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A distinct adipose tissue distribution pattern was observed in patients with methylmalonyl-CoA mutase deficiency, an inborn error of branched-chain amino acid (BCAA) metabolism, characterized by centripetal obesity with proximal upper and lower extremity fat deposition and paucity of visceral fat, that resembles familial multiple lipomatosis syndrome. To explore brown and white fat physiology in methylmalonic acidemia (MMA), body composition, adipokines, and inflammatory markers were assessed in 46 patients with MMA and 99 matched controls. Fibroblast growth factor 21 levels were associated with acyl-CoA accretion, aberrant methylmalonylation in adipose tissue, and an attenuated inflammatory cytokine profile. In parallel, brown and white fat were examined in a liver-specific transgenic MMA mouse model (Mmut-/- TgINS-Alb-Mmut). The MMA mice exhibited abnormal nonshivering thermogenesis with whitened brown fat and had an ineffective transcriptional response to cold stress. Treatment of the MMA mice with bezafibrates led to clinical improvement with beiging of subcutaneous fat depots, which resembled the distribution seen in the patients. These studies defined what we believe to be a novel lipodystrophy phenotype in patients with defects in the terminal steps of BCAA oxidation and demonstrated that beiging of subcutaneous adipose tissue in MMA could readily be induced with small molecules.
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Erros Inatos do Metabolismo dos Aminoácidos , Fatores de Crescimento de Fibroblastos , Lipodistrofia , Animais , Humanos , Camundongos , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Erros Inatos do Metabolismo dos Aminoácidos/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Camundongos TransgênicosRESUMO
Melanocortin 4 receptor (MC4R) mutations are the most common cause of human monogenic obesity and are associated with hyperphagia and increased linear growth. While MC4R is known to activate Gsα/cAMP signaling, a substantial proportion of obesity-associated MC4R mutations do not affect MC4R/Gsα signaling. To further explore the role of specific MC4R signaling pathways in the regulation of energy balance, we examined the signaling properties of one such mutant, MC4R (F51L), as well as the metabolic consequences of MC4RF51L mutation in mice. The MC4RF51L mutation produced a specific defect in MC4R/Gq/11α signaling and led to obesity, hyperphagia, and increased linear growth in mice. The ability of a melanocortin agonist to acutely inhibit food intake when delivered to the paraventricular nucleus (PVN) was lost in MC4RF51L mice, as well as in WT mice in which a specific Gq/11α inhibitor was delivered to the PVN; this provided evidence that a Gsα-independent signaling pathway, namely Gq/11α, significantly contributes to the actions of MC4R on food intake and linear growth. These results suggest that a biased MC4R agonist that primarily activates Gq/11α may be a potential agent to treat obesity with limited untoward cardiovascular and other side effects.
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Hiperfagia , Receptor Tipo 4 de Melanocortina , Humanos , Camundongos , Animais , Receptor Tipo 4 de Melanocortina/metabolismo , Hiperfagia/genética , Hiperfagia/metabolismo , Obesidade/metabolismo , Transdução de Sinais/fisiologia , MutaçãoRESUMO
A1 adenosine receptor (A1AR) agonists have cerebroprotective, cardioprotective, antinociceptive, and other pharmaceutical applications. We explored the structure-activity relationship of 5-arylethynyl aminothiophenes as A1AR positive allosteric modulators (PAMs). The derivatives were compared in binding and functional assays at the human A1AR, indicating that some fluoro-substituted analogues have enhanced PAM activity. We identified substitution of the terminal phenyl ring in 12 (2-F-Ph), 15 (3,4-F2-Ph, MRS7935), and 21 (2-CF3-Ph) as particularly enhancing the PAM activity. 15 was also shown to act as an A1 ago-PAM with EC50 ≈ 2 µM, without activity (30 µM) at other ARs. Molecular modeling indicated that both the 5-arylethynyl and the 4-neopentyl groups are located in a region outside the receptor transmembrane helix bundle that is in contact with the phospholipid bilayer, consistent with the preference for nonpolar substitution of the aryl moiety. Although they are hydrophobic, these PAMs could provide potential drug candidate molecules for engaging protective A1ARs.
