RESUMEN
Endoplasmic reticulum (ER) stress is closely associated with type 2 diabetes (T2D). Activating transcription factor 5 (ATF5) is a member of the ATF/cAMP response element binding protein (CREB) family whose levels are increased upon stress in pancreatic islets from mice. Intriguingly, ATF5 deficiency has been shown to contribute to increased ER stress and apoptosis in mouse islet micro-organs. We hypothesized that either deficiency or overexpression of ATF5 is equally deleterious for pancreatic islets in terms of ER stress and apoptosis. To test this, we used a number of in vitro and in vivo models whereby ATF5 levels were overexpressed. We also determined the regulation of ATF5 in the context of metabolic derangements by using various mouse models of obesity and T2D. Our in vitro results show that ATF5 overexpression promoted palmitic acid (PA)-induced lipotoxic apoptosis. In vivo, global ATF5 overexpression in mice was lethal and pancreas-specific ATF5 overexpressing mice exhibit increased ß-cell apoptosis. Interestingly, ATF5 is downregulated in all mouse models of severe obesity and T2D used in the current study. In conclusion, a tight control on ATF5 levels might be considered when developing novel agents targeting ATF5 for prevention and treatment of metabolic diseases.
Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Ratones , Animales , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Factores de Transcripción Activadores/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Apoptosis/fisiología , Modelos Animales de Enfermedad , Dieta , Obesidad/metabolismo , Células Secretoras de Insulina/metabolismo , Estrés del Retículo Endoplásmico/fisiologíaRESUMEN
Apolipoprotein CIII (apoCIII) is increased in obesity-induced insulin resistance and type-2 diabetes. Emerging evidences support the advantages of small interfering RNAs (siRNAs) to target disease-causing genes. The aim of this study was to develop siRNAs for in vivo silencing of apoCIII and investigate if this results in metabolic improvements comparable to what we have seen using antisense oligonucelotides against apoCIII. Twenty-four siRNAs were synthesized and tested in a dual luciferase reporter assay. The eight best were selected, based on knockdown at 20 nM, and of these, two were selected based on IC50 values. In vivo experiments were performed in ob/ob mice, an obese animal model for diabetes. To determine the dose-dependency, efficacy, duration of effect and therapeutic dose we used a short protocol giving the apoCIII-siRNA mix for three days. To evaluate long-term metabolic effects mice were treated for three days, every second week for eight weeks. The siRNA mix effectively and selectively reduced expression of apoCIII in liver in vivo. Treatment had to be repeated every two weeks to maintain a suppression of apoCIII. The reduction of apoCIII resulted in increased LPL activity, lower triglycerides, reduced liver fat, ceased weight gain, enhanced insulin sensitivity, and improved glucose homeostasis. No off-target or side effects were observed during the eight-week treatment period. These results suggest that in vivo silencing of apoCIII with siRNA, is a promising approach with the potential to be used in the battle against obesity-induced metabolic disorders.
Asunto(s)
Diabetes Mellitus Tipo 2 , Síndrome Metabólico , Ratones , Animales , Apolipoproteína C-III/genética , Apolipoproteína C-III/metabolismo , Apolipoproteína C-III/farmacología , ARN Interferente Pequeño , ObesidadRESUMEN
Increased levels of apolipoprotein CIII (apoCIII), a key regulator of lipid metabolism, result in obesity-related metabolic derangements. We investigated mechanistically whether lowering or preventing high-fat diet (HFD)-induced increase in apoCIII protects against the detrimental metabolic consequences. Mice, first fed HFD for 10 weeks and thereafter also given an antisense (ASO) to lower apoCIII, already showed reduced levels of apoCIII and metabolic improvements after 4 weeks, despite maintained obesity. Prolonged ASO treatment reversed the metabolic phenotype due to increased lipase activity and receptor-mediated hepatic uptake of lipids. Fatty acids were transferred to the ketogenic pathway, and ketones were used in brown adipose tissue (BAT). This resulted in no fat accumulation and preserved morphology and function of liver and BAT. If ASO treatment started simultaneously with the HFD, mice remained lean and metabolically healthy. Thus, lowering apoCIII protects against and reverses the HFD-induced metabolic phenotype by promoting physiological insulin sensitivity.
