The role of glucagon : GCGR axis in pancreatic neuroendocrine

Abstract Cancer metabolism research has studied the relationship between cellular bioenergetics, biosynthesis, and tumour progression. However, the impact of systemic metabolism and diet on tumour evolution is less understood. This thesis delves into the role of glucagon, a key hormone in systemic metabolism, particularly its influence on pancreatic neuroendocrine tumours (pNETs). Glucagon ability to bind to its natural receptor, GCGR, and its regulator, GLP-1R, introduces intricate dynamics to cancer biology. Our research focused on the role of glucagon in the regulation of cancer cell features and the metabolic remodelling in the presence and absence of glucose. To fulfil the experiments pNETs cell lines (BON-1 and QGP-1) and non-malignant pancreatic α-TC1 cell line were used as models. Results showed pNETs cells responded differently to glucose deprivation than α-TC1 cells. Specifically, pNETs cells upregulated GCGR in the absence of glucose, while α-TC1 cells did so in high-glucose conditions. Glucagon activated the MAPK/ERK pathway, especially in pNETs cells without glucose and α-TC1 cells with high glucose. Tests revealed that glucagon enhanced metabolic viability, proliferation, and migration in pNETs cells under glucose deprived conditions and hyperglucagonemia. Meanwhile, in α-TC1 cell line, glucagon modulated these features under high-glucose conditions and physiological glucagon levels. The study also explored the effects of blocking GCGR using an inhibitor, LGD-6972. The results varied across cell lines and glucagon conditions. Metabolic analysis using NMR revealed differences in amino acid levels and metabolic processes based on glucose availability between cell lines. Interestingly, QGP-1 and α-TC1 produced glucose in no-glucose conditions, and glucagon upregulated glucose production in α-TC1. Furthermore, gene expression analysis showed that glucose conditions largely influenced gene expression patterns in both pNETs and non-malignant α-cells. The increased levels of alanine, and the ability of QGP-1 and α-TC1 cells to produce glucose together with the upregulation of gluconeogenesis related genes it was clear that the synthesis of glucose is a core pathway in α-TC1 cell biology and QGP-1 cells. Hence gluconeogenesis may be beneficial for some pNETs subsets and its open new perspectives to novel metabolism-based strategies to clinically manage pNETs. Furthermore, the production of glucose by α-cells was not described so far and is a step forward in endocrinology and systemic metabolism. Lastly, the study examined the association between GCGR and disease parameters in pNETs cases. Findings revealed a negative correlation between GCGR expression and tumour grading. Additionally, GLP-1R expression was lower in pNETs than in healthy tissue. This underscores the significant, yet not fully understood, role of glucagon signalling in pNETs progression. The complex relationship between glucose conditions, glucagon signalling, and cellular characteristics warrants further exploration for future therapeutic strategies for pNETs and related diseases.Resumo À medida que a investigação sobre o metabolismo em cancro aprofunda a sua compreensão da relação entre bioenergética e biossíntese celular e progressão tumoral, as influências do metabolismo sistémico e da dieta na evolução tumoral permanecem amplamente inexploradas. Esta tese pretende explorar a relevância do glucagon, uma hormona vital no metabolismo sistémico, investigando o seu papel na biologia do cancro e progressão da doença, especificamente em tumores neuroendócrinos pancreáticos (pNETs). A capacidade do glucagon ligar-se tanto ao seu recetor natural, GCGR, como ao recetor do seu controlador GLP-1 (GLP-1R) introduz dinâmicas complexas no contexto da biologia do cancro. O nosso estudo centrou-se no papel do glucagon na regulação das características das células malignas e na remodelação metabólica na presença e ausência de glucose. Para tal, foram usadas como modelo as linhas celulares de pNETs (BON-1 e QGP-1) e