Featured ArticlesVolume 5 | No. 10 | October 2016
|α-Melanocyte stimulating hormone promotes muscle glucose uptakeEnriori, Chen et al. set out to determine if peripheral α-melanocyte stimulating hormone (α-MSH) plays a role in glucose homeostasis and test the hypothesis that the pituitary is able to sense a physiological increase in circulating glucose and responds by secreting α-MSH. They show that glucose stimulates α-MSH production and increases circulating concentrations, which increases muscle glucose uptake through the activation of the melanocortin 5 receptor (MC5R)-PKA pathway.|
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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]
|Estrogens modulate ventrolateral ventromedial hypothalamic glucose-inhibited neuronsSantiago and colleagues show a sexual dimorphism in nonadapting ventrolateral ventromedial hypothalamic nucleus (VL-VMN) glucose-inhibited (GI) neurons, which is apparently due to organizational effects since differences are present in the absence of estrogens. 17β-estradiol (17βE) affects the glucose sensing machinery (AMP-activated protein kinase) in both non-adapting GI and adapting GI (AdGI) neurons of both sexes. The data suggest that sex differences observed in VL-VMN nonadapting GI neurons and 17βE effects on the glucose sensitivity of both nonadaptive GI and AdGI neurons may underlie the observed sex differences in hypoglycemia detection and counterregulation.|
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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]
|The satiating hormone amylin enhances neurogenesis in the area postremaAmylin is a pancreatic hormone whose role in the control of food intake and energy metabolism is well characterized. Liberini and colleagues demonstrate that amylin regulates genes involved in pathways and processes that drive neurogenesis in the adult mammalian brain. Their results also show an in vivo increase in the number of newly proliferating area postrema (AP)-cells after chronic amylin treatment. Amylin has the potential to commit the AP adult-born cells to a neuronal fate.|
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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]
|Defining a novel leptin-melanocortin-kisspeptin pathwayRecent reports documented that earlier puberty is linked to a higher risk of cardio-metabolic disease, including hypertension and type-2 diabetes. The data of Manfredi-Lozano, Roa et al. in wild type and genetically modified rodent models provide a novel mechanistic insight into the melanocortin regulation of pubertal maturation in the female by documenting a discernible leptin → α-melanocyte stimulating hormone → kisspeptin → gonadotropin-releasing hormone pathway, which appears to play an important role in the metabolic control of puberty.|
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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]
|Mechanisms underlying prorenin actions on hypothalamic neurons implicated in cardiometabolic controlPitra and colleagues provide novel evidence that indicates that prorenin stimulates hypothalamic magnocellular neurosecretory cells and presympathetic paraventricular nucleus neuronal activity via distinct, angiotensin II-independent and dependent mechanisms, respectively, and that these actions involve suppression of a voltage gated K+ channel in a Ca2+-dependent manner. Elucidating the basic cellular targets and mechanisms by which prorenin and its receptor regulate neuronal activity within the hypothalamus is fundamental information required for a more comprehensive understanding of how the central RAS influences sympatho-humoral regulation of cardiovascular and metabolic functions.|
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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]
|Reprogramming the body weight set point reverts extreme obesityChhabra and colleagues demonstrate that proopiomelanocortin (Pomc)-deficiency in the arcuate (Arc) nucleus increases fat mass and the ratio of fat to lean mass, and it impairs the function of leptin to reduce body weight and food intake independently of body weight. Rescue of ArcPomc expression in weight-matched mice with ArcPomc silencing, whether or not there is a history of previous obesity, reestablishes normal energy homeostasis and body weight set point under ad libitum feeding conditions by correcting the abnormal body composition and restoring leptin sensitivity. Massive hyperleptinemia induced by PASylated leptin administration prevents this normalization by blocking the complete restoration of ArcPomc expression. A strong reciprocal association between leptin levels and hypothalamic Pomc in the regulation of energy homeostasis is revealed.|
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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]
|Adiponectin potentiates the acute effects of leptin in arcuate Pomc neuronsSun, Gao, Yao et al. test the hypothesis that adiponectin and leptin may synergistically activate melanocortin neurons. They utilize transgenic and Cre-Lox technology to identify neuropeptide Y/Agouti-related peptide (NPY/AgRP) and proopiomelanocortin (Pomc) neurons, which express leptin receptors. They show that leptin and adiponectin modulate neuronal excitability of melanocortin neurons in an additive manner, an activity that requires phosphoinositide-3-kinase (PI3K) signaling. The data provide evidence for PI3K as a substrate for both leptin and adiponectin to regulate metabolism via melanocortin activity.|