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OBJECTIVE: Otopetrin 1 (OTOP1) is a proton channel that is highly expressed in brown adipose tissue. We examined the physiology of Otop1-/- mice, which lack functional OTOP1. METHODS: Mice were studied by indirect calorimetry and telemetric ambulatory body temperature monitoring. Mitochondrial function was measured as oxygen consumption and extracellular acidification. RESULTS: Otop1-/- mice had similar body temperatures as control mice at baseline and in response to cold and hot ambient temperatures. However, in response to fasting the Otop1-/- mice exhibited an exaggerated hypothermia and hypometabolism. Similarly, in ex vivo tests of Otop1-/- brown adipose tissue mitochondrial function, there was no change in baseline oxygen consumption, but the oxygen consumption was reduced after maximal uncoupling with FCCP and increased upon stimulation with the ß3-adrenergic agonist CL316243. Mast cells also express Otop1, and Otop1-/- mice had intact, possibly greater hypothermia in response to mast cell activation by the adenosine A3 receptor agonist MRS5698. No increase in insulin resistance was observed in the Otop1-/- mice. CONCLUSIONS: Loss of OTOP1 does not change basal function of brown adipose tissue but affects stimulated responses.
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Hipotermia , Animais , Camundongos , Tecido Adiposo Marrom , Temperatura Corporal , Regulação da Temperatura Corporal , Jejum , Camundongos KnockoutRESUMO
Neuropeptides may exert trophic effects during development, and then neurotransmitter roles in the developed nervous system. One way to associate peptide-deficiency phenotypes with either role is first to assess potential phenotypes in so-called constitutive knockout mice, and then proceed to specify, regionally and temporally, where and when neuropeptide expression is required to prevent these phenotypes. We have previously demonstrated that the well-known constellation of behavioral and metabolic phenotypes associated with constitutive pituitary adenylate cyclase-activating peptide (PACAP) knockout mice are accompanied by transcriptomic alterations of two types: those that distinguish the PACAP-null phenotype from wild-type (WT) in otherwise quiescent mice (cPRGs), and gene induction that occurs in response to acute environmental perturbation in WT mice that do not occur in knockout mice (aPRGs). Comparing constitutive PACAP knockout mice to a variety of temporally and regionally specific PACAP knockouts, we show that the prominent hyperlocomotor phenotype is a consequence of early loss of PACAP expression, is associated with Fos overexpression in hippocampus and basal ganglia, and that a thermoregulatory effect previously shown to be mediated by PACAP-expressing neurons of medial preoptic hypothalamus is independent of PACAP expression in those neurons in adult mice. In contrast, PACAP dependence of weight loss/hypophagia triggered by restraint stress, seen in constitutive PACAP knockout mice, is phenocopied in mice in which PACAP is deleted after neuronal differentiation. Our results imply that PACAP has a prominent role as a trophic factor early in development determining global central nervous system characteristics, and in addition a second, discrete set of functions as a neurotransmitter in the fully developed nervous system that support physiological and psychological responses to stress.
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Neurotransmissores , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Animais , Camundongos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Neurônios/metabolismo , Fenótipo , Camundongos KnockoutRESUMO
OBJECTIVE: Physical activity is a major component of total energy expenditure (TEE) that exhibits extreme variability in mice. Our objective was to construct a general, physiology-based model of TEE to accurately quantify the energy cost of physical activity. METHODS: Spontaneous home cage physical activity, body temperature, TEE, and energy intake were measured with frequent sampling. The energy cost of activity was modeled considering six contributors to TEE (basal metabolic rate, thermic effect of food, body temperature, cold induced thermogenesis, physical activity, and body weight). An ambient temperature of 35 °C was required to remove the contribution from cold induced thermogenesis. Basal metabolic rate was adjusted for body temperature using a Q10 temperature coefficient. RESULTS: We developed a TEE model that robustly explains 70-80% of the variance in TEE at 35 °C while fitting only two parameters, the basal metabolic rate and the mass-specific energy cost per unit of physical activity, which averaged 60 cal/km/g body weight. In Ucp1-/- mice the activity cost was elevated by 60%, indicating inefficiency and increased muscle thermogenesis. The diurnal rhythm in TEE was quantitatively explained by the combined diurnal differences in physical activity, body temperature, and energy intake. Incorporating body temperature into human basal metabolic rate measurements significantly reduced the inter-individual variation. CONCLUSIONS: The physiology-based model of TEE allows quantifying the energy cost of physical activity. While applied here to mice, the model should be generally valid across species. Due to the effect of body temperature, we suggest that basal metabolic rate measurements be corrected to a reference body temperature, including in humans. Having an accurate cost of physical activity allows mechanistic dissection of disorders of energy homeostasis, including obesity.
