RESUMO
Metformin (MET) is the most prescribed antidiabetic drug, but its mechanisms of action remain elusive. Recent data point to the gut as MET's primary target. Here, we explored the effect of MET on the gut glucose transport machinery. Using human enterocytes (Caco-2/TC7 cells) in vitro, we showed that MET transiently reduced the apical density of sodium-glucose transporter 1 (SGLT1) and decreased the absorption of glucose, without changes in the mRNA levels of the transporter. Administered 1 h before a glucose challenge in rats (Wistar, GK), C57BL6 mice and mice pigs, oral MET reduced the post-prandial glucose response (PGR). This effect was abrogated in SGLT1-KO mice. MET also reduced the luminal clearance of 2-(18F)-fluoro-2-deoxy-D-glucose after oral administration in rats. In conclusion, oral metformin transiently lowers post-prandial glucose response by reducing the apical expression of SGLT1 in enterocytes, which may contribute to the clinical effects of the drug.
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BACKGROUND: Aortic valve stenosis involves inflammation, excess deposition of a collagen-rich extracellular matrix, and calcification. Recent studies have shown that M1 or inflammatory macrophages derived from infiltrating monocytes promote calcification of valvular interstitial cells, the most prevalent cell type of the aortic valve. We hypothesized that valvular interstitial cells could modulate inflammatory macrophages phenotype. METHODS: We first assessed macrophage phenotype in human aortic valve stenosis and control aortic valves from donors. Then, we examined profibrotic and inflammatory-related gene expression in valves and valvular interstitial cells. Finally, we investigated whether valvular interstitial cells can modify the phenotype of inflammatory macrophages. RESULTS: Circulating monocytes and plasma transforming growth factor beta-1 levels of patients with aortic valve stenosis were significantly higher compared with patients without aortic valve stenosis. Histologic analysis of thickened spongiosa of the aortic valve from patients with aortic valve stenosis showed a high macrophage infiltration but a low matrix metalloproteinase-9 expression compared with control aortic valves. On the other hand, valvular interstitial cell culture of aortic valve stenosis exhibited a profibrotic phenotype with a high expression of transforming growth factor beta-1 and transforming growth factor beta-1/transforming growth factor beta-3 ratio but a decreased expression of the peroxisome proliferator-activated receptor gamma nuclear receptor. Valvular interstitial cell-conditioned media of aortic valve stenosis led to a decrease in enzymatic activity of matrix metalloproteinase-9 and an increase in production of collagen in inflammatory macrophages compared with valvular interstitial cell-conditioned media from control aortic valve donors. CONCLUSIONS: These findings indicate that profibrotic valvular interstitial cells promote the imbalance of extracellular matrix remodeling by reducing matrix metalloproteinase-9 production on inflammatory macrophages that lead to excessive collagen deposition observed in aortic valve stenosis. Further investigation is needed to clarify the role of transforming growth factor beta-1/proliferator-activated receptor gamma nuclear receptor/matrix metalloproteinase-9 in aortic valve stenosis.
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The alimentary limb has been proposed to be a key driver of the weight-loss-independent metabolic improvements that occur upon bariatric surgery. However, the one anastomosis gastric bypass (OAGB) procedure, consisting of one long biliary limb and a short common limb, induces similar beneficial metabolic effects compared to Roux-en-Y Gastric Bypass (RYGB) in humans, despite the lack of an alimentary limb. The aim of this study was to assess the role of the length of biliary and common limbs in the weight loss and metabolic effects that occur upon OAGB. OAGB and sham surgery, with or without modifications of the length of either the biliary limb or the common limb, were performed in Gottingen minipigs. Weight loss, metabolic changes, and the effects on plasma and intestinal bile acids (BAs) were assessed 15 days after surgery. OAGB significantly decreased body weight, improved glucose homeostasis, increased postprandial GLP-1 and fasting plasma BAs, and qualitatively changed the intestinal BA species composition. Resection of the biliary limb prevented the body weight loss effects of OAGB and attenuated the postprandial GLP-1 increase. Improvements in glucose homeostasis along with changes in plasma and intestinal BAs occurred after OAGB regardless of the biliary limb length. Resection of only the common limb reproduced the glucose homeostasis effects and the changes in intestinal BAs. Our results suggest that the changes in glucose metabolism and BAs after OAGB are mainly mediated by the length of the common limb, whereas the length of the biliary limb contributes to body weight loss.NEW & NOTEWORTHY Common limb mediates postprandial glucose metabolism change after gastric bypass whereas biliary limb contributes to weight loss.
