Role of osteopontin and its regulation in pancreatic islet.

Osteopontin (OPN) is involved in various physiological processes and also implicated in multiple pathological states. It has been suggested that OPN may have a role in type 2 diabetes (T2D) by protecting pancreatic islets and interaction with incretins. However, the regulation and function of OPN in islets, especially in humans, remains largely unexplored. In this study, we performed our investigations on both diabetic mouse model SUR1-E1506K+/+ and islets from human donors. We demonstrated that OPN protein, secretion and gene expression was elevated in the diabetic SUR1-E1506K+/+ islets. We also showed that high glucose and incretins simultaneously stimulated islet OPN secretion. In islets from human cadaver donors, OPN gene expression was elevated in diabetic islets, and externally added OPN significantly increased glucose-stimulated insulin secretion (GSIS) from diabetic but not normal glycemic donors. The increase in GSIS by OPN in diabetic human islets was Ca2+ dependent, which was abolished by Ca2+-channel inhibitor isradipine. Furthermore, we also confirmed that OPN promoted cell metabolic activity when challenged by high glucose. These observations provided evidence on the protective role of OPN in pancreatic islets under diabetic condition, and may point to novel therapeutic targets for islet protection in T2D.

SIRT1/HSF1/HSP pathway is essential for exenatide-alleviated, lipid-induced hepatic endoplasmic reticulum stress.

Recent studies have indicated that lipid-induced endoplasmic reticulum (ER) stress is a major contributor to the progression of hepatic steatosis. Exenatide (exendin-4), a glucagon-like peptide-1 receptor agonist, is known to improve hepatic steatosis, with accumulating evidence. In this study, we investigated whether exenatide could alleviate lipid-induced hepatic ER stress through mammal sirtuin 1 (SIRT1) and illustrated the detailed mechanisms. Male C57BL/6J mice challenged with a high-fat diet (HFD) were treated with exenatide or normal saline by intraperitoneal injection for 4 weeks. We observed that HFD feeding induced hepatic ER stress as indicated by increased expression of glucose-regulated protein 78, phosphorylated protein kinase-like ER kinase, and phosphorylated eukaryotic initiation factor 2α, while these increases were significantly inhibited by exenatide. Exenatide notably decreased the liver weight and hepatic steatosis induced by HFD challenge. Consistently, in human HepG2 cells and primary murine hepatocytes, exendin-4 also significantly alleviated the ER stress and lipid accumulation induced by palmitate. Importantly, further studies showed that exendin-4 enhanced the binding of heat shock factor 1 to the promoter of heat shock protein (HSP) genes through SIRT1-mediated deacetylation, which then increased the expression of molecular chaperones HSP70 and HSP40 to alleviate hepatic ER stress. Finally, inhibition of SIRT1 by genetic whole-body heterozygous knockout or by lentiviral short hairpin RNA knockdown greatly diminished the effect of exenatide on deacetylating heat shock factor 1, increasing HSP expression and alleviating ER stress and hepatic steatosis in HFD-fed mice. CONCLUSION: The SIRT1/heat shock factor 1/HSP pathway is essential for exenatide-alleviated, lipid-induced ER stress and hepatic steatosis, which provides evidence for a molecular mechanism to support exenatide and incretin mimetics as promising therapeutics for obesity-induced hepatic steatosis. (Hepatology 2017;66:809-824).

Epigenetic regulation of the thioredoxin-interacting protein (TXNIP) gene by hyperglycemia in kidney.

Diabetic kidney disease is the leading cause of end-stage renal disease. Genetic factors have been suggested to contribute to its susceptibility. However, results from genetic studies are disappointing possibly because the role of glucose in diabetic kidney disease predisposed by epigenetic mechanisms has not been taken into account. Since thioredoxin-interacting protein (TXNIP) has been shown to play an important role in the pathogenesis of diabetic kidney disease, we tested whether glucose could induce expression of TXNIP in the kidney by epigenetic mechanisms. In kidneys from diabetic Sur1-E1506K(+/+) mice, hyperglycemia-induced Txnip expression was associated with stimulation of activating histone marks H3K9ac, H3K4me3, and H3K4me1, as well as decrease in the repressive histone mark H3K27me3 at the promoter region of the gene. Glucose also coordinated changes in histone marks and TXNIP gene expression in mouse SV40 MES13 mesangial cells and the normal human mesangial cell line NHMC. The involvement of histone acetylation in glucose-stimulated TXNIP expression was confirmed by reversing or enhancing acetylation using the histone acetyltransferase p300 inhibitor C646 or the histone deacetylase inhibitor trichostatin A. Thus, glucose is a potent inducer of histone modifications, which could drive expression of proinflammatory genes and thereby predispose to diabetic kidney disease.

