Methods: Electrophysiological recordings of VL-VMN glucose sensing neurons in brain slices isolated from age- and weight-matched female and male mice were performed in the presence and absence of 17βE.
Results: We found a new class of VL-VMN GI neurons whose response to low glucose was transient despite continued exposure to low glucose. Heretofore, we refer to these newly identified VL-VMN GI neurons as ‘adapting’ or AdGI neurons. We found a sexual dimorphic response to low glucose, with male nonadapting GI neurons, but not AdGI neurons, responding more robustly to low glucose than those from females. 17βE blunted the response of both nonadapting GI and AdGI neurons to low glucose in both males and females, which was mediated by activation of estrogen receptor β and inhibition of AMP-activated kinase. In contrast, 17βE had no impact on GE or non-glucose sensing neurons in either sex.
Conclusions: These data suggest sex differences and estrogenic regulation of VMN hypothalamic glucose sensing may contribute to the sexual dimorphism in glucose homeostasis.[Hide abstract]
Objective: Adult neurogenesis in the subgranular zone and subventricular zone is generally accepted, but its existence in other brain areas is still controversial. Circumventricular organs, such as the area postrema (AP) have recently been described as potential neurogenic niches in the adult brain. The AP is the major site of action of the satiating hormone amylin. Amylin has been shown to promote the formation of neuronal projections originating from the AP in neonatal rodents but the role of amylin in adult neurogenesis remains unknown.
Methods: To test this, we first performed an RNA-sequencing of the AP of adult rats acutely injected with either amylin (20 µg/kg), amylin plus the amylin receptor antagonist AC187 (500 µg/kg) or vehicle. Second, animals were subcutaneously equipped with minipumps releasing either amylin (50 µg/kg/day) or vehicle for 3 weeks to assess cell proliferation and differentiation with the 5'-bromo-2-deoxyuridine (BrdU) technique.
Results: Acute amylin injections affected genes involved in pathways and processes that control adult neurogenesis. Amylin consistently upregulated NeuroD1 transcript and protein in the adult AP, and this effect was blocked by the co-administration of AC187. Further, chronic amylin treatment increased the number of newly proliferated AP-cells and significantly promoted their differentiation into neurons rather than astrocytes.
Conclusions: Our findings revealed a novel role of the satiating hormone amylin in promoting neurogenesis in the AP of adult rats.[Hide abstract]
Objective: Puberty is a key developmental phenomenon highly sensitive to metabolic modulation. Worrying trends of changes in the timing of puberty have been reported in humans. These might be linked to the escalating prevalence of childhood obesity and could have deleterious impacts on later (cardio-metabolic) health, but their underlying mechanisms remain unsolved. The neuropeptide α-MSH, made by POMC neurons, plays a key role in energy homeostasis by mediating the actions of leptin and likely participates in the control of reproduction. However, its role in the metabolic regulation of puberty and interplay with kisspeptin, an essential puberty-regulating neuropeptide encoded by Kiss1, remain largely unknown. We aim here to unveil the potential contribution of central α-MSH signaling in the metabolic control of puberty by addressing its role in mediating the pubertal effects of leptin and its potential interaction with kisspeptin.
Methods: Using wild type and genetically modified rodent models, we implemented pharmacological studies, expression analyses, electrophysiological recordings, and virogenetic approaches involving DREADD technology to selectively inhibit Kiss1 neurons, in order to interrogate the physiological role of a putative leptin → α-MSH → kisspeptin pathway in the metabolic control of puberty.
Results: Stimulation of central α-MSH signaling robustly activated the reproductive axis in pubertal rats, whereas chronic inhibition of melanocortin receptors MC3/4R, delayed puberty, and prevented the permissive effect of leptin on puberty onset. Central blockade of MC3/4R or genetic elimination of kisspeptin receptors from POMC neurons did not affect kisspeptin effects. Conversely, congenital ablation of kisspeptin receptors or inducible, DREADD-mediated inhibition of arcuate nucleus (ARC) Kiss1 neurons resulted in markedly attenuated gonadotropic responses to MC3/4R activation. Furthermore, close appositions were observed between POMC fibers and ARC Kiss1 neurons while blockade of α-MSH signaling suppressed Kiss1 expression in the ARC of pubertal rats.