Asunto(s)
Dieta Alta en Grasa , Enfermedades Metabólicas , Tejido Adiposo Pardo/metabolismo , Animales , Apolipoproteína C-III/metabolismo , Dieta Alta en Grasa/efectos adversos , Enfermedades Metabólicas/metabolismo , Ratones , Ratones Endogámicos C57BL , Obesidad/etiología , Obesidad/metabolismo , Obesidad/prevención & controlRESUMEN
It is well known that type-2 diabetes mellitus (T2D) is increasing worldwide, but also the autoimmune form, type-1 diabetes (T1D), is affecting more people. The latest estimation from the International Diabetes Federation (IDF) is that 1.1 million children and adolescents below 20 years of age have T1D. At present, we have no primary, secondary or tertiary prevention or treatment available, although many efforts testing different strategies have been made. This review is based on the findings that apolipoprotein CIII (apoCIII) is increased in T1D and that in vitro studies revealed that healthy ß-cells exposed to apoCIII became apoptotic, together with the observation that humans with higher levels of the apolipoprotein, due to mutations in the gene, are more susceptible to developing T1D. We have summarized what is known about apoCIII in relation to inflammation and autoimmunity in in vitro and in vivo studies of T1D. The aim is to highlight the need for exploring this field as we still are only seeing the top of the iceberg.
Asunto(s)
Apolipoproteína C-III/genética , Diabetes Mellitus Tipo 1/genética , Inflamación/genética , Células Secretoras de Insulina/metabolismo , Adulto , Calcio/metabolismo , Diabetes Mellitus Tipo 1/epidemiología , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patología , Regulación de la Expresión Génica/genética , Humanos , Inflamación/patología , Células Secretoras de Insulina/patologíaRESUMEN
Apolipoprotein CIII (apoCIII) is proinflammatory and increases in high-fat diet (HFD)-induced obesity and insulin resistance. We have previously shown that reducing apoCIII improves insulin sensitivity in vivo by complex mechanisms involving liver and brown adipose tissue. In this study the focus was on subcutaneous (SAT) and visceral (VAT) white adipose tissue (WAT). Mice were either given HFD for 14 weeks and directly from start also treated with antisense oligonucleotide (ASO) against apoCIII or given HFD for 10 weeks and HFD+ASO for an additional 14 weeks. Both groups had animals treated with inactive (Scr) ASO as controls and in parallel chow-fed mice were injected with saline. Preventing an increase or lowering apoCIII in the HFD-fed mice decreased adipocytes' size, reduced expression of inflammatory cytokines and increased expression of genes related to thermogenesis and beiging. Isolated adipocytes from both VAT and SAT from the ASO-treated mice had normal insulin-induced inhibition of lipolysis compared to cells from Scr-treated mice. In conclusion, the HFD-induced metabolic derangements in WATs can be prevented and reversed by lowering apoCIII.
Asunto(s)
Tejido Adiposo Blanco/metabolismo , Apolipoproteína C-III/metabolismo , Inflamación/metabolismo , Resistencia a la Insulina/fisiología , Obesidad/metabolismo , Adipocitos/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Dieta Alta en Grasa , Insulina/metabolismo , Lipólisis/fisiología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Termogénesis/fisiologíaRESUMEN
A Picornavirus (Ljungan virus [LV]) originally found in bank voles has been associated with type 1 diabetes (T1D) in its wild rodent reservoir, but also associated with T1D in a laboratory rat model for the disease, the diabetes prone (DP) Bio Breeding (BB) rat. Successful treatment of diabetes in this rat model, using experimental antiviral compounds directed against picornavirus, has been reported. In the present study we show significant clinical response in DP-BB rats using antiviral compounds available for human use (Pleconaril, Efavirenz, and Ribavirin). Presence of LV picornavirus antigen has been detected in islets of Langerhans from both human and the T1D rat model with clear morphological similarity. Based on these data it would be of interest to test antiviral treatment in patients with newly diagnosed T1D. Successful outcome will offer both proof of concept regarding the role of virus involvement in the disease and possibly a first generation treatment interrupting a persistent infection and stopping ß-cell destruction.