Epigenetic regulation of glucose-stimulated osteopontin (OPN) expression in diabetic kidney.

Diabetes nephropathy (DN) is the leading cause of end stage renal disease and it affects up to 40% of diabetic patients. In addition to hyperglycemia, genetic factors are thought to contribute to the development of DN, but few if any genetic factors have been convincingly linked to DN. Other possible mechanisms may involve epigenetic regulation of glucose-stimulated gene activity which was suggested to explain long-term effects of poor glycemic control on risk of diabetic complications, often referred to as metabolic memory. Osteopontin (OPN) is one of the genes upregulated in kidneys from diabetic mouse models as well as humans with DN, and suggested to play an important role in the pathogenesis of DN. In this study, we demonstrated that OPN gene expression is upregulated in the kidneys of a hyperglycemia diabetes mouse model SUR1-E1506K, and glucose-stimulated OPN gene expression is strongly associated with increases in activating histone marks H3K9ac, H3K4me1 and H3K4me3 and decrease in inactivating mark H3K27me3 in the promoter region of OPN gene. These findings were replicated in human mesangial cells treated with high glucose. Further proof for the involvement of histone acetylation and methylation in glucose-induced changes in OPN gene expression was obtained by manipulating histone modifications thereby OPN gene expression by histone deacetylase (HDAC) inhibitor trichostatin A and histone methyltransferase (HMT) inhibitor MM-102. We conclude that glucose is a potent inducer of histone acetylation and methylation, which in turn leads to upregulation of OPN gene expression. Treatment targeting histone marks may therefore represent an alternative method to protect kidneys from deleterious effects of glucose.

The SRE Motif in the Human PNPLA3 Promoter (-97 to -88 bp) Mediates Transactivational Effects of SREBP-1c.

Patatin-like phospholipase domain containing 3 (PNPLA3) is a non-secreted protein primarily expressed in liver and adipose tissue. Recently, numerous genetic studies have shown that PNPLA3 is a major susceptibility gene for nonalcoholic fatty liver disease (NAFLD). However, the mechanism involved in transcriptional regulation of the PNPLA3 gene remains unknown. We performed a detailed analysis of the human PNPLA3 gene promoter and identified two novel cis-acting elements (SRE and NFY binding motifs) located at -97/-88 and -26/-22 bp, respectively. Overexpression of SREBP-1c in HepG2 cells significantly increased PNPLA3 promoter activity. Mutation of either of the putative SRE or NFY binding motifs blocked the transactivation effects of SREBP-1c on the promoter. Overexpression of SREBP-1c and NFY together increased PNPLA3 promoter activity twice as much as that of SREBP-1c or NFY expression alone. This result suggests that SREBP-1c and NFY synergistically transactivate the human PNPLA3 gene. The ability of SREBP-1c and NFY to bind these cis-elements was confirmed using gel shift analysis. Putative SRE and NFY motifs also mediated synergistic insulin-induced transactivation of the PNPLA3 promoter in HepG2 cells. Additionally, the ability of SREBP-1c to bind to the PNPLA3 promoter was increased by insulin in a dose-dependent manner. Moreover, the treatment of HepG2 cells with the PI3K inhibitor LY294002 led to reduced insulin promoter-activating ability accompanied by a decrease in PNPLA3 and SREBP-1c protein expression. These results demonstrate that SREBP-1c is a direct activator of the human PNPLA3 gene and insulin transactivates the PNPLA3 gene via the PI3K-SREBP-1c/NFY pathway in HepG2 cells.

The many faces of diabetes: a disease with increasing heterogeneity.

Diabetes is a much more heterogeneous disease than the present subdivision into types 1 and 2 assumes; type 1 and type 2 diabetes probably represent extremes on a range of diabetic disorders. Both type 1 and type 2 diabetes seem to result from a collision between genes and environment. Although genetic predisposition establishes susceptibility, rapid changes in the environment (ie, lifestyle factors) are the most probable explanation for the increase in incidence of both forms of diabetes. Many patients have genetic predispositions to both forms of diabetes, resulting in hybrid forms of diabetes (eg, latent autoimmune diabetes in adults). Obesity is a strong modifier of diabetes risk, and can account for not only a large proportion of the epidemic of type 2 diabetes in Asia but also the ever-increasing number of adolescents with type 2 diabetes. With improved characterisation of patients with diabetes, the range of diabetic subgroups will become even more diverse in the future.