Conclusions: Our physiological, virogenetic, and functional genomic studies document a novel α-MSH → kisspeptin → GnRH neuronal signaling pathway involved in transmitting the permissive effects of leptin on pubertal maturation, which is relevant for the metabolic (and, eventually, pharmacological) regulation of puberty onset.[Hide abstract]
Background: Hypertension and obesity are highly interrelated diseases, being critical components of the metabolic syndrome. Despite the growing prevalence of this syndrome in the world population, efficient therapies are still missing. Thus, identification of novel targets and therapies are warranted. An enhanced activity of the hypothalamic renin-angiotensin system (RAS), including the recently discovered prorenin (PR) and its receptor (PRR), has been implicated as a common mechanism underlying aberrant sympatho-humoral activation that contributes to both metabolic and cardiovascular dysregulation in the metabolic syndrome. Still, the identification of precise neuronal targets, cellular mechanisms and signaling pathways underlying PR/PRR actions in cardiovascular- and metabolic related hypothalamic nuclei remain unknown.
Methods and results: Using a multidisciplinary approach including patch-clamp electrophysiology, live calcium imaging and immunohistochemistry, we aimed to elucidate cellular mechanisms underlying PR/PRR actions within the hypothalamic supraoptic (SON) and paraventricular nucleus (PVN), key brain areas previously involved in cardiometabolic regulation. We show for the first time that PRR is expressed in magnocellular neurosecretory cells (MNCs), and to a lesser extent, in presympathetic PVN neurons (PVNPS). Moreover, we show that while PRR activation efficiently stimulates the firing activity of both MNCs and PVNPS neurons, these effects involved AngII-independent and AngII-dependent mechanisms, respectively. In both cases however, PR excitatory effects involved an increase in intracellular Ca2+ levels and a Ca2+-dependent inhibition of a voltage-gated K+ current.
Conclusions: We identified novel neuronal targets and cellular mechanisms underlying PR/PRR actions in critical hypothalamic neurons involved in cardiometabolic regulation. This fundamental mechanistic information regarding central PR/PRR actions is essential for the development of novel RAS-based therapeutic targets for the treatment of cardiometabolic disorders in obesity and hypertension.[Hide abstract]
Objective: A major challenge for obesity treatment is the maintenance of reduced body weight. Diet-induced obese mice are resistant to achieving normoweight once the obesogenic conditions are reversed, in part because lowered circulating leptin leads to a reduction in metabolic rate and a rebound of hyperphagia that defend the previously elevated body weight set point. Because hypothalamic POMC is a central leptin target, we investigated whether changes in circulating leptin modify Pomc expression to maintain normal energy balance in genetically predisposed obese mice.
Methods: Mice with reversible Pomc silencing in the arcuate nucleus (ArcPomc-/-) become morbidly obese eating low-fat chow. We measured body composition, food intake, plasma leptin, and leptin sensitivity in ArcPomc-/- mice weight-matched to littermate controls by calorie restriction, either from weaning or after developing obesity. Pomc was reactivated by tamoxifen-dependent Cre recombinase transgenes. Long acting PASylated leptin was administered to weight-reduced ArcPomc-/- mice to mimic the super-elevated leptin levels of obese mice.
Results: ArcPomc-/- mice had increased adiposity and leptin levels shortly after weaning. Despite chronic calorie restriction to achieve normoweight, ArcPomc-/- mice remained moderately hyperleptinemic and resistant to exogenous leptin's effects to reduce weight and food intake. However, subsequent Pomc reactivation in weight-matched ArcPomc-/- mice normalized plasma leptin, leptin sensitivity, adiposity, and food intake. In contrast, extreme hyperleptinemia induced by PASylated leptin blocked the full restoration of hypothalamic Pomc expression in calorie restricted ArcPomc-/- mice, which consequently regained 30% of their lost body weight and attained a metabolic steady state similar to that of tamoxifen treated obese ArcPomc-/- mice.
Conclusions: Pomc reactivation in previously obese, calorie-restricted ArcPomc-/- mice normalized energy homeostasis, suggesting that their body weight set point was restored to control levels. In contrast, massively obese and hyperleptinemic ArcPomc-/- mice or those weight-matched and treated with PASylated leptin to maintain extreme hyperleptinemia prior to Pomc reactivation converged to an intermediate set point relative to lean control and obese ArcPomc-/- mice. We conclude that restoration of hypothalamic leptin sensitivity and Pomc expression is necessary for obese ArcPomc-/- mice to achieve and sustain normal metabolic homeostasis; whereas deficits in either parameter set a maladaptive allostatic balance that defends increased adiposity and body weight.[Hide abstract]
Objective: Adiponectin receptors (AdipoRs) are located on neurons of the hypothalamus involved in metabolic regulation – including arcuate proopiomelanocortin (Pomc) and Neuropeptide Y/Agouti-related peptide (NPY/AgRP) neurons. AdipoRs play a critical role in regulating glucose and fatty acid metabolism by initiating several signaling cascades overlapping with Leptin receptors (LepRs). However, the mechanism by which adiponectin regulates cellular activity in the brain remains undefined.