Assuntos
Ácidos e Sais Biliares/metabolismo , Sistema Biliar/patologia , Ducto Colédoco/patologia , Derivação Gástrica/métodos , Glucose/metabolismo , Anastomose Cirúrgica/métodos , Animais , Ácidos e Sais Biliares/sangue , Sistema Biliar/metabolismo , Procedimentos Cirúrgicos do Sistema Biliar/métodos , Glicemia/metabolismo , Ducto Colédoco/metabolismo , Ducto Colédoco/cirurgia , Feminino , Modelos Animais , Obesidade Mórbida/metabolismo , Obesidade Mórbida/cirurgia , Período Pós-Prandial , Distribuição Aleatória , Suínos , Porco Miniatura , Redução de Peso/fisiologiaRESUMO
The newest classes of anti-diabetic agents include sodium-glucose cotransporter 2 (SGLT2) inhibitors and glucagon-like peptide 1 receptor (GLP1R) agonists. The SGLT2 inhibitor dapagliflozin reduces glucotoxicity by glycosuria but elevates glucagon secretion. The GLP1R agonist liraglutide inhibits glucagon; therefore, we hypothesize that the cotreatment of dapagliflozin with liraglutide could reduce hyperglucagonemia and hyperglycemia. Here we use five complementary models: human islet cultures, healthy mice, db/db mice, diet-induced obese (DIO) mice, and somatostatin receptor-2 (SSTR2) KO mice. A single administration of liraglutide and dapagliflozin in combination improves glycemia and reduces dapagliflozin-induced glucagon secretion in diabetic mice. Chronic treatment with liraglutide and dapagliflozin produces a sustainable reduction of glycemia compared with each drug alone. Moreover, liraglutide reduces dapagliflozin-induced glucagon secretion by enhancing somatostatin release, as demonstrated by SSTR2 inhibition in human islets and in mice. Collectively, these data provide mechanistic insights into how intra-islet GLP1R activation is critical for the regulation of glucose homeostasis.
Assuntos
Compostos Benzidrílicos/efeitos adversos , Diabetes Mellitus Experimental/tratamento farmacológico , Glucagon/efeitos dos fármacos , Glucosídeos/efeitos adversos , Liraglutida/uso terapêutico , Somatostatina/efeitos dos fármacos , Animais , Humanos , Liraglutida/farmacologia , Masculino , CamundongosRESUMO
OBJECTIVE: A minipig model was employed to explore the changes in endogenous leptin transport into the central nervous system and in hypothalamic sensitivity to exogenous leptin when individuals are placed on high-fat diet (HFD) compared with standard diet. METHODS: Serum and cerebrospinal fluid (CSF) leptin concentrations during 10 weeks of HFD versus standard diet and exogenous leptin-induced STAT3 phosphorylation in the hypothalamus of minipigs were assessed, and the hypothalamic leptin-sensitive cells were characterized by immunofluorescence. RESULTS: The efficiency of the passage of endogenous blood-borne leptin into the CSF (measured as the log [CSF:serum leptin ratio]) decreased over time in minipigs fed a HFD (ß = -0.04 ± 0.005 per kilogram of weight gain in HFD; P < 0.0001), while it remained stable in minipigs fed a standard diet. However, the ability of peripherally administered leptin to activate its receptor in hypothalamic neurons was preserved in obese minipigs at 10 weeks of HFD. CONCLUSIONS: Together, these data are consistent with the existence of an early-onset tranport deficiency for endogenous circulating leptin into the brain in individuals developing obesity, preceding the acquisition of hypothalamic leptin resistance. Although additional studies are required to identify the underlying mechanisms, our study paves the way for the development of new preclinical pharmacological models targeting the restoration of the shuttling of peripheral leptin into the central nervous system to manage obesity.