Role for sterol regulatory element binding protein-1c activation in mediating skeletal muscle insulin resistance via repression of rat insulin receptor substrate-1 transcription.

AIMS/HYPOTHESIS: Sterol regulatory element binding protein-1c (SREBP-1c) is a master regulator of fatty acid synthase and controls lipogenesis. IRS-1 is the key insulin signalling mediator in skeletal muscle. In the present study, we investigated the role of SREBP-1c in the regulation of IRS-1 in skeletal muscle cells. METHODS: L6 muscle cells were treated with palmitic acid (PA) or metformin. Adenovirus vectors expressing Srebp-1c (also known as Srebf1) and small interfering RNA (siRNA) against Srebp-1c were transfected into the L6 cells. Protein-DNA interactions were assessed by luciferase reporter analysis, electrophoretic mobility shift assay and chromatin immunoprecipitation assay. RESULTS: We found that both gene and protein expression of SREBP-1c was increased in contrast to IRS-1 expression in PA-treated L6 cells. SREBP-1c overproduction decreased Irs-1 mRNA and IRS-1 protein expression in a dose-dependent manner, and suppressed the resultant insulin signalling, whereas SERBP-1c knockdown by Serbp-1c siRNA blocked the downregulation of IRS-1 induced by PA. Protein-DNA interaction studies demonstrated that SREBP-1c was able to bind to the rat Irs-1 promoter region, thereby repressing its gene transcription. Of particular importance, we found that metformin treatment downregulated Srebp-1c promoter activity, decreased the specific binding of SREBP-1c to Irs-1 promoter and upregulated Irs-1 promoter activity in PA-cultured L6 cells. CONCLUSIONS/INTERPRETATION: Our data indicate for the first time that SREBP-1c activation participates in skeletal muscle insulin resistance through a direct effect of suppressing Irs-1 transcription. These findings imply that SREBP-1c could serve as an attractive therapeutic target for insulin resistance.

Role of liver FGF21-KLB signaling in ketogenic diet-induced amelioration of hepatic steatosis.

BACKGROUND: The effectiveness of ketogenic diet (KD) in ameliorating fatty liver has been established, although its mechanism is under investigation. Fibroblast growth factor 21 (FGF21) positively regulates obesity-associated metabolic disorders and is elevated by KD. FGF21 conventionally initiates its intracellular signaling via receptor ß-klotho (KLB). However, the mechanistic role of FGF21-KLB signaling for KD-ameliorated fatty liver remains unknown. This study aimed to delineate the critical role of FGF21 signaling in the ameliorative effects of KD on hepatic steatosis. METHODS: Eight-week-old C57BL/6 J mice were fed a chow diet (CD), a high-fat diet (HFD), or a KD for 16 weeks. Adeno-associated virus-mediated liver-specific KLB knockdown mice and control mice were fed a KD for 16 weeks. Phenotypic assessments were conducted during and after the intervention. We investigated the mechanism underlying KD-alleviated hepatic steatosis using multi-omics and validated the expression of key genes. RESULTS: KD improved hepatic steatosis by upregulating fatty acid oxidation and downregulating lipogenesis. Transcriptional analysis revealed that KD dramatically activated FGF21 pathway, including KLB and fibroblast growth factor receptor 1 (FGFR1). Impairing liver FGF21 signaling via KLB knockdown diminished the beneficial effects of KD on ameliorating fatty liver, insulin resistance, and regulating lipid metabolism. CONCLUSION: KD demonstrates beneficial effects on diet-induced metabolic disorders, particularly on hepatic steatosis. Liver FGF21-KLB signaling plays a critical role in the KD-induced amelioration of hepatic steatosis.

SGLT2 inhibitor empagliflozin downregulates miRNA-34a-5p and targets GREM2 to inactivate hepatic stellate cells and ameliorate non-alcoholic fatty liver disease-associated fibrosis.

