The Molecular Mechanisms Of MKP-5 Regulate Metabolic Abnormalities Of Glucolipid And Compromised Islet Β Cells In Diabetes

Type 2 diabetes(T2DM)is a chronic metabolic disorder characterized by a relative insufficiency in insulin secretion,insulin resistance,and elevated blood glucose levels.Insulin resistance plays a pivotal role in the pathogenesis of T2 DM,while islet β-cells attempt to maintain normal blood glucose levels through compensatory insulin secretion.However,this compensatory mechanism leads to the occurrence of Endoplasmic Reticulum Stress(ERS),oxidative stress,inhibition of autophagic flux,pyroptosis,dysfunction in insulin secretion and ultimately apoptosis.Lipotoxicity is the primary etiological factor responsible for islet β-cell impairment in individuals with T2 DM,whereby elevated levels of free fatty acids(FFA)result in perturbations in the physiological and metabolic processes of islet β-cells,ultimately culminating in cellular dysfunction and death.Type 1 diabetes mellitus(T1DM)is characterized by an immune system deficiency,wherein autoimmune antibodies inflict damage upon islet β-cells,leading to an absolute deficiency in insulin secretion.Notably,both T2 DM and T1 DM exhibit islet β-cell death during the intermediate and advanced stages of the diseases.The role of MKP-5,also known as Mitogen activated protein phosphatase 5,as a negative regulator of the MAPK family,has been implicated in metabolic disorders,including immune regulation and pulmonary fibrosis.However,the specific involvement in islet β-cells of diabetic mice remains to be elucidated.Objectives:1.To investigate the impact of MKP-5 overexpression/suppression on lipotoxicity induced defect of islet β-cells and primary islet cells.2.To examine and compare the effects of MKP-5 knockout on glycolipid metabolism disorders and compromised islet β-cell in T2 DM and T1 DM mice.3.To analyze the differential gene changes in islet cells following MKP-5knockout and elucidate the molecular mechanism through which MKP-5 regulates islet cell failure in diabetic mice.Methods and results:1.MKP-5 regulated lipotoxic compromise of islet β-cellsThe results obtained through Real-time PCR and Western blot demonstrated that overexpression of MKP-5 effectively suppressed the activation of inflammation,oxidative stress,ERS and apoptosis induced by PA in Rin-m5 f cells.Additionally,MKP-5 mitigated the PA-induced inhibition of cell proliferation.Furthermore,the glucose-stimulated insulin secretion(GSIS)assay,along with real-time PCR and Western blot,revealed that MKP-5 alleviated the dysfunction of insulin secretion induced by PA in Rin-m5 f cells.In contrast,the downregulation of MKP-5 in Rin-m5 f cells by si-RNA resulted in the exacerbation of mitochondrial pathway apoptosis,ERS and dysfunction in insulin secretion induced by PA.The expression of MKP-5 in primary islet cells of obese mice exhibited a significant decrease,accompanied by a notable increase in apoptosis and ERS-related proteins,as well as a suppression in the expression of functional proteins of β-cells.The overexpression /suppression of MKP-5 demonstrated a mitigating/exacerbating effect respectively on the deficit of primary islet cells induced by lipotoxicity.MKP-5 developed a mitigating effect on the activation of the MAPK pathway induced by PA.Additionally,JNK and P38 inhibitors alleviated MKP-5-regulated PA-induced mitochondrial pathway apoptosis and functional impairment in islet β-cells,respectively.2.The impact of MKP-5 knockout on metabolic abnormalities and compromised islet cells in diabetic mice.The knockout of MKP-5 resulted in further increased weight gain in mice with T2 DM and weight loss in mice with T1 DM.Furthermore,it exacerbated blood glucose levels in both T2 DM and T1 DM mice.The intraperitoneal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)revealed that MKP-5knockout worsened glucose tolerance and insulin resistance in diabetic mice.Additionally,MKP-5 knockout further deteriorated certain lipid indices in both T2 DM and T1 DM mice.The results of immunohistochemistry and enzyme-linked immunosorbent assay(ELISA)demonstrated that the depletion of MKP-5 in streptozotocin(STZ)treated mice exacerbated the infiltration of immune cells in islets and the release of serum inflammatory factors.Additionally,histological examination(HE staining)revealed that MKP-5 knockout further increased the size of islets in mice fed a high-fat diet(HFD),while also further inhibiting the size of primary islets in STZ-injected mice.Furthermore,immunohistochemistry analysis indicated that MKP-5 knockout further suppressed ratio of insulin intensity and islet area in male mice fed an HFD,but had no effect on the ratio in female mice.The suppression of ratio of insulin intensity and islet area in islets of STZ-injected mice was further exacerbated by MKP-5 knockout.Additionally,in MKP-5 KO HFD female mice,the ratio