Asunto(s)
Antivirales/uso terapéutico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Oxadiazoles/uso terapéutico , Oxazoles/uso terapéutico , Ribavirina/uso terapéutico , Adulto , Alquinos/uso terapéutico , Animales , Benzoxazinas/uso terapéutico , Ciclopropanos/uso terapéutico , Humanos , Masculino , Prueba de Estudio Conceptual , RatasRESUMEN
Replacement of the insulin-secreting beta cells through transplantation of pancreatic islets to the liver is a promising treatment for type-1 diabetes. However, low oxygen tension, shear stress, and the induction of inflammation lead to significant islet dysfunction and loss. The anterior chamber of the eye (ACE) has gained considerable interest and represents an alternative therapeutic islet transplantation site because of its accessibility, high oxygen tension, and immune-privileged milieu. We have previously demonstrated the feasibility of intraocular islet transplant in mouse and nonhuman primate models of type-1 diabetes and are now assessing its efficacy on glucose homeostasis in a nonhuman primate model of type-2 diabetes. We transplanted allogeneic donor islets (1,500 islet equivalents/kg) into the anterior chamber of one eye in a cynomolgus monkey with high-fat-diet-induced type-2 diabetes. Repeated examinations of the anterior and posterior segments of both eyes were done to monitor the engrafted islets and assess the overall ocular health. Fasting blood glucose level, blood biochemistry, and other metabolic parameters were routinely evaluated to determine the function of the islet graft and diabetes status. The transplanted islets were rapidly engrafted onto the iris and became vascularized 1 month after transplantation. We did not detect changes in intraocular pressure, cataract formation, ophthalmitis, or retinal vessel deformation. A significant lower fasting blood glucose level was observed while the graft was in place, and the transplantation reverts the progression of diabetes. The metabolic markers, hemoglobin A1C and fructosamine, demonstrated improvement following islet transplantation. As a conclusion, intraocular islet transplantation in one eye of a cynomolgus monkey with type-2 diabetes improved its overall plasma glucose homeostasis, as evidenced by short-term measures and long-term metabolic markers. These results further support the future application of the ACE as an alternative site for clinical islet transplants in the context of type-2 diabetes.
Asunto(s)
Cámara Anterior/metabolismo , Células Secretoras de Insulina/citología , Trasplante de Islotes Pancreáticos , Islotes Pancreáticos/metabolismo , Animales , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Insulinas/metabolismo , Trasplante de Islotes Pancreáticos/métodos , Macaca fascicularis/metabolismoRESUMEN
Apolipoprotein CIII (apoCIII) is associated with triglyceride (TG)-rich particles like VLDL and exerts an inhibitory effect of lipoprotein lipase. Increased levels are related to cardiovascular diseases and diabetes and therefore apoCIII has been proposed as a useful biomarker. Even if several commercial assays for measuring apoCIII in human plasma/serum are available, data is scarce concerning their reliability and none is used clinically. In the present study a comparative investigation has been done. Two ELISA-based methods (Cusabio Biotech and Assay Pro) and one nephelometric assay (Siemens Healthcare) were investigated. Serum and plasma samples were obtained from healthy volunteers and from samples sent to the Laboratory of Clinical Chemistry, preferably with higher levels of TGs. The Cusabio Biotech assay did not yield any valid results. However, both the methods from Assay Pro and Siemens Healthcare showed good performance with similar dynamic ranges. The latter assay had lower CV and required less work. In healthy individuals, apoCIII levels were not affected by fasting, freezing or thawing, nor did we find any gender differences. Individuals with elevated levels of TG displayed higher apoCIII values. Females with oral intake of contraceptives had higher levels. In conclusion, the nephelometric assay showed the best performance with the lowest CV, was less labor intensive than an assay based on ELISA and could therefore be suitable for clinical use.