BACKGROUND AND RATIONALE: Activation of hepatic stellate cells (HSCs), the central event of fibrosis, indicates the severe stage of non-alcoholic fatty liver disease (NAFLD). MicroRNAs (miRNAs) participate in this process. Treatment with a sodium-glucose cotransporter 2 inhibitor (SGLT2i) alleviates liver fibrosis in patients with type 2 diabetes and NAFLD; however, the role of SGLT2i in ameliorating liver fibrosis in NAFLD by regulating miRNAs remains unclear. APPROACH AND RESULTS: We monitored the expression of NAFLD-associated miRNAs in the livers of two NAFLD models and observed high expression of miR-34a-5p. miR-34a-5p was highly expressed in mouse primary liver non-parenchymal cells and LX-2 HSCs, and this miRNA was positively correlated with alanine transaminase levels in NAFLD models. Overexpression of miR-34a-5p enhanced LX-2 activation, whereas its inhibition prevented HSCs activation by regulating the TGFß signaling pathway. The SGLT2i empagliflozin significantly downregulated miR-34a-5p, inhibited the TGFß signaling pathway, and ameliorated hepatic fibrosis in NAFLD models. Subsequently, GREM2 was identified as a direct target of miR-34a-5p through database prediction and a dual-luciferase reporter assay. In LX-2 HSCs, the miR-34a-5p mimic and inhibitor directly downregulated and upregulated GREM2, respectively. Overexpressing GREM2 inactivated the TGFß pathway whereas GREM2 knockdown activated it. Additionally, empagliflozin upregulated Grem2 expression in NAFLD models. In methionine- and choline-deficient diet-fed ob/ob mice, a fibrosis model, empagliflozin downregulated miR-34a-5p and upregulated Grem2 to improve liver fibrosis. CONCLUSIONS: Empagliflozin ameliorates NAFLD-associated fibrosis by downregulating miR-34a-5p and targeting GREM2 to inhibit the TGFß pathway in HSCs.

SIRT1-dependent deacetylation of Txnip H3K9ac is critical for exenatide-improved diabetic kidney disease.

Glucagon-like peptide 1 receptor agonist exenatide (exendin-4) has potential protective capabilities against diabetic kidney disease (DKD). However, the underlying mechanism has not been fully elucidated. The expression of thioredoxin-interacting protein (Txnip) is upregulated during DKD progression by histone acetylation. Sirtuin 1 (SIRT1) is a deacetylase and is decreased in DKD, which indicates that it may regulate Txnip in this disease. Here, we used whole-body heterozygous Sirt1 knockout (Sirt1+/-) and kidney-specific Sirt1 knockout (KSK) mice to investigate whether SIRT1 regulates Txnip via histone deacetylation in DKD and exenatide-alleviated DKD. Exenatide substantially improved renal pathological damage, decreased the albumin-to-creatinine ratio (ACR), upregulated SIRT1 expression, and downregulated Txnip expression in kidneys of high-fat diet-treated C57BL/6J mice. However, these effects diminished in Sirt1+/- and KSK mice under exenatide treatment. The downregulation of Txnip expression by exendin-4 in high-glucose-treated SV40 MES13 cells was hampered during Sirt1 knockdown. These results demonstrate that kidney SIRT1 is indispensable in exenatide-improved DKD and downregulation of Txnip expression. Exendin-4 mechanistically downregulated Txnip histone 3 lysine 9 acetylation (H3K9ac) in a SIRT1-dependent manner and decreased spliced X-box binding protein 1 (XBP1s) recruitment to the Txnip promoter. These findings provide epigenetic evidence elucidating the specific mechanism for exenatide-mediated DKD alleviation and highlight the importance of Txnip as a promising therapeutic target for DKD.

Malonylation of Acetyl-CoA carboxylase 1 promotes hepatic steatosis and is attenuated by ketogenic diet in NAFLD.

Protein post-translational modifications (PTMs) participate in important bioactive regulatory processes and therefore can help elucidate the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Here, we investigate the involvement of PTMs in ketogenic diet (KD)-improved fatty liver by multi-omics and reveal a core target of lysine malonylation, acetyl-coenzyme A (CoA) carboxylase 1 (ACC1). ACC1 protein levels and Lys1523 malonylation are significantly decreased by KD. A malonylation-mimic mutant of ACC1 increases its enzyme activity and stability to promote hepatic steatosis, whereas the malonylation-null mutant upregulates the ubiquitination degradation of ACC1. A customized Lys1523ACC1 malonylation antibody confirms the increased malonylation of ACC1 in the NAFLD samples. Overall, the lysine malonylation of ACC1 is attenuated by KD in NAFLD and plays an important role in promoting hepatic steatosis. Malonylation is critical for ACC1 activity and stability, highlighting the anti-malonylation effect of ACC1 as a potential strategy for treating NAFLD.

ER stress aggravates diaphragm weakness through activating PERK/JNK signaling in obesity hypoventilation syndrome.

