Glycolytic enzyme expression and pyruvate kinase activity in cultured fibroblasts from type 1 diabetic patients with and without nephropathy

Abstract

Since type 1 diabetes mellitus (T1DM) patients with nephropathy (DN+) are insulin-resistant, we aimed to identify (new) potential molecular sites involved in the alterations of glucose metabolism in these patients. We examined the expression of glycolytic enzymes in cultured fibroblasts from T1DM(DN+) patients as compared to those from T1DM patients without nephropathy (DN-) and from controls. Pyruvate kinase (PK) activity was also determined. Human skin fibroblasts were grown in normal glucose (6 mM). RNAs and proteins were analyzed, respectively, using cRNA microarray and two-dimensional electrophoresis followed by identification with mass spectrometry. PK activity was measured using a spectrophotometric assay. As compared to controls, increases in the gene expression of hexokinase, phosphoglucomutase, phosphofructokinase, aldolase and triosephosphate isomerase were found in T1DM(DN+) patients, but not in T1DM(DN-) patients. In T1DM(DN+) patients, the protein analysis showed an altered expression of three glycolytic enzymes: triosophosphate isomerase, enolase and PK. In addition, PK activity in fibroblasts from T1DM(DN+) patients was lower than that in T1DM(DN-) and in controls. In conclusion, this study reports novel alterations of enzymes involved in glucose metabolism that may be associated with the pathophysiology of insulin resistance and of renal damage in T1DM(DN+) patients.

Early urinary biomarkers of diabetic nephropathy in type 1 diabetes mellitus show involvement of kallikrein-kinin system

“…Kidney has been established as an important site of glucose metabolism and increased oxidative stress due to impaired glycolysis is considered the major culprit responsible for development of angiopathy and organ complications [16–18]. Glycolytic flux is blocked upstream from glyceraldehyde-3-phosphate dehydrogenase (G3P) which is supposed to play a central role in the dysregulation.…”

Proteomics Reveals Novel Oxidative and Glycolytic Mechanisms in Type 1 Diabetic Patients' Skin Which Are Normalized by Kidney-Pancreas Transplantation

“…In recent years, the number of novel proteins identified by genomic and proteomic research projects has dramatically increased, with a concomitant more rapid characterization of molecular processes of living cells through large-scale studies in specific biological contexts. We reasoned that the proteomic approach might offer a powerful tool to assess differences in protein expression associated with diabetes and renal complications, as few papers have described the use of this technique in diabetes [18], [19].…”

Stable Isotope Labeling with Amino Acids (SILAC)-Based Proteomics of Primary Human Kidney Cells Reveals a Novel Link between Male Sex Hormones and Impaired Energy Metabolism in Diabetic Kidney Disease

“…As an example, in cultured fibroblasts from type 1 diabetic patients with and without nephropathy, mRNA levels of GPI and other enzymes of the glycolytic pathway were found to be increased in the context of DKD (62). In turn, HADHA and HADHB, as subunits of the mitochondrial trifunctional protein, are involved not only in FAO but also in molecular events relevant to kidney disease, such as lysine acetylation (63), and modulation of renin expression (64), respectively.…”