Methods: In order to resolve this issue, we utilized neuron-specific transgenic mouse models to identify Pomc and NPY/AgRP neurons which express LepRs for patch-clamp electrophysiology experiments.
Results: We found that leptin and adiponectin synergistically activated melanocortin neurons in the arcuate nucleus. Conversely, NPY/AgRP neurons were inhibited in response to adiponectin. The adiponectin-induced depolarization of arcuate Pomc neurons occurred via activation of Phosphoinositide-3-kinase (PI3K) signaling, independent of 5' AMP-activated protein kinase (AMPK) activity. Adiponectin also activated melanocortin neurons at various physiological glucose levels.
Conclusions: Our results demonstrate a requirement for PI3K signaling in the acute adiponectin-induced effects on the cellular activity of arcuate melanocortin neurons. Moreover, these data provide evidence for PI3K as a substrate for both leptin and adiponectin to regulate energy balance and glucose metabolism via melanocortin activity.[Hide abstract]
Objective: To investigate the role played by leptin in thermoregulation, we studied the effects of physiological leptin replacement in leptin-deficient ob/ob mice on determinants of energy balance, thermogenesis and heat retention under 3 different ambient temperatures.
Methods: The effects of housing at 14 °C, 22 °C or 30 °C on core temperature (telemetry), energy expenditure (respirometry), thermal conductance, body composition, energy intake, and locomotor activity (beam breaks) were measured in ob/ob mice implanted subcutaneously with osmotic minipumps at a dose designed to deliver a physiological replacement dose of leptin or its vehicle-control.
Results: As expected, the hypothermic phenotype of ob/ob mice was partially rescued by administration of leptin at a dose that restores plasma levels into the physiological range. This effect of leptin was not due to increased energy expenditure, as cold exposure markedly and equivalently stimulated energy expenditure and induced activation of brown adipose tissue irrespective of leptin treatment. Instead, the effect of physiological leptin replacement to raise core body temperature of cold-exposed ob/ob mice was associated with reduced thermal conductance, implying a physiological role for leptin in heat conservation. Finally, both leptin- and vehicle-treated ob/ob mice failed to match energy intake to expenditure during cold exposure, resulting in weight loss.
Conclusions: The physiological effect of leptin to reduce thermal conductance contributes to maintenance of core body temperature under sub-thermoneutral conditions.[Hide abstract]
Objective: Obesity and type 2 diabetes (T2D) lead to various life-threatening diseases such as coronary heart disease, stroke, osteoarthritis, asthma, and neurodegeneration. Therefore, extensive research is ongoing to identify novel pathways that can be targeted in obesity/T2D. Deletion of the inositol pyrophosphate (5-IP7) biosynthetic enzyme, inositol hexakisphosphate kinase-1 (IP6K1), protects mice from high fat diet (HFD) induced obesity (DIO) and insulin resistance. Yet, whether this pathway is a valid pharmacologic target in obesity/T2D is not known. Here, we demonstrate that TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine], a pan-IP6K inhibitor, has strong anti-obesity and anti-diabetic effects in DIO mice.
Methods: Q-NMR, GTT, ITT, food intake, energy expenditure, QRT-PCR, ELISA, histology, and immunoblot studies were conducted in short (2.5-week)- and long (10-week)-term TNP treated DIO C57/BL6 WT and IP6K1-KO mice, under various diet and temperature conditions.
Results: TNP, when injected at the onset of HFD-feeding, decelerates initiation of DIO and insulin resistance. Moreover, TNP facilitates weight loss and restores metabolic parameters, when given to DIO mice. However, TNP does not reduce weight gain in HFD-fed IP6K1-KO mice. TNP specifically enhances insulin sensitivity in DIO mice via Akt activation. TNP decelerates weight gain primarily by enhancing thermogenic energy expenditure in the adipose tissue. Accordingly, TNP's effect on body weight is partly abolished whereas its impact on glucose homeostasis is preserved at thermoneutral temperature.
Conclusion: Pharmacologic inhibition of the inositol pyrophosphate pathway has strong therapeutic potential in obesity, T2D, and other metabolic diseases.[Hide abstract]
Objective: Characterizing specific metabolites in sub-clinical phases preceding the onset of type 2 diabetes to enable efficient preventive and personalized interventions.
