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1.
JCI Insight ; 8(19)2023 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-37669115

RESUMO

Tregs expressing chimeric antigen receptors (CAR-Tregs) are a promising tool to promote transplant tolerance. The relationship between CAR structure and Treg function was studied in xenogeneic, immunodeficient mice, revealing advantages of CD28-encoding CARs. However, these models could underrepresent interactions between CAR-Tregs, antigen-presenting cells (APCs), and donor-specific Abs. We generated Tregs expressing HLA-A2-specific CARs with different costimulatory domains and compared their function in vitro and in vivo using an immunocompetent model of transplantation. In vitro, the CD28-encoding CAR had superior antigen-specific suppression, proliferation, and cytokine production. In contrast, in vivo, Tregs expressing CARs encoding CD28, ICOS, programmed cell death 1, and GITR, but not 4-1BB or OX40, all extended skin allograft survival. To reconcile in vitro and in vivo data, we analyzed effects of a CAR encoding CD3ζ but no costimulatory domain. These data revealed that exogenous costimulation from APCs can compensate for the lack of a CAR-encoded CD28 domain. Thus, Tregs expressing a CAR with or without CD28 are functionally equivalent in vivo, mediating similar extension of skin allograft survival and controlling the generation of anti-HLA-A2 alloantibodies. This study reveals a dimension of CAR-Treg biology and has important implications for the design of CARs for clinical use in Tregs.


Assuntos
Receptores de Antígenos Quiméricos , Camundongos , Animais , Antígenos CD28 , Linfócitos T Reguladores , Transplante Homólogo , Aloenxertos/metabolismo
2.
Eur J Immunol ; 52(9): 1482-1497, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35746855

RESUMO

Regulatory T-cell (Treg) therapy is under clinical investigation for the treatment of transplant rejection, autoimmune disease, and graft-versus-host disease. With the advent of genome editing, attention has turned to reinforcing Treg function for therapeutic benefit. A hallmark of Tregs is dampened activation of PI3K-AKT signaling, of which PTEN is a major negative regulator. Loss-of-function studies of PTEN, however, have not conclusively shown a requirement for PTEN in upholding Treg function and stability. Using CRISPR-based genome editing in human Tregs, we show that PTEN ablation does not cause a global defect in Treg function and stability; rather, it selectively blocks their ability to suppress antigen-presenting cells. PTEN-KO Tregs exhibit elevated glycolytic activity, upregulate FOXP3, maintain a Treg phenotype, and have no discernible defects in lineage stability. Functionally, PTEN is dispensable for human Treg-mediated inhibition of T-cell activity in vitro and in vivo but is required for suppression of costimulatory molecule expression by antigen-presenting cells. These data are the first to define a role for a signaling pathway in controlling a subset of human Treg activity. Moreover, they point to the functional necessity of PTEN-regulated PI3K-AKT activity for optimal human Treg function.


Assuntos
Doenças Autoimunes , PTEN Fosfo-Hidrolase , Linfócitos T Reguladores , Fatores de Transcrição Forkhead/metabolismo , Humanos , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
3.
Sci Transl Med ; 12(557)2020 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-32817364

RESUMO

Antigen-specific regulatory T cells (Tregs) engineered with chimeric antigen receptors (CARs) are a potent immunosuppressive cellular therapy in multiple disease models and could overcome shortcomings of polyclonal Treg therapy. CAR therapy was initially developed with conventional T cells, which have different signaling requirements than do Tregs To date, most of the CAR Treg studies used second-generation CARs, encoding a CD28 or 4-1BB co-receptor signaling domain and CD3ζ, but it was not known if this CAR design was optimal for Tregs Using a human leukocyte antigen-A2-specific CAR platform and human Tregs, we compared 10 CARs with different co-receptor signaling domains and systematically tested their function and CAR-stimulated gene expression profile. Tregs expressing a CAR encoding CD28wt were markedly superior to all other CARs tested in an in vivo model of graft-versus-host disease. In vitro assays revealed stable expression of Helios and an ability to suppress CD80 expression on dendritic cells as key in vitro predictors of in vivo function. This comprehensive study of CAR signaling domain variants in Tregs can be leveraged to optimize CAR design for use in antigen-specific Treg therapy.


