RESUMEN
The Wnt signalling pathway in beta-cells has been linked to the development of type 2 diabetes. Investigating the impact of a non-canonical Wnt ligand, Wnt4, on beta-cell function we found that in INS-1 cells, Wnt4 was able to completely block Wnt3a stimulated cell growth and insulin secretion. However, despite high levels of Wnt4 protein being detected in INS-1 cells, reducing the expression of Wnt4 had no impact on cell growth or Wnt3a signalling. As such, the role of the endogenously expressed Wnt4 in beta-cells is unclear, but the data showing that Wnt4 can act as a negative regulator of canonical Wnt signalling in beta-cells suggests that this pathway could be a potential target for modulating beta-cell function.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Proteína Wnt3A/metabolismo , Proteína Wnt4/metabolismo , Animales , Línea Celular , Proliferación Celular , Glucosa/metabolismo , Glucosa/farmacología , Humanos , Inmunohistoquímica , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Ratas , Vía de Señalización Wnt , Proteína Wnt3A/antagonistas & inhibidores , Proteína Wnt3A/farmacología , Proteína Wnt4/genética , Proteína Wnt4/farmacología , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Exposure of pancreatic beta-cells to long-chain free fatty acids leads to differential responses according to the chain length and degree of unsaturation. In particular, long-chain saturated molecules such as palmitate (C16:0) cause apoptosis, whereas equivalent mono-unsaturated species (for example, palmitoleate (C16:1)) are not overtly toxic. Moreover, mono-unsaturates exert a powerful cytoprotective response against a range of proapoptotic stimuli. However, the structural requirements that determine cytoprotection have not been determined and form the basis of the present study. EXPERIMENTAL APPROACH: BRIN-BD11 and INS-1 beta-cells were exposed either to the saturated fatty acid palmitate, or to serum withdrawal, to mediate cytotoxicity. The protective effects of a wide range of mono-unsaturated fatty acid derivatives were tested in cytotoxicity assays. Effector caspase activity was also measured and correlated with viability. KEY RESULTS: The cytotoxic actions of palmitate were inhibited dose-dependently by long-chain mono-unsaturated fatty acids with a defined potency order C18:1>C16:1>>C14:1. The configuration of the double bond was also important with cis forms being more potent than trans forms. Alkylated mono-unsaturated fatty-acid derivates were also cytoprotective, although their efficacy declined as the alkyl chain length increased. Cytoprotection was achieved rapidly on addition of mono-unsaturates and correlated with a rapid and dramatic inhibition of caspase-3/7 activity in palmitate-treated cells. CONCLUSIONS AND IMPLICATIONS: The data reveal the structural requirements that dictate the cytoprotective actions of mono-unsaturated fatty acids in pancreatic beta-cells. Metabolic activation is not required and the data point at the potential involvement of a fatty acid receptor in mediating cytoprotection.
Asunto(s)
Ácidos Grasos Monoinsaturados/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Animales , Inhibidores de Caspasas , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ácidos Grasos Monoinsaturados/administración & dosificación , Células Secretoras de Insulina/metabolismo , Ratas , Relación Estructura-ActividadRESUMEN
Chronic exposure of pancreatic beta-cells to saturated fatty acids leads to loss of viability, an effect that has been implicated in the process of beta-cell 'lipotoxicity' associated with the progression of type 2 diabetes. The mechanisms involved are unknown but recent evidence has implicated the delta isoform of protein kinase C (PKCdelta) in mediating fatty acid toxicity. We have investigated this proposition in the clonal insulin-secreting cell line, BRIN-BD11. BRIN-BD11 cells were found to undergo apoptosis when exposed to palmitate and this response was attenuated by the purportedly selective inhibitor of PKCdelta, rottlerin. However, activation of PKCdelta with the phorbol ester, phorbol-12-myristate-13-acetate (PMA), failed to promote cell death and down-regulation of PKCdelta did not prevent the cytotoxic effects of palmitate. Moreover, rottlerin remained effective as a blocker of the palmitate response in cells depleted of PKCdelta. Since rottlerin can inhibit various other kinases in addition to PKCdelta, a range of additional kinase inhibitors was also tested. Of these, only the putative Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) inhibitor, KN-62, was found to inhibit palmitate-induced cell death. However, this effect was not reproduced by a more selective pseudo-substrate inhibitor of CaM kinase II. Therefore, the present results reveal that palmitate induces cell death in BRIN-BD11 cells and suggest that this may involve the activation of a rottlerin (and KN-62)-sensitive kinase. However, it is clear that PKCdelta is not required for this response.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Palmitatos/toxicidad , Proteína Quinasa C/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Acetofenonas/farmacología , Animales , Benzopiranos/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Ésteres del Forbol/farmacología , Fosforilación/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C-delta , Transducción de Señal/efectos de los fármacosRESUMEN
Melanin-concentrating hormone (MCH) is a hypothalamic orexigenic peptide. Recently, an orphan G-protein-coupled receptor (SLC-1) was identified that binds MCH with high affinity. Here, we demonstrate the mRNA expression of this receptor in insulin-producing cells including CRI-G1 and RINm5F cells, and in rat islets of Langerhans. Immunofluorescence studies in CRI-G1 and RINm5F cell-lines demonstrated cell-surface expression of the receptor. Rat MCH significantly stimulated insulin secretion in both cell-lines. The potency and the efficacy of MCH were significantly increased in the simultaneous presence of forskolin, suggesting that MCH may amplify the insulinotropic effect of cyclic AMP elevating stimuli. Salmon MCH, which differs from rat/human MCH by six amino acids, was less efficacious than rat/human MCH in stimulating insulin release. The data provide evidence for the expression of MCH receptors in insulin producing cells. The insulinotropic effect of MCH may contribute to the regulation of metabolism and energy balance by this peptide.