OBJECTIVE: Obesity hypoventilation syndrome is associated with diaphragmatic dysfunction. This study aimed to explore the role of endoplasmic reticulum (ER) stress in mediating obesity-induced diaphragmatic dysfunction. METHODS: A pulmonary function test and ultrasound were applied to evaluate diaphragmatic function and magnetic resonance imaging was applied to measure diaphragmatic lipid deposition in human patients. For the mechanistic study, obese mice were introduced to a high-fat diet for 24 weeks, followed by diaphragmatic ultrasound measurement, transcriptomic sequencing, and respective biochemical analysis. Automatic force mapping was applied to measure the mechanical properties of C2C12 myotubes. RESULTS: People with obesity showed significant diaphragm weakness and lipid accumulation, which was further confirmed in obese mice. Consistently, diaphragms from obese mice showed altered gene expression profile in lipid metabolism and activation of ER stress response, indicated by elevated protein kinase R-like ER kinase (PERK) and c-Jun NH2 -terminal kinase (JNK) activation. In C2C12 myotubes, inhibition of PERK or JNK signaling abrogated lipotoxicity-induced intracellular lipid deposition and insulin resistance. Inhibition of JNK signaling reversed lipotoxicity-induced impairment of elasticity in C2C12 myotubes. CONCLUSIONS: These data suggest that ectopic lipid deposition impairs the diaphragmatic function of people with obesity. Activation of PERK/JNK signaling is involved in the pathogenesis of lipotoxicity-induced diaphragm weakness in obesity hypoventilation syndrome.

SBC (Sanhuang Xiexin Tang combined with Baihu Tang plus Cangzhu) alleviates NAFLD by enhancing mitochondrial biogenesis and ameliorating inflammation in obese patients and mice.

In the context of non-alcoholic fatty liver disease (NAFLD), characterized by dysregulated lipid metabolism in hepatocytes, the quest for safe and effective therapeutics targeting lipid metabolism has gained paramount importance. Sanhuang Xiexin Tang (SXT) and Baihu Tang (BHT) have emerged as prominent candidates for treating metabolic disorders. SXT combined with BHT plus Cangzhu (SBC) has been used clinically for Weihuochisheng obese patients. This retrospective analysis focused on assessing the anti-obesity effects of SBC in Weihuochisheng obese patients. We observed significant reductions in body weight and hepatic lipid content among obese patients following SBC treatment. To gain further insights, we investigated the effects and underlying mechanisms of SBC in HFD-fed mice. The results demonstrated that SBC treatment mitigated body weight gain and hepatic lipid accumulation in HFD-fed mice. Pharmacological network analysis suggested that SBC may affect lipid metabolism, mitochondria, inflammation, and apoptosis-a hypothesis supported by the hepatic transcriptomic analysis in HFD-fed mice treated with SBC. Notably, SBC treatment was associated with enhanced hepatic mitochondrial biogenesis and the inhibition of the c-Jun N-terminal kinase (JNK)/nuclear factor-kappa B (NF-κB) and extracellular signal-regulated kinase (ERK)/NF-κB pathways. In conclusion, SBC treatment alleviates NAFLD in both obese patients and mouse models by improving lipid metabolism, potentially through enhancing mitochondrial biogenesis. These effects, in turn, ameliorate inflammation in hepatocytes.

[The effects of insulin and gliclazide therapy on endoplasmic reticulum stress and insulin sensitivity in liver of type 2 diabetic rats].