Assuntos
Receptores de Antígenos Quiméricos , Antígenos CD28 , Humanos , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais , Linfócitos T Reguladores
4.
Am J Transplant ; 20(6): 1562-1573, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31957209

RESUMO

Cell therapy with autologous donor-specific regulatory T cells (Tregs) is a promising strategy to minimize immunosuppression in transplant recipients. Chimeric antigen receptor (CAR) technology has recently been used successfully to generate donor-specific Tregs and overcome the limitations of enrichment protocols based on repetitive stimulations with alloantigens. However, the ability of CAR-Treg therapy to control alloreactivity in immunocompetent recipients is unknown. We first analyzed the effect of donor-specific CAR Tregs on alloreactivity in naive, immunocompetent mice receiving skin allografts. Tregs expressing an irrelevant or anti-HLA-A2-specific CAR were administered to Bl/6 mice at the time of transplanting an HLA-A2+ Bl/6 skin graft. Donor-specific CAR-Tregs, but not irrelevant-CAR Tregs, significantly delayed skin rejection and diminished donor-specific antibodies (DSAs) and frequencies of DSA-secreting B cells. Donor-specific CAR-Treg-treated mice also had a weaker recall DSA response, but normal responses to an irrelevant antigen, demonstrating antigen-specific suppression. When donor-specific CAR Tregs were tested in HLA-A2-sensitized mice, they were unable to delay allograft rejection or diminish DSAs. The finding that donor-specific CAR-Tregs restrain de novo but not memory alloreactivity has important implications for their use as an adoptive cell therapy in transplantation.


Assuntos
Receptores de Antígenos Quiméricos , Aloenxertos , Animais , Rejeição de Enxerto/prevenção & controle , Humanos , Isoantígenos , Camundongos , Linfócitos T Reguladores , Doadores de Tecidos
5.
iScience ; 23(1): 100775, 2020 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-31962237

RESUMO

Macrophages play a dynamic role in tissue repair following injury. Here we found that following streptozotocin (STZ)-induced beta-cell death, mouse islet macrophages had increased Igf1 expression, decreased proinflammatory cytokine expression, and transcriptome changes consistent with macrophages undergoing efferocytosis and having an enhanced state of metabolism. Macrophages were the major, if not sole, contributors to islet insulin-like growth factor-1 (IGF-1) production. Adoptive transfer experiments showed that macrophages can maintain insulin secretion in vivo following beta-cell death with no effects on islet cell turnover. IGF-1 neutralization during STZ treatment decreased insulin secretion without affecting islet cell apoptosis or proliferation. Interestingly, high-fat diet (HFD) combined with STZ further skewed islet macrophages to a reparative state. Finally, islet macrophages from db/db mice also expressed decreased proinflammatory cytokines and increased Igf1 mRNA. These data have important implications for islet biology and pathology and show that islet macrophages preserve their reparative state following beta-cell death even during HFD feeding and severe hyperglycemia.

6.
JCI Insight ; 4(6)2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30753169

RESUMO

Chimeric antigen receptor (CAR) technology can be used to engineer the antigen specificity of regulatory T cells (Tregs) and improve their potency as an adoptive cell therapy in multiple disease models. As synthetic receptors, CARs carry the risk of immunogenicity, particularly when derived from nonhuman antibodies. Using an HLA-A*02:01-specific CAR (A2-CAR) encoding a single-chain variable fragment (Fv) derived from a mouse antibody, we developed a panel of 20 humanized A2-CARs (hA2-CARs). Systematic testing demonstrated variations in expression, and ability to bind HLA-A*02:01 and stimulate human Treg suppression in vitro. In addition, we developed a new method to comprehensively map the alloantigen specificity of CARs, revealing that humanization reduced HLA-A cross-reactivity. In vivo bioluminescence imaging showed rapid trafficking and persistence of hA2-CAR Tregs in A2-expressing allografts, with eventual migration to draining lymph nodes. Adoptive transfer of hA2-CAR Tregs suppressed HLA-A2+ cell-mediated xenogeneic graft-versus-host disease and diminished rejection of human HLA-A2+ skin allografts. These data provide a platform for systematic development and specificity testing of humanized alloantigen-specific CARs that can be used to engineer specificity and homing of therapeutic Tregs.


