Jianxiang Xu

β-cell death and proliferation after intermittent hypoxia: Role of oxidative stress

Intermittent hypoxia (IH), such as occurs in sleep apnea, induces increased oxidative stress and is associated with altered glucose homeostasis. Because pancreatic beta cells are very sensitive to oxidative stress we tested whether they could be affected by IH. The effects of IH exposure (24 h/day, 5.7 and 21% O(2) alternation) in mice on beta-cell proliferation and beta-cell death were tested using Ki67 staining and TUNEL staining, respectively. To assess the role of oxidative stress in these processes, transgenic mice with beta-cell-specific overexpression of the antioxidant protein MnSOD were exposed to IH. After 4 days of IH exposure, beta-cell proliferation was increased almost fourfold. Coinciding with the increase in proliferation, the subcellular localization of the cell cycle regulator cyclin D2 was increased in the nucleus. In addition, beta-cell death was increased approximately fourfold. MnSOD transgene did not alter the effects of IH on beta-cell proliferation, but completely abrogated the IH effects on cell death. Thus, IH exposure that mimics sleep apnea can lead to increased beta-cell proliferation and cell death. Furthermore, the cell death response seems to be due to oxidative stress.

The role of pyruvate carboxylase in insulin secretion and proliferation in rat pancreatic beta cells.

Aims/hypothesisPyruvate carboxylase (PC) or pyruvate dehydrogenase (PDH) is required to transfer carbons from pyruvate into the Krebs cycle. PC activity is preserved in the islets of obese animals, but it is reduced in the islets of animal models of type 2 diabetes, suggesting that PC is important in beta cell adaptation to insulin resistance and that PC reduction may lead to beta cell failure.MethodsTo confirm the significance of PC, we first lowered activity using Pc (now known as Pcx) small interfering RNA (siRNA) in INS-1 cells and in dispersed rat islet cells. Second, we overexpressed PC in INS-1 cells, and third, we inhibited PDH by overexpressing the gene encoding pyruvate dehydrogenase kinase 4 (Pdk4) in INS-1 cells.ResultsTreatment of INS-1 cells or dispersed rat islet cells with Pc siRNA resulted in a significant reduction in insulin secretion in both cell types and reduced proliferation in INS-1 cells. This treatment also reduced the content of oxaloacetate, malate and ATP, as well as the NADPH:NADP+ ratio and activity of the pyruvate–malate shuttle. Overexpression of PC in INS-1 cells led to an elevation of insulin secretion and cell proliferation, whereas inhibition of PDH activity by overexpressing Pdk4 in INS-1 cells did not reduce insulin secretion.Conclusions/interpretationOur findings indicate that the PC pathway in beta cells might play a key role in pyruvate metabolism, insulin secretion and cell proliferation.

Long-term effect of maternal obesity on pancreatic beta cells of offspring: reduced beta cell adaptation to high glucose and high-fat diet challenges in adult female mouse offspring

Aim/hypothesisObesity is a global problem with high risks of cardiovascular diseases, stroke and type 2 diabetes. It is well known that maternal obesity affects offspring by inducing malformation, functional abnormalities in many organs and cells, and by increased risk of obesity and type 2 diabetes. However, little is known about abnormalities induced by maternal obesity in pancreatic beta cells of offspring.MethodsWe used mouse mothers with the Agouti yellow modification on a C57BL/6 background as a maternal model of normoglycaemic obesity, and produced Agouti-negative offspring. Half of the offspring were fed a high-fat diet. Offspring glucose tolerance was tested at different ages, and animals were killed at 50 weeks of age for islet function analysis.ResultsMaternal obesity impaired glucose tolerance in female offspring fed a high-fat diet, and significantly reduced insulin secretion at 50 weeks of age in female offspring that had been fed a normal diet and high-fat diet. Insulin secretion and glucose potentiation from these islets were significantly reduced. Islet protein, DNA and insulin contents were increased while glyceraldehyde-3-phosphate dehydrogenase and transketolase activities were reduced in female offspring.Conclusions/interpretationOur results indicate that maternal obesity has a long-term effect on the beta cells of female, but not of male, offspring, and leads to increased risk of gestational diabetes and type 2 diabetes in the offspring’s later lives.