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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]
|Physiological role for leptin in the control of thermal conductanceKaiyala and colleagues report that physiological leptin levels play a role in conferring the ability to defend normothermia during cold exposure. This effect does not stem from increased energy expenditure, the induction of BAT, or increased ambulatory activity levels, but is due, in part, to an effect of leptin to reduce heat loss. These findings identify a new physiological role for leptin as a mediator of reduced heat dissipation elicited by cold exposure, an efficient and economical homeostatic strategy for maintenance of core temperature in light of the extremely high metabolic cost of matching energy intake to markedly elevated energy expenditure levels during cold exposure in small mammals.|
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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]
|TNP ameliorates diet induced obesity and insulin resistanceGoshal and colleagues show that pharmacologic inhibition of the inositol pyrophosphate pathway ameliorates obesity and insulin resistance in diet induced obesity (DIO) mice. TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine] fails to reduce weight gain in high fat diet-fed inositol hexakisphosphate kinase-1 (IP6K1)-knock out mice. This suggests that the compound reduces body weight specifically via IP6K1 inhibition. Even a single-dose of TNP enhances insulin sensitivity in DIO mice by increasing Akt activity in metabolic tissues. TNP reduces body weight, at least in part, by augmenting adipose tissue browning/thermogenesis mediated energy expenditure.|
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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]
|Impact of statistical models on the prediction of type 2 diabetes Although epidemiological studies have reported numerous risk factors for type 2 diabetes, the predictive performances of statistical models based on these predictors still need to be improved. The study of Yengo and colleagues highlights that few biomarkers with an efficient combination as risk scores can improve the identification of incident type 2 diabetes cases, especially in those poorly recognized by classical clinical risk factors. The clinical use of such biomarkers is important for the development of early interventions for the prevention of type 2 diabetes, involving changes in life style and pharmacotherapy.|
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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]
|Defects in muscle branched-chain amino acid oxidation contribute to impaired lipid metabolismBranched-chain amino acids (BCAAs) have been identified as potential contributors to insulin resistance. The data of Lerin and colleagues suggest that perturbations in BCAA metabolism may contribute to metabolic phenotypes associated with insulin resistance and type 2 diabetes risk. The study also suggests that searching for metabolic phenotypes in carriers of methylmalonyl-CoA mutase mutations would be relevant for understanding disease pathogenesis and guiding clinical management strategies.|
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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]
|Depot specific differences in the adipogenic potential of precursorsThe extracellular matrix (ECM) is known to play an important role in terminal differentiation of preadipocytes. Given this and the known differences in the response of different adipose depots, Grandl and colleagues employ a strategy for deriving the extracellular matrix from primary cells of different adipose depots to investigate its role in adipose precursor differentiation. They demonstrate that the potential for depot-specific adipocyte precursors to form mature adipocytes is mainly dependent on the ECM. They also find that integration of the necessary ECM to cell signaling cues might in part be mediated through transmembrane protein Flotillin 2.|
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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]
|Genetic backgrounds determine brown remodeling of white fatGenetic background is a major contributing factor in the susceptibility to diet induced obesity and type 2 diabetes. The study of Ferrannini, Namwanje et al. reveals that genetic background is a significant factor in regulating brown remodeling of white adipose tissue in a strain specific and depot-specific manner. They identify novel candidates in regulating adipose tissue plasticity, particularly Hoxc10 as a “browning brake” in subcutaneous white adipose tissue. The expectation of the authors is that the coordination between activators and repressors in brown remodeling determines the physiological consequences of adipose tissue and thus may provide novel insights for obesity treatment. |
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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]
