Yumi Takiyama

Tubular Injury in a Rat Model of Type 2 Diabetes Is Prevented by Metformin: A Possible Role of HIF-1α Expression and Oxygen Metabolism

OBJECTIVE Chronic hypoxia has been recognized as a key regulator in renal tubulointerstitial fibrosis, as seen in diabetic nephropathy, which is associated with the activation of hypoxia-inducible factor (HIF)-1α. We assess here the effects of the biguanide, metformin, on the expression of HIF-1α in diabetic nephropathy using renal proximal tubular cells and type 2 diabetic rats. RESEARCH DESIGN AND METHODS We explored the effects of metformin on the expression of HIF-1α using human renal proximal tubular epithelial cells (HRPTECs). Male Zucker diabetic fatty (ZDF; Gmi-fa/fa) rats were treated from 9 to 39 weeks with metformin (250 mg ⋅ kg−1 ⋅ day−1) or insulin. RESULTS Metformin inhibited hypoxia-induced HIF-1α accumulation and the expression of HIF-1–targeted genes in HRPTECs. Although metformin activated the downstream pathways of AMP-activated protein kinase (AMPK), neither the AMPK activator, AICAR, nor the mTOR inhibitor, rapamycin, suppressed hypoxia-induced HIF-1α expression. In addition, knockdown of AMPK-α did not abolish the inhibitory effects of metformin on HIF-1α expression. The proteasome inhibitor, MG-132, completely eradicated the suppression of hypoxia-induced HIF-1α accumulation by metformin. The inhibitors of mitochondrial respiration similarly suppressed hypoxia-induced HIF-1α expression. Metformin significantly decreased ATP production and oxygen consumption rates, which subsequently led to increased cellular oxygen tension. Finally, metformin, but not insulin, attenuated tubular HIF-1α expression and pimonidazole staining and ameliorated tubular injury in ZDF rats. CONCLUSIONS Our data suggest that hypoxia-induced HIF-1α accumulation in diabetic nephropathy could be suppressed by the antidiabetes drug, metformin, through the repression of oxygen consumption.

Reduction of both beta cell death and alpha cell proliferation by dipeptidyl peptidase-4 inhibition in a streptozotocin-induced model of diabetes in mice

Aims/hypothesisIncretins stimulate insulin secretion in a glucose-dependent manner but also promote pancreatic beta cell protection. Dipeptidyl peptidase-4 (DPP-4) inhibitors are a new glucose-lowering treatment that blocks incretin degradation by DPP-4. We assessed whether DPP-4 inhibition suppresses the progression to hyperglycaemia in a low-dose streptozotocin (STZ)-induced diabetic mouse model, and then investigated how DPP-4 inhibition affects islet function and morphology.MethodsThe DPP-4 inhibitor, des-fluoro-sitagliptin (SITA), was administered to mice during and after STZ injections, and in some mice also before STZ.ResultsIn control mice, STZ resulted in hyperglycaemia associated with impaired insulin secretion and excess glucagon secretion. In SITA-treated STZ mice, these metabolic abnormalities were improved, particularly when SITA administration was initiated before STZ injections. We observed beta cell loss and dramatic alpha cell expansion associated with decreased insulin content and increased glucagon content after STZ administration. In SITA-treated mice, islet architecture and insulin content were preserved, and no significant increase in glucagon content was observed. After STZ exposure, beta cell apoptosis increased before hyperglycaemia, and SITA treatment reduced the number of apoptotic beta cells. Interestingly, alpha cell proliferation was observed in non-treated mice after STZ injection, but the proliferation was not observed in SITA-treated mice.Conclusions/interpretationOur results suggest that the ability of DPP-4 inhibition to suppress the progression to STZ-induced hyperglycaemia involves both alleviation of beta cell death and alpha cell proliferation.