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Metabolismo Basal , Metabolismo Energético , Humanos , Animais , Camundongos , Metabolismo Energético/fisiologia , Peso Corporal/fisiologia , Metabolismo Basal/fisiologia , Obesidade , Termogênese/fisiologiaRESUMO
Some non-adenosinergic drugs are reported to also act through adenosine receptors (ARs). We used mouse hypothermia, which can be induced by agonism at any of the four ARs, as an in vivo screen for adenosinergic effects. An AR contribution was identified when a drug caused hypothermia in wild type mice that was diminished in mice lacking all four ARs (quadruple knockout, QKO). Alternatively, an adenosinergic effect was identified if a drug potentiated adenosine-induced hypothermia. Four drugs (dipyridamole, nimodipine, cilostazol, cyclosporin A) increased the hypothermia caused by adenosine. Dipyridamole and nimodipine probably achieved this by inhibition of adenosine clearance via ENT1. Two drugs (cannabidiol, canrenoate) did not cause hypothermia in wild type mice. Four other drugs (nifedipine, ranolazine, ketamine, ethanol) caused hypothermia, but the hypothermia was unchanged in QKO mice indicating non-adenosinergic mechanisms. Zinc chloride caused hypothermia and hypoactivity; the hypoactivity was blunted in the QKO mice. Interestingly, the antidepressant amitriptyline caused hypothermia in wild type mice that was amplified in the QKO mice. Thus, we have identified adenosine-related effects for some drugs, while other candidates do not affect adenosine signaling by this in vivo assay. The adenosine-modulating drugs could be considered for repurposing based on predicted effects on AR activation.
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Adenosina , Hipotermia , Camundongos , Animais , Adenosina/farmacologia , Hipotermia/induzido quimicamente , Nimodipina/efeitos adversos , Receptores Purinérgicos P1 , Dipiridamol/efeitos adversosRESUMO
Following our study of 4'-truncated (N)-methanocarba-adenosine derivatives that displayed unusually high mouse (m) A3AR affinity, we incorporated dopamine-related N6 substituents in the full agonist 5'-methylamide series. N6-(2-(4-Hydroxy-3-methoxy-phenyl)ethyl) derivative MRS7618 11 displayed Ki (nM) 0.563 at hA3AR (â¼20,000-fold selective) and 1.54 at mA3AR. 2-Alkyl ethers maintained A3 affinity, but with less selectivity than 2-alkynes. Parallel functional assays of G protein-dependent and ß-arrestin 2 (ßarr2)-dependent pathways indicate these are full agonists but not biased. Through use of computational modeling, we hypothesized that phenyl OH/OMe groups interact with polar residues, particularly Gln261, on the mA3AR extracellular loops as the basis for the affinity enhancement. Although the pharmacokinetics indicated facile clearance of parent O-methyl catechol nucleosides 21 and 31, prolonged mA3AR activation in vivo was observed in a hypothermia model, suggested potential formation of active metabolites through demethylation. Selected analogues induced mouse hypothermia following i.p. injection, indicative of peripheral A3AR agonism in vivo.
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Agonistas do Receptor A3 de Adenosina/farmacologia , Dopamina/farmacologia , Receptor A3 de Adenosina/metabolismo , Agonistas do Receptor A3 de Adenosina/síntese química , Agonistas do Receptor A3 de Adenosina/química , Dopamina/síntese química , Dopamina/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
MYC is a transcription factor with broad biological functions, notably in the control of cell proliferation. Here, we show that intestinal MYC regulates systemic metabolism. We find that MYC expression is increased in ileum biopsies from individuals with obesity and positively correlates with body mass index. Intestine-specific reduction of MYC in mice improves high-fat-diet-induced obesity, insulin resistance, hepatic steatosis and steatohepatitis. Mechanistically, reduced expression of MYC in the intestine promotes glucagon-like peptide-1 (GLP-1) production and secretion. Moreover, we identify Cers4, encoding ceramide synthase 4, catalysing de novo ceramide synthesis, as a MYC target gene. Finally, we show that administration of the MYC inhibitor 10058-F4 has beneficial effects on high-fat-diet-induced metabolic disorders, and is accompanied by increased GLP-1 and reduced ceramide levels in serum. This study positions intestinal MYC as a putative drug target against metabolic diseases, including non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.