Assuntos
Isoantígenos/imunologia , Isoantígenos/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Transferência Adotiva , Aloenxertos , Animais , Feminino , Antígenos HLA-A , Antígeno HLA-A2/imunologia , Humanos , Tolerância Imunológica , Imunoterapia , Imunoterapia Adotiva , Células K562 , Camundongos , Camundongos Transgênicos , Anticorpos de Cadeia Única , Pele/patologia , Transplante de Pele , Imunologia de Transplantes , Transplante Homólogo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Diabetes ; 63(9): 2984-95, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24812426

RESUMO

Dysregulated glucagon secretion accompanies islet inflammation in type 2 diabetes. We recently discovered that interleukin (IL)-6 stimulates glucagon secretion from human and rodent islets. IL-6 family cytokines require the glycoprotein 130 (gp130) receptor to signal. In this study, we elucidated the effects of α-cell gp130 receptor signaling on glycemic control in type 2 diabetes. IL-6 family cytokines were elevated in islets in rodent models of this disease. gp130 receptor activation increased STAT3 phosphorylation in primary α-cells and stimulated glucagon secretion. Pancreatic α-cell gp130 knockout (αgp130KO) mice showed no differences in glycemic control, α-cell function, or α-cell mass. However, when subjected to streptozotocin plus high-fat diet to induce islet inflammation and pathophysiology modeling type 2 diabetes, αgp130KO mice had reduced fasting glycemia, improved glucose tolerance, reduced fasting insulin, and improved α-cell function. Hyperinsulinemic-euglycemic clamps revealed no differences in insulin sensitivity. We conclude that in a setting of islet inflammation and pathophysiology modeling type 2 diabetes, activation of α-cell gp130 receptor signaling has deleterious effects on α-cell function, promoting hyperglycemia. Antagonism of α-cell gp130 receptor signaling may be useful for the treatment of type 2 diabetes.


Assuntos
Receptor gp130 de Citocina/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Células Secretoras de Glucagon/metabolismo , Animais , Receptor gp130 de Citocina/antagonistas & inibidores , Dieta Hiperlipídica , Glucagon/metabolismo , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Ratos , Fator de Transcrição STAT3/metabolismo
8.
Diabetologia ; 57(8): 1645-54, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24816367

RESUMO

AIMS/HYPOTHESIS: Inflammation contributes to pancreatic beta cell dysfunction in type 2 diabetes. Toll-like receptor (TLR)-2 and -4 ligands are increased systemically in recently diagnosed type 2 diabetes patients, and TLR2- and TLR4-deficient mice are protected from the metabolic consequences of a high-fat diet. Here we investigated the role of macrophages in TLR2/6- and TLR4-mediated effects on islet inflammation and beta cell function. METHODS: Genetic and pharmacological approaches were used to determine the effects of TLR2/6 and TLR4 ligands on mouse islets, human islets and purified rat beta cells. Islet macrophages were depleted and sorted by flow cytometry and the effects of TLR2/6- and TLR4-activated bone-marrow-derived macrophages (BMDMs) on beta cell function were assessed. RESULTS: Macrophages contributed to TLR2/6- and TLR4-induced islet Il1a/IL1A and Il1b/IL1B mRNA expression in mouse and human islets and IL-1ß secretion from human islets. TLR2/6 and TLR4 ligands also reduced insulin gene expression; however, this occurred in a non-beta cell autonomous manner. TLR2/6- and TLR4-activated BMDMs reduced beta cell insulin secretion partly via reducing Ins1, Ins2, and Pdx1 mRNA expression. Antagonism of the IL-1 receptor and neutralisation of IL-6 completely reversed the effects of activated macrophages on beta cell gene expression. CONCLUSIONS/INTERPRETATION: We conclude that islet macrophages are major contributors to islet IL-1ß secretion in response to TLR2/6 and TLR4 ligands. BMDMs stimulated with TLR2/6 and TLR4 ligands reduce insulin secretion from pancreatic beta cells, partly via IL-1ß- and IL-6-mediated decreased insulin gene expression.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/genética , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Ilhotas Pancreáticas/metabolismo , Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Insulina/metabolismo , Camundongos Knockout , Ratos , Receptores Toll-Like/genética
9.
J Immunol ; 192(2): 623-9, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24323581