Assuntos
Líquido Cefalorraquidiano/química , Dieta Hiperlipídica/efeitos adversos , Leptina/metabolismo , Obesidade/sangue , Animais , Humanos , Masculino , Suínos , Porco MiniaturaRESUMO
BACKGROUND AND STUDY AIMS : Endoscopic techniques have demonstrated their effectiveness in metabolic surgery, notably through a gastrointestinal (GI) liner, with a less invasive approach than conventional surgery. Our study evaluates the safety and efficacy of endoscopic GI anastomosis (EGIA) using a lumen-apposing stent to secure the anastomosis. MATERIALS AND METHODS : EGIA was performed using the transgastric approach with a two-channel endoscope with a novel stent (Cousin Biotech). First, a safety study with a follow-up of 12 months was performed on five piglets. Then, metabolic changes were investigated in a minipig model (nâ=â10) before and after EGIA or open GIA (OGIA). RESULTS: EGIA was technically successful with no complications observed during clinical monitoring. Endoscopic and postmortem examinations during the second part of study showed a secure anastomosis between the stomach and the intestinal limb in all except one minipig. Both minipigs subjected to EGIA and those in the control group (OGIA) exhibited increased postprandial glucagon-like peptide-1 (GLP-1) production (incretin secretion) and impaired D-xylose absorption (malabsorption effect). CONCLUSION : Performing EGIA with this dedicated stent appears safe, technically feasible, durable, and reproducible in providing a simple and effective endoscopic GI bypass capable of ensuring metabolic effect.
Assuntos
Endoscopia Gastrointestinal/instrumentação , Peptídeo 1 Semelhante ao Glucagon/sangue , Jejuno/cirurgia , Stents Metálicos Autoexpansíveis , Estômago/cirurgia , Anastomose Cirúrgica/instrumentação , Anastomose Cirúrgica/métodos , Animais , Glicemia , Endoscopia Gastrointestinal/métodos , Estudos de Viabilidade , Feminino , Absorção Intestinal , Masculino , Período Pós-Prandial , Suínos , Xilose/metabolismoRESUMO
RATIONALE: Vascular calcification is a process similar to bone formation leading to an inappropriate deposition of calcium phosphate minerals in advanced atherosclerotic plaques. Monocyte-derived macrophages, located in atherosclerotic lesions and presenting heterogeneous phenotypes, from classical proinflammatory M1 to alternative anti-inflammatory M2 macrophages, could potentially display osteoclast-like functions. OBJECTIVE: To characterize the phenotype of macrophages located in areas surrounding the calcium deposits in human atherosclerotic plaques. METHODS AND RESULTS: Macrophages near calcium deposits display an alternative phenotype being both CD68 and mannose receptor-positive, expressing carbonic anhydrase type II, but relatively low levels of cathepsin K. In vitro interleukin-4-polarization of human primary monocytes into macrophages results in lower expression and activity of cathepsin K compared with resting unpolarized macrophages. Moreover, interleukin-4 polarization lowers expression levels of the osteoclast transcriptional activator nuclear factor of activated T cells type c-1, associated with increased gene promoter levels of the transcriptional repression mark H3K27me3 (histone 3 lysine 27 trimethylation). Despite higher expression of the receptor activator of nuclear factor κB receptor, receptor activator of nuclear factor κB ligand/macrophage colony-stimulating factor induction of nuclear factor of activated T cells type c-1 and cathepsin K expression is defective in these macrophages because of reduced Erk/c-fos-mediated downstream signaling resulting in impaired bone resorption capacity. CONCLUSIONS: These results indicate that macrophages surrounding calcium deposits in human atherosclerotic plaques are phenotypically defective being unable to resorb calcification.