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Mucosa Intestinal/metabolismo , Obesidade/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Biomarcadores , Dieta Hiperlipídica , Modelos Animais de Doenças , Suscetibilidade a Doenças , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Humanos , Ílio/metabolismo , Resistência à Insulina , Camundongos , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/etiologia , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
Obesity is the major driver of the worldwide epidemic in type 2 diabetes (T2D). In the obese state, chronically elevated plasma free fatty acid levels contribute to peripheral insulin resistance, which can ultimately lead to the development of T2D. For this reason, drugs that are able to regulate lipolytic processes in adipocytes are predicted to have considerable therapeutic potential. Gi-coupled P2Y14 receptor (P2Y14R; endogenous agonist, UDP-glucose) is abundantly expressed in both mouse and human adipocytes. Because activated Gi-type G proteins exert an antilipolytic effect, we explored the potential physiological relevance of adipocyte P2Y14Rs in regulating lipid and glucose homeostasis. Metabolic studies indicate that the lack of adipocyte P2Y14R enhanced lipolysis only in the fasting state, decreased body weight, and improved glucose tolerance and insulin sensitivity. Mechanistic studies suggested that adipocyte P2Y14R inhibits lipolysis by reducing lipolytic enzyme activity, including ATGL and HSL. In agreement with these findings, agonist treatment of control mice with a P2Y14R agonist decreased lipolysis, an effect that was sensitive to inhibition by a P2Y14R antagonist. In conclusion, we demonstrate that adipose P2Y14Rs were critical regulators of whole-body glucose and lipid homeostasis, suggesting that P2Y14R antagonists might be beneficial for the therapy of obesity and T2D.
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Glucose/metabolismo , Lipólise/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND AND AIMS: 17-Beta hydroxysteroid dehydrogenase 13 (HSD17B13) is genetically associated with human nonalcoholic fatty liver disease (NAFLD). Inactivating mutations in HSD17B13 protect humans from NAFLD-associated and alcohol-associated liver injury, fibrosis, cirrhosis, and hepatocellular carcinoma, leading to clinical trials of anti-HSD17B13 therapeutic agents in humans. We aimed to study the in vivo function of HSD17B13 using a mouse model. APPROACH AND RESULTS: Single-cell RNA-sequencing and quantitative RT-PCR data revealed that hepatocytes are the main HSD17B13-expressing cells in mice and humans. We compared Hsd17b13 whole-body knockout (KO) mice and wild-type (WT) littermate controls fed regular chow (RC), a high-fat diet (HFD), a Western diet (WD), or the National Institute on Alcohol Abuse and Alcoholism model of alcohol exposure. HFD and WD induced significant weight gain, hepatic steatosis, and inflammation. However, there was no difference between genotypes with regard to body weight, liver weight, hepatic triglycerides (TG), histological inflammatory scores, expression of inflammation-related and fibrosis-related genes, and hepatic retinoid levels. Compared to WT, KO mice on the HFD had hepatic enrichment of most cholesterol esters, monoglycerides, and certain sphingolipid species. Extended feeding with the WD for 10 months led to extensive liver injury, fibrosis, and hepatocellular carcinoma, with no difference between genotypes. Under alcohol exposure, KO and WT mice showed similar hepatic TG and liver enzyme levels. Interestingly, chow-fed KO mice showed significantly higher body and liver weights compared to WT mice, while KO mice on obesogenic diets had a shift toward larger lipid droplets. CONCLUSIONS: Extensive evaluation of Hsd17b13 deficiency in mice under several fatty liver-inducing dietary conditions did not reproduce the protective role of HSD17B13 loss-of-function mutants in human NAFLD. Moreover, mouse Hsd17b13 deficiency induces weight gain under RC. It is crucial to understand interspecies differences prior to leveraging HSD17B13 therapies.