RESUMO

Chronic inflammation is known to promote metabolic dysregulation in obesity and type 2 diabetes. Although the precise origin of the unchecked inflammatory response in obesity is unclear, it is known that overproduction of proinflammatory cytokines by innate immune cells affects metabolism. For example, TNF-α contributes to the inability of cells to respond to insulin and to the increase in levels of insulin. Whether this hyperinsulinemia itself is part of a feedback loop that affects the progression of chronic adipose inflammation is unknown. In this article, we show that regulatory T cells (Tregs) express the insulin receptor, and that high levels of insulin impair the ability of Tregs to suppress inflammatory responses via effects on the AKT/mTOR signaling pathway. Insulin activated AKT signaling in Tregs, leading to inhibition of both IL-10 production and the ability of Tregs to suppress the production of TNF-α by macrophages in a contact-independent manner. The effect of insulin on Treg suppression was limited to IL-10 production and it did not alter the expression of other proteins associated with Treg function, including CTLA-4, CD39, and TGF-ß. In a model of diet-induced obesity, Tregs from the visceral adipose tissue of hyperinsulinemic, obese mice showed a similar specific decrease in IL-10 production, as well as a parallel increase in production of IFN-γ. These data suggest that hyperinsulinemia may contribute to the development of obesity-associated inflammation via a previously unknown effect of insulin on the IL-10-mediated function of Tregs.


Assuntos
Insulina/imunologia , Insulina/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Obesidade/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Apirase/imunologia , Apirase/metabolismo , Antígeno CTLA-4/imunologia , Antígeno CTLA-4/metabolismo , Células Cultivadas , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Epitélio/imunologia , Epitélio/metabolismo , Hiperinsulinismo/imunologia , Hiperinsulinismo/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/antagonistas & inibidores , Gordura Intra-Abdominal/imunologia , Gordura Intra-Abdominal/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismo , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor de Insulina/imunologia , Receptor de Insulina/metabolismo , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/imunologia , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
10.
J Biomed Mater Res B Appl Biomater ; 100(4): 1017-28, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22323400

RESUMO

Encapsulation of insulin-producing cells in alginate beads could improve the treatment of type 1 diabetes by reducing or eliminating the need for immunosuppression. We have recently adapted an emulsion and internal gelation process to ß-cell encapsulation. This process has the advantages of being well suited for m(3)/h production rates and allowing the use of increased alginate concentrations. Compared with 1.5% alginate beads generated by a standard extrusion process, 5% alginate emulsion-generated beads demonstrated greater in vitro stability and greater volumetric exclusion of antibody-sized pullulan. When ßTC3 cells were transplanted into streptozotocin-induced allogeneic diabetic mice, a significant decrease in the blood glucose levels was seen within 2 days with the 5% emulsion-generated beads but not until >16 days with the 1.5% extrusion-generated beads. This was correlated with higher cell survival and lower graft-specific plasma immunoglobulin levels. These results suggest that higher-concentration alginate beads generated by emulsion and internal gelation have improved graft immunoprotection. The emulsion process is a promising and scalable technology for cellular therapies requiring immune isolation.