Asunto(s)
Apolipoproteína C-III/sangre , Química Clínica/métodos , Diabetes Mellitus/sangre , Nefelometría y Turbidimetría/normas , Enfermedad del Hígado Graso no Alcohólico/sangre , Triglicéridos/sangre , Adulto , Anciano , Biomarcadores/sangre , Índice de Masa Corporal , Estudios de Casos y Controles , Química Clínica/normas , HDL-Colesterol/sangre , Anticonceptivos Orales/administración & dosificación , Diabetes Mellitus/diagnóstico , Ensayo de Inmunoadsorción Enzimática , Ayuno/sangre , Humanos , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/diagnóstico , Manejo de EspecímenesRESUMEN
OBJECTIVE: Neuroendocrine tumors of the pancreas (Pan-NETs) are rare, but among the most common neuroendocrine neoplasias. They are mostly slowly growing with a capacity to metastasize, but transition to a higher grade occurs, which lead to a more aggressive tumor phenotype. Very seldom, non-functional tumors can become hormonally active. Here we present four patients with originally non-functional Pan-NETs that subsequently started to produce insulin or its precursors, causing severe hypoglycemia. METHODS: We reviewed the medical files, biochemistry and radiological investigations. Pathology tissue samples were re-examined, and additional immunohistochemical analyses were performed. RESULTS: Four patients; three women and one man, aged 51, 61, 65 and 68 years at diagnosis developed malignant insulinomas 2, 5, 6 and 7 years respectively after initial diagnosis of non-secreting Pan-NETs. They had all metastatic disease at diagnosis. Ki-67 was initially 2, 5 and 6% and progressed to 25, 17 and 45%, respectively. In one patient the initial Ki-67 was 5% but was not reexamined. All four patients died due to their cancer disease within 12, 6, 19 and 29 months after treatment for hypoglycemia commenced. The clinical profile and/or review of the histopathology confirmed all original lesions as non-functional Pan-NETs with subsequent transformation into insulin-producing tumors. CONCLUSIONS: Non-functional, metastatic Pan-NETs may transform to insulin secreting lesions, with negative impact on prognosis. Therefore, if symptoms as hypoglycemia develops continuous follow-up of clinical parameters, biochemical profiles of pancreatic hormones and histopathological evaluation of proliferation is suggested to detect changes in characteristics of these malignant neoplasms.
Asunto(s)
Transformación Celular Neoplásica/patología , Insulinoma/patología , Tumores Neuroendocrinos/patología , Neoplasias Pancreáticas/patología , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , PronósticoRESUMEN
Serum levels of the pro-inflammatory apolipoprotein CIII (apoCIII) are increased in type-1 diabetic (T1D) patients and when ß-cells are exposed to apoCIII they undergo apoptosis, which can be prevented by an antibody against apoCIII. We have previously investigated the BB rat, an animal model that develops a human-like T1D at the age of around 60 days, and found that apoCIII was also increased in sera from pre-diabetic rats and this promoted ß-cell death. Lowering apoCIII with an oligonucleotide antisense during a phase of the pre-diabetic period prolonged the time to onset of T1D. In order to find other ways to lower apoCIII we in this study tested non-alcoholic red wine with medium and high concentrations of polyphenols and the lipid-lowering drug, fenofibrate, both reported to decrease the expression of apoCIII by activating peroxisome proliferator-activated receptors. Pre-diabetic BB-rats were treated orally for one month prior to the expected onset of diabetes with the two different wines or fenofibrate. None of the treatments prevented or prolonged the time to onset of diabetes and the expression of apoCIII was unaffected in this animal model for T1D. However, it must be emphasized that this does not exclude that other species can show a response to these substances.
RESUMEN
PURPOSE OF REVIEW: Type-1 and type-2 diabetes are diseases with an increasing number of patients and a complex, multifactorial pathogenesis. Apolipoprotein (apo) CIII is increased in both types of diabetes and interventions preventing the increase have effects on the development of diabetes. RECENT FINDINGS: ApoCIII affects intracellular Ca-handling by activating voltage-gated Ca-channels. ApoCIII is produced within the pancreatic islets and it increases in parallel with the development of insulin resistance and type-2 diabetes. Preventing the increase maintains a normal glucose tolerance as well as Ca-handling and no signs of inflammation can be seen in islets wherein the augmented local production of the apolipoprotein is absent. SUMMARY: ApoCIII has been found to interfere with both function and survival of the ß-cell and thereby promote the development of diabetes. Increased levels of this apolipoprotein affects intracellular Ca-handling and insulin sensitivity, which finally results in impaired glucose homeostasis and diabetes. Interestingly, in a type-1 diabetes rat model lowering of apoCIII delays onset of diabetes. In type-2 diabetes insulin resistance within the pancreatic islets leads to a local increase in apoCIII that promotes inflammation and ß-cell dysfunction. Hence, targeting apoCIII may constitute a novel pharmacological strategy to treat both type-1 and type-2 diabetes.