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17-Hidroxiesteroide Desidrogenases/deficiência , Dieta Hiperlipídica/efeitos adversos , 17-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Dieta Ocidental/efeitos adversos , Etanol/efeitos adversos , Fígado Gorduroso/etiologia , Lipídeos/análise , Fígado/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Aumento de PesoRESUMO
BACKGROUND AND AIMS: Iron is essential yet also highly chemically reactive and potentially toxic. The mechanisms that allow cells to use iron safely are not clear; defects in iron management are a causative factor in the cell-death pathway known as ferroptosis. Poly rC binding protein 1 (PCBP1) is a multifunctional protein that serves as a cytosolic iron chaperone, binding and transferring iron to recipient proteins in mammalian cells. Although PCBP1 distributes iron in cells, its role in managing iron in mammalian tissues remains open for study. The liver is highly specialized for iron uptake, utilization, storage, and secretion. APPROACH AND RESULTS: Mice lacking PCBP1 in hepatocytes exhibited defects in liver iron homeostasis with low levels of liver iron, reduced activity of iron enzymes, and misregulation of the cell-autonomous iron regulatory system. These mice spontaneously developed liver disease with hepatic steatosis, inflammation, and degeneration. Transcriptome analysis indicated activation of lipid biosynthetic and oxidative-stress response pathways, including the antiferroptotic mediator, glutathione peroxidase type 4. Although PCBP1-deleted livers were iron deficient, dietary iron supplementation did not prevent steatosis; instead, dietary iron restriction and antioxidant therapy with vitamin E prevented liver disease. PCBP1-deleted hepatocytes exhibited increased labile iron and production of reactive oxygen species (ROS), were hypersensitive to iron and pro-oxidants, and accumulated oxidatively damaged lipids because of the reactivity of unchaperoned iron. CONCLUSIONS: Unchaperoned iron in PCBP1-deleted mouse hepatocytes leads to production of ROS, resulting in lipid peroxidation (LPO) and steatosis in the absence of iron overload. The iron chaperone activity of PCBP1 is therefore critical for limiting the toxicity of cytosolic iron and may be a key factor in preventing the LPO that triggers the ferroptotic cell-death pathway.
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Proteínas de Ligação a DNA/metabolismo , Fígado Gorduroso/etiologia , Compostos de Ferro/metabolismo , Peroxidação de Lipídeos , Metalochaperonas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos Knockout , Estresse OxidativoRESUMO
The G-protein subunits Gqα and G11α (Gq/11α) couple receptors to phospholipase C, leading to increased intracellular calcium. In this study we investigated the consequences of Gq/11α deficiency in the dorsomedial hypothalamus (DMH), a critical site for the control of energy homeostasis. Mice with DMH-specific deletion of Gq/11α (DMHGq/11KO) were generated by stereotaxic injection of adeno-associated virus (AAV)-Cre-green fluorescent protein (GFP) into the DMH of Gqαflox/flox:G11α-/- mice. Compared with control mice that received DMH injection of AAV-GFP, DMHGq/11KO mice developed obesity associated with reduced energy expenditure without significant changes in food intake or physical activity. DMHGq/11KO mice showed no defects in the ability of the melanocortin agonist melanotan II to acutely stimulate energy expenditure or to inhibit food intake. At room temperature (22°C), DMHGq/11KO mice showed reduced sympathetic nervous system activity in brown adipose tissue (BAT) and heart, accompanied with decreased basal BAT uncoupling protein 1 (Ucp1) gene expression and lower heart rates. These mice were cold intolerant when acutely exposed to cold (6°C for 5 h) and had decreased cold-stimulated BAT Ucp1 gene expression. DMHGq/11KO mice also failed to adapt to gradually declining ambient temperatures and to develop adipocyte browning in inguinal white adipose tissue although their BAT Ucp1 was proportionally stimulated. Consistent with impaired cold-induced thermogenesis, the onset of obesity in DMHGq/11KO mice was significantly delayed when housed under thermoneutral conditions (30°C). Thus our results show that Gqα and G11α in the DMH are required for the control of energy homeostasis by stimulating energy expenditure and thermoregulation.NEW & NOTEWORTHY This paper demonstrates that signaling within the dorsomedial hypothalamus via the G proteins Gqα and G11α, which couple cell surface receptors to the stimulation of phospholipase C, is critical for regulation of energy expenditure, thermoregulation by brown adipose tissue and the induction of white adipose tissue browning.