a linha celular pancreática não maligna α-TC1. Identificámos uma resposta adaptativa nas linhas celulares de pNETs sob privação de glucose com o aumento da expressão do GCGR. Em contraste, células α-TC1mostraram um aumento da expressão do GCGR em condições de elevados níveis de glucose. O glucagon ativou a via MAPK/ERK, observado pelo aumento dos níveis de pERK, especialmente nas células celulares de pNETs sob privação de glucose e em células α-TC1 em condições ricas em glucose. Foi realizada uma análise intensiva para compreender os efeitos do glucagon nas características celulares como a viabilidade metabólica, a proliferação e a migração celular. Observou-se que, em condições de privação de glucose e hiperglucagonemia, o glucagon aumentou estas atividades nas linhas BON-1 e QGP-1. Entretanto, na linha celular αTC1, o glucagon teve o mesmo efeito, mas sob condições ricas em glucose e níveis fisiológicos de glucagon. O estudo também explorou os efeitos do bloqueio do GCGR usando um inibidor competitivo, LGD-6972. Os resultados variaram entre linhas celulares e condições de glucagon. Ao aplicar a técnica de espetroscopia de ressonância magnética nuclear (RMN), observamos diferenças nos níveis de aminoácidos e remodelação metabólica com base na disponibilidade de glucose entre as linhas celulares. Curiosamente, as linhas celulares QGP-1 e α-TC1 produziram glucose em condições sem glucose, e o glucagon aumentou a produção de glucose nas α-TC1. Além disso, a análise da expressão genética mostrou que as condições de glucose influenciaram amplamente os padrões de expressão genética em ambas as linhas celulares de pNETs e células α não malignas. A capacidade das células QGP-1 e α-TC1 de produzir glucose, juntamente com regulação positiva de genes relacionados com a gluconeogénese, tornou claro que a síntese de glucose é uma via central na biologia celular α-TC1 e nas células QGP-1. Além disso, a produção de glucose por células α não foi descrita até agora e representa um avanço na endocrinologia e no metabolismo sistémico. Por último, o estudo avaliou a associação entre a expressão de GCGR e parâmetros da doença em casos de pNETs. Os resultados revelaram uma correlação negativa entre a expressão de GCGR o grau da doença. Além disso, a expressão de GLP-1R foi menor nos pNETs do que em tecido saudável. Isto sublinha o papel significativo, mas ainda não totalmente compreendido, da sinalização do glucagon na progressão dos pNETs. A relação complexa entre condições de glucose, sinalização de glucagon e características celulares exige mais investigação para futuras estratégias terapêuticas para pNETs e outras patologias relacionadas

Molecular regulation of hormone secretion, growth and apoptosis of GLP-1-producing cells

Type 2 diabetes (T2D) spreads like an epidemic in today’s society, and there is a great need for new and improved treatments. T2D is characterized by hyperglycemia, resulting from impaired insulin production and insulin resistance in peripheral tissues. Incretin hormones, such as glucagon-like peptide-1 (GLP-1), secreted from L-cells dispersed along the intestinal tract, potentiate meal-stimulated insulin secretion in a glucose-dependent manner. Defective GLP-1 secretion has been indicated in T2D and administration of GLP-1 to T2D patients restores glucose-induced insulin secretion and normalizes glycemia, making stable analogs of GLP-1 among the best available treatments for T2D today. However, enhancing endogenous GLP-1 production/secretion by direct stimulation of GLP-1 secretion/promotion of growth and viability of L-cells may be a novel and more physiological option in incretin-based diabetes therapy. The aim of this work was to determine the effect of diabetic conditions and anti-diabetic agents on GLP-1-producing cells, in order to unravel some of the mechanisms regulating growth, survival and function of this cell type. Studies I-III were performed in vitro using the murine GLUTag cell line as a model. In study I, direct effects of metformin on apoptosis, and function of GLP-1-secreting cells were determined. Simulated