Assuntos
Reabsorção Óssea/metabolismo , Macrófagos/metabolismo , Osteoclastos/metabolismo , Placa Aterosclerótica/metabolismo , Ligante RANK/metabolismo , Calcificação Vascular/metabolismo , Reabsorção Óssea/patologia , Células Cultivadas , Humanos , Microdissecção e Captura a Laser/métodos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Macrófagos/patologia , Osteoclastos/patologia , Placa Aterosclerótica/patologia , Calcificação Vascular/patologiaAssuntos
Derivação Gástrica , Glucose , Anastomose em-Y de Roux , Humanos , Intestinos , Laparoscopia , Obesidade Mórbida/cirurgiaRESUMO
Active sodium-glucose transporters play a role to glucose homeostasis and represent novels targets for the management of type 2 diabetes (T2D). Sodium-glucose cotransporter 1 (SGLT1) is essential for intestinal glucose absorption from the lumen into enterocytes, whereas glucose reabsorption by the kidney is mainly mediated by sodium-glucose cotransporter 2 (SGLT2). SGLT2 inhibitors were developed to occlude SGLT2 glucose reabsorption pathway and cause glycosuria, thereby reducing plasma glucose concentrations. This new class of antidiabetic drugs has been shown to be effective in reducing cardiovascular morbidity and mortality in patients with T2D. Initial clinical studies also suggest that SGLT1 inhibition increases glucagon-like peptide 1 (GLP-1) secretion and decreases postchallenge blood glucose excursion, resulting in a dose-dependent improvement of glucose control. In parallel, we recently identified a previously unknown effect of bile diversion in gastric bypass on sodium glucose transport and postprandial glucose homeostasis, through the modulation of intestinal trafficking of endogenous sodium. This mechanism is consistent with available clinical evidence, and opens up new perspectives in metabolic surgery. More generally, the modulation of intestinal sodium-glucose cotransport appears to be a promising avenue to prevent or treat T2D.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus Tipo 2/cirurgia , Derivação Gástrica/métodos , Proteínas de Transporte de Sódio-Glucose/fisiologia , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/fisiopatologia , Homeostase/fisiologia , Humanos , Insulina/fisiologia , Mucosa Intestinal/metabolismo , Proteínas de Transporte de Sódio-Glucose/antagonistas & inibidoresRESUMO
Gastro-intestinal exclusion by Roux-en-Y gastric bypass (RYGB) improves glucose metabolism, independent of weight loss. Although changes in intestinal bile trafficking have been shown to play a role, the underlying mechanisms are unclear. We performed RYGB in minipigs and showed that the intestinal uptake of ingested glucose is blunted in the bile-deprived alimentary limb (AL). Glucose uptake in the AL was restored by the addition of bile, and this effect was abolished when active glucose intestinal transport was blocked with phlorizin. Sodium-glucose cotransporter 1 remained expressed in the AL, while intraluminal sodium content was markedly decreased. Adding sodium to the AL had the same effect as bile on glucose uptake. It also increased postprandial blood glucose response in conscious minipigs following RYGB. The decrease in intestinal uptake of glucose after RYGB was confirmed in humans. Our results demonstrate that bile diversion affects postprandial glucose metabolism by modulating sodium-glucose intestinal cotransport.
Assuntos
Ácidos e Sais Biliares/fisiologia , Derivação Gástrica , Glucose/metabolismo , Jejuno/metabolismo , Animais , Transporte Biológico , Feminino , Humanos , Absorção Intestinal , Masculino , Obesidade/cirurgia , Sódio/metabolismo , Proteínas de Transporte de Sódio-Glucose/metabolismo , Suínos , Porco MiniaturaRESUMO
Bile acids are signalling molecules, which activate the transmembrane receptor TGR5 and the nuclear receptor FXR. BA sequestrants (BAS) complex bile acids in the intestinal lumen and decrease intestinal FXR activity. The BAS-BA complex also induces glucagon-like peptide-1 (GLP-1) production by L cells which potentiates ß-cell glucose-induced insulin secretion. Whether FXR is expressed in L cells and controls GLP-1 production is unknown. Here, we show that FXR activation in L cells decreases proglucagon expression by interfering with the glucose-responsive factor Carbohydrate-Responsive Element Binding Protein (ChREBP) and GLP-1 secretion by inhibiting glycolysis. In vivo, FXR deficiency increases GLP-1 gene expression and secretion in response to glucose hence improving glucose metabolism. Moreover, treatment of ob/ob mice with the BAS colesevelam increases intestinal proglucagon gene expression and improves glycaemia in a FXR-dependent manner. These findings identify the FXR/GLP-1 pathway as a new mechanism of BA control of glucose metabolism and a pharmacological target for type 2 diabetes.