Asunto(s)
Apolipoproteína C-III/metabolismo , Diabetes Mellitus/metabolismo , Animales , HumanosRESUMEN
A picornavirus (Ljungan virus) has been associated with diabetes in its wild rodent reservoir and in diabetes-prone biobreeding (DP-BB) rats. We attempted to alter the development of diabetes in DP-BB rats using two anti-picornavirus compounds (pleconaril and APO-N039), singly or in combination. Antiviral therapy was initiated 2 weeks before expected onset of diabetes. Pleconaril or APO-N039 alone did not affect the debut of diabetes. However, animals receiving a combination of both compounds were protected for at least the entire period of treatment (4 weeks after expected time of diabetes onset). Immunohistochemistry demonstrated that the presence and distribution of virus antigen in the pancreatic islets coincided with the clinical status of the animal. Data indicate that a treatable picornavirus can be involved in the cellular assault resulting in diabetes and in these cases the disease mechanism appears to involve a virus present in the pancreatic beta cell mass itself.
RESUMEN
Cathelicidins are pleiotropic antimicrobial peptides largely described for innate antimicrobial defenses and, more recently, immunomodulation. They are shown to modulate a variety of immune or nonimmune host cell responses. However, how cathelicidins are expressed by ß cells and modulate ß-cell functions under steady-state or proinflammatory conditions are unknown. We find that cathelicidin-related antimicrobial peptide (CRAMP) is constitutively expressed by rat insulinoma ß-cell clone INS-1 832/13. CRAMP expression is inducible by butyrate or phenylbutyric acid and its secretion triggered upon inflammatory challenges by IL-1ß or LPS. CRAMP promotes ß-cell survival in vitro via the epidermal growth factor receptor (EGFR) and by modulating expression of antiapoptotic Bcl-2 family proteins: p-Bad, Bcl-2, and Bcl-xL. Also via EGFR, CRAMP stimulates glucose-stimulated insulin secretion ex vivo by rat islets. A similar effect is observed in diabetes-prone nonobese diabetic (NOD) mice. Additional investigation under inflammatory conditions reveals that CRAMP modulates inflammatory responses and ß-cell apoptosis, as measured by prostaglandin E2 production, cyclooxygenases (COXs), and caspase activation. Finally, CRAMP-deficient cnlp(-/-) mice exhibit defective insulin secretion, and administration of CRAMP to prediabetic NOD mice improves blood glucose clearance upon glucose challenge. Our finding suggests that cathelicidins positively regulate ß-cell functions and may be potentially used for intervening ß-cell dysfunction-associated diseases.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Células Secretoras de Insulina/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/genética , Apoptosis/genética , Línea Celular Tumoral , Dinoprostona/genética , Dinoprostona/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Wistar , Proteína Letal Asociada a bcl/genética , Proteína Letal Asociada a bcl/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismo , CatelicidinasRESUMEN
Insulin resistance and ß-cell failure are the major defects in type 2 diabetes mellitus. However, the molecular mechanisms linking these two defects remain unknown. Elevated levels of apolipoprotein CIII (apoCIII) are associated not only with insulin resistance but also with cardiovascular disorders and inflammation. We now demonstrate that local apoCIII production is connected to pancreatic islet insulin resistance and ß-cell failure. An increase in islet apoCIII causes promotion of a local inflammatory milieu, increased mitochondrial metabolism, deranged regulation of ß-cell cytoplasmic free Ca(2+) concentration ([Ca(2+)]i) and apoptosis. Decreasing apoCIII in vivo results in improved glucose tolerance, and pancreatic apoCIII knockout islets transplanted into diabetic mice, with high systemic levels of the apolipoprotein, demonstrate a normal [Ca(2+)]i response pattern and no hallmarks of inflammation. Hence, under conditions of islet insulin resistance, locally produced apoCIII is an important diabetogenic factor involved in impairment of ß-cell function and may thus constitute a novel target for the treatment of type 2 diabetes mellitus.