Research design and methods: We developed predictive models of type 2 diabetes using two strategies. One strategy focused on the probability of incidence only and was based on logistic regression (MRS1); the other strategy accounted for the age at diagnosis of diabetes and was based on Cox regression (MRS2). We assessed 293 metabolites using non-targeted metabolomics in fasting plasma samples of 1,044 participants (including 231 incident cases over 9 years) used as training population; and fasting serum samples of 128 participants (64 incident cases versus 64 controls) used as validation population. We applied a LASSO-based variable selection aiming at maximizing the out-of-sample area under the receiver operating characteristic curve (AROC) and integrated AROC.
Results: Sixteen and 17 metabolites were selected for MRS1 and MRS2, respectively, with AROC = 90% and 73% in the training and validation populations, respectively for MRS1. MRS2 had a similar performance and was significantly associated with a younger age of onset of type 2 diabetes (β = −3.44 years per MRS2 SD in the training population, p = 1.56 × 10−7; β = −4.73 years per MRS2 SD in the validation population, p = 4.04 × 10−3).
Conclusions: Overall, this study illustrates that metabolomics improves prediction of type 2 diabetes incidence of 4.5% on top of known clinical and biological markers, reaching 90% in total AROC, which is considered the threshold for clinical validity, suggesting it may be used in targeting interventions to prevent type 2 diabetes.[Hide abstract]
Objective: Plasma levels of branched-chain amino acids (BCAA) are consistently elevated in obesity and type 2 diabetes (T2D) and can also prospectively predict T2D. However, the role of BCAA in the pathogenesis of insulin resistance and T2D remains unclear.
Methods: To identify pathways related to insulin resistance, we performed comprehensive gene expression and metabolomics analyses in skeletal muscle from 41 humans with normal glucose tolerance and 11 with T2D across a range of insulin sensitivity (SI, 0.49 to 14.28). We studied both cultured cells and mice heterozygous for the BCAA enzyme methylmalonyl-CoA mutase (Mut) and assessed the effects of altered BCAA flux on lipid and glucose homeostasis.
Results: Our data demonstrate perturbed BCAA metabolism and fatty acid oxidation in muscle from insulin resistant humans. Experimental alterations in BCAA flux in cultured cells similarly modulate fatty acid oxidation. Mut heterozygosity in mice alters muscle lipid metabolism in vivo, resulting in increased muscle triglyceride accumulation, increased plasma glucose, hyperinsulinemia, and increased body weight after high-fat feeding.
Conclusions: Our data indicate that impaired muscle BCAA catabolism may contribute to the development of insulin resistance by perturbing both amino acid and fatty acid metabolism and suggest that targeting BCAA metabolism may hold promise for prevention or treatment of T2D.[Hide abstract]
Objective: Adipose tissue shows a high degree of plasticity, and adipocyte hyperplasia is an important mechanism for adipose tissue expansion. Different adipose depots respond differently to an increased demand for lipid storage. Orchestrating cellular expansion in vivo requires extracellular matrix (ECM) remodeling and a high degree of interaction between cells and ECM.
Methods: We studied decellularized primary adipose stromal cell derived ECM of different adipose depots and reseeded them with primary adipose precursors. We tested ECM effect on adipocyte differentiation and analyzed ECM composition using proteomic and immunohistochemical approaches to identify factors in the ECM influencing adipogenesis.
Results: We show that the ECM of an adipose depot is the major determinant for the differentiation capacity of primary preadipocytes. Visceral adipose tissue stromal cells differentiate less than subcutaneous cells, which, in turn, are less adipogenic than BAT-derived cells. This effect is based on the ECM composition of the respective depot and not dependent on the precursor origin. Addition of vitamin C pronounces the pro-adipogenic effects of the ECM, indicating the importance of collagenous ECM in mediating the effect. Using a proteomic global and a targeted downstream analysis, we identify Flotillin 2 as a protein enriched in pro-adipogenic ECM, which is involved in orchestrating ECM to preadipocyte signaling.
Conclusions: We show that adipose tissue SVF secretes collagenous ECM, which directly modulates terminal differentiation of adipocyte precursors in a depot specific manner. These data demonstrate the importance of the tissue microenvironment in preadipocyte differentiation.[Hide abstract]
Objective: Genetic background largely contributes to the complexity of metabolic responses and dysfunctions. Induction of brown adipose features in white fat, known as brown remodeling, has been appreciated as a promising strategy to offset the positive energy balance in obesity and further to improve metabolism. Here we address the effects of genetic background on this process.
Methods: We investigated browning remodeling in a depot-specific manner by comparing the response of C57BL/6J, 129/Sv and FVB/NJ mouse strains to cold.