Assuntos
Alginatos/química , Diabetes Mellitus/terapia , Animais , Glicemia/metabolismo , Sobrevivência Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Microscopia Crioeletrônica/métodos , Emulsões , Géis/química , Hidrogéis/química , Imunossupressores/farmacologia , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura/métodos , Permeabilidade , Estresse Mecânico , Transplante Homólogo/métodos , Viscosidade
11.
Endocrinology ; 152(9): 3385-95, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21750049

RESUMO

Leptin, an adipocyte-derived hormone, plays an essential role in the maintenance of normal body weight and energy expenditure, as well as glucose homeostasis. Indeed, leptin-deficient ob/ob mice are obese with profound hyperinsulinemia, insulin resistance, and often hyperglycemia. Interestingly, low doses of exogenous leptin can reverse the hyperinsulinemia and hyperglycemia in these animals without altering body weight. The hyperinsulinemia in ob/ob mice may result directly from the absence of leptin signaling in pancreatic ß-cells and, in turn, contribute to both obesity and insulin resistance. Here, we acutely attenuated endogenous leptin signaling in normal mice with a polyethylene glycol (PEG)ylated mouse leptin antagonist (PEG-MLA) to determine the contribution of leptin signaling in the regulation of glucose homeostasis. PEG-MLA was either injected or continuously administered via osmotic minipumps for several days, and various metabolic parameters were assessed. PEG-MLA-treated mice had increased fasting and glucose-stimulated plasma insulin levels, decreased whole-body insulin sensitivity, elevated hepatic glucose production, and impaired insulin-mediated suppression of hepatic glucose production. Moreover, PEG-MLA treatment resulted in increased food intake and increased respiratory quotient without significantly altering energy expenditure or body composition as assessed by the lean:lipid ratio. Our findings indicate that alterations in insulin sensitivity occur before changes in the lean:lipid ratio and energy expenditure during the acute disruption of endogenous leptin signaling.


Assuntos
Glucose/metabolismo , Resistência à Insulina/fisiologia , Insulina/sangue , Leptina/antagonistas & inibidores , Leptina/metabolismo , Transdução de Sinais/fisiologia , Animais , Calorimetria Indireta , Colesterol/metabolismo , Metabolismo Energético/fisiologia , Jejum/metabolismo , Técnica Clamp de Glucose , Bombas de Infusão Implantáveis , Fígado/metabolismo , Masculino , Camundongos , Triglicerídeos/metabolismo
12.
Am J Physiol Endocrinol Metab ; 300(5): E923-32, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21304061

RESUMO

Dramatic improvement of type 2 diabetes is commonly observed after bariatric surgery. However, the mechanisms behind the alterations in glucose homeostasis are still elusive. We examined the effect of duodenal-jejunal bypass (DJB), which maintains the gastric volume intact while bypassing the entire duodenum and the proximal jejunum, on glycemic control, ß-cell mass, islet morphology, and changes in enteroendocrine cell populations in nonobese diabetic Goto-Kakizaki (GK) rats and nondiabetic control Wistar rats. We performed DJB or sham surgery in GK and Wistar rats. Blood glucose levels and glucose tolerance were monitored, and the plasma insulin, glucagon-like peptide-1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) levels were measured. ß-Cell area, islet fibrosis, intestinal morphology, and the density of enteroendocrine cells expressing GLP-1 and/or GIP were quantified. Improved postprandial glycemia was observed from 3 mo after DJB in diabetic GK rats, persisting until 12 mo after surgery. Compared with the sham-GK rats, the DJB-GK rats had an increased ß-cell area and a decreased islet fibrosis, increased insulin secretion with increased GLP-1 secretion in response to a mixed meal, and an increased population of cells coexpressing GIP and GLP-1 in the jejunum anastomosed to the stomach. In contrast, DJB impaired glucose tolerance in nondiabetic Wistar rats. In conclusion, although DJB worsens glucose homeostasis in normal nondiabetic Wistar rats, it can prevent long-term aggravation of glucose homeostasis in diabetic GK rats in association with changes in intestinal enteroendocrine cell populations, increased GLP-1 production, and reduced ß-cell deterioration.