Asunto(s)
Apolipoproteína C-III/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Resistencia a la Insulina/fisiología , Células Secretoras de Insulina/patología , Análisis de Varianza , Animales , Apolipoproteína C-III/genética , Western Blotting , Calcio/metabolismo , Línea Celular Tumoral , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Confocal , Mitocondrias/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
The voltage-gated Ca(2+) (CaV) channel acts as a key player in ß cell physiology and pathophysiology. ß cell CaV channels undergo hyperactivation subsequent to exposure to type 1 diabetic (T1D) serum resulting in increased cytosolic free Ca(2+) concentration and thereby Ca(2+)-triggered ß cell apoptosis. The present study was aimed at revealing the subtypes of CaV1 channels hyperactivated by T1D serum as well as the biophysical mechanisms responsible for T1D serum-induced hyperactivation of ß cell CaV1 channels. Patch-clamp recordings and single-cell RT-PCR analysis were performed in pancreatic ß cells from CaV1 channel knockout and corresponding control mice. We now show that functional CaV1.3 channels are expressed in a subgroup of islet ß cells from CaV1.2 knockout mice (CaV1.2(-/-)). T1D serum enhanced whole-cell CaV currents in islet ß cells from CaV1.3 knockout mice (CaV1.3(-/-)). T1D serum increased the open probability and number of functional unitary CaV1 channels in CaV1.2(-/-) and CaV1.3(-/-) ß cells. These data demonstrate that T1D serum hyperactivates both CaV1.2 and CaV1.3 channels by increasing their conductivity and number. These findings suggest CaV1.2 and CaV1.3 channels as potential targets for anti-diabetes therapy.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Diabetes Mellitus Tipo 1/sangre , Células Secretoras de Insulina/metabolismo , Animales , Canales de Calcio Tipo L/genética , Células Cultivadas , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Masculino , Ratones , Ratones NoqueadosRESUMEN
Little is known about the molecular mechanisms underlying age-dependent deterioration in ß-cell function. We now demonstrate that age-dependent impairment in insulin release, and thereby glucose homeostasis, is associated with subtle changes in Ca(2+) dynamics in mouse ß-cells. We show that these changes are likely to be accounted for by impaired mitochondrial function and to involve phospholipase C/inositol 1,4,5-trisphosphate-mediated Ca(2+) mobilization from intracellular stores as well as decreased ß-cell Ca(2+) influx over the plasma membrane. We use three mouse models, namely, a premature aging phenotype, a mature aging phenotype, and an aging-resistant phenotype. Premature aging is studied in a genetically modified mouse model with an age-dependent accumulation of mitochondrial DNA mutations. Mature aging is studied in the C57BL/6 mouse, whereas the 129 mouse represents a model that is more resistant to age-induced deterioration. Our data suggest that aging is associated with a progressive decline in ß-cell mitochondrial function that negatively impacts on the fine tuning of Ca(2+) dynamics. This is conceptually important since it emphasizes that even relatively modest changes in ß-cell signal transduction over time lead to compromised insulin release and a diabetic phenotype.
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Envejecimiento/metabolismo , Glucemia/metabolismo , Calcio/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Transporte de Electrón/fisiología , Células Secretoras de Insulina/metabolismo , Mitocondrias/metabolismo , Animales , Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Ratones Endogámicos C57BL , Mitocondrias/genética , Fosfolipasas de Tipo C/metabolismoRESUMEN
Apolipoprotein CIII (ApoCIII) not only serves as an inhibitor of triglyceride hydrolysis but also participates in diabetes-related pathological events such as hyperactivation of voltage-gated Ca(2+) (CaV) channels in the pancreatic ß cell. However, nothing is known about the molecular mechanisms whereby ApoCIII hyperactivates ß cell CaV channels. We now demonstrate that ApoCIII increased CaV1 channel open probability and density. ApoCIII enhanced whole-cell Ca(2+) currents and the CaV1 channel blocker nimodipine completely abrogated this enhancement. The effect of ApoCIII was not influenced by individual inhibition of PKA, PKC, or Src. However, combined inhibition of PKA, PKC, and Src counteracted the effect of ApoCIII, similar results obtained by coinhibition of PKA and Src. Moreover, knockdown of ß1 integrin or scavenger receptor class B type I (SR-BI) prevented ApoCIII from hyperactivating ß cell CaV channels. These data reveal that ApoCIII hyperactivates ß cell CaV1 channels through SR-BI/ß1 integrin-dependent coactivation of PKA and Src.