Results: Surprisingly, 129/Sv and FVB/NJ mice showed distinct brown remodeling features despite their similar resistance to metabolic disorders in comparison to the obesity-prone C57BL/6J mice. FVB/NJ mice demonstrated a preference of brown remodeling in inguinal subcutaneous white adipose tissue (iWAT), whereas 129/Sv mice displayed robust brown remodeling in visceral epididymal fat (eWAT). We further compared gene expression in different depots by RNA-sequencing and identified Hoxc10 as a novel “brake” of brown remodeling in iWAT.
Conclusion: Rodent genetic background determines the brown remodeling of different white fat depots. This study provides new insights into the role of genetic variation in fat remodeling in susceptibility to metabolic diseases.[Hide abstract]
Objective: We previously demonstrated that the handle-region peptide, a prorenin/renin receptor [(P)RR] blocker, reduces body weight and fat mass and may improve insulin sensitivity in high-fat fed mice. We hypothesized that knocking out the adipose tissue (P)RR gene would prevent weight gain and insulin resistance.
Methods: An adipose tissue-specific (P)RR knockout (KO) mouse was created by Cre-loxP technology using AP2-Cre recombinase mice. Because the (P)RR gene is located on the X chromosome, hemizygous males were complete KO and had a more pronounced phenotype on a normal diet (ND) diet compared to heterozygous KO females. Therefore, we challenged the female mice with a high-fat diet (HFD) to uncover certain phenotypes. Mice were maintained on either diet for 9 weeks.
Results: KO mice had lower body weights compared to wild-types (WT). Only hemizygous male KO mice presented with lower total fat mass, higher total lean mass as well as smaller adipocytes compared to WT mice. Although food intake was similar between genotypes, locomotor activity during the active period was increased in both male and female KO mice. Interestingly, only male KO mice had increased O2 consumption and CO2 production during the entire 24-hour period, suggesting an increased basal metabolic rate. Although glycemia during a glucose tolerance test was similar, KO males as well as HFD-fed females had lower plasma insulin and C-peptide levels compared to WT mice, suggesting improved insulin sensitivity. Remarkably, all KO animals exhibited higher circulating adiponectin levels, suggesting that this phenotype can occur even in the absence of a significant reduction in adipose tissue weight, as observed in females and, thus, may be a specific effect related to the (P)RR.
Conclusion: (P)RR may be an important therapeutic target for the treatment of obesity and its associated complications such as type 2 diabetes.[Hide abstract]
Objective: Arising from common progenitors in the bone marrow, adipogenesis and osteogenesis are closely associated yet mutually exclusive during bone marrow mesenchymal stem cell (BMSC) development. Previous studies have shown that morphological changes can affect the early commitment of pluripotent BMSCs to the adipose versus osteoblastic lineage via modulation of RhoA activity. The RhoA pathway regulates actin polymerization to promote the incorporation of globular actin (G-actin) into filamentous actin (F-actin). In doing so, myocardin-related transcription factors (MRTFs) dissociate from bound G-actin and enter the nucleus to co-activate serum response factor (SRF) target gene expression. In this study, we investigated whether MRTFA/SRF is acting downstream of the RhoA pathway to regulate BMSC commitment in mice.
Methods: The effects of knocking out MRTFA on skeletal homeostasis was studied in MRTFA KO mice using micro-CT, QPCR and western blot assays. To determine how MRTFA affects the mechanisms regulating BMSC fate decisions, primary bone marrow stromal cells from WT and MRTFA KO mice as well as C3H10T1/2 cell lines were analyzed in vitro.
Results: Global MRTFA KO mice have lower whole body weight, shorter femoral and tibial lengths as well as significantly decreased bone mass in their femurs. BMSCs isolated from the KO mice show increased adipogenesis and reduced osteogenesis when compared to WT littermates. KO mice, particularly females, develop osteopenia with age, and this was enhanced by a high fat diet. Over-expression of MRTFA or SRF enhances osteogenesis in CH310T1/2 cell lines. Sca1+, CD45− cells from KO marrow express lower amounts of smooth muscle actin (SMA) and TAZ/YAP target genes compared to WT counterparts.
Conclusion: This study identified MRTFA as a novel regulator of skeletal homeostasis by regulating the balance between adipogenic and osteogenic differentiation of BMSCs. We propose that MRTFA promotes the osteogenic activity of TAZ/YAP by maintaining SMA production in BMSCs.[Hide abstract]
Objective: Glucose-stimulated insulin secretion in pancreatic beta cells requires metabolic signals including the generation of glucose-derived short chain acyl-CoAs in the cytosol from mitochondrially-derived metabolites. One concept of insulin secretion is that ATP citrate lyase generates short chain acyl-CoAs in the cytosol from mitochondrially-derived citrate. Of these, malonyl-CoA, is believed to be an important signal in insulin secretion. Malonyl-CoA is also a precursor for lipids. Our recent evidence suggested that, in the mitochondria of beta cells, glucose-derived pyruvate can be metabolized to acetoacetate that is exported to the cytosol and metabolized to the same short chain acyl-CoAs and fatty acids that can be derived from citrate. We tested for redundancy of the citrate pathway.
