The Mechanism Of Mof Regulating Pancreatic ? Cell Mass And Function

BackgroundType 2 diabetes mellitus(T2DM)has become the main public health crisis in China.Characterized by its high incidence,difficulty to be cured and severe complications,T2DM is a great threat to patients' quality of life and a heavy economic burden to the patients and the nation.According to current understanding about the etiology,not only pancreatic ? cells play a role in pathogenesis of T2DM,but the dysfunction of other tissues,including pancreatic ? cells,liver,adipose tissue,skeletal muscles and intestine,play nonnegligible roles.Pancreatic ? cells secret glucagon,elevating blood glucose levels via promoting hepatic glucose output when hypoglycemic occurs to maintain the glucose homeostasis.In T2DM patients,due to the hyperfunction of pancreatic ? cells,their fasting and postprandial glucagon levels are higher,but the ratios of insulin/glucagon are lower than normal control.Inhibiting glucagon secretion or glucagon signal pathway effectively reduces glycemia.As indicated by recent researches,there exist a small number of pancreatic ? cells converting preproglucagon into glucagon-like peptide-1(GLP-1)instead of glucagon.The a cells-derived GLP-1 exerts intra-islet paracrine effect,which promotes ? cell proliferation and insulin secretion but inhibits glucagon secretion.The ratio of pancreatic ? cells producing GLP-1 can achieve 90%in developing stage,but with the process of developing and ? cell maturation,these cells gradually disappear.In pregnant individuals and T2DM patients,the percentage of a cells producing GLP-1 becomes higher,and in T2DM patients,this ratio will increase in accordance with the progression of disease.These phenomena suggest that in adult,the increase of ? cells producing GLP-1 aims to meet the individuals' demand for more insulin.Transplanting ? cells producing GLP-1 to T2DM mice effectively reduces their blood sugar.Therefore,reducing ? cells producing glucagon,increasing ? cells producing GLP-1 and inhibiting glucagon secretion are potent strategies to achieve glucose homeostasis in T2DM.Histone acetylation is an evolutionally conversed histone modification,which can loosen the regional chromatin structure via adding acetyl group to certain lysine and provide space for RNA polymerase to initiate transcription.Histone acetyltransferase Mof is one member of MYST family,which substrate-specifically acetylate histone 4 lysine 16(H4K16).In mammal cells,Mof has profound influence on cell proliferation,apoptosis,the self-renewal of stem cells,autophagy,DNA damage repair and the initiation and development of tumors.Latest evidence showed that Mof was closely related with metabolic process and metabolic disorders.In human population,Mof variation is related with waists of North Han males.In animals,Mof knockdown in macrophage accelerates the healing of diabetic wound.In cells,Mof and H4K16ac directly regulate the hepatic accumulation of reactive oxygen species under fatty acid stimulation and the expressions of genes involved in fatty acid ? oxidation,tricarboxylic acid cycle and glycolysis.However,it is unknown whether Mof is involved in glucose homeostasis.In this paper,we used tamoxifen-induced global Mof deficiency mice to investigate its role in glucose regulation and in which tissue it exerted its effect.Then we utilized tissue-specific Mof deficiency mice and cell line to study the mechanism.Ultimately,we investigated whether Mof acetyltransferase activity inhibition could ameliorate blood glucose levels in T2DM mice.Part ? Mof deficiency mice exhibited different glucose tolerance and altered a cell mass and functionObjectiveTo investigate whether Mof is involved in glucose homeostasis regulation and the physiological mechanism.Methods1.We established global Mof deficient Mof flox/flox;ERT Cre mice and Mof flox/+;ERT Cre mice via tamoxifen.Then we performed intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test to observe the effect of Mof on glucose metabolism.Via immunohistochemistry(IHC)and immunofluorescence(IF)staining,we confirmed the tissue where Mof exerted its effects.2.HE staining was conducted to analyze the areas of pancreatic islets.IF staining was performed to analyze a cell ratio.Enzyme linked immunosorbent assay(ELISA)was used to measure serum insulin and glucagon levels.3.We established a cell-specific Mof deficient Mof flox/+;Glucagon(Glu)Cre mice.Then,IPGTT and IPITT were performed to verify that Mof regulated blood glucose via? cells.HE staining,IF staining and ELISA were conducted to confirm the effect of a cell Mof deficiency in islet size,? and ? cell mass and islet function.4.Primary islet of Mof flox/+;Glu Cre and Mof flox/+ mice were isolated.q-PCR were utilized to analyze difference in gene expressions,ELISA was performed to measure intra-islet GLP-1 and IF staining was conducted to show the co-staining of glucagon and PC1/3 or PC2.Results1.Compared with control,global Mof deficient mice exhibited dramatically decreased glucose levels.The insulin sensitivity was impaired,indicating Mof functioned in pancreatic islets.IHC and IF staining showed that Mof was highly expressed in pancreatic ? cells.2.Global Mof deficient mice exhibited enlarged islets,decreased ? cell ratio,lower fasting glucagon levels and higher 30 min post-challenge insulin levels.3.Compared with Mofflox/+ mice,Mofflox/+;Glu Cre mice showed lower fasting blood glucose and small areas under the IPGTT curves.Mofflox/+;Glu Cre also exhibited enlarged islet sizes,decreased ? cell mass,increased ? cell proliferation and mass,declined fasting glucagon levels and higher 30 min post-challenge insulin levels.4.Islets of Mofflox/+;Glu Cre showed enhanced ? cell but decreased ? cell-specific gene expressions and more GLP-1 secretion.IF staining indicated that despite greatly a cell loss,more PC1/3 positive ? cells,which were a cell producing GLP-1 and more PC2 negative ? cells,which were ? cells not producing glucagon,appeared.Conclusion1.Mof deficient mice showed lower blood sugar,mediated by the role of Mof in ? cells.2.Mof deficient mice exhibited less a cells and glucagon secretion,more ? cells and post-challenge insulin levels.3.Mof deficient in ? cells led to decreased ? cell mass but increased PC 1/3 positive a cells,which promoted ? cell proliferation and insulin secretion via intra-islet GLP-1.Part ? The Effect and mechanism of Mof on regulating ? cell number and function in vitroObjectiveTo investigate the regulatory mechanism of Mof in pancreatic ? cells in vitro.Methods1.We knocked down Mof expression in ?-TC1-6 cell line via small inference RNA(siRNA),and performed western blotting to measure the expression of apoptosis,autophagy,metabolism and a cell terminal differentiation markers.TUNEL staining were adapted to show apoptotic cells.IF staining were used to show DNA damage.2.Transcriptome sequence were performed to show mRNA alterations.Furthermore,GO and KEGG analyses based on it demonstrated how Mof regulated ? cell function.3.Mus-Mof were overexpressed in ?-TC1-6 and Min6,then q-PCR were performed to investigate the effect of Mof on regulating transcriptional factor expressions.4.Chromatin immunoprecipitation sequence(ChIP-seq)based on Mof-mediated H4K16ac were performed to demonstrate which gene promoters Mof-mediated histone acetylation directly bind to.GO and KEGG analyses based on ChIP-seq data showed which biological behaviors and pathways were regulated via H4K16ac.5.Mof acetyltransferase activity was inhibited by mg149.Then,Western blotting was utilized to detect the expressions of markers of apoptosis,metabolism and a cell function.TUNEL staining were used to show apoptotic cells.Results1.Mof knockdown led to increased apoptosis(increased Bax/Bcl2,cleaved-caspase3/caspase3,cleaved-PARP/PARP and more TUNEL positive cells).Increased LC3 ?