Assuntos
Cirurgia Bariátrica , Diabetes Mellitus Tipo 2/cirurgia , Duodeno/fisiologia , Sistema Endócrino/citologia , Polipeptídeo Inibidor Gástrico/biossíntese , Peptídeo 1 Semelhante ao Glucagon/biossíntese , Hiperglicemia/sangue , Células Secretoras de Insulina/efeitos dos fármacos , Jejuno/fisiologia , Animais , Glicemia/metabolismo , Composição Corporal/fisiologia , Peso Corporal/fisiologia , Diabetes Mellitus Tipo 2/patologia , Sistema Endócrino/efeitos dos fármacos , Sistema Endócrino/metabolismo , Ensaio de Imunoadsorção Enzimática , Fibrose , Teste de Tolerância a Glucose , Imuno-Histoquímica , Incretinas/sangue , Ilhotas Pancreáticas/patologia , Masculino , Ratos , Ratos Wistar
13.
Diabetes ; 59(12): 3032-40, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20876720

RESUMO

OBJECTIVE: The liver plays a critical role in integrating and controlling glucose metabolism. Thus, it is important that the liver receive and react to signals from other tissues regarding the nutrient status of the body. Leptin, which is produced and secreted from adipose tissue, is a hormone that relays information regarding the status of adipose depots to other parts of the body. Leptin has a profound influence on glucose metabolism, so we sought to determine if leptin may exert this effect in part through the liver. RESEARCH DESIGN AND METHODS: To explore this possibility, we created mice that have disrupted hepatic leptin signaling using a Cre-lox approach and then investigated aspects of glucose metabolism in these animals. RESULTS: The loss of hepatic leptin signaling did not alter body weight, body composition, or blood glucose levels in the mild fasting or random-fed state. However, mice with ablated hepatic leptin signaling had increased lipid accumulation in the liver. Further, as male mice aged or were fed a high-fat diet, the loss of hepatic leptin signaling protected the mice from glucose intolerance. Moreover, the mice displayed increased liver insulin sensitivity and a trend toward enhanced glucose-stimulated plasma insulin levels. Consistent with increased insulin sensitivity, mice with ablated hepatic leptin signaling had increased insulin-stimulated phosphorylation of Akt in the liver. CONCLUSIONS: These data reveal that unlike a complete deficiency of leptin action, which results in impaired glucose homeostasis, disruption of leptin action in the liver alone increases hepatic insulin sensitivity and protects against age- and diet-related glucose intolerance. Thus, leptin appears to act as a negative regulator of insulin action in the liver.


Assuntos
Intolerância à Glucose/prevenção & controle , Leptina/fisiologia , Fígado/fisiologia , Envelhecimento/fisiologia , Animais , Diabetes Mellitus Tipo 2/genética , Feminino , Glucose/farmacologia , Técnica Clamp de Glucose/métodos , Teste de Tolerância a Glucose , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/fisiologia , Leptina/deficiência , Leptina/genética , Masculino , Camundongos , Camundongos Transgênicos , Obesidade/genética , Reação em Cadeia da Polimerase , Receptores para Leptina/deficiência , Receptores para Leptina/genética , Receptores para Leptina/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
14.
Am J Physiol Endocrinol Metab ; 296(3): E473-9, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19106249

