RESUMO
Energy metabolism and reproduction are closely linked and reciprocally regulated. The detrimental effect of underweight on reproduction complicates the safety evaluation of anti-obesity drugs, making it challenging to distinguish pathological changes mediated through the intended drug-induced weight loss from direct drug effects on reproductive organs. Four-weeks dosing of normal weight Sprague Dawley rats with a glucagon-like peptide 1 (GLP-1)/glucagon receptor co-agonist induced a robust weight loss, accompanied by histological findings in prostate, seminal vesicles, mammary glands, uterus/cervix and vagina. Characterization of the hypothalamus-pituitary-gonadal (HPG) axis in male rats revealed reduced hypothalamic Kiss1 mRNA levels and decreased serum luteinizing hormone (LH) and testosterone concentrations following co-agonist dosing. These alterations resemble hypogonadotropic hypogonadism typically seen in adverse energy deprived conditions, like chronic food restriction. Concomitant daily administration of kisspeptin-52 from day 21 to the end of the four-week co-agonist dosing period evoked LH and testosterone responses without normalizing histological findings. This incomplete rescue by kisspeptin-52 may be due to the rather short kisspeptin-52 treatment period combined with a desensitization observed on testosterone responses. Concomitant leptin treatment from day 21 did not reverse co-agonist induced changes in HPG axis activity. Furthermore, a single co-agonist injection in male rats slightly elevated LH levels but left testosterone unperturbed, thereby excluding a direct acute inhibitory effect on the HPG axis. Our data suggest that the reproductive phenotype after repeated co-agonist administration was driven by the intended weight loss, however, we cannot exclude a direct organ related effect in chronically treated rats.
Assuntos
Fármacos Antiobesidade/farmacologia , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Kisspeptinas/farmacologia , Testículo/efeitos dos fármacos , Animais , Kisspeptinas/administração & dosagem , Masculino , Ratos , Ratos Sprague-Dawley , Testículo/metabolismo , Magreza , Redução de Peso/efeitos dos fármacosRESUMO
The possibility to use leptin therapeutically for lowering glucose levels in patients with type 1 diabetes has attracted interest. However, earlier animal models of type 1 diabetes are severely catabolic with very low endogenous leptin levels, unlike most patients with diabetes. Here, we aim to test glucose-lowering effects of leptin in novel, more human-like murine models. We examined the glucose-lowering potential of leptin in diabetic models of two types: streptozotocin-treated mice and mice treated with the insulin receptor antagonist S961. To prevent hypoleptinemia, we used combinations of thermoneutral temperature and high-fat feeding. Leptin fully normalized hyperglycemia in standard chow-fed streptozotocin-treated diabetic mice. However, more humanized physiological conditions (high-fat diets or thermoneutral temperatures) that increased adiposity - and thus also leptin levels - in the diabetic mice abrogated the effects of leptin, i.e., the mice developed leptin resistance also in this respect. The glucose-lowering effect of leptin was not dependent on the presence of the uncoupling protein-1 and was not associated with alterations in plasma insulin, insulin-like growth factor 1, food intake or corticosterone but fully correlated with decreased plasma glucagon levels and gluconeogenesis. An important implication of these observations is that the therapeutic potential of leptin as an additional treatment in patients with type 1 diabetes is probably limited. This is because such patients are treated with insulin and do not display low leptin levels. Thus, the potential for a glucose-lowering effect of leptin would already have been attained with standard insulin therapy, and further effects on blood glucose level through additional leptin cannot be anticipated.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Leptina/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Glicemia/metabolismo , Corticosterona/metabolismo , Modelos Animais de Doenças , Ingestão de Alimentos , Glucagon/metabolismo , Gluconeogênese , Humanos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Leptina/farmacologia , Camundongos , Camundongos Knockout , Consumo de Oxigênio , Peptídeos/farmacologia , Ácido Pirúvico/metabolismo , Receptor de Insulina/antagonistas & inibidores , Transcriptoma , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
Cell-autonomous and cell-nonautonomous mechanisms of neurodegeneration appear to occur in the proteinopathies, including Alzheimer's and Parkinson's diseases. However, how neuronal toxicity is generated from misfolding-prone proteins secreted by nonneuronal tissues and whether modulating protein aggregate levels at distal locales affects the degeneration of postmitotic neurons remains unknown. We generated and characterized animal models of the transthyretin (TTR) amyloidoses that faithfully recapitulate cell-nonautonomous neuronal proteotoxicity by expressing human TTR in the Caenorhabditis elegans muscle. We identified sensory neurons with affected morphological and behavioral nociception-sensing impairments. Nonnative TTR oligomer load and neurotoxicity increased following inhibition of TTR degradation in distal macrophage-like nonaffected cells. Moreover, reducing TTR levels by RNAi or by kinetically stabilizing natively folded TTR pharmacologically decreased TTR aggregate load and attenuated neuronal dysfunction. These findings reveal a critical role for in trans modulation of aggregation-prone degradation that directly affects postmitotic tissue degeneration observed in the proteinopathies.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Pré-Albumina/metabolismo , Agregados Proteicos , Neuropatias Amiloides/genética , Neuropatias Amiloides/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Humanos , Pré-Albumina/genética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismoRESUMO
Several proteins have been identified as amyloid forming in humans, and independent of protein origin, the fibrils are morphologically similar. Therefore, there is a potential for structures with amyloid seeding ability to induce both homologous and heterologous fibril growth; thus, molecular interaction can constitute a link between different amyloid forms. Intravenous injection with preformed fibrils from islet amyloid polypeptide (IAPP), proIAPP, or amyloid-beta (Aß) into human IAPP transgenic mice triggered IAPP amyloid formation in pancreas in 5 of 7 mice in each group, demonstrating that IAPP amyloid could be enhanced through homologous and heterologous seeding with higher efficiency for the former mechanism. Proximity ligation assay was used for colocalization studies of IAPP and Aß in islet amyloid in type 2 diabetic patients and Aß deposits in brains of patients with Alzheimer disease. Aß reactivity was not detected in islet amyloid although islet ß cells express AßPP and convertases necessary for Aß production. By contrast, IAPP and proIAPP were detected in cerebral and vascular Aß deposits, and presence of proximity ligation signal at both locations showed that the peptides were <40 nm apart. It is not clear whether IAPP present in brain originates from pancreas or is locally produced. Heterologous seeding between IAPP and Aß shown here may represent a molecular link between type 2 diabetes and Alzheimer disease.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Amiloidose/metabolismo , Encéfalo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Amiloidose/patologia , Animais , Encéfalo/patologia , Diabetes Mellitus Tipo 2/patologia , Feminino , Humanos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-IdadeRESUMO
AIMS/HYPOTHESIS: Islet amyloid polypeptide (IAPP) is a beta cell hormone secreted together with insulin upon glucose stimulation. IAPP participates in normal glucose regulation, but IAPP is also known for its ability to misfold and form islet amyloid. Amyloid fibrils form through smaller cell toxic intermediates and deposited amyloid disrupts normal islet architecture. Even though IAPP and amyloid formation are much discussed in type 2 diabetes, our aim was to study the significance of IAPP in type 1 diabetes. RESULTS: Plasma IAPP levels in children and adolescents with newly diagnosed type 1 diabetes (nâ=â224) were analysed and concentrations exceeding 100 pmol/L (127.2-888.7 pmol/L) were found in 11% (25/224). The IAPP increase did not correlate with C-peptide levels. CONCLUSIONS/INTERPRETATION: Plasma levels of IAPP and insulin deviate in a subpopulation of young with newly-diagnosed type 1 diabetes. The determined elevated levels of IAPP might increase the risk for IAPP misfolding and formation of cell toxic amyloid in beta cells. This finding add IAPP-aggregation to the list over putative pathological factors causing type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/sangue , Polipeptídeo Amiloide das Ilhotas Pancreáticas/sangue , Adolescente , Autoanticorpos/sangue , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Insulina/imunologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/imunologia , Masculino , Estudos ProspectivosRESUMO
The gut hormone peptideâ YY (PYY) is postprandially secreted from enteroendocrine Lâ cells and is involved in the regulation of energy homeostasis. The N-terminal truncated version PYY(3-36) decreases food intake and has potential as an anti-obesity agent. The anorectic effect of PYY(3-36) is mediated through Y2 receptors in the hypothalamus, vagus, and brainstem regions, and it is well known that the C-terminal tetrapeptide sequence of PYY(3-36) is crucial for Y2 receptor activation. The aim of this work was to develop a semisynthetic methodology for the generation of a library of C-terminally modified PYY(3-36) analogues. By using an intein-based expression system, PYY(3-29) was generated as a C-terminal peptide α-thioester. Heptapeptides bearing an N-terminal cysteine and modifications at one of the four C-terminal positions were synthesized in a 96-well plate by parallel solid-phase synthesis. In the plate format, an array of [Ala30]PYY(3-36) analogues were generated by ligation, desulfurization, and subsequent solid-phase extraction. The generated analogues, in which either Arg33, Gln34, Arg35, or Tyr36 had been substituted with proteinogenic or non-proteinogenic amino acids, were tested in a functional Y2 receptor assay. Generally, substitutions of Tyr36 were better tolerated than modifications of Arg33, Gln34, and Arg35. Two analogues showed significantly improved Y2 receptor selectivity; therefore, these results could be used to design new drug candidates for the treatment of obesity.