OBJECTIVE: To investigate the effect of insulin and gliclazide therapy on endoplasmic reticulum (ER) stress and insulin sensitivity in the liver of type 2 diabetic rats. METHODS: A high fat diet plus a low-dose of streptozotocin was implemented to create a type 2 diabetic rats which were randomly divided into diabetes mellitus (DM) group, insulin treatment (INS) group and gliclazide treatment (GT) group; and healthy rats were as normal control group. Diabetic rats in INS and GT groups were given neutral protamine hagedorn (NPH) insulin and gliclazide respectively for 3 weeks. Protein expression levels of immunoglobulin binding protein (Bip), spliced X-box binding protein 1 (XBP-1s), phosphorylated c-Jun on serine 73 (p-c-Jun), phosphorylated insulin receptor substrate 1 on serine 307 (p-IRS-1), and glucose-6-phosphatase (G6Pase) in liver homogenate were detected by Western blotting. RESULTS: Compared with the normal rats, Bip and XBP-1s in the DM group were up-regulated (0.28 ± 0.07 vs 0.90 ± 0.10 for Bip; 0.41 ± 0.07 vs 0.95 ± 0.07 for XBP-1s; both P < 0.01); p-c-Jun (0.59 ± 0.18 vs 1.94 ± 0.03), p-IRS-1 (1.73 ± 0.18 vs 5.32 ± 0.22) and G6Pase(0.11 ± 0.01 vs 0.45 ± 0.01) were increased (all P values < 0.01). In the INS group, all of aforementioned changes were reversed (0.90 ± 0.10 vs 0.25 ± 0.04 for Bip; 0.95 ± 0.07 vs 0.47 ± 0.01 for XBP-1s; 1.94 ± 0.03 vs 0.50 ± 0.10 for p-c-Jun; 5.32 ± 0.22 vs 1.59 ± 0.32 for p-IRS-1; 0.45 ± 0.01 vs 0.15 ± 0.02 for G6Pase, all P values < 0.01). In the GT group, all of aforementioned changes were also attenuated (0.90 ± 0.10 vs 0.53 ± 0.02 for Bip; 0.95 ± 0.07 vs 0.78 ± 0.02 for XBP-1s; 1.94 ± 0.03 vs 1.33 ± 0.11 for p-c-Jun; 5.32 ± 0.22 vs 3.13 ± 0.02 for p-IRS-1; 0.45 ± 0.01 vs 0.25 ± 0.01 for G6Pase, all P values < 0.05). Furthermore, all of aforementioned protein levels were down-regulated more obviously in the INS group comparing to the GT group (all P values < 0.01). CONCLUSIONS: Both insulin and gliclazide therapy could relieve ER stress and c-Jun N-terminal kinase activity and improved insulin sensitivity. The effect of insulin on Bip, XBP-1s, p-c-Jun, p-IRS-1 and G6Pase protein expressions is more obvious than that of gliclazide, which indicates besides lowering glucose, insulin might have protective effects of anti-inflammation, anti-oxidative stress or stimulation of lipid redistribution.

Dysregulated hepatic lipid metabolism and gut microbiota associated with early-stage NAFLD in ASPP2-deficiency mice.

Background: Growing evidence indicates that lipid metabolism disorders and gut microbiota dysbiosis were related to the progression of non-alcoholic fatty liver disease (NAFLD). Apoptosis-stimulating p53 protein 2 (ASPP2) has been reported to protect against hepatocyte injury by regulating the lipid metabolism, but the mechanisms remain largely unknown. In this study, we investigate the effect of ASPP2 deficiency on NAFLD, lipid metabolism and gut microbiota using ASPP2 globally heterozygous knockout (ASPP2+/-) mice. Methods: ASPP2+/- Balb/c mice were fed with methionine and choline deficient diet for 3, 10 and 40 day to induce an early and later-stage of NAFLD, respectively. Fresh fecal samples were collected and followed by 16S rRNA sequencing. HPLC-MRM relative quantification analysis was used to identify changes in hepatic lipid profiles. The expression level of innate immunity-, lipid metabolism- and intestinal permeability-related genes were determined. A spearman's rank correlation analysis was performed to identify possible correlation between hepatic medium and long-chain fatty acid and gut microbiota in ASPP2-deficiency mice. Results: Compared with the WT control, ASPP2-deficiency mice developed moderate steatosis at day 10 and severe steatosis at day 40. The levels of hepatic long chain omega-3 fatty acid, eicosapentaenoic (EPA, 20:5 n-3) and docosahexaenoic (DHA, 22:6 n-3), were decreased at day 10 and increased at day 40 in ASPP+/- mice. Fecal microbiota analysis showed significantly increased alpha and beta diversity, as well as the composition of gut microbiota at the phylum, class, order, family, genus, species levels in ASPP2+/- mice. Moreover, ASPP-deficiency mice exhibited impaired intestinal barrier function, reduced expression of genes associated with chemical barrier (REG3B, REG3G, Lysozyme and IAP), and increased expression of innate immune components (TLR4 and TLR2). Furthermore, correlation analysis between gut microbiota and fatty acids revealed that EPA was significantly negatively correlated with Bifidobacterium family. Conclusion: Our findings suggested that ASPP2-deficiency promotes the progression of NAFLD, alterations in fatty acid metabolism and gut microbiota dysbiosis. The long chain fatty acid EPA was significantly negatively correlated with Bifidobacterial abundance, which is a specific feature of NAFLD in ASPP2-deficiency mice. Totally, the results provide evidence for a mechanism of ASPP2 on dysregulation of fatty acid metabolism and gut microbiota dysbiosis.