diabetic hyperlipidemia resulted in increased caspase-3 activity and DNA fragmentation, indicating lipoapoptosis. Metformin treatment significantly decreased this lipoapoptosis in conjunction with increased phosphorylation of AMPK. In addition, metformin treatment stimulated GLP-1 secretion. In study II, we determined molecular mechanisms mediating lipotoxicity and metformininduced lipoprotection in GLP-1-secreting cells. Diabetic hyperlipidemia was simulated in this cell system by addition of the fatty acid palmitate. Palmitate increased ROS production in GLP- 1-secreting cells, and the lipotoxic effects of palmitate were abolished in the presence of the antioxidant Trolox. Further, palmitate phosphorylated p38 MAPK and inhibition of this enzyme significantly reduced lipoapoptosis. Pre-incubation with metformin further increased palmitate- induced ROS production, while significantly reducing the expression of p38 MAPK. Study III focused on direct effects of insulin and exendin-4/GLP-1 on lipoapoptosis and function of GLP-1-secreting cells. The GLP-1R was found to be expressed in the GLUTag cells, and diabetic lipotoxicity was partially inhibited by pre-incubation with insulin or the stable GLP-1 analog exendin-4. The lipoprotective effect of exendin-4 was GLP-1R-dependent, while independent of PKA activity. In addition, both insulin and exendin-4 significantly stimulated acute and long term GLP-1 secretion in the presence of glucose. In study IV, we investigated if a high fat diet (HFD) reduces the number of enteroendocrine GLP-1-secreting Lcells in C57/Bl6 mice. We also determined the effects of a HFD on GLP-1 plasma levels and possible effects on these parameters by metformin treatment. A HFD rapidly induced a diabetic phenotype with increased HbA1c levels, as well as fasting plasma insulin levels in conjunction with reduced oral glucose tolerance – indicating the manifestation of insulin resistance. A 14 day oral administration of metformin reduced HbA1c, fasting insulin and prandial FFA levels. The number of L-cells was significantly reduced after 12 weeks on a HFD, while -- in contrast - - there was a clear trend toward increased prandial plasma GLP-1 levels despite reduced food intake in HFD-fed mice. These findings may be of pathogenic significance not only in understanding mechanisms of the impaired incretin response characterizing T2D patients, but may also be harnessed to therapeutic advantage in efforts to enhance endogenous GLP-1 production. Such an approach has hitherto received little attention but may be superior to contemporary incretin-based antidiabetic therapy, which does not faithfully mimick physiologic GLP-1 release in for instance terms of secretory pattern (e.g. pulsatility) and actions on topographically adjacent hormone receptors (e.g. in the portal vein)

Investigation of molecular and cellular events associated with beta cell function and elucidation of extracellular RNAs as potential biomarker for diabetes

Diabetes is a chronic disorder of glucose metabolism and a major cause of premature mortality. The potential use of replacement beta cells as therapy for diabetes requires an ability to culture such cells while maintaining their functional status. Glucose stimulated insulin secretion (GSIS) is lost in long-term cultured MIN6 heterogeneous cells. MIN6 B1, a clonal sub-line derived from MIN6, has been described as highly glucose-responsive. This study aimed to investigate the GSIS function, changes in gene expression and, subsequently, to develop possible experimental approaches to overcome this loss. Understanding the molecular basis for loss of GSIS may contribute to better culture conditions for islets in transplantation programmes; it may also add to our understanding of beta cell insensitivity to high blood glucose in Type 2 diabetes. Whole genome microarray analysis on biological triplicate samples of glucoseresponsive low passage (p19) (MIN6 B1(GSIS)) and non glucose-responsive high passage (p23) (MIN6 B1(Non-GSIS)) was performed. 