Assuntos
Oligopeptídeos/química , Peptídeo YY/química , Sequência de Aminoácidos , Escherichia coli/metabolismo , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Peptídeo YY/genética , Peptídeo YY/metabolismo , Ligação Proteica , Receptores de Neuropeptídeo Y/química , Receptores de Neuropeptídeo Y/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Técnicas de Síntese em Fase Sólida , Relação Estrutura-AtividadeRESUMO
It has been hypothesized that amphipathic peptides might bind to membranes prior to activating their cognate receptors, but this has proven difficult to test. The peptide hormone PYY3-36 is believed to perform its appetite-suppressing actions through binding to hypothalamic Y2 receptors. It has been proposed that PYY3-36 via its amphipathic α-helix binds to the plasma membrane prior to receptor docking. Here, our aim was to study the implication of this hypothesis using new analogs of PYY3-36. We first studied membrane binding of PYY3-36. Next, we designed a series of PYY3-36 analogs to increase membrane-binding affinity by substituting the N-terminal segment with a de novo designed α-helical, amphipathic sequence. These 2-helix variants of PYY3-36 were assembled by solid-phase peptide synthesis. Pharmacological studies demonstrated that even though the native peptide sequence was radically changed, highly active Y2 receptor agonists were generated. A potent analog, with a Kd of 4 nM for membranes, was structurally characterized by NMR in the membrane-bound state, which clearly showed that it formed the expected 2-helix. The topology of the peptide-micelle association was studied by paramagnetic relaxation enhancement using a spin label, which confirmed that the hydrophobic residues bound to the membrane. Our studies further support the hypothesis that PYY3-36 associates with the membrane and indicate that this can be used in the design of novel molecules with high receptor binding potency. These observations are likely to be generally important for peptide hormones and biopharmaceutical drugs derived from them. This new 2-helix variant of PYY3-36 will be useful as a tool compound for studying peptide-membrane interactions.
Assuntos
Membrana Celular/metabolismo , Hormônios Peptídicos/síntese química , Hormônios Peptídicos/metabolismo , Peptídeo YY/química , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Hormônios Peptídicos/química , Ligação Proteica , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Diabetes in the domestic ferret (Mustela putorius furo) has previously been described and the purpose of this study was to evaluate if the ferret could serve as a model for the study of ß-cell degeneration associated with formation of islet amyloid. The nucleotide and amino acid sequence of ferret islet amyloid polypeptide (IAPP) 1-37 was identified and the synthesized peptide was studied with regards to in vitro amyloidogenicity and potential cellular toxicity in a comparative approach to human, cat and the nonamyloidogenic rat IAPP. Ferret IAPP forms amyloid-like fibrils, but with a longer lag phase than human and cat IAPP and the aggregation process was shown to reduce cell viability of cultured ß-cells, but with less potency than these two amyloidogenic counterparts. Immunohistochemistry of ferret pancreas confirmed IAPP expression in the islets of Langerhans, but no islet amyloid was found in a very limited sample size of one diabetic and five healthy ferrets. Islet amyloid has never been described in ferrets, and it is not possible to determine if it is due to lack of studies/material or to the fact that the ferret's life span is too short to present with such pathology.