Diabetes causes multiple genetic alterations and downregulates expression of DNA repair genes in the prostate.

The molecular impact of diabetes mellitus on prostate gland has not been elucidated. In this study, we performed a whole-genome cDNA microarray analysis using a streptozotocin-induced diabetic rat model to identify the effects of diabetes on the gene expression profiles in prostate. Our study shows that diabetes causes changes in the expression of multiple genes, particularly those related to cell proliferation and differentiation, oxidative stress, DNA damage repair, cell cycle checkpoints, angiogenesis and apoptosis. These findings were confirmed by real-time polymerase chain reaction and immunohistochemical staining using rat and human prostate tissue. We also used a cell culture model (human normal prostatic RWPE-1 cell line) to study the direct effect of high glucose. We found that high glucose caused increased intracellular oxidative stress and DNA damage, as well as downregulation of anti-oxidative enzymes and DNA damage repair genes MRE11 and XRCC3. Our findings provide important insights into understanding the pathogenesis of the diabetes-induced changes in prostate as well as identifying potential therapeutic targets for future studies.

Insight into the biochemical characteristics of a novel glucokinase gene mutation.

Glucokinase (GCK) acts as a glucose sensor and regulates ß-cell insulin secretion. The heterozygous mutations in the gene encoding GCK cause a reduction of the enzyme activity, which results in a monogenic form of diabetes, maturity-onset diabetes of the young. In the present study, we identified and functionally characterized a novel missense mutation in the GCK gene, which results in a protein mutation Glu(339)→Lys (E339K), from a Chinese family with hyperglycemia. The same GCK mutation that co-segregated with diabetes phenotype was identified in five members of this family but was not found in 200 healthy control individuals. We expressed and affinity-purified the GCK proteins from bacterial expression system that carries mutation (E339K) and fused to glutathione S-transferase. The expressed GCK protein was subjected to the measurement of its biochemical effects of the missense mutation on GCK activity. Our results showed that the mutation reduced the GCK protein yield. The enzymatic kinetics and the thermal stability analysis on the recombinant GCK proteins revealed that the mutation inactivates enzyme kinetics and severely impaired the GCK protein stability.

[Effects of early insulin therapy on sterol regulatory element binding protein 1 pathway and lipid accumulation in liver of type 2 diabetic rats].

OBJECTIVE: To explore the effect of early insulin therapy on sterol regulatory element binding protein 1 (SREBP1) pathway and lipid accumulation in liver of type 2 diabetic rats (DM). METHODS: A high-fat diet plus a low-dose of streptozotocin (STZ) was administered to the Sprague-Dawley (SD) rats to create a type 2 diabetic animal model. Then the rats were divided into 3 groups: normal control (NC), DM (untreated diabetic rats) and INS (a 3-week treatment of NPH insulin initiated from day 3 of STZ injection). Insulin was delivered daily by a 3-week subcutaneous injection (6 - 8 U/day). Liver homogenate was prepared. The protein levels of ER stress marker immunoglobulin binding protein (Bip), oxygen-regulated protein 150 (ORP150), insulin-induced gene 1 (Insig1), SREBP1 and nuclear SREBP1 (nSREBP1) were assayed by Western blot. Adipose tissue mass was measured. RESULTS: In the DM group, ER (endoplasmic reticulum) stress marker Bip and ORP150 were up-regulated (0.67 ± 0.02 vs 0.43 ± 0.01 for Bip; 1.11 ± 0.04 vs 1.83 ± 0.03 for ORP150, P < 0.05 for both) and Insig1 decreased (0.25 ± 0.02 vs 0.80 ± 0.07, P < 0.05). And the expressions of SREBP1 and nSREBP1 were elevated (1.03 ± 0.14 vs 0.41 ± 0.01 for SREBP1; 3.63 ± 0.77 vs 0.96 ± 0.20 for nSREBP1, P < 0.05 for both) in comparison with the normal control rats. In the INS group, all aforementioned changes became attenuated or reversed (0.41 ± 0.04 vs 0.67 ± 0.02 for Bip; 1.83 ± 0.03 vs 1.11 ± 0.04 for ORP150; 0.43 ± 0.02 vs 0.25 ± 0.02 for Insig1; 0.46 ± 0.01 vs 1.03 ± 0.14 for SREBP1; 1.65 ± 0.18 vs 3.63 ± 0.77 for nSREBP1, P < 0.05 for all). Furthermore, adipose tissue mass increased (22.4 g ± 3.6 g vs 12.0 g ± 2.6 g, P < 0.05). CONCLUSION: The early insulin therapy induces a fat redistribution from liver to adipose tissue. The mechanism is probably through a reduction of ER stress and a down-regulated pathway of SREBP1 in liver of diabetic rats.