of glucagon intensity and islet area of was further elevated,whereas MKP-5knockout had no significant impact on above ration in islets of STZ-injected mice.The results of GSIS demonstrated that both Chow-diet and HFD-fed mice with MKP-5 knockout developed an increase in their serum insulin levels.Meanwhile,the suppression of GSIS in primary islets by STZ was intensified by the knockout of MKP-5.Western blot and immunofluorescence revealed that HFD resulted in an upregulation of apoptosis-related proteins in islet cells of WT mice.Conversely,the injection of STZ had no discernible effect on those proteins.Furthermore,the expression levels of apoptotic proteins were significantly elevated in islets of MKP-5KO mice when subjected to either HFD or STZ treatment,as compared to WT mice.In WT mouse islets,HFD led to an increase in cleaved GSDMD expression,while STZ injection resulted in elevated levels of cleaved Caspase-1 and cleaved GSDMD expression.Significantly,the aforementioned pyroptosis proteins exhibited substantial increases in islets derived from the MKP-5 KO mice.In WT mice islets,HFD solely resulted in an elevation of p-JNK/JNK levels,while having no impact on p-P38/P38 and p-ERK/ERK levels.However,both p-P38/P38 and p-ERK/ERK expression levels were significantly augmented in the MKP-5 KO HFD group.In the islets of WT mice injected with STZ,p-ERK/ERK levels were significantly increased,whereas pP38/P38 and p-JNK/JNK levels remained unaffected.Nevertheless,all of these ratios experienced significant increases in the MKP-5 KO STZ group.3.MKP-5 attenuates lipotoxic defect including autophagic suppression of islet β-cells via GRP-78The transcriptome sequencing results revealed significant variations in autophagy,ERS,insulin pathway,energy metabolism,and other pathways within the islets of WT and MKP-5 KO mice.Additionally,Western blot demonstrated that the protein expression of LC3-II and p-m TOR was significantly elevated while the protein expression of p-AMPK and Beclin-1 was markedly suppressed in primary islets of HFD KO mice compared to HFD WT mice.Moreover,the injection of STZ further suppressed the expression of p-AMPK in islets from MKP-5 KO mice,while exhibiting no discernible impact on other autophagy-related proteins.Immunofluorescence analysis indicated that HFD had a suppressive effect on the expression of Beclin-1 protein in islet β-cells of WT mice,while STZ did not have the same effect.Furthermore,the absence of MKP-5 further failed to inhibit the expression of Beclin-1 in islet β-cells of HFD-fed mice.The utilization of laser confocal microscopy,transmission electron microscopy,and Western blot techniques collectively provided evidence that the overexpression of MKP-5 alleviated the inhibition of autophagic flux in islet β-cells induced by lipotoxicity.This alleviation occurred through the involvement of JNK and P38 signaling pathways.Furthermore,the overexpression of MKP-5 demonstrated a reduction in apoptosis,ERS,oxidative stress,and inhibition of functional proteins induced by the autophagy inhibitor 3-Methyladenine(3-MA)with PA in Rin-m5 f cells.However,upon the knockdown of the autophagy gene AMPK in Rin-m5 f cells,MKP-5 no longer exhibited its capacity to alleviate the inhibition of autophagy and expression levels of functional proteins in islet β-cells.The luciferase reporter gene assay provided that MKP-5 may exert the regulatory effects by modulating AMPK transcript levels.Consistent with the results from transcriptome sequencing,the expression of GRP-78 was significantly increased in the islet cells of MKP-5 KO mice.Furthermore,the MKP-5 KO mice exhibited exacerbated expression of GRP-78 and ERS proteins in their islet cells induced by HFD.In contrast,there was no change in the expression of GRP-78 and ERS proteins in WT mice after being injected with STZ.Therefore,in this study,we constructed a Retro Q-sh GRP78 retrovirus and observed that it further intensified lipotoxicity by inhibiting autophagic flux in Rin-m5 f cells.This effect was achieved by suppressing autophagosomal synthesis in addition to inhibiting the autophagy gene AMPK in primary islet cells.Furthermore,sh GRP-78 exacerbated the elevated levels of ERS,pyroptosis,apoptotic proteins,and diminished levels of functional proteins of islet β-cells within the MKP-5 KO HFD group.Lastly,Coimmunoprecipitation(Co-IP)experiments indicated that MKP-5 proteins potentially mitigate the lipotoxic defect upon islet β cells by interacting with GRP-78 proteins.Conclusion:(1)MKP-5 knockout exacerbated glucolipid metabolism disorders and abnormal function of islet cells in mice with T1 DM or T2 DM.(2)MKP-5 partially mitigated the lipotoxic defect in islet β-cells by regulating autophagic flow through AMPK.(3)MKP-5 alleviated the lipotoxic compromise including autophagic suppression in islet β-cells by interacting with the endoplasmic reticulum molecular chaperone GRP-78.