Assuntos
Amiloidose/veterinária , Diabetes Mellitus Tipo 2/veterinária , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Sequência de Aminoácidos , Amiloide/química , Amiloide/metabolismo , Amiloide/ultraestrutura , Amiloidose/etiologia , Amiloidose/patologia , Animais , Gatos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Tipo 2/complicações , Modelos Animais de Doenças , Furões , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Masculino , Dados de Sequência Molecular , Pâncreas/metabolismo , Pâncreas/patologia , Fragmentos de Peptídeos/farmacologia , Multimerização Proteica , Homologia de Sequência de AminoácidosRESUMO
Accumulation of amyloid beta-peptide (Abeta) and tau aggregates, possibly linked to age-associated deficiencies in protein homeostasis, appear to cause Alzheimer's disease. Schiff-base formation between Abeta and the aldehyde-bearing cholesterol oxidation product 3-beta-hydroxy-5-oxo-5,6-secocholestan-6-al is known to increase Abeta amyloidogenicity. Here, we synthesized Abeta variants site-specifically modified with the cholesterol aldehyde at Asp-1, Lys-16, or Lys-28, rather than studying mixtures. These distinct modifications have a similar effect on the thermodynamic propensity for aggregation, enabling aggregation at low concentrations. In contrast, the modification site differentially influences the aggregation kinetics; Lys-16-modified Abeta formed amorphous aggregates fastest and at the lowest concentration (within 2 h at a concentration of 20 nM), followed by the Lys-28 and Asp-1 conjugates. Also, the aggregates resulting from Abeta Lys-16 cholesterol aldehyde conjugation were more toxic to primary rat cortical neurons than treatment with unmodified Abeta under identical conditions and at the same concentration. Our results show that Abeta modification by cholesterol derivatives, especially at Lys-16, renders it kinetically and thermodynamically competent to form neurotoxic aggregates at concentrations approaching the physiologic concentration of Abeta.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Colesterol/metabolismo , Mutagênese Sítio-Dirigida , Neurônios/patologia , Peptídeos/química , Peptídeos/toxicidade , Sequência de Aminoácidos , Peptídeos beta-Amiloides/toxicidade , Peptídeos beta-Amiloides/ultraestrutura , Animais , Benzotiazóis , Células Cultivadas , Fluorescência , Cinética , Luz , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Estrutura Quaternária de Proteína , Ratos , Espalhamento de Radiação , Tiazóis/metabolismoRESUMO
Our aim was to study, at the same glycemic control, how treatment with either the insulin secretagogue repaglinide or exogenous insulin aspart affects endogenous insulin secretion, plasma insulin and IAPP (islet amyloid polypeptide) levels, GH-IGF (growth hormone-insulin-like growth factor) axis and plasma lipoprotein concentrations in patients with type 2 diabetes. Five patients, age 65.0+/-4.1 years (mean+/-SE), body weight 82.5+/-5.0 kg, BMI (body mass index) 27.7+/-1.5 kg/m(2) were treated for 10 weeks with repaglinide or insulin aspart in a randomized, cross-over study. At the end of each treatment a 24-h metabolic profile was performed. Blood glucose, C-peptide, free human insulin, free total (human and analogue) insulin, proinsulin, IAPP, IGF-I, IGFBP-1 (IGF binding protein-1), GHBP (growth hormone binding protein) and plasma lipoprotein concentrations were measured. Similar 24-h blood glucose profiles were obtained with repaglinide and insulin aspart treatment. During the repaglinide treatment, the meal related peaks of C-peptide and free human insulin were about twofold higher than during treatment with insulin aspart. Proinsulin, GHBP were higher and IAPP levels tended to be higher during repaglinide compared to insulin aspart. Postprandial plasma total cholesterol, triglycerides and apolipoprotein B concentrations were higher on repaglinide than on insulin aspart treatment. Our results show that, at the same glycemic control, treatment with exogenous insulin aspart in comparison with the insulin secretagogue repaglinide result in a lower endogenous insulin secretion, and a tendency towards a less atherogenic postprandial lipid profile.