Methods: We inhibited ATP citrate lyase activity using hydroxycitrate as well as studying a stable cell line generated with shRNA knockdown of ATP citrate lyase in the pancreatic beta cell line INS-1 832/13.
Results: In both instances glucose-stimulated insulin release was not inhibited. Mass spectrometry analysis showed that the flux of carbon from [U-13C]glucose and/or [U-13C]α-ketoisocaproic acid (KIC) into short chain acyl-CoAs in cells with hydroxycitrate-inhibited ATP citrate lyase or in the cell line with stable severe (>90%) shRNA knockdown of ATP citrate lyase was similar to the controls. Both 13C-glucose and 13C-KIC introduced substantial 13C labeling into acetyl-CoA, malonyl-CoA, and HMG-CoA under both conditions. Glucose flux into fatty acids was not affected by ATP citrate lyase knockdown.
Conclusion: The results establish the involvement of the acetoacetate pathway in insulin secretion in pancreatic beta cells.[Hide abstract]
Objective: G protein-coupled receptor (GPCR) signaling regulates insulin secretion and pancreatic β cell-proliferation. While much knowledge has been gained regarding how GPCRs are activated in β cells, less is known about the mechanisms controlling their deactivation. In many cell types, termination of GPCR signaling is controlled by the family of Regulators of G-protein Signaling (RGS). RGS proteins are expressed in most eukaryotic cells and ensure a timely return to the GPCR inactive state upon removal of the stimulus. The aims of this study were i) to determine if RGS16, the most highly enriched RGS protein in β cells, regulates insulin secretion and β-cell proliferation and, if so, ii) to elucidate the mechanisms underlying such effects.
Methods: Mouse and human islets were infected with recombinant adenoviruses expressing shRNA or cDNA sequences to knock-down or overexpress RGS16, respectively. 60 h post-infection, insulin secretion and cAMP levels were measured in static incubations in the presence of glucose and various secretagogues. β-cell proliferation was measured in infected islets after 72 h in the presence of 16.7 mM glucose ± somatostatin and various inhibitors.
Results: RGS16 mRNA levels are strongly up-regulated in islets of Langerhans under hyperglycemic conditions in vivo and ex vivo. RGS16 overexpression stimulated glucose-induced insulin secretion in isolated mouse and human islets while, conversely, insulin secretion was impaired following RGS16 knock-down. Insulin secretion was no longer affected by RGS16 knock-down when islets were pre-treated with pertussis toxin to inactivate Gαi/o proteins, or in the presence of a somatostatin receptor antagonist. RGS16 overexpression increased intracellular cAMP levels, and its effects were blocked by an adenylyl cyclase inhibitor. Finally, RGS16 overexpression prevented the inhibitory effect of somatostatin on insulin secretion and β-cell proliferation.
Conclusions: Our results identify RGS16 as a novel regulator of β-cell function that coordinately controls insulin secretion and proliferation by limiting the tonic inhibitory signal exerted by δ-cell-derived somatostatin in islets.[Hide abstract]
Objective: Cardiovascular diseases (CVDs) are the leading cause of mortality in Western countries. Atherosclerosis is a multi-step inflammatory disease characterized at early stages by accumulation of cholesterol in the arterial wall followed by recruitment of immune cells. We sought to determine if pharmacological suppression of RORα/γ activity is beneficial in treatment of atherosclerosis.
Methods: To identify the role of RORα and RORγ in atherosclerosis, we used the LDL-R−/− mouse model of atherosclerosis placed on a high cholesterol diet treated with SR1001, a RORα/γ inverse agonist, for four weeks.
Results: Our results demonstrate that treatment with the ROR inverse agonist substantially decreases plaque formation in vivo. The mechanism of the anti-atherogenic activity of the inhibition of RORα/γ activity appeared to be due to targeting two distinct pathways. SR1001 treatment reduced plasma low density lipoprotein (LDL) level without affecting high density lipoprotein (HDL) via increasing intestinal cholesterol excretion. Treatment with SR1001 also induced an anti-atherogenic immune profile that was characterized by a reduction in Th17 cells and an increase in Treg and Th2 cells. Our data suggest that RORα and RORγ play a critical role in atherosclerosis development by regulating at least two major pathways important in the pathology of this disease: cholesterol flux and inflammation.