Tubular Injury in a Rat Model of Type 2 Diabetes Is Prevented by Metformin: A Possible Role of Hypoxia Inducible Factor−1α Expression and Oxygen Metabolism

OBJECTIVE Chronic hypoxia has been recognized as a key regulator in renal tubulointerstitial fibrosis, as seen in diabetic nephropathy, which is associated with the activation of hypoxia-inducible factor (HIF)-1α. We assess here the effects of the biguanide, metformin, on the expression of HIF-1α in diabetic nephropathy using renal proximal tubular cells and type 2 diabetic rats. RESEARCH DESIGN AND METHODS We explored the effects of metformin on the expression of HIF-1α using human renal proximal tubular epithelial cells (HRPTECs). Male Zucker diabetic fatty (ZDF; Gmi-fa/fa) rats were treated from 9 to 39 weeks with metformin (250 mg ⋅ kg−1 ⋅ day−1) or insulin. RESULTS Metformin inhibited hypoxia-induced HIF-1α accumulation and the expression of HIF-1–targeted genes in HRPTECs. Although metformin activated the downstream pathways of AMP-activated protein kinase (AMPK), neither the AMPK activator, AICAR, nor the mTOR inhibitor, rapamycin, suppressed hypoxia-induced HIF-1α expression. In addition, knockdown of AMPK-α did not abolish the inhibitory effects of metformin on HIF-1α expression. The proteasome inhibitor, MG-132, completely eradicated the suppression of hypoxia-induced HIF-1α accumulation by metformin. The inhibitors of mitochondrial respiration similarly suppressed hypoxia-induced HIF-1α expression. Metformin significantly decreased ATP production and oxygen consumption rates, which subsequently led to increased cellular oxygen tension. Finally, metformin, but not insulin, attenuated tubular HIF-1α expression and pimonidazole staining and ameliorated tubular injury in ZDF rats. CONCLUSIONS Our data suggest that hypoxia-induced HIF-1α accumulation in diabetic nephropathy could be suppressed by the antidiabetes drug, metformin, through the repression of oxygen consumption.

High glucose activates HIF-1-mediated signal transduction in glomerular mesangial cells through a carbohydrate response element binding protein

High glucose evokes a variety of signals in mesangial cells that alter cellular functions responsible for the development of diabetic glomerulopathy. The hypoxia-inducible factor-1alpha (HIF-1alpha) regulates cellular homeostasis under hypoxic conditions, but it also has pleiotropic effects in response to cellular stresses at normoxia. Here we determined whether HIF-1alpha has a role in the regulation of mesangial cells in hyperglycemia. In the streptozotocin-induced diabetic mouse model, glomerular mesangial cells had a significant increase in HIF-1alpha expression in the nucleus. In cultured mesangial cells, high glucose enhanced the expression of HIF-1alpha and its target genes known to be involved in the development of diabetic glomerulopathy. A glucose-responsive carbohydrate response element binding protein (ChREBP) was found to have a critical role in the transcriptional upregulation of HIF-1alpha and downstream gene expression in mesangial cells exposed to high glucose. Knockdown of HIF-1alpha or ChREBP in mesangial cells abrogated the high glucose-mediated perturbation of gene expression. Our results show that ChREBP and HIF-1alpha mediate gene regulation in mesangial cells. Further studies will be needed to find out whether these findings are relevant to the development of the diabetic nephropathy.

Upregulated IL-18 expression in type 2 diabetic subjects with nephropathy : TGF-β1 enhanced IL-18 expression in human renal proximal tubular epithelial cells

AIM In order to clarify the importance of Interleukin (IL)-18 in the development of diabetic nephropathy (DN), we evaluated the expressions of IL-18 in diabetic kidney. METHODS We performed immmunohistochemical analysis of IL-18 and IL-18 receptor (IL-18 R) in human kidney tissue derived from 12 subjects with type 2 diabetes and overt nephropathy, and compared with those in 7 subjects with minimal change nephrotic syndrome (MCNS). In addition, we examined the regulation of IL-18 expression using human renal proximal tubular epithelial cells (HRPTECs) in culture. RESULTS IL-18 expression in tubular cells was observed in higher rate (83%) in patients with diabetes, whereas one positive specimen (14.3%) for IL-18 in patients with MCNS. In contrast, IL-18 R was expressed in glomerular mesangial cells and endothelial cells as well as tubular cells, similarly in almost of both groups. Exposure to transforming growth factor beta (TGF-beta(1)) led to two-fold increase in IL-18 gene expression in HRPTECs, and mitogen-activated protein kinases (MAPK) inhibitors abolished TGF-beta(1)-induced IL-18 mRNA expression. Western blot analysis showed the IL-18 protein in HRPTECs. CONCLUSION The present data indicate that IL-18 is overexpressed in human tubular epithelial cells in diabetic nephropathy, probably through the activation of MAPK pathways induced by TGF-beta(1).