Assuntos
Glicemia/metabolismo , Carbamatos/uso terapêutico , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Insulina/análogos & derivados , Lipídeos/sangue , Piperidinas/uso terapêutico , Idoso , Apolipoproteínas/sangue , Glicemia/efeitos dos fármacos , Peptídeo C/sangue , Colesterol/sangue , Estudos Cross-Over , Esquema de Medicação , Feminino , Humanos , Insulina/sangue , Insulina/uso terapêutico , Insulina Aspart , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina/sangue , Fator de Crescimento Insulin-Like I/metabolismo , Lipoproteínas/sangue , Masculino , Pessoa de Meia-Idade , Proinsulina/sangueRESUMO
Amyloid formation is cytotoxic and can activate the caspase cascade. Here, we monitor caspase-3-like activity as reduction of fluorescence resonance energy transfer (FRET) using the contstruct pFRET2-DEVD containing enhanced cyan fluorescent protin (EYFP) linked by the caspase-3 specific cleavage site residues DEVD. Beta-TC-6 cells were transfected, and the fluoorescence was measured at 440 nm excitation and 535 nm (EYFP) and 480 nm (ECFP) emission wavelength. Cells were incubated with recombinant pro lset Amyloid Polypeptide (rec prolAPP) or the processing metabolites of prolAPP; the N-terminal flanking peptide withIAPP (recN+IAPP); IAPP with the C-terminal flanking peptied (recIAPP+C) and lslet Amyloid Polypeptide (recIAPP) . Peptides were added in solubilized from (50 microM) or as performed amyloid-like fibrils, or as a combination of these. FRET was measured and incubation with a mixture of solubilized peptide and performed fibrils resulted in loss of FRET and apoptosis was determined to occure in cells incubated with recproIAPP (49%), recN+IAPP (46%), recIAPP (72%) and recIAPP+C (59%). These results show that proIAPP and the processing intermediates reside the same cell toxic capacity as IAPP, and they can all have a central role in the reduction of beta-cell number in type 2 diabetes.
Assuntos
Amiloide/metabolismo , Apoptose , Caspase 3/metabolismo , Insulinoma/metabolismo , Insulinoma/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Células Cultivadas , Diabetes Mellitus Tipo 2/metabolismo , Transferência Ressonante de Energia de Fluorescência , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Following the encouraging report of the Edmonton group, there was a rejuvenation of the islet transplantation field. After that, more pessimistic views spread when long-term results of the clinical outcome were published. A progressive loss of the beta-cell function meant that almost all patients were back on insulin therapy after 5 years. More than 10 years ago, we demonstrated that amyloid deposits rapidly formed in human islets and in mouse islets transgenic for human IAPP when grafted into nude mice. It is, therefore, conceivable to consider amyloid formation as one potential candidate for the long-term failure. The present paper reviews attempts in our laboratories to elucidate the dynamics of and mechanisms behind the formation of amyloid in transplanted islets with special emphasis on the impact of long-term hyperglycemia.
Assuntos
Amiloide/metabolismo , Transplante das Ilhotas Pancreáticas/efeitos adversos , Sequência de Aminoácidos , Amiloide/genética , Animais , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/metabolismo , Humanos , Hiperglicemia/patologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Transplante das Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The amyloid present in the islets of Langerhans in type 2 diabetes is polymerized islet amyloid polypeptide (IAPP). The precursor protein proIAPP is posttranslationally modified, a process involving the removal of NH2- and COOH-terminal flanking peptides. This step is performed by the prohormone convertases PC2 and PC1/3. PC2 processes proIAPP preferably at the NH2-terminal processing site, and PC1/3 processes proIAPP exclusively at the COOH-terminal site. Little is known regarding the exact circumstances leading to islet amyloid formation. In this study, we have examined the possible significance of aberrant processing of proIAPP on amyloid formation in several in vitro cellular systems. In our studies, human (h)-proIAPP was transfected into beta-TC-6 cells expressing both prohormone convertases and in which proIAPP is processed into IAPP. Additionally, h-proIAPP was transfected into three different pituitary-derived cell lines with different prohormone convertase profiles: AtT-20 cells (deficient in PC2), GH3 cells (deficient in PC1/3), and GH4C1 cells (deficient in both convertases). We followed the processing of h-proIAPP with antibodies specific for the respective cleavage sites and stained the cells with Congo red to verify the accumulation of amyloid. Incomplete processing of h-proIAPP that occurs in AtT-20 and GH4C1 cells resulted in the formation of intracellular amyloid. No amyloid developed in beta-TC-6 and GH3 cells lines with full processing of proIAPP. An intracellular increase in proIAPP and/or its metabolic products may thus promote intracellular amyloid formation, thereby causing cell death. When extracellularly exposed, this amyloid might act as template for continuing amyloid formation from processed IAPP released from the surrounding beta-cells.