111 differentially-regulated genes were identified and 16 gene transcripts were selected based on p-value <0.05; fold change 1.2; difference 100 and validated using qRT-PCR including Txnip, Gcg and Pcsk9. Txnip was up-regulated whereas Gcg and Pcsk9 were down-regulated in MIN6 B1(Non-GSIS) compared to MIN6 B1(GSIS). siRNA and shRNA silencing of Txnip in MIN6(H) (non-responsive) significantly increased the GSIS. Over-expression of Txnip cDNA in MIN6(L) (glucose-responsive) caused significant loss of GSIS. siRNA silencing of Gcg and Pcsk9 caused a significant loss of GSIS in MIN6(L) compared to scrambled-transfected cells. Over-expression of Gcg cDNA in MIN6(H) increased GSIS. In parallel, in an attempt to identify more reliable biomarkers for diabetes, we also investigated if extracellular mRNAs are reproducibly detectable in conditioned medium (CM) from a range of insulin-producing cell types and in serum specimens from Type 2 diabetes and controls. Pdx1, Npy, Egr1, Pld1, Chgb, Ins1, Ins2, and betaactin from MIN6(L), MIN6(H), and MIN6 B1 cells and their CM suggests that beta cells transcribe and release these mRNAs into their culture environment.This study was subsequently translated to analysis of serum from people with Type 2 diabetes and controls to help establish the clinical relevance of these findings. The result from this clinical phase of the study indicated Txnip to be up-regulated and Egr1 to be down-regulated in Type 2 diabetes compared to controls

Hyperlipidemia-induced MicroRNA155-5p improves β-cell Function by targeting Mafb

A high-fat diet increases intestinal permeability and promotes leakage of LPS into the circulation, where it primarily binds to lipoproteins and may promote GLP-1-mediated insulin secretion. Notably, patients with familial hypercholesterolemia have a reduced risk for T2DM. However, chronically elevated circulating LPS levels during HFD feeding also induce adipose tissue inflammation and obesity-induced insulin resistance. In macrophages, LPS and hyperlipidemia selectively induce miR-155-5p expression and trigger inflammatory activation. However, the role of miR-155-5p in obesity-related metabolic and cardiovascular diseases is poorly understood. Therefore, the aim of the current study was to determine whether miR-155-5p mediates the effects of hyperlipidemia on obesity and glucose homeostasis. Hyperlipidemia-associated endotoxemia increased deposition of oxLDL and induced miR-155-5p expression in pancreatic islets of Ldlr–/– mice. Mild oxidative modification of LDL led to increased endotoxin activity and increased miR-155-5p expression in pancreatic β-cells. In Mir155–/–Ldlr–/–mice, glucose and plasma glucagon levels were increased, whereas plasma insulin and GLP-1 levels were reduced compared with Mir155+/+Ldlr–/– mice. The α-to-β-cell ratio and the glucagon protein level were higher, whereas the insulin and GLP-1 protein content was reduced in islets from Mir155–/–Ldlr–/–mice. Treatment with low-dose LPS up-regulated islet miR-155-5p expression, increased insulin and GLP-1 plasma levels and lowered the glucose levels following intraperitoneal glucose injection in Ldlr–/– mice, and the effects of LPS on glucose metabolism were partially abolished by Mir155 knockout. Microarray analysis revealed inhibition of insulin, GLP-1, and IL-6 signaling pathways and upregulation of putative miR-155 targets, including Mafb, Sema5a, Med12l, Auh, and Stmn2 in islets from Mir155–/–Ldlr–/–mice. In murine β-cells, overexpression of miR-155-5p enriched Mafb mRNA in the RISC. Moreover, luciferase reporter assays showed that MafB suppressed Il6 expression by binding to the Il6 promoter region. In Ldlr–/–mice, blocking the interaction between miR-155-5p and MafB increased FBG levels and decreased the expression of Il6, the GLP-1 producing enzyme Pcsk1, and insulin production in islets. Probably due to elevated plasma GLP-1 levels, miR-155-5p limited the progression of obesity and adipose tissue inflammation, and reduced hyperlipidemia and atherosclerosis in HFD-fed Ldlr–/– mice. Taken together, hyperlipidemia-associated endotoxemia can improve glucose homeostasis by upregulating miR-155-5p