The Role of the DAP12 Signal in Mouse Myeloid Differentiation

DAP12 is a recently cloned, immunoreceptor tyrosine-based activation motif-bearing transmembrane adapter molecule that is associated with the NK-activating receptors. Previous reports showed that the DAP12 message could be detected not only in NK cells but also in granulocytes, monocytes, dendritic cells, and macrophages. In this study we found a significant level of DAP12 protein expression in macrophage-related cell lines and organs. Additionally, we observed increased expression of DAP12 after LPS-induced differentiation of M1 cells into macrophages. To examine the role of DAP12 in the myeloid cell lineage, we established M1 FLAG-DAP12 transfectants (FDAP-M1) and demonstrated the marked morphological changes in FDAP-M1 cells caused by signaling through DAP12. Cell surface phenotypic analysis showed up-regulation of macrophage markers CD11b, 2.4G2, and adhesion molecule B7-2. Additionally, after stimulation through DAP12, phosphorylated FLAG -DAP12 could be immunoprecipitated using anti-phosphotyrosine mAbs. Collectively, these findings indicate that direct DAP12 signaling has an important role in macrophage differentiation.

Modulation of Hepatic Granulomatous Responses by Transgene Expression of DAP12 or TREM-1-Ig Molecules

DAP12 (also known as KARAP) is a novel ITAM-bearing transmembrane adapter molecule that is expressed on the cell surface of natural killer cells, monocytes, dendritic cells, and macrophages. Several myeloid cell-specific DAP12-associating receptors, such as TREM receptor family, SIRP-beta1, and MDL-1 have been identified. The in vivo function of DAP12 and its associating molecules in inflammation has remained primarily unknown. To investigate DAP12 signaling during chronic inflammation, we constructed two adenoviral gene transfer vectors to express FLAG/DAP12 (Ad-FDAP12) and the extracellular domain of mouse TREM-1 and the Fc portion of human IgG1 (Ad-TREM-1 Ig), respectively, and observed their modulatory activities in a mouse model of hepatic granulomatous inflammation elicited by zymosan A. Mice were injected with zymosan A intravenously and 24 hours after zymosan A, they were injected with Ad-FDAP12 or Ad-TREM-1 Ig. Zymosan A-induced hepatic granuloma formation peaked at day 7 and markedly declined by day 10. Although adenoviral-mediated DAP12 gene transfer did not enhance granuloma formation by day 7, it sustained and enhanced granuloma formation beyond day 7. However, an anti-FLAG monoclonal antibody used to potentiate the signaling of adenoviral-derived DAP12, enhanced granuloma formation at day 7. In sharp contrast to the effect by Ad-FDAP12, transgene expression in the liver of soluble form of extracellular domain of TREM-1 as an antagonist of DAP12 signaling, remarkably inhibited zymosan A-induced granuloma formation at all time points examined. Our findings thus suggest that both DAP12 and TREM-1 are involved in the development of granulomatous responses in the liver.

Loss of HIF-1α impairs GLUT4 translocation and glucose uptake by the skeletal muscle cells.

Defects in glucose uptake by the skeletal muscle cause diseases linked to metabolic disturbance such as type 2 diabetes. The molecular mechanism determining glucose disposal in the skeletal muscle in response to cellular stimuli including insulin, however, remains largely unknown. The hypoxia-inducible factor-1α (HIF-1α) is a transcription factor operating in the cellular adaptive response to hypoxic conditions. Recent studies have uncovered pleiotropic actions of HIF-1α in the homeostatic response to various cellular stimuli, including insulin under normoxic conditions. Thus we hypothesized HIF-1α is involved in the regulation of glucose metabolism stimulated by insulin in the skeletal muscle. To this end, we generated C2C12 myocytes in which HIF-1α is knocked down by short-hairpin RNA and examined the intracellular signaling cascade and glucose uptake subsequent to insulin stimulation. Knockdown of HIF-1α expression in the skeletal muscle cells resulted in abrogation of insulin-stimulated glucose uptake associated with impaired mobilization of glucose transporter 4 (GLUT4) to the plasma membrane. Such defect seemed to be caused by reduced phosphorylation of the protein kinase B substrate of 160 kDa (AS160). AS160 phosphorylation and GLUT4 translocation by AMP-activated protein kinase activation were abrogated as well. In addition, expression of the constitutively active mutant of HIF-1α (CA-HIF-1α) or upregulation of endogenous HIF-1α in C2C12 cells shows AS160 phosphorylation comparable to the insulin-stimulated level even in the absence of insulin. Accordingly GLUT4 translocation was increased in the cells expressing CA-HIF1α. Taken together, HIF-1α is a determinant for GLUT4-mediated glucose uptake in the skeletal muscle cells thus as a possible target to alleviate impaired glucose metabolism in, e.g., type 2 diabetes.