FVB mouse genotype confers susceptibility to OVE26 diabetic albuminuria

OVE26 (OVE) diabetic mice on the inbred strain FVB are a valuable model of diabetic nephropathy that excretes the highest amount of urine albumin of all diabetic mouse models. Crossing of OVE mice to C57BL6 or DBA2 mice reduced albuminuria 17-fold in F1 diabetic offspring without reducing diabetes. When comparing renal histology of OVE mice on the FVB background to F1 C57BL6 crosses, we found that the F1 kidneys had significantly smaller glomeruli, much less albumin accumulation in tubules, reduced mesangial matrix expansion, and less interstitial fibrosis. A genome scan of 108 OVE-positive N2 offspring for albuminuria revealed one significant peak on chromosome 11 and nearly significant peaks on chromosomes 9, 13, and 19. Homozygosity for the FVB genotype for peaks on chromosomes 11, 13, or 19 increased albuminuria. Homozygosity for the chromosome 9 peak reduced albuminuria. Combined homozyogosity for the peaks on chromosomes 11, 13, and 19 increased albuminuria over 12-fold and accounted for >70% of the difference between OVE mice on the FVB vs. the F1 background. These loci contain sequences important to susceptibility to diabetic albuminuria.

Malic enzyme is present in mouse islets and modulates insulin secretion

Aims/hypothesisThe pyruvate–malate shuttle is a metabolic cycle in pancreatic beta cells and is important for beta cell function. Cytosolic malic enzyme (ME) carries out an essential step in the shuttle by converting malate to pyruvate and generating NADPH. In rat islets the pyruvate–malate shuttle may regulate insulin secretion and it has been shown to play a critical role in adaptation to obesity and insulin resistance. However, ME has not been demonstrated in mouse islets and three reports indicate that mouse islets contain no ME activity. If mouse islets lack ME, rat and mouse islets must regulate insulin secretion by different mechanisms.MethodsWe measured ME activity by a fluorometric enzymatic assay and Me mRNA by real-time PCR. ME activity was also measured in streptozotocin-treated mouse islets. FACS-purified beta cells were obtained from MIP-GFP mouse islets, agouti-L obese mouse islets and mouse beta cell line MIN-6. Insulin secretion and NADPH/NADP+ ratios were measured in Me siRNA-treated beta cells.ResultsME activity and Me mRNA were present in C57BL/6 mouse islets. ME activity was reduced in streptozotocin-treated mouse islets. ME activity was also measurable in FACS-purified mouse beta cells. In addition, ME activity was significantly increased in obese agouti-L mouse islets and the mouse MIN-6 cell line. Me siRNA inhibited ME activity and reduced glucose-stimulated insulin secretion and also inhibited NADPH products.Conclusions/interpretationMouse islets contain ME, which plays a significant role in regulating insulin secretion.

Uninephrectomy of Diabetic OVE26 Mice Greatly Accelerates Albuminuria, Fibrosis, Inflammatory Cell Infiltration and Changes in Gene Expression

Background/Aims: OVE26 (OVE) mice provide a valuable model of advanced diabetic nephropathy (DN), but they take 8 months to develop moderate interstitial fibrosis and reduced glomerular filtration rate (GFR). The aim of this project was to produce a more rapid and advanced model of DN. Methods: Uninephrectomy was applied to OVE and FVB mice at 2 months of age. Albuminuria, GFR, glomerulosclerosis, interstitial fibrosis, gene expression and monocyte infiltration were evaluated as a function of diabetes and uninephrectomy. Results: Albuminuria, monocyte infiltration, mesangial matrix expansion and renal fibrosis were greatly accelerated in uninephrectomized mice. DN was more advanced 10 weeks after uninephrectomy than in untreated OVE mice at 8 months of age. Uninephrectomy had almost no effect on these characteristics in non-diabetic mice. Microarray studies indicated that the accelerated fibrosis and cell infiltration in nephrectomized OVE mice were accompanied by corresponding gene expression changes in canonical pathways for fibrosis and inflammation. Conclusion: Uninephrectomy greatly accelerates all features of diabetic renal damage. This procedure provides a 10-week period after surgery to examine very large changes in the pathology of DN. The model may be particularly useful for testing new therapies and for analysis of the progression of albuminuria and fibrosis in DN.