expression in pancreatic β-cells. Targeting of MafB by miR-155-5p may limit β-to-α-cell reprogramming and promotes β-cell function likely through IL-6-induced GLP-1 production in α-cells. Hence, hyperlipidemia-induced miR-155-5p improves the adaptation of β-cells to insulin resistance. Increasing miR-155-5p levels in β-cell may be a valuable therapeutic strategy against diabetes and the MetS.Die Verfütterung eines „high fat“ Futters führt zu einer erhöhten Darmpermeabilität, die zu einer Steigerung der LPS Konzentration in der Zirkulation führt. Dort bindet LPS an Lipoproteine und stimuliert dadurch die GLP-1-vermittelte Sekretion von Insulin. Patienten mit familiären Hypercholesterinämie leiden seltener an T2DM. Die durch das „high fat“ Futter chronisch erhöhte LPS Konzentration in der Zirkulation induziert die Entzündung im adipösen Gewebe und die Adipositas-bedingte Insulinresistenz. LPS und Hyperlipidämie induzieren die Expression von miR-155-5p und rufen eine inflammatorische Aktivierung in Makrophagen hervor. Allerdings ist über die Rolle von miR-155-5p in mit Adipositas verbundenen metabolischen und kardiovaskulären Erkrankungen wenig bekannt. Das Ziel dieses Vorhabens ist die Effekte von miR-155-5p in Hyperlipidämie-vermittelter Adipositas und Glukose-Homöostase zu untersuchen. Die Hyperlipidämie-assoziierte Endotoxämie erhöhte die Akkumulation von oxLDL und die Expression von miR-155-5p in den Langerhans-Inseln von Ldlr–/– Mäusen. Während die Glukose- und Glukagon Plasmawerte verglichen mit den Werten in Mir155+/+Ldlr–/– Mäusen erhöht waren, wurde eine Reduktion der Insulin- und GLP-1 Plasmawerte in Mir155–/–Ldlr–/– Mäusen detektiert. Das Verhältnis von α-zu-β Zellen und die Glukagon-Proteinwerte waren erhöht, wohingegen die Insulin- und GLP-1 Proteinwerte in den Langerhans-Inseln der Mir155–/–Ldlr–/– Mäusen reduziert waren. Eine Behandlung mit niedrig dosiertem LPS erhöhte die Expression von miR-155-5p, steigerte die Insulin- und die GLP-1 Plasmawerte, reduzierte jedoch die Glukosewerte nach einer intraperitonealen Glukose-Injektion in Ldlr–/– Mäusen. Ferner ist die LPS-assoziierte Wirkung teilweise durch den Mir155 Knockout aufgehoben. In den Langerhans-Inseln derMir155–/–Ldlr–/– Mäusen zeigte die Microarray-Analyse eine Inhibierung von Insulin, GLP-1 und dem IL-6 Signalweg sowie eine Erhöhung vermeintlicher miR-155 Targets, wie z.B. Mafb, Sema5a, Med12l, Auh und Stmn2. Die Überexpression von miR-155-5p führte zur Anreicherung der Mafb mRNA im RISC. Die Luciferase Reporter-Assay zeigte, dass MafB die Expression von Il6 durch die Bindung an den Il6 Promoter supprimiert. In Ldlr–/– Mäusen führte die Hemmung der Interaktion zwischen miR-155-5p und MafB zu erhöhten FBG Werten und zu reduzierten Expressionen von Il6, GLP-1 produziertem Enzym Pcsk1 und zur verringerten Insulin Produktion in den Langerhans-Inseln. MiR-155-5p reduzierte die Adipositas und die Inflammation im adipösen Gewebe wahrscheinlich aufgrund der erhöhten GLP-1 Konzentration im Plasma. Nach Verfütterung des „high fat“ Futters inhibierte miR-155-5p die Hyperlipidämie und die Entwicklung der Atherosklerose in Ldlr–/– Mäusen. Zusammenfassend verbessert die Hyperlipidämie-assoziierte Endotoxämie die Glukose-Homöostase durch die gesteigerte Expression von miR-155-5p in den β-Zellen. Die Hemmung von MafB durch miR-155-5p reduzierte die β-α Zell Umprogrammierung und verbesserte die β-Zell Funktion durch eine IL-6-induzierte GLP-1 Produktion in den α-Zellen. Die Hyperlipidämie-vermittelte Induktion von miR-155-5p trägt zur Anpassung von β-Zellen an die Insulinresistenz bei. Die erhöhte Expression von miR-155-5p in den β-Zellen stellt einen vielversprechenden Ansatz in der Therapie des Diabetes und des MetS dar

Hyperlipidemia-induced MicroRNA155-5p improves β-cell Function by targeting Mafb