Hypoxia in Diabetic Kidneys

Diabetic nephropathy (DN) is now a leading cause of end-stage renal disease. In addition, DN accounts for the increased mortality in type 1 and type 2 diabetes, and then patients without DN achieve long-term survival compatible with general population. Hypoxia represents an early event in the development and progression of DN, and hypoxia-inducible factor- (HIF-) 1 mediates the metabolic responses to renal hypoxia. Diabetes induces the “fraternal twins” of hypoxia, that is, pseudohypoxia and hypoxia. The kidneys are susceptible to hyperoxia because they accept 20% of the cardiac output. Therefore, the kidneys have specific vasculature to avoid hyperoxia, that is, AV oxygen shunting. The NAD-dependent histone deacetylases (HDACs) sirtuins are seven mammalian proteins, SIRTs 1–7, which are known to modulate longevity and metabolism. Recent studies demonstrated that some isoforms of sirtuins inhibit the activation of HIF by deacetylation or noncatalyzing effects. The kidneys, which have a vascular system that protects them against hyperoxia, unfortunately experience extraordinary hypernutrition today. Then, an unexpected overload of glucose augments the oxygen consumption, which ironically results in hypoxia. This review highlights the primary role of HIF in diabetic kidneys for the metabolic adaptation to diabetes-induced hypoxia.

Dipeptidyl peptidase-4 inhibitor treatment induces a greater increase in plasma levels of bioactive GIP than GLP-1 in non-diabetic subjects

Objective Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) possess multiple bioactive isoforms that are rendered non-insulinotropic by the enzyme dipeptidyl peptidase-4 (DPP-4). Recently, some ELISA kits have been developed to specifically measure “active” GIP and GLP-1, but it is unclear if these kits can accurately quantify all bioactive forms. Therefore, it remains uncertain to what extent treatment with a DPP-4 inhibitor boosts levels of biologically active GIP and GLP-1. Thus, we evaluated our novel receptor-mediated incretin bioassays in comparison to commercially available ELISA kits using plasma samples from healthy subjects before and after DPP-4 inhibitor administration. Methods We utilized cell lines stably co-transfected with human GIP or GLP-1 receptors and a cAMP-inducible luciferase expression construct for the bioassays and commercially available ELISA kits. Assays were tested with synthetic GIP and GLP-1 receptor agonists and plasma samples collected from subjects during a 75 g oral glucose tolerance test (OGTT) performed before or following 3-day administration of a DPP-4 inhibitor. Results A GIP isoform GIP(1–30)NH2 increased luciferase activity similarly to GIP(1–42) in the GIP bioassay but was not detectable by either a total or active GIP ELISA kit. During an OGTT, total GIP levels measured by ELISA rapidly increased from 0 min to 15 min, subsequently reaching a peak of 59.2 ± 8.3 pmol/l at 120 min. In contrast, active GIP levels measured by the bioassay peaked at 15 min (43.4 ± 6.4 pmol/l) and then progressively diminished at all subsequent time points. Strikingly, at 15 min, active GIP levels as determined by the bioassay reached levels approximately 20-fold higher after the DPP-4 inhibitor treatment, while total and active GIP levels determined by ELISA were increased just 1.5 and 2.1-fold, respectively. In the absence of DPP-4 inhibition, total GLP-1 levels measured by ELISA gradually increased up to 90 min, reaching 23.5 ± 2.4 pmol/l, and active GLP-1 levels determined by the bioassay did not show any apparent peak. Following administration of a DPP-4 inhibitor there was an observable peak of active GLP-1 levels as determined by the bioassay at 15 min after oral glucose load, reaching 11.0 ± 0.62 pmol/l, 1.4-fold greater than levels obtained without DPP-4 inhibitor treatment. In contrast, total GLP-1 levels determined by ELISA were decreased after DPP-4 inhibitor treatment. Conclusion Our results using bioassays indicate that there is a greater increase in plasma levels of bioactive GIP than GLP-1 in subjects treated with DPP-4 inhibitors, which may be unappreciated using conventional ELISAs.