Efficacy and Safety of a Decision Support Intervention for Basal Insulin Self-Titration Assisted by the Nurse in Outpatients with T2DM: A Randomized Controlled Trial.

OBJECTIVE: The main aim of this study was to evaluate a combined fasting blood glucose based dosage self-titration setting and decision supported telephone coaching intervention on glycemic control and diabetes self-management skills, compared to the conventional care. METHODS: A 12-week, single-blinded, randomized controlled trial was conducted on adults with type 2 diabetes (T2DM) primarily treated with basal insulin. After randomization, the intervention group (IG, n = 426) received a basal insulin self-titration decision support intervention administered by the Diabetes Specialty Nurses while the control group (CG, n = 423) received conventional care for 12 weeks, both included five telephone interviews. The primary efficacy endpoint was the effect of intervention on glycemic control, measured as the change in glycated hemoglobin (HbA1c) from baseline to Week 12 (after intervention) compared to the control group. Other endpoints included comparisons of the effects of intervention on fasting plasma glucose (FPG), postprandial plasma glucose (PPG), body weight, Michigan diabetes knowledge test (MDKT), diabetes empowerment scale-short Form (DES-DSF), and summary of diabetes self-care activities (SDSCA). Changes in the primary and secondary outcomes were compared using the t-test for continuous variables with a normal distribution and χ 2-test for categorical variables. RESULTS: The IG showed more improvements on mean HbA1c, compared to the CG (-2.8% vs -1.8%), so did the FPG, PPG, MDKT, DES-DSF and SDSCA (all P<0.01) after the 12-week follow up. Though the final mean insulin dose in the IG was higher than the CG at the end of the study (0.32 U/kg vs 0.28 U/kg), the changes of body weight were similar between the two groups (0.46kg vs 0.40kg, P=0.246), and the proportion of patients with hypoglycemia events during the whole trial were similar (20.65% vs 17.73%, P=0.279). CONCLUSION: Decision supporting of basal insulin glargine self-titration assisted by Diabetes Specialty Nurses is effective and safe in patients with T2DM. Decision supported telephone coaching intervention offers ongoing encouragement, guidance, and determination of relevant sources of decisional conflict, facilitating adjusting the insulin dose.

Dietary Knowledge, Attitude and Practice (KAP) Among the Family Members of Patients with Type 2 Diabetes Mellitus (T2DM) and Its Influence on the KAP of T2DM Patients.

PURPOSE: To investigate the dietary knowledge, attitude and practice (KAP) among the family members (FMs) of Chinese type 2 diabetes mellitus (T2DM) patients and its influence on the KAP of T2DM patients. PATIENTS AND METHODS: Two hundred thirty-six pairs of hospitalized T2DM patients and their FMs (472 in total) in our hospital were enrolled. A pair of self-designed questionnaires on dietary KAP (Cronbach's α ≥ 0.763, I-CVI ≥ 0.857, S-CVI = 0.964, 0.958) were used to collect data and assess the KAP towards diabetes diets. RESULTS: The mean score for dietary KAP of T2DM patients was 2.33 ± 0.60, 3.03 ± 0.44 and 2.77 ± 0.38, whereas that of their FMs was 2.37 ± 0.55, 3.08 ± 0.48 and 2.82 ± 0.61, respectively. Pearson's correlation analysis showed that the glycosylated hemoglobin (HbA1c) of T2DM patients was negatively correlated to their dietary practice (r = -0.218, P < 0.01). There was a positive correlation between T2DM patients and their FMs for dietary KAP (r = 0.306, P < 0.05). The dietary practice of T2DM patients was positively correlated with the dietary KAP of their FMs (r = 0.305, 0.252 and 0.136, respectively, P < 0.01). Logistic regression analysis revealed that the score for dietary knowledge and attitude, occupation, residence, family history, complications of the T2DM patient, and the sex and dietary knowledge score of the FM were significantly associated with dietary practice for T2DM patients. CONCLUSION: The dietary attitude of FMs was moderate but dietary knowledge and practice were poor. Dietary KAP was positively correlated with T2DM patients and their FMs.