Inflammatory Gene Expression in OVE26 Diabetic Kidney during the Development of Nephropathy

Aim: To define renal gene expression during the development of severe albuminuria in OVE26 diabetic mice. Methods: Kidney microarray analysis was performed at 2, 4 and 8 months. Data were validated by RT-PCR, in situ hybridization and immunohistochemistry. Results: Gene expression differences between control and diabetic mice increased 10-fold from 2 to 8 months. This change was most obvious for inflammatory genes. Three inflammatory genes, complement C3, VCAM1 and CD44 were upregulated more than 4-fold. Inflammatory gene expression correlated with albuminuria and C3 and CD44 increased in tubules that accumulated albumin. VCAM1 was induced in different tubules that were neither dilated nor accumulated albumin. Six of 8 genes previously reported to be markers of human advanced diabetic nephropathy and the NF-ĸB_IFN_x promoter module were elevated in the oldest diabetic mice. Vitamin D inhibits diabetic renal inflammation. Vitamin D and mRNA for vitamin D synthetic enzyme CYP2B1 were elevated in kidneys of young OVE26 mice. Conclusions: OVE26 mice induce inflammatory genes consistent with advanced renal disease, associated with severe albuminuria and to a greater extent than reported in other diabetic models. They provide an excellent model of diabetic nephropathy to assess the effect of induction of inflammatory proteins.

Reduced Cardiac Fructose 2,6 Bisphosphate Increases Hypertrophy and Decreases Glycolysis following Aortic Constriction

This study was designed to test whether reduced levels of cardiac fructose-2,6-bisphosphate (F-2,6-P2) exacerbates cardiac damage in response to pressure overload. F-2,6-P2 is a positive regulator of the glycolytic enzyme phosphofructokinase. Normal and Mb transgenic mice were subject to transverse aortic constriction (TAC) or sham surgery. Mb transgenic mice have reduced F-2,6-P2 levels, due to cardiac expression of a transgene for a mutant, kinase deficient form of the enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) which controls the level of F-2,6-P2. Thirteen weeks following TAC surgery, glycolysis was elevated in FVB, but not in Mb, hearts. Mb hearts were markedly more sensitive to TAC induced damage. Echocardiography revealed lower fractional shortening in Mb-TAC mice as well as larger left ventricular end diastolic and end systolic diameters. Cardiac hypertrophy and pulmonary congestion were more severe in Mb-TAC mice as indicated by the ratios of heart and lung weight to tibia length. Expression of α-MHC RNA was reduced more in Mb-TAC hearts than in FVB-TAC hearts. TAC produced a much greater increase in fibrosis of Mb hearts and this was accompanied by 5-fold more collagen 1 RNA expression in Mb-TAC versus FVB-TAC hearts. Mb-TAC hearts had the lowest phosphocreatine to ATP ratio and the most oxidative stress as indicated by higher cardiac content of 4-hydroxynonenal protein adducts. These results indicate that the heart’s capacity to increase F-2,6-P2 during pressure overload elevates glycolysis which is beneficial for reducing pressure overload induced cardiac hypertrophy, dysfunction and fibrosis.

Zinc rescues obesity-induced cardiac hypertrophy via stimulating metallothionein to suppress oxidative stress-activated BCL10/CARD9/p38 MAPK pathway

Obesity often leads to obesity‐related cardiac hypertrophy (ORCH), which is suppressed by zinc‐induced inactivation of p38 mitogen‐activated protein kinase (p38 MAPK). In this study, we investigated the mechanisms by which zinc inactivates p38 MAPK to prevent ORCH.

Diabetes Induced Changes in Podocyte Morphology and Gene Expression Evaluated Using GFP Transgenic Podocytes

The effect of diabetes in vivo has not been examined on isolated podocytes. To achieve this, GFP was expressed constitutively in podocytes of PGFP transgenic mice which were bred to OVE mice to produce diabetic OVE-GFP mice. Viewing GFP fluorescence, foot processes of OVE-GFP podocytes were visually and measurably effaced, which did not occur with less severe STZ diabetes. Over 300,000 podocytes were purified from each PGFP mouse but only 49,000 podocytes per diabetic OVE-GFP mouse. The low yield from OVE-GFP mice appeared to be due to more fragile state of most OVE-GFP diabetic podocytes which did not survive the isolation process. Diabetic podocytes that were isolated had high levels of the lipid peroxidation product 4-HNE and they were more sensitive to death due to oxidative stress. Gene array analysis of OVE-GFP podocytes showed strong diabetes induction of genes involved in inflammation. Four CXC chemokines were induced at least 3-fold and the chemokine CXCL1 was shown for the first time to be specifically induced in podocytes by OVE, dbdb and STZ diabetes.