A high-fat diet increases intestinal permeability and promotes leakage of LPS into the circulation, where it primarily binds to lipoproteins and may promote GLP-1-mediated insulin secretion. Notably, patients with familial hypercholesterolemia have a reduced risk for T2DM. However, chronically elevated circulating LPS levels during HFD feeding also induce adipose tissue inflammation and obesity-induced insulin resistance. In macrophages, LPS and hyperlipidemia selectively induce miR-155-5p expression and trigger inflammatory activation. However, the role of miR-155-5p in obesity-related metabolic and cardiovascular diseases is poorly understood. Therefore, the aim of the current study was to determine whether miR-155-5p mediates the effects of hyperlipidemia on obesity and glucose homeostasis. Hyperlipidemia-associated endotoxemia increased deposition of oxLDL and induced miR-155-5p expression in pancreatic islets of Ldlr–/– mice. Mild oxidative modification of LDL led to increased endotoxin activity and increased miR-155-5p expression in pancreatic β-cells. In Mir155–/–Ldlr–/–mice, glucose and plasma glucagon levels were increased, whereas plasma insulin and GLP-1 levels were reduced compared with Mir155+/+Ldlr–/– mice. The α-to-β-cell ratio and the glucagon protein level were higher, whereas the insulin and GLP-1 protein content was reduced in islets from Mir155–/–Ldlr–/–mice. Treatment with low-dose LPS up-regulated islet miR-155-5p expression, increased insulin and GLP-1 plasma levels and lowered the glucose levels following intraperitoneal glucose injection in Ldlr–/– mice, and the effects of LPS on glucose metabolism were partially abolished by Mir155 knockout. Microarray analysis revealed inhibition of insulin, GLP-1, and IL-6 signaling pathways and upregulation of putative miR-155 targets, including Mafb, Sema5a, Med12l, Auh, and Stmn2 in islets from Mir155–/–Ldlr–/–mice. In murine β-cells, overexpression of miR-155-5p enriched Mafb mRNA in the RISC. Moreover, luciferase reporter assays showed that MafB suppressed Il6 expression by binding to the Il6 promoter region. In Ldlr–/–mice, blocking the interaction between miR-155-5p and MafB increased FBG levels and decreased the expression of Il6, the GLP-1 producing enzyme Pcsk1, and insulin production in islets. Probably due to elevated plasma GLP-1 levels, miR-155-5p limited the progression of obesity and adipose tissue inflammation, and reduced hyperlipidemia and atherosclerosis in HFD-fed Ldlr–/– mice. Taken together, hyperlipidemia-associated endotoxemia can improve glucose homeostasis by upregulating miR-155-5p expression in pancreatic β-cells. Targeting of MafB by miR-155-5p may limit β-to-α-cell reprogramming and promotes β-cell function likely through IL-6-induced GLP-1 production in α-cells. Hence, hyperlipidemia-induced miR-155-5p improves the adaptation of β-cells to insulin resistance. Increasing miR-155-5p levels in β-cell may be a valuable therapeutic strategy against diabetes and the MetS.Die Verfütterung eines „high fat“ Futters führt zu einer erhöhten Darmpermeabilität, die zu einer Steigerung der LPS Konzentration in der Zirkulation führt. Dort bindet LPS an Lipoproteine und stimuliert dadurch die GLP-1-vermittelte Sekretion von Insulin. Patienten mit familiären Hypercholesterinämie leiden seltener an T2DM. Die durch das „high fat“ Futter chronisch erhöhte LPS Konzentration in der Zirkulation induziert die Entzündung im adipösen Gewebe und die Adipositas-bedingte Insulinresistenz. LPS und Hyperlipidämie induzieren die Expression von miR-155-5p und rufen eine inflammatorische Aktivierung in Makrophagen hervor. Allerdings ist über die Rolle von miR-155-5p in mit Adipositas verbundenen metabolischen und kardiovaskulären Erkrankungen wenig bekannt. Das Ziel dieses Vorhabens ist die Effekte von miR-155-5p in Hyperlipidämie-vermittelter Adipositas und Glukose-Homöostase zu untersuchen. Die Hyperlipidämie-assoziierte Endotoxämie erhöhte die Akkumulation von oxLDL und die Expression von miR-155-5p in den Langerhans-Inseln von Ldlr–/– Mäusen. Während die Glukose- und Glukagon Plasmawerte verglichen mit den Werten in Mir155+/+Ldlr–/– Mäusen erhöht waren, wurde eine Reduktion der Insulin- und GLP-1 Plasmawerte in Mir155–/–Ldlr–/– Mäusen detektiert. Das Verhältnis von α-zu-β Zellen und die Glukagon-Proteinwerte waren erhöht, wohingegen die Insulin- und