Asunto(s)
Apolipoproteína C-III/farmacología , Antígenos CD36/metabolismo , Canales de Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Secretoras de Insulina/metabolismo , Integrina beta1/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Animales , Apolipoproteína C-III/metabolismo , Apolipoproteína C-III/fisiología , Antígenos CD36/genética , Calcio/metabolismo , Células Cultivadas , Electrofisiología , Femenino , Técnicas de Silenciamiento del Gen , Integrina beta1/genética , Integrina beta1/fisiología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Interferencia de ARN , Regulación hacia ArribaRESUMEN
BACKGROUND: Lipoprotein lipase (LPL) hydrolyzes triglycerides in plasma lipoproteins and enables uptake of lipolysis products for energy production or storage in tissues. Our aim was to study the localization of LPL and its endothelial anchoring protein glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) in mouse pancreas, and effects of diet and leptin deficiency on their expression patterns. For this, immunofluorescence microscopy was used on pancreatic tissue from C57BL/6 mouse embryos (E18), adult mice on normal or high-fat diet, and adult ob/ob-mice treated or not with leptin. The distribution of LPL and GPIHBP1 was compared to insulin, glucagon and CD31. Heparin injections were used to discriminate between intracellular and extracellular LPL. RESULTS: In the exocrine pancreas LPL was found in capillaries, and was mostly co-localized with GPIHBP1. LPL was releasable by heparin, indicating localization on cell surfaces. Within the islets, most of the LPL was associated with beta cells and could not be released by heparin, indicating that the enzyme remained mostly within cells. Staining for LPL was found also in the glucagon-producing alpha cells, both in embryos (E18) and in adult mice. Only small amounts of LPL were found together with GPIHBP1 within the capillaries of islets. Neither a high fat diet nor fasting/re-feeding markedly altered the distribution pattern of LPL or GPIHBP1 in mouse pancreas. Islets from ob/ob mice appeared completely deficient of LPL in the beta cells, while LPL-staining was normal in alpha cells and in the exocrine pancreas. Leptin treatment of ob/ob mice for 12 days reversed this pattern, so that most of the islets expressed LPL in beta cells. CONCLUSIONS: We conclude that both LPL and GPIHBP1 are present in mouse pancreas, and that LPL expression in beta cells is dependent on leptin.
Asunto(s)
Leptina/deficiencia , Leptina/metabolismo , Lipoproteína Lipasa/metabolismo , Páncreas/metabolismo , Receptores de Lipoproteína/metabolismo , Animales , Capilares/metabolismo , Dieta Alta en Grasa/métodos , Células Endoteliales/metabolismo , Glucagón/metabolismo , Células Secretoras de Glucagón/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Heparina/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismoRESUMEN
Pancreatic alpha cells contribute to glucose homeostasis by the regulated secretion of glucagon, which increases glycogenolysis and hepatic gluconeogenesis in response to hypoglycemia. Alterations of glucagon secretion are observed in diabetic patients and exacerbate the disease. The restricted availability of purified primary alpha cells has limited our understanding of their function in health and disease. This study was designed to establish convenient protocols for the purification of viable alpha cells from rat and human pancreatic islets by FACS, using intrinsic cellular properties. Islets were isolated from the pancreata of Wistar rats or deceased human organ donors. Dispersed islet cells were separated by FACS based on light scatter and autofluorescence. Purity of sorted cells was evaluated by immunocytochemistry using hormone specific antibodies. Relative hormone expression was further determined by quantitative RT-PCR. Viability was determined by Annexin V and propidium iodide staining and function was assessed by monitoring cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) using Fura-2/AM. We developed species-specific FACS gating strategies that resulted in populations consisting mainly of alpha cells (96.6 ± 1.4%, n = 3 for rat; 95.4 ± 1.7%, n = 4 for human, mean ± SEM). These cell fractions showed ~5-fold and ~4-fold enrichment (rat and human, respectively) of glucagon mRNA expression compared to total ungated islet cells. Most of the sorted cells were viable and functional, as they responded with an increase in [Ca(2+)](i) upon stimulation with L-arginine (10 mM). The majority of the sorted human alpha cells responded also to stimulation with kainate (100 µM), whereas this response was infrequent in rat alpha cells. Using the same sample preparation, but a different gating strategy, we were also able to sort rat and human populations enriched in beta cells. In conclusion, we have simplified and optimized a method for the purification of rat alpha cells, as well as established a novel approach to separate human alpha cells using neither antibodies nor dyes possibly interfering with cellular functions.