|Adipose tissue (P)RR regulates insulin sensitivity, fat mass and body weightThe study of patients suffering from obesity and obese rodent models has shed light on the importance of the renin-angiotensin system (RAS) in the development of obesity. The prorenin/renin receptor [(P)RR] is a component of the RAS where its main role is to increase the catalytic activity of renin. The results of Shamansurova, Tan et al. suggest that adipose tissue (P)RR is involved in the development of obesity and its associated complications. Suppression of the (P)RR, specifically in adipose tissue, produces a clear phenotype of reduced body weight and fat masses as well as improved adipose tissue structure and insulin sensitivity. |
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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]
|Myocardin-related transcription factor A (MRTFA) regulates the fate of bone marrow mesenchymal stem cellsAdipogenesis and osteogenesis are mutually exclusive processes during mesenchymal stem cell differentiation, and appropriate balance between them is essential for maintaining homeostasis in bone marrow. Once the precisely regulated balance is disturbed, various metabolic-related diseases develop. The study of Bian and colleagues identifies myocardin-related transcription factor A as a novel regulator of skeletal homeostasis by controlling the balance between adipogenic and osteogenic differentiation in bone marrow stem cells, and thus holds promise as a potential target for therapeutic intervention for osteoporosis.|
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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]
|Knockdown of ATP citrate lyase in pancreatic beta cells does not inhibit insulin secretion or glucose fluxIn addition to ATP production, an important role of mitochondria in the pancreatic beta cell is the net synthesis of citric acid cycle intermediates that are exported to the cytosol where they are converted to numerous other metabolites that stimulate and/or support insulin secretion. Azzouny and colleagues show that the flux of glucose into cytosolic short chain acyl-CoAs is maintained in pure beta cells in the presence of inhibition of ATP citrate lyase. The only known pathway other than through the ATP citrate lyase reaction leading to the formation of short chain acyl-CoAs in the cytosol is the acetoacetate pathway. The results establish the role of the acetoacetate pathway in insulin secretion.|
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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]
|The regulator of G-protein signaling RGS16 promotes insulin secretion and β-cell proliferationThe results of Vivot and colleagues demonstrate that in pancreatic β-cells, RGS16 limits the inhibitory effect of somatostatin (SST) on insulin secretion and β-cell proliferation by dampening Gαi/o activity downstream of SST receptor and, consequently, increasing intracellular cAMP levels. Since RGS16 expression is strongly stimulated by glucose, these observations suggest a role for RGS16 in β-cell compensation to increased metabolic demand whereby releasing a tonic “break” on insulin secretion and β-cell proliferation enable the β-cell to quickly adjust to a changing metabolic environment.|
Abstract | PDF
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]
|Inhibition of RORα/γ suppresses atherosclerosis via inhibition of both cholesterol absorption and inflammationRAR-related orphan receptors (ROR) are members of the NR superfamily that are known to be involved in inflammatory and metabolic processes. The data of Billon and colleagues demonstrate that pharmacological suppression of RORα and RORγ activity suppresses atherosclerosis in a mouse model. RORα and RORγ can be targeted to modulate the immune system and cholesterol excretion, two main risk factors that lead to atherosclerosis. |
Abstract | PDF
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]
|Roux-en-Y gastric bypass surgery is effective in fibroblast growth factor-21 deficient miceFibroblast growth factor 21 (FGF21) has profound beneficial effects on body weight and metabolism in preclinical obesity models. Morrison and colleagues show that FGF21-signaling is not a critical single factor required for Roux-en-Y gastric bypass surgery (RYGB) to lower body weight and improve glycemic control but may play a minor role in food choice and locomotor activity. The findings in this study do not rule out the possibility that FGF21 acts as an important co-factor with other putative mechanisms. If FGF21 is not directly involved in RYGB’s effects on energy balance, it might be useful as an adjuvant future therapy in patients with failed or suboptimal bariatric surgery outcomes.|
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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]
|Fibroblast activation protein (FAP) as a novel metabolic targetFibroblast activation protein (FAP) is a serine protease belonging to a S9B prolyl oligopeptidase subfamily and has been recently reported to regulate degradation of FGF21, a potent metabolic hormone. Sánchez-Garrido and colleagues demonstrate that the FAP inhibitor, talabostat (TB), as a single agent, provides body weight reduction, glucose control, insulin sensitization, cholesterol lowering, and increased energy expenditure associated with elevated plasma fibroblast growth factor 21 (FGF21) levels. The absence of any effect on glucose or weight reduction in FGF21-deficient mice supports their hypothesis that FGF21 is a central mediator of the efficacy delivered by TB therapy. All of these effects are substantially attenuated in lean mice suggesting the ability to selectively inhibit FAP without overt toxicity. |
Abstract | PDF
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]