GLP-1 Proteinwerte in den Langerhans-Inseln der Mir155–/–Ldlr–/– Mäusen reduziert waren. Eine Behandlung mit niedrig dosiertem LPS erhöhte die Expression von miR-155-5p, steigerte die Insulin- und die GLP-1 Plasmawerte, reduzierte jedoch die Glukosewerte nach einer intraperitonealen Glukose-Injektion in Ldlr–/– Mäusen. Ferner ist die LPS-assoziierte Wirkung teilweise durch den Mir155 Knockout aufgehoben. In den Langerhans-Inseln derMir155–/–Ldlr–/– Mäusen zeigte die Microarray-Analyse eine Inhibierung von Insulin, GLP-1 und dem IL-6 Signalweg sowie eine Erhöhung vermeintlicher miR-155 Targets, wie z.B. Mafb, Sema5a, Med12l, Auh und Stmn2. Die Überexpression von miR-155-5p führte zur Anreicherung der Mafb mRNA im RISC. Die Luciferase Reporter-Assay zeigte, dass MafB die Expression von Il6 durch die Bindung an den Il6 Promoter supprimiert. In Ldlr–/– Mäusen führte die Hemmung der Interaktion zwischen miR-155-5p und MafB zu erhöhten FBG Werten und zu reduzierten Expressionen von Il6, GLP-1 produziertem Enzym Pcsk1 und zur verringerten Insulin Produktion in den Langerhans-Inseln. MiR-155-5p reduzierte die Adipositas und die Inflammation im adipösen Gewebe wahrscheinlich aufgrund der erhöhten GLP-1 Konzentration im Plasma. Nach Verfütterung des „high fat“ Futters inhibierte miR-155-5p die Hyperlipidämie und die Entwicklung der Atherosklerose in Ldlr–/– Mäusen. Zusammenfassend verbessert die Hyperlipidämie-assoziierte Endotoxämie die Glukose-Homöostase durch die gesteigerte Expression von miR-155-5p in den β-Zellen. Die Hemmung von MafB durch miR-155-5p reduzierte die β-α Zell Umprogrammierung und verbesserte die β-Zell Funktion durch eine IL-6-induzierte GLP-1 Produktion in den α-Zellen. Die Hyperlipidämie-vermittelte Induktion von miR-155-5p trägt zur Anpassung von β-Zellen an die Insulinresistenz bei. Die erhöhte Expression von miR-155-5p in den β-Zellen stellt einen vielversprechenden Ansatz in der Therapie des Diabetes und des MetS dar

Studies on the effects of short-chain fatty acids on beta cell differentiation and maturation in neonatal porcine islet-like cell clusters

Cell replacement therapy is a realistic option for the treatment and cure of type 1 diabetes. Neonatal porcine islets cell clusters (NPICCs) are considered to represent an unlimited cell source but face some challenges including the strong immunogenicity and the need for the generation of high numbers of excellent quality NPICCs. Since NPICCs are composed of progenitor cells and immature insulin-secreting beta cells, novel strategies to improve in vitro generation of matured beta cells and/or to increase islet numbers is a prerequisite for the near future transfer of xenotransplantation to the clinic. In the present study, NPICCs were isolated from 2-3 days-old piglets to evaluate the effects of three main short-chain fatty acids (SCFA), acetate, propionate and butyrate, on in vitro islet differentiation and beta cell function assessed by real-time quantitative PCR, FACS analysis, immunohistology and glucose-stimulated insulin secretion (GSIS). Butyrate promoted a significant time- and dose-dependent up-regulation in insulin gene expression and an increased beta cell number, whereas acetate or propionate only marginally influenced the beta cell phenotype. The treatment with specific inhibitors of SCFA receptors such as G-protein-coupled receptor GPR41 (β-hydroxybutyrate) and GPR43 (GPLG0974) did not suppress the butyrate-mediated increase of insulin expression. However, the application of specific class I histone deacetylase (HDAC) inhibitors mocetinostat, MS275, FK228 and RGFP996 mimicked butyrate on beta cell differentiation, whereas selective class II HDAC inhibitors (TMP269, MC1568) displayed no effects. These data suggest that the pro-endocrine impact of butyrate is mainly mediated through its HDAC inhibitory activity. The data of the present study suggest that butyrate and class I HDAC inhibitors are important agents to study the mechanisms of beta cell differentiation in porcine islets and produce optimized NPICC cell products for novel cell replacement therapies