RESUMO

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are released during meals from endocrine cells located in the gut mucosa and stimulate insulin secretion from pancreatic beta-cells in a glucose-dependent manner. Although the gut epithelium senses luminal sugars, the mechanism of sugar sensing and its downstream events coupled to the release of the incretin hormones are not clearly elucidated. Recently, it was reported that sucralose, a sweetener that activates the sweet receptors of taste buds, triggers incretin release from a murine enteroendocrine cell line in vitro. We confirmed that immunoreactivity of alpha-gustducin, a key G-coupled protein involved in taste sensing, is sometimes colocalized with GIP in rat duodenum. We investigated whether secretion of incretins in response to carbohydrates is mediated via taste receptors by feeding rats the sweet-tasting compounds saccharin, acesulfame potassium, d-tryptophan, sucralose, or stevia. Oral gavage of these sweeteners did not reduce the blood glucose excursion to a subsequent intraperitoneal glucose tolerance test. Neither oral sucralose nor oral stevia reduced blood glucose levels in Zucker diabetic fatty rats. Finally, whereas oral glucose increased plasma GIP levels approximately 4-fold and GLP-1 levels approximately 2.5-fold postadministration, none of the sweeteners tested significantly increased levels of these incretins. Collectively, our findings do not support the concept that release of incretins from enteroendocrine cells is triggered by carbohydrates via a pathway identical to the sensation of "sweet taste" in the tongue.


Assuntos
Sacarose Alimentar/farmacologia , Duodeno/metabolismo , Peptídeo 1 Semelhante ao Glucagon/sangue , Incretinas/sangue , Edulcorantes/farmacologia , Administração Oral , Animais , Polipeptídeo Inibidor Gástrico/sangue , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Wistar , Ratos Zucker , Sacarina/farmacologia , Stevia , Sacarose/análogos & derivados , Sacarose/farmacologia , Tiazinas/farmacologia , Transducina/metabolismo , Triptofano/farmacologia
15.
Peptides ; 29(8): 1354-61, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18508160

RESUMO

Obestatin is purported to be a peptide hormone encoded in preproghrelin. We studied the metabolic effects of continuous infusion of obestatin via subcutaneously implanted osmotic mini-pumps. Administration of up to 500nmol/kg body weight/day obestatin did not change 24h cumulative food intake or body weight in rats. Similarly, no effects were observed when obestatin was infused at 1000nmol/kg body weight/day for seven days. This dose of obestatin infused during a 24h fast did not alter weight loss, suggesting that obestatin has no effect on energy expenditure, and this dose did not alter glucose or insulin responses during an IPGTT. Obestatin was originally proposed to interact with GPR39 and subsequently the receptor for GLP-1. While both receptors are expressed in pancreatic islets, incubation with obestatin did not alter insulin release from islets in vitro. Moreover, obestatin did not bind to INS-1 beta-cells or HEK cells overexpressing GLP-1 receptors or displace GLP-1 binding to these cells. Our findings do not support the concept that obestatin is a hormone with metabolic actions.


Assuntos
Grelina/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Células Cultivadas , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Grelina/administração & dosagem , Receptor do Peptídeo Semelhante ao Glucagon 1 , Teste de Tolerância a Glucose , Infusões Parenterais , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Lipídeos/sangue , Masculino , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores de Glucagon/biossíntese , Receptores de Glucagon/efeitos dos fármacos , Receptores de Glucagon/genética , Tempo
16.
Biochem Biophys Res Commun ; 362(4): 1007-12, 2007 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-17803965

RESUMO

In type 2 diabetes (T2DM) beta-cell responsiveness to glucose-dependent insulinotropic polypeptide (GIP) is reduced. In a model of T2DM, the VDF Zucker rat, GIP receptor mRNA and protein levels were shown to be down-regulated. Possible restoration of responsiveness to GIP in Zucker rats by reducing hyperglycemia has been examined. ZDF rats with extreme hyperglycemia demonstrated greater islet GIP receptor mRNA down-regulation (94.3+/-3.8%) than ZF rats (48.8+/-22.8%). GIP receptor mRNA levels in ZDF rats returned to 83.0+/-17.9% of lean following normalization of hyperglycemia by phlorizin treatment and pancreas perfusions demonstrated markedly improved GIP responsiveness. Treatment of VDF rats with a DP IV inhibitor (P32/98) resulted in improved glucose tolerance and restored sensitivity to GIP in isolated pancreata. These findings support the proposal that GIP receptor down-regulation in rodent T2DM is secondary to chronic hyperglycemia and that normalization of glycemia can restore GIP sensitivity.


Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Inibidor Gástrico/administração & dosagem , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Animais , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Resistência a Medicamentos , Resistência à Insulina , Ratos , Ratos Zucker , Resultado do Tratamento
17.
Cell Metab ; 4(4): 291-302, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17011502

RESUMO

The hormone leptin plays a crucial role in maintenance of body weight and glucose homeostasis. This occurs through central and peripheral pathways, including regulation of insulin secretion by pancreatic beta cells. To study this further in mice, we disrupted the signaling domain of the leptin receptor gene in beta cells and hypothalamus. These mice develop obesity, fasting hyperinsulinemia, impaired glucose-stimulated insulin release, and glucose intolerance, similar to leptin receptor null mice. However, whereas complete loss of leptin function causes increased food intake, this tissue-specific attenuation of leptin signaling does not alter food intake or satiety responses to leptin. Moreover, unlike other obese models, these mice have reduced fasting blood glucose. These results indicate that leptin regulation of glucose homeostasis extends beyond insulin sensitivity to influence beta cell function, independent of pathways controlling food intake. These data suggest that defects in this adipoinsular axis could contribute to diabetes associated with obesity.


Assuntos
Glucose/metabolismo , Homeostase , Células Secretoras de Insulina/metabolismo , Leptina/metabolismo , Animais , Ingestão de Alimentos , Feminino , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores para Leptina , Sensibilidade e Especificidade , Transdução de Sinais/fisiologia , Fatores de Tempo
18.
Life Sci ; 75(15): 1857-70, 2004 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-15302229

RESUMO

Gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal regulator of insulin release and glucose homeostasis following a meal. Strategies have been undertaken to delineate the bioactive domains of GIP with the intention of developing small molecular weight GIP mimetics. The molecular cloning of receptors for GIP and the related hormone GLP-1 (glucagon-like peptide-1) has allowed examination of the characteristics of incretin analogs in transfected cell models. The current report examines the N-terminal bioactive domain of GIP residing in residues 1-14 by alanine scanning mutagenesis and N-terminal substitution/modification. Further studies examined peptide chimeras of GIP and GLP-1 designed to localize bioactive determinants of the two hormones. The alanine scan of the GIP(1-14) sequence established that the peptide was extremely sensitive to structural perturbations. Only replacement of amino acids 2 and 13 with those found in glucagon failed to dramatically reduce receptor binding and activation. Of four GIP(1-14) peptides modified by the introduction of DP IV-resistant groups, a peptide with a reduced bond between Ala2 and Glu3 demonstrated improved receptor potency compared to native GIP(1-14). The peptide chimera studies supported recent results on the importance of a mid-region helix for bioactivity of GIP, and confirmed existence of two separable regions with independent intrinsic receptor binding and activation properties. Furthermore, peptide chimeras showed that binding of GLP-1 also involves both N- and C-terminal domains, but that it apparently contains only a single bioactive domain in its N-terminus. Together, these results should facilitate development of incretin based therapies using rational drug design for potential treatment of diabetes.


Assuntos
Polipeptídeo Inibidor Gástrico/farmacologia , Fragmentos de Peptídeos/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Células CHO , Cricetinae , AMP Cíclico/biossíntese , Relação Dose-Resposta a Droga , Feminino , Polipeptídeo Inibidor Gástrico/química , Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Peso Molecular , Fragmentos de Peptídeos/química , Plasmídeos/genética , Precursores de Proteínas/farmacologia , Ensaio Radioligante , Receptores dos Hormônios Gastrointestinais/efeitos dos fármacos , Estimulação Química
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