/? and decreased mTOR,Atg5 and Beclinl expressions indicated dysregulated autophagy.The expressions of Irs1,Irs2,Gck,Akt and p-Akt were decreased.About a cell terminal differentiation,decreased Arx,Nkx2.2 and Nkx6.1 but enhanced Brn4 expressions indicated impaired ? cell terminal differentiation status.2.Mof knockdown contributed to impaired calcium and cAMP signal pathways,which were closely related with glucagon secretion.3.Overexpression of Mof in ?-TC1-6 and Min6 led to enhanced expressions of many transcriptional factors and glucagon,but not insl and ins2.4.ChIP-seq data showed that H4K16ac directly regulate expressions of Pax6 and Foxa2,thus regulating a cell maturation,number and function.GO and KEGG indicated that H4K16ac also regulate glucose and lipid metabolism,apoptosis and autophagy in ?-TC1-6.5.Inhibiting H4K16 acetylation led to increased a cell apoptosis and decreased Pax6,Foxa2,p-mTOR/mTOR and glucagon expressions.Conclusion1.Mof knockdown led to increased DNA damage and dysregulated autophagy,therefore promoting a cell apoptosis.Impaired calcium and cAMP signal pathway explained impaired glucagon secretion in vivo.2.Mof activated a cell-specific transcriptional network in islet endocrine cells.3.Mof inhibition and decreased H4K16ac led to a cell apoptosis and impaired a cell function.Part ? The Effect of inhibiting Mof acetyltransferase activity in vivo on glucose tolerance in T2DM miceObjectiveTo explore whether Mof inhibition could be a therapeutic target of T2DMMethods1.8-week-old male C57BL/6J mice were injected with 1mg/kg mg149.Body weight were monitored in the whole process.IPGTT were performed before,and 7 d,14 d post mg 149 administration.In 21 d,fasting and 30 min post-challenge insulin levels were measured.IPITT were performed after 14 days' administration.Then mice were sacrificed.Liver tissues were used to examine Mof knockdown efficacy via western blotting.Serums were used to measure fasting insulin,glucagon and GLP-1 levels.Pancreases were used to analyze islet size,? cell ratio,co-staining of glucagon and PC 1/3 or PC2 via HE and IF staining.2.8-week-old male T2DM model db/db mice were injected with 1mg/kg mg149.Above tests except IPITT were performed.Before mice being sacrificed,urines were collected using metabolic cages,and 24 h urine albumin were measured by ELISA.3.4-week-old male WT mice were fed with 60%fat diet for 10 weeks to establish prediabetic DIO models.DIO mice were injected with lmg/kg mg149.Identical tests with WT mice were performed.Results1.mg 149 administration had no effect on body weight or Mof expression,but can reduce H4K16ac.2.When mg 149 was used,no difference in IPGTT was shown in wild type mice.IPITT indicated impaired elevating glucose,which may due to decreased glucagon receptor expression.No difference was observed in fasting insulin,glucagon,GLP-1 and post-challenge insulin levels,but less a cell ratio and increased PC 1/3 positive ? cells were shown.3.Administation of mg 149 in db/db led to decreased glucose levels,mg 149 administration resulted in less fasting glucagon and more post-challenge insulin levels,but no difference in fasting insulin and GLP-1 levels.Similarly,less a cell ratio and increased PC 1/3 positive ? cells were shown.24 h urine albumin excretion was decreased in mg149 group.4.Administration of mg149 in DIO resulted in ameliorated glucose levels and unaltered insulin tolerance.mg 149 group exhibited lower fasting glucagon,enhanced post-challenge insulin levels,less a cell ratio and increased PC 1/3 positive ? cells.Conclusion1.Mof inhibition had no effect on body weight and glucose handling of wild type mice.2.Mof inhibition effectively ameliorated glucose tolerance of T2DM mice.Mof inhibition contributed to decreased ? cell ratio and increased PC 1/3 positive a cells,which further resulted in lower fasting glucagon but higher post-challenge insulin levels.