Assuntos
Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/genética , Mucosa Intestinal/metabolismo , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Animais , Anticolesterolemiantes/farmacologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ácidos e Sais Biliares/metabolismo , Glicemia/metabolismo , Cloridrato de Colesevelam/farmacologia , Colo/citologia , Colo/metabolismo , Dieta Hiperlipídica , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Glicólise , Humanos , Íleo/citologia , Íleo/metabolismo , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Intestinos/citologia , Jejuno/citologia , Jejuno/metabolismo , Camundongos , Camundongos Knockout , Camundongos Obesos , Proteínas Nucleares/metabolismo , Obesidade/genética , Obesidade/metabolismo , Proglucagon/efeitos dos fármacos , Proglucagon/genética , Proglucagon/metabolismo , Receptores Acoplados a Proteínas G/genética , Sequestrantes/farmacologia , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
RATIONALE: In atherosclerotic plaques, iron preferentially accumulates in macrophages where it can exert pro-oxidant activities. OBJECTIVE: The objective of this study was, first, to better characterize the iron distribution and metabolism in macrophage subpopulations in human atherosclerotic plaques and, second, to determine whether iron homeostasis is under the control of nuclear receptors, such as the liver X receptors (LXRs). METHODS AND RESULTS: Here we report that iron depots accumulate in human atherosclerotic plaque areas enriched in CD68 and mannose receptor (MR)-positive (CD68(+)MR(+)) alternative M2 macrophages. In vitro IL-4 polarization of human monocytes into M2 macrophages also resulted in a gene expression profile and phenotype favoring iron accumulation. However, M2 macrophages on iron exposure acquire a phenotype favoring iron release, through a strong increase in ferroportin expression, illustrated by a more avid oxidation of extracellular low-density lipoprotein by iron-loaded M2 macrophages. In line, in human atherosclerotic plaques, CD68(+)MR(+) macrophages accumulate oxidized lipids, which activate LXRα and LXRß, resulting in the induction of ABCA1, ABCG1, and apolipoprotein E expression. Moreover, in iron-loaded M2 macrophages, LXR activation induces nuclear factor erythroid 2-like 2 expression, thereby increasing ferroportin expression, which, together with a decrease of hepcidin mRNA levels, promotes iron export. CONCLUSIONS: These data identify a role for M2 macrophages in iron handling, a process regulated by LXR activation.
Assuntos
Ferro/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Receptores Nucleares Órfãos/fisiologia , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Apolipoproteínas E/metabolismo , Transporte Biológico/fisiologia , Células Cultivadas , Homeostase/fisiologia , Humanos , Técnicas In Vitro , Lectinas Tipo C/metabolismo , Receptores X do Fígado , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Fenótipo , Receptores de Superfície Celular/metabolismoRESUMO
Obesity is associated with a significantly increased risk for cancer suggesting that adipose tissue dysfunctions might play a crucial role therein. Macrophages play important roles in adipose tissue as well as in cancers. Here, we studied whether human adipose tissue macrophages (ATM) modulate cancer cell function. Therefore, ATM were isolated and compared with monocyte-derived macrophages (MDM) from the same obese patients. ATM, but not MDM, were found to secrete factors inducing inflammation and lipid accumulation in human T47D and HT-29 cancer cells. Gene expression profile comparison of ATM and MDM revealed overexpression of functional clusters, such as cytokine-cytokine receptor interaction (especially CXC-chemokine) signaling as well as cancer-related pathways, in ATM. Comparison with gene expression profiles of human tumor-associated macrophages showed that ATM, but not MDM resemble tumor-associated macrophages. Indirect co-culture experiments demonstrated that factors secreted by preadipocytes, but not mature adipocytes, confer an ATM-like phenotype to MDM. Finally, the concentrations of ATM-secreted factors related to cancer are elevated in serum of obese subjects. In conclusion, ATM may thus modulate the cancer cell phenotype.