Conclusion: Our data suggest that pharmacological targeting of RORα/γ may be an effective method for treatment of atherosclerosis offering a distinct mechanism of action relative to statins.[Hide abstract]
Objective: The mechanisms by which bariatric surgeries so effectively and lastingly reduce body weight and normalize metabolic dysfunction are not well understood. Fibroblast growth fator-21 (FGF21) is a key regulator of metabolism and is currently considered for treatment of obesity. Although elevated by acute food deprivation, it is downregulated after weight loss induced by chronic calorie restriction but not after Roux-en-Y gastric bypass surgery. Therefore, the goal of the present study was to assess the role of FGF21-signaling in the beneficial effects of Roux-en-Y gastric bypass surgery (RYGB).
Methods: High-fat diet-induced obese FGF21-deficient (FGF21−/−) and wildtype (WT) mice were subjected to RYGB, sham surgery, or caloric restriction to match body weight of RYGB mice. Body weight, body composition, food intake, energy expenditure, glucose tolerance, and insulin sensitivity, as well as plasma levels and hepatic mRNA expression of FGF21 were measured.
Results: Hepatic expression and plasma levels of FGF21 are higher after RYGB compared with similar weight loss induced by caloric restriction, suggesting that elevated FGF21 might play a role in preventing increased hunger and weight regain after RYGB. However, although the body weight differential between RYGB and sham surgery was significantly reduced in FGF21−/− mice, RYGB induced similarly sustained body weight and fat mass loss, initial reduction of food intake, increased energy expenditure, and improvements in glycemic control in FGF21−/− and WT mice.
Conclusions: FGF21 signaling is not a critical single factor for the beneficial metabolic effects of RYGB. This may open up the possibility to use FGF21 as adjuvant therapy in patients with ineffective bariatric surgeries.[Hide abstract]
Objective: Fibroblast activation protein (FAP) is a serine protease belonging to a S9B prolyl oligopeptidase subfamily. This enzyme has been implicated in cancer development and recently reported to regulate degradation of FGF21, a potent metabolic hormone. Using a known FAP inhibitor, talabostat (TB), we explored the impact of FAP inhibition on metabolic regulation in mice.
Methods: To address this question we evaluated the pharmacology of TB in various mouse models including those deficient in FGF21, GLP1 and GIP signaling. We also studied the ability of FAP to process FGF21 in vitro and TB to block FAP enzymatic activity.
Results: TB administration to diet-induced obese (DIO) animals led to profound decreases in body weight, reduced food consumption and adiposity, increased energy expenditure, improved glucose tolerance and insulin sensitivity, and lowered cholesterol levels. Total and intact plasma FGF21 were observed to be elevated in TB-treated DIO mice but not lean animals where the metabolic impact of TB was significantly attenuated. Furthermore, and in stark contrast to naïve DIO mice, the administration of TB to obese FGF21 knockout animals demonstrated no appreciable effect on body weight or any other measures of metabolism. In support of these results we observed no enzymatic degradation of human FGF21 at either end of the protein when FAP was inhibited in vitro by TB.
Conclusions: We conclude that pharmacological inhibition of FAP enhances levels of FGF21 in obese mice to provide robust metabolic benefits not observed in lean animals, thus validating this enzyme as a novel drug target for the treatment of obesity and diabetes.[Hide abstract]
Objective: Compared to men, postmenopausal women suffer from a disproportionate burden of many co-morbidities associated with obesity, e.g. cardiovascular disease, cancer, and dementia. The underlying mechanism for this sex difference is not well understood but is believed to relate to absence of the protective effect of estrogen through the action of estrogen receptor alpha (ERα) in the central nervous system. With the recently developed neuron-specific lipoprotein lipase deficient mice (NEXLPL−/−) (Wang et al., Cell Metabolism, 2011 ), we set to explore the possible role of lipid sensing in sex differences in obesity development.
Methods: Both male and female NEXLPL−/− mice and littermate WT controls were subjected to pair feeding (pf) where daily food amount given was adjusted according to body weight to match the food intake of ad libitum (ad) fed control WT mice. Food intake and body weight were measured daily, and pair feeding was maintained to 42 wk in male mice and to 38 wk in female mice. Various brain regions of the mice were harvested, and ERα gene expression was examined in both male and female NEXLPL−/− and WT control mice under both ad- and pf-fed conditions.