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Doenças do Sistema Nervoso Autônomo/genética , Metabolismo Energético/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Hipotálamo/metabolismo , Obesidade/genética , Animais , Doenças do Sistema Nervoso Autônomo/metabolismo , Doenças do Sistema Nervoso Autônomo/fisiopatologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/deficiência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Obesidade/fisiopatologia , Especificidade de Órgãos/genética , Sistema Nervoso Simpático/metabolismo , Sistema Nervoso Simpático/fisiopatologiaRESUMO
Extracellular adenosine, a danger signal, can cause hypothermia. We generated mice lacking neuronal adenosine A1 receptors (A1AR, encoded by the Adora1 gene) to examine the contribution of these receptors to hypothermia. Intracerebroventricular injection of the selective A1AR agonist (Cl-ENBA, 5'-chloro-5'-deoxy-N6-endo-norbornyladenosine) produced hypothermia, which was reduced in mice with deletion of A1AR in neurons. A non-brain penetrant A1AR agonist [SPA, N6-(p-sulfophenyl) adenosine] also caused hypothermia, in wild type but not mice lacking neuronal A1AR, suggesting that peripheral neuronal A1AR can also cause hypothermia. Mice expressing Cre recombinase from the Adora1 locus were generated to investigate the role of specific cell populations in body temperature regulation. Chemogenetic activation of Adora1-Cre-expressing cells in the preoptic area did not change body temperature. In contrast, activation of Adora1-Cre-expressing dorsomedial hypothalamus cells increased core body temperature, concordant with agonism at the endogenous inhibitory A1AR causing hypothermia. These results suggest that A1AR agonism causes hypothermia via two distinct mechanisms: brain neuronal A1AR and A1AR on neurons outside the blood-brain barrier. The variety of mechanisms that adenosine can use to induce hypothermia underscores the importance of hypothermia in the mouse response to major metabolic stress or injury.
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Hipotermia/metabolismo , Receptor A1 de Adenosina/metabolismo , Agonistas do Receptor A1 de Adenosina/farmacologia , Animais , Hipotálamo/metabolismo , Hipotálamo/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nervos Periféricos/metabolismo , Nervos Periféricos/fisiopatologiaRESUMO
Dopamine-derived N6-substituents, compared to N6-(2-phenylethyl), in truncated (N)-methanocarba (bicyclo[3.1.0]hexyl) adenosines favored high A3 adenosine receptor (AR) affinity/selectivity, e.g., C2-phenylethynyl analogue 15 (MRS7591, Ki = 10.9/17.8 nM, at human/mouse A3AR). 15 was a partial agonist in vitro (hA3AR, cAMP inhibition, 31% Emax; mA3AR, [35S]GTP-γ-S binding, 16% Emax) and in vivo and also antagonized hA3AR in vitro. Distal H-bonding substitutions of the N6-(2-phenylethyl) moiety particularly enhanced mA3AR affinity by polar interactions with the extracellular loops, predicted using docking and molecular dynamics simulation with newly constructed mA3AR and hA3AR homology models. These hybrid models were based on an inactive antagonist-bound hA1AR structure for the upper part of TM2 and an agonist-bound hA2AAR structure for the remaining TM portions. These species-independent A3AR-selective nucleosides are low efficacy partial agonists and novel, nuanced modulators of the A3AR, a drug target of growing interest.
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Agonistas do Receptor A3 de Adenosina/química , Agonistas do Receptor A3 de Adenosina/metabolismo , Nucleosídeos/química , Nucleosídeos/metabolismo , Receptor A3 de Adenosina/química , Receptor A3 de Adenosina/metabolismo , Agonistas do Receptor A3 de Adenosina/farmacologia , Animais , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleosídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Secundária de ProteínaRESUMO
The blood hormone erythropoietin (EPO), upon binding to its receptor (EpoR), modulates high-fat diet-induced (HFD-induced) obesity in mice, improves glucose tolerance, and prevents white adipose tissue inflammation. Transgenic mice with constitutive overexpression of human EPO solely in the brain (Tg21) were used to assess the neuroendocrine EPO effect without increasing the hematocrit. Male Tg21 mice resisted HFD-induced weight gain; showed lower serum adrenocorticotropic hormone, corticosterone, and C-reactive protein levels; and prevented myeloid cell recruitment to the hypothalamus compared with WT male mice. HFD-induced hypothalamic inflammation (HI) and microglial activation were higher in male mice, and Tg21 male mice exhibited a lower increase in HI than WT male mice. Physiological EPO function in the brain also showed sexual dimorphism in regulating HFD response. Female estrogen production blocked reduced weight gain and HI. Targeted deletion of EpoR gene expression in neuronal cells worsened HFD-induced glucose intolerance in both male and female mice but increased weight gain and HI in the hypothalamus in male mice only. Both male and female Tg21 mice kept on normal chow and HFD showed significantly improved glycemic control. Our data indicate that cerebral EPO regulates weight gain and HI in a sex-dependent response, distinct from EPO regulation of glycemic control, and independent of erythropoietic EPO response.