Assuntos
Adipócitos/citologia , Tecido Adiposo/metabolismo , Regulação Neoplásica da Expressão Gênica , Macrófagos/citologia , Neoplasias/metabolismo , Compostos Azo/farmacologia , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Progressão da Doença , Humanos , Imuno-Histoquímica/métodos , Inflamação , Macrófagos/metabolismo , Obesidade/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , FenótipoRESUMO
OBJECTIVE: Bile acids (BA) participate in the maintenance of metabolic homeostasis acting through different signaling pathways. The nuclear BA receptor farnesoid X receptor (FXR) regulates pathways in BA, lipid, glucose, and energy metabolism, which become dysregulated in obesity. However, the role of FXR in obesity and associated complications, such as dyslipidemia and insulin resistance, has not been directly assessed. RESEARCH DESIGN AND METHODS: Here, we evaluate the consequences of FXR deficiency on body weight development, lipid metabolism, and insulin resistance in murine models of genetic and diet-induced obesity. RESULTS: FXR deficiency attenuated body weight gain and reduced adipose tissue mass in both models. Surprisingly, glucose homeostasis improved as a result of an enhanced glucose clearance and adipose tissue insulin sensitivity. In contrast, hepatic insulin sensitivity did not change, and liver steatosis aggravated as a result of the repression of ß-oxidation genes. In agreement, liver-specific FXR deficiency did not protect from diet-induced obesity and insulin resistance, indicating a role for nonhepatic FXR in the control of glucose homeostasis in obesity. Decreasing elevated plasma BA concentrations in obese FXR-deficient mice by administration of the BA sequestrant colesevelam improved glucose homeostasis in a FXR-dependent manner, indicating that the observed improvements by FXR deficiency are not a result of indirect effects of altered BA metabolism. CONCLUSIONS: Overall, FXR deficiency in obesity beneficially affects body weight development and glucose homeostasis.
Assuntos
Glucose/metabolismo , Obesidade/metabolismo , Receptores Citoplasmáticos e Nucleares/deficiência , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Ácidos e Sais Biliares/sangue , Modelos Animais de Doenças , Homeostase , Hipertrigliceridemia/etiologia , Insulina/farmacologia , Resistência à Insulina , Metabolismo dos Lipídeos , Masculino , Camundongos , Camundongos Obesos , Aumento de Peso/fisiologiaRESUMO
BACKGROUND & AIMS: Glucagon-like peptide (GLP)-1, an intestinal incretin produced by L cells through proglucagon processing, is secreted after nutrient ingestion and acts on endocrine pancreas beta cells to enhance insulin secretion. Peroxisome proliferator-activated receptor (PPAR) ß/δ is a nuclear receptor that improves glucose homeostasis and pancreas islet function in diabetic animal models. Here, we investigated whether PPARß/δ activation regulates L cell GLP-1 production. METHODS: Proglucagon regulation and GLP-1 release were evaluated in murine GLUTag and human NCI-H716 L cells and in vivo using wild-type, PPARß/δ-null, and ob/ob C57Bl/6 mice treated with the PPARß/δ synthetic agonists GW501516 or GW0742. RESULTS: PPARß/δ activation increased proglucagon expression and enhanced glucose- and bile acid-induced GLP-1 release by intestinal L cells in vitro and ex vivo in human jejunum. In vivo treatment with GW0742 increased proglucagon messenger RNA levels in the small intestine in wild-type but not in PPARß/δ-deficient mice. Treatment of wild-type and ob/ob mice with GW501516 enhanced the increase in plasma GLP-1 level after an oral glucose load and improved glucose tolerance. Concomitantly, proglucagon and GLP-1 receptor messenger RNA levels increased in the small intestine and pancreas, respectively. Finally, PPARß/δ agonists activate the proglucagon gene transcription by interfering with the ß-catenin/TCF-4 pathway. CONCLUSIONS: Our data show that PPARß/δ activation potentiates GLP-1 production by the small intestine. Pharmacologic targeting of PPARß/δ is a promising approach in the treatment of patients with type 2 diabetes mellitus, especially in combination with dipeptidyl peptidase IV inhibitors.