Results: Although both male and female NEXLPL−/− mice developed obesity similarly on standard chow, male NEXLPL−/− mice still developed obesity under with pair feeding, but on a much delayed time course, while female NEXLPL−/− mice were protected from extra body weight and fat mass gain compared to pair-fed WT control mice. Pair feeding alone induced extra fat mass gain in both male and female WT mice, and this was mostly driven by the reduction in physical activity. LPL deficiency resulted in an increase in ERα mRNA in the hypothalamus of ad-fed female mice, while pair feeding alone also resulted in an increase of ERα in both female WT control and NEXLPL−/− mice. The effect on increasing ERα by pair feeding and LPL deficiency was additive in pair-fed female NEXLPL−/− mice. ERα mRNA levels were not significantly modified in other brain regions examined, nor in the hypothalamus of male NEXLPL−/− mice compared to control mice.
Conclusions: These results suggest that the mechanism underlying ERα regulation of body weight interacts with the LPL-dependent lipid processing in the hypothalamus in a sex specific way. ERα could provide the link between brain lipid sensing and sex differences in obesity development. This study has the potential important clinical implication to provide better management for women who suffer from obesity and obesity-related co-morbidities.[Hide abstract]
Objective: DNA methylation may be a stable epigenetic contributor to defining fat cell lineage.
Methods: We performed reduced representation bisulfite sequencing (RRBS) and RNA-seq to depict a genome-wide integrative view of the DNA methylome and transcriptome during brown and white adipogenesis.
Results: Our analysis demonstrated that DNA methylation is a stable epigenetic signature for brown and white cell lineage before, during, and after differentiation. We identified 31 genes whose promoters were significantly differentially methylated between white and brown adipogenesis at all three time points of differentiation. Among them, five genes belong to the Hox family; their expression levels were anti-correlated with promoter methylation, suggesting a regulatory role of DNA methylation in transcription. Blocking DNA methylation with 5-Aza-cytidine increased the expression of these genes, with the most prominent effect on Hoxc10, a repressor of BAT marker expression.
Conclusions: Our data suggest that DNA methylation may play an important role in lineage-specific development in adipocytes.[Hide abstract]
Objective: A decrease in muscle protein turnover and therefore in muscle mass is a hallmark of aging. Because the circulating levels of the bone-derived hormone osteocalcin decline steeply during aging in mice, monkeys and humans we asked here whether this hormone might regulate muscle mass as mice age.
Methods: We examined muscle mass and strength in mice lacking osteocalcin (Ocn−/−) or its receptor in all cells (Gprc6a−/−) or specifically in myofibers (Gprc6aMck−/−) as well as in 9 month-old WT mice receiving exogenous osteocalcin for 28 days. We also examined protein synthesis in WT and Gprc6a−/− mouse myotubes treated with osteocalcin.
Results: We show that osteocalcin signaling in myofibers is necessary to maintain muscle mass in older mice in part because it promotes protein synthesis in myotubes without affecting protein breakdown. We further show that treatment with exogenous osteocalcin for 28 days is sufficient to increase muscle mass of 9-month-old WT mice.
Conclusions: This study uncovers that osteocalcin is necessary and sufficient to prevent age-related muscle loss in mice.[Hide abstract]
Objective: The sodium-glucose transporter 2 (SGLT2) inhibitors Canagliflozin and Dapagliflozin are recently approved medications for type 2 diabetes. Recent studies indicate that SGLT2 inhibitors may inhibit the growth of some cancer cells but the mechanism(s) remain unclear.
Methods: Cellular proliferation and clonogenic survival were used to assess the sensitivity of prostate and lung cancer cell growth to the SGLT2 inhibitors. Oxygen consumption, extracellular acidification rate, cellular ATP, glucose uptake, lipogenesis, and phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase, and the p70S6 kinase were assessed. Overexpression of a protein that maintains complex-I supported mitochondrial respiration (NDI1) was used to establish the importance of this pathway for mediating the anti-proliferative effects of Canagliflozin.
Results: Clinically achievable concentrations of Canagliflozin, but not Dapagliflozin, inhibit cellular proliferation and clonogenic survival of prostate and lung cancer cells alone and in combination with ionizing radiation and the chemotherapy Docetaxel. Canagliflozin reduced glucose uptake, mitochondrial complex-I supported respiration, ATP, and lipogenesis while increasing the activating phosphorylation of AMPK. The overexpression of NDI1 blocked the anti-proliferative effects of Canagliflozin indicating reductions in mitochondrial respiration are critical for anti-proliferative actions.
Conclusions: These data indicate that like the biguanide metformin, Canagliflozin not only lowers blood glucose but also inhibits complex-I supported respiration and cellular proliferation in prostate and lung cancer cells. These observations support the initiation of studies evaluating the clinical efficacy of Canagliflozin on limiting tumorigenesis in pre-clinical animal models as well epidemiological studies on cancer incidence relative to other glucose lowering therapies in clinical populations.[Hide abstract]