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Encéfalo/metabolismo , Eritropoetina/metabolismo , Hipotálamo/patologia , Inflamação/metabolismo , Fatores Sexuais , Animais , Glicemia/metabolismo , Comportamento Alimentar , Feminino , Hipotálamo/metabolismo , Resistência à Insulina , Masculino , Camundongos , Camundongos Transgênicos , Receptores da Eritropoetina/genéticaRESUMO
During infection, hepatocytes must undergo a reprioritization of metabolism, termed metabolic reprogramming. Hepatic metabolic reprogramming in response to infection begins within hours of infection, suggesting a mechanism closely linked to pathogen recognition. Following injection with polyinosinic:polycytidylic acid, a mimic of viral infection, a robust hepatic innate immune response could be seen involving the TNFα pathway at 2 h. Repeated doses led to the adoption of Warburg-like metabolism in the liver as determined by in vivo metabolic imaging, expression analyses, and metabolomics. Hepatic macrophages, Kupffer cells, were able to induce Warburg-like metabolism in hepatocytes in vitro via TNFα. Eliminating macrophages in vivo or blocking TNFα in vitro or in vivo resulted in abrogation of the metabolic phenotype, establishing an immune-metabolic axis in hepatic metabolic reprogramming. Overall, we suggest that macrophages, as early sensors of pathogens, instruct hepatocytes via TNFα to undergo metabolic reprogramming to cope with challenges to homeostasis initiated by infection. This work not only addresses a key component of end-organ physiology, but also raises questions about the side effects of biologics in the treatment of inflammatory diseases. KEY MESSAGES: ⢠Hepatocytes develop Warburg-like metabolism in vivo during viral infection. ⢠Macrophage TNFα promotes expression of glycolytic enzymes in hepatocytes. ⢠Blocking this immune-metabolic axis abrogates Warburg-like metabolism in the liver. ⢠Implications for patients being treated for inflammatory diseases with biologics.
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Hepatócitos/metabolismo , Fígado/metabolismo , Macrófagos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Hepatócitos/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Fígado/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: Gsα couples multiple receptors, including the melanocortin 4 receptor (MC4R), to intracellular cAMP generation. Germline inactivating Gsα mutations lead to obesity in humans and mice. Mice with brain-specific Gsα deficiency also develop obesity with reduced energy expenditure and locomotor activity, and impaired adaptive thermogenesis, but the underlying mechanisms remain unclear. METHODS: We created mice (DMHGsKO) with Gsα deficiency limited to the dorsomedial hypothalamus (DMH) and examined the effects on energy balance and thermogenesis. RESULTS: DMHGsKO mice developed severe, early-onset obesity associated with hyperphagia and reduced energy expenditure and locomotor activity, along with impaired brown adipose tissue thermogenesis. Studies in mice with loss of MC4R in the DMH suggest that defective DMH MC4R/Gsα signaling contributes to abnormal energy balance but not to abnormal locomotor activity or cold-induced thermogenesis. Instead, DMHGsKO mice had impaired leptin signaling along with increased expression of the leptin signaling inhibitor protein tyrosine phosphatase 1B in the DMH, which likely contributes to the observed hyperphagia and reductions in energy expenditure, locomotor activity, and cold-induced thermogenesis. CONCLUSIONS: DMH Gsα signaling is critical for energy balance, thermogenesis, and leptin signaling. This study provides insight into how distinct signaling pathways can interact to regulate energy homeostasis and temperature regulation.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Hiperfagia/metabolismo , Hipotálamo/metabolismo , Leptina/metabolismo , Obesidade/metabolismo , Transdução de Sinais/fisiologia , Termogênese/fisiologia , Tecido Adiposo Marrom/metabolismo , Animais , Metabolismo Energético/fisiologia , Regulação da Expressão Gênica , Predisposição Genética para Doença/genética , Glucose/metabolismo , Homeostase/fisiologia , Masculino , Camundongos , Camundongos Knockout , Obesidade/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Receptor Tipo 4 de Melanocortina/metabolismo , Sistema Nervoso Simpático/metabolismoRESUMO