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Células Enteroendócrinas/metabolismo , Regulação da Expressão Gênica , Peptídeo 1 Semelhante ao Glucagon/biossíntese , PPAR beta/metabolismo , RNA Mensageiro/genética , Animais , Glicemia/metabolismo , Western Blotting , Células Cultivadas , Diabetes Mellitus Experimental/patologia , Modelos Animais de Doenças , Células Enteroendócrinas/patologia , Peptídeo 1 Semelhante ao Glucagon/genética , Humanos , Masculino , Camundongos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RatosRESUMO
In its native form, the chemokine CX3CL1 is a firmly adhesive molecule promoting leukocyte adhesion and migration and hence involved, along with its unique receptor CX3CR1, in various inflammatory processes. Here we investigated the role of molecular aggregation in the CX3CL1 adhesiveness. Assays of bioluminescence resonance energy transfer (BRET) and homogeneous time-resolved fluorescence (HTRF) in transfected cell lines and in primary cells showed specific signals indicative of CX3CL1 clustering. Truncation experiments showed that the transmembrane domain played a central role in this aggregation. A chimera with mutations of the 12 central transmembrane domain residues had significantly reduced BRET signals and characteristics of a non-clustering molecule. This mutant was weakly adhesive according to flow and dual pipette adhesion assays and was less glycosylated than CX3CL1, although, as we demonstrated, loss of glycosylation did not affect the CX3CL1 adhesive potency. We postulate that cell surfaces express CX3CL1 as a constitutive oligomer and that this oligomerization is essential for its adhesive potency. Inhibition of CX3CL1 self-assembly could limit the recruitment of CX3CR1-positive cells and may be a new pathway for anti-inflammatory therapies.
Assuntos
Quimiocina CX3CL1/metabolismo , Mutação , Sequência de Aminoácidos , Anti-Inflamatórios/farmacologia , Adesão Celular , Linhagem Celular , Deleção de Genes , Glicosilação , Humanos , Inflamação , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
OBJECTIVE: We investigated the role of monocyte-recruiting chemokines in cerebrovascular diseases among the subjects of the GENIC case-control study of brain infarction (BI). METHODS AND RESULTS: Of the genotypes tested, only homozygosity for the rare CX3CR1 alleles was more frequent in cases than in controls: the I249 and M280 alleles were associated with an increased risk of BI (OR, 1.66 and OR, 2.62 with P<0.05, respectively). This effect was independent of other established risk factors and uncorrelated with disease severity. The study confirmed previous reports of a dominant protective association between CX3CR1-I249 allele and the risk of cardiovascular history. The risk of BI associated with homozygosity for the rare CX3CR1 alleles was enhanced in patients with no previous cardiovascular events. Ex vivo studies showed that the number of monocytes adhering to immobilized CX3CL1, the CX3CR1 ligand, increased proportionally to the number of CX3CR1 mutated alleles carried by the individual. CONCLUSIONS: The rare CX3CR1 alleles were associated with an increased risk of BI and with reduced frequency of cardiovascular history. We propose that the extra adhesion of monocytes observed in individuals carrying rare alleles of CX3CR1 may favor mechanisms leading to stroke.