Adenosine is a constituent of many molecules of life; increased free extracellular adenosine indicates cell damage or metabolic stress. The importance of adenosine signaling in basal physiology, as opposed to adaptive responses to danger/damage situations, is unclear. We generated mice lacking all four adenosine receptors (ARs), Adora1-/-;Adora2a-/-;Adora2b-/-;Adora3-/- (quad knockout [QKO]), to enable investigation of the AR dependence of physiologic processes, focusing on body temperature. The QKO mice demonstrate that ARs are not required for growth, metabolism, breeding, and body temperature regulation (diurnal variation, response to stress, and torpor). However, the mice showed decreased survival starting at about 15 weeks of age. While adenosine agonists cause profound hypothermia via each AR, adenosine did not cause hypothermia (or bradycardia or hypotension) in QKO mice, indicating that AR-independent signals do not contribute to adenosine-induced hypothermia. The hypothermia elicited by adenosine kinase inhibition (with A134974), inosine, or uridine also required ARs, as each was abolished in the QKO mice. The proposed mechanism for uridine-induced hypothermia is inhibition of adenosine transport by uridine, increasing local extracellular adenosine levels. In contrast, adenosine 5'-monophosphate (AMP)-induced hypothermia was attenuated in QKO mice, demonstrating roles for both AR-dependent and AR-independent mechanisms in this process. The physiology of the QKO mice appears to be the sum of the individual knockout mice, without clear evidence for synergy, indicating that the actions of the four ARs are generally complementary. The phenotype of the QKO mice suggests that, while extracellular adenosine is a signal of stress, damage, and/or danger, it is less important for baseline regulation of body temperature.
Assuntos
Hipotermia/metabolismo , Receptor A1 de Adenosina/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Animais , Pressão Sanguínea/genética , Pressão Sanguínea/fisiologia , Temperatura Corporal/genética , Temperatura Corporal/fisiologia , Cafeína/farmacologia , Feminino , Genótipo , Frequência Cardíaca/genética , Frequência Cardíaca/fisiologia , Hipotermia/induzido quimicamente , Hipotermia/genética , Inosina/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Receptor A1 de Adenosina/genética , Receptor A2A de Adenosina/genética , Receptor A2B de Adenosina/genética , Receptor A3 de Adenosina/genética , Uridina/toxicidadeRESUMO
(N)-Methanocarba ([3.1.0]bicyclohexyl) adenosines and corresponding ribosides were synthesized to identify novel A1 adenosine receptor (A1AR) agonists for CNS or peripheral applications. Human and mouse AR binding was determined to assess the constrained ring system's A1AR compatibility. N6-Dicyclobutylmethyl ribose agonist (9, MRS7469, >2000-fold selective for A1AR) and known truncated N6-dicyclopropylmethyl methanocarba 7 (MRS5474) were drug-like. The pure diastereoisomer of known riboside 4 displayed high hA1AR selectivity. Methanocarba modification reduced A1AR selectivity of N6-dicyclopropylmethyl and endo-norbornyladenosines but increased ribavirin selectivity. Most analogues tested (ip) were inactive or weak in inducing mouse hypothermia, despite mA1AR full agonism and variable mA3AR efficacy, but strong hypothermia by 9 depended on A1AR, which reflects CNS activity (determined using A1AR or A3AR null mice). Conserved hA1AR interactions were preserved in modeling of 9 and methanocarba equivalent 24 (â¼400-fold A1AR-selective). Thus, we identified, and characterized in vivo, ribose and methanocarba nucleosides, including with A1AR-enhancing N6-dicyclobutylmethyl-adenine and 1,2,4-triazole-3-carboxamide (40, MRS7451) nucleobases.