Assuntos
Arteriosclerose/epidemiologia , Arteriosclerose/genética , Proteínas de Membrana/genética , Polimorfismo Genético , Receptores de Quimiocinas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Arteriosclerose/imunologia , Receptor 1 de Quimiocina CX3C , Estudos de Casos e Controles , Infarto Cerebral/epidemiologia , Infarto Cerebral/genética , Infarto Cerebral/imunologia , Feminino , Predisposição Genética para Doença/epidemiologia , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Infarto do Miocárdio/epidemiologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/imunologia , Fatores de Risco , Acidente Vascular Cerebral/epidemiologia , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/imunologiaRESUMO
It was recently shown that individuals carrying the naturally occurring mutant CX3CR1-Ile(249)-Met(280) (hereafter called CX3CR1-IM) have a lower risk of cardiovascular disease than individuals homozygous for the wild-type CX3CR1-Val(249)-Thr(280) (CX3CR1-VT). We report here that peripheral blood mononuclear cells (PBMC) from individuals with the CX3CR1-IM haplotype adhered more potently to membrane-bound CX3CL1 than did PBMC from homozygous CX3CR1-VT donors. Similar excess adhesion was observed with CX3CR1-IM-transfected human embryonic kidney (HEK) cell lines tested with two different methods: the parallel plate laminar flow chamber and the dual pipette aspiration technique. Suppression of the extra adhesion in the presence of pertussis toxin indicates that G-protein mediated the underlying transduction pathway, in contrast to the G-protein-independent adhesion previously described for CX3CR1-VT. Surprisingly, HEK and PBMC that expressed CX3CR1-IM and -VT were indistinguishable when tested with the soluble form of CX3CL1 for chemotaxis, calcium release, and binding capacity. In conclusion, only the membrane-anchored form of CX3CL1 functionally discriminated between these two allelic isoforms of CX3CR1. These results suggest that each form of this ligand may lead to a different signaling pathway. The extra adhesion of CX3CR1-IM may be related to immune defenses and to atherogenesis, both of which depend substantially on adhesive intercellular events.
Assuntos
Isoleucina/química , Proteínas de Membrana/química , Proteínas de Membrana/genética , Metionina/química , Receptores de Quimiocinas/química , Receptores de Quimiocinas/genética , Western Blotting , Receptor 1 de Quimiocina CX3C , Cálcio/metabolismo , Adesão Celular , Linhagem Celular , Movimento Celular , Quimiotaxia , Citometria de Fluxo , Homozigoto , Humanos , Leucócitos Mononucleares/metabolismo , Mutação , Toxina Pertussis/farmacologia , Ligação Proteica , Transdução de Sinais , Fatores de Tempo , TransfecçãoRESUMO
We identified two novel isoforms of the human chemokine receptor CX3CR1, produced by alternative splicing and with N-terminal regions extended by 7 and 32 aa. Expression of the messengers coding these isoforms, compared with that of previously described V28 messengers, is lower in monocytes and NK cells, but higher in CD4(+) T lymphocytes. CX3CR1 and its extended isoforms were expressed in HEK-293 cells and compared for expression, ligand binding, and cellular responses. In steady state experiments, all three CX3CR1 isoforms bound CX3CL1 with similar affinity. In kinetic binding studies, however, k(on) and k(off) were significantly greater for the extended CX3CR1 isoforms, thereby suggesting that the N-terminal extensions may alter the functions induced by CX3CL1. In signaling studies, all three CX3CR1 isoforms mediated agonist-dependent calcium mobilization, but the EC(50) was lower for the extended than for the standard isoforms. In addition, chemotactic responses for these extended isoforms shifted left, also indicating a more sensitive response. Finally, the longer variants appeared to be more potent HIV coreceptors when tested in fusion and infection assays. In conclusion, we identified and characterized functionally two novel isoforms of CX3CR1 that respond more sensitively to CX3CL1 and HIV viral envelopes. These data reveal new complexity in CX3CR1 cell activation and confirm the critical role of the N-terminal domain of the chemokine receptors in ligand recognition and cellular response.