Combined transcriptome and metabolome analyses of metformin effects reveal novel links between metabolic networks in steroidogenic systems.

Metformin is an antidiabetic drug, which inhibits mitochondrial respiratory-chain-complex I and thereby seems to affect the cellular metabolism in many ways. It is also used for the treatment of the polycystic ovary syndrome (PCOS), the most common endocrine disorder in women. In addition, metformin possesses antineoplastic properties. Although metformin promotes insulin-sensitivity and ameliorates reproductive abnormalities in PCOS, its exact mechanisms of action remain elusive. Therefore, we studied the transcriptome and the metabolome of metformin in human adrenal H295R cells. Microarray analysis revealed changes in 693 genes after metformin treatment. Using high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS-NMR), we determined 38 intracellular metabolites. With bioinformatic tools we created an integrated pathway analysis to understand different intracellular processes targeted by metformin. Combined metabolomics and transcriptomics data analysis showed that metformin affects a broad range of cellular processes centered on the mitochondrium. Data confirmed several known effects of metformin on glucose and androgen metabolism, which had been identified in clinical and basic studies previously. But more importantly, novel links between the energy metabolism, sex steroid biosynthesis, the cell cycle and the immune system were identified. These omics studies shed light on a complex interplay between metabolic pathways in steroidogenic systems

Generation and utilization of NADPH in the endoplasmic reticulum : novel insight into the role of luminal NADPH in pathophysiological processes

Increasing evidence emphasizes the importance of the redox balance in the endoplasmic reticulum (ER). Disturbance of redox regulation can cause ER stress and contribute to the development of metabolic disease, cancer and neurodegenerative disorders. Nevertheless, the mechanisms underlying the well-regulated NADPH balance, and the generation and utilization of pyridine nucleotides in the luminal compartment are insufficiently understood. The aim of this work is to identify novel components involved in NADPH regulation in the ER. Due to the observation that fructose-6-phosphate stimulates luminal NADPH generation, and enhances 11ß-hydroxysteroid dehydrogenase 1 dependent glucocorticoid activation, we hypothesized the existence of a luminal hexose-6-phosphate isomerase. Using microsomal fractions, we characterized a novel luminal hexose-6-phosphate isomerase, which converts fructose-6-phosphate to glucose-6-phosphate. By further purification and protein sequencing, we try to identify the gene encoding this enzyme. In order to identify additional genes encoding luminal enzymes involved in NAPDH generation in the ER (potential enzymes of the luminal pentose-phosphate pathway), we decided to apply a combination of classical activity-guided purification, mass-spectrometric analysis and sequence analysis. Furthermore, for promising candidate proteins, we attempt to confirm their intracellular localization and investigate their impact on luminal NADPH balance. To determine whether ER-associated and membrane proteins are facing the cytoplasmic or luminal compartment, we optimized the methods to determine membrane topology and intracellular localization. We used selective semi-permeabilization analysis using digitonin, followed by immunodetection and confocal microscopy, proteinase protection assays of microsomal preparations as well as glycosylation assays. Furthermore, we determined the membrane topology of 17ß-hydroxysteroid dehydrogenase 3, an enzyme responsible for the oxoreduction of androstenedione. We provide information on the functional impact of hexose-6-phosphate dehydrogenase, as well as the nutritional state of the cell on the formation of testosterone. The findings are relevant regarding the understanding of the coupling between the cellular energy state, hormonal regulation, ER redox regulation and oxidative stress-induced damage in a cell

Vitamin D-Dependent Rickets Type 1 Caused by Mutations in CYP27B1 Affecting Protein Interactions With Adrenodoxin

Abstract Context: CYP27B1 converts 25-hydroxyvitamin D3 to active 1,25-dihydroxyvitamin D3, playing a vital role in calcium homeostasis and bone growth. Vitamin D-dependent rickets type 1 (VDDR-1) is a rare autosomal recessive disorder caused by mutations in CYP27B1. Objective: The objective of the study was an enzymatic and structural analysis of mutations in a patient with calcipenic rickets. Design, Setting, Patient, and Intervention: Two siblings presented with calcipenic rickets and normal 1,25-dihydroxyvitamin D3 levels. CYP27B1 gene analysis showed compound heterozygous mutations confirming VDDR-1. We studied wild-type CYP27B1 and mutations H441Y and R459L by computational homology modeling, molecular dynamics simulations, and functional studies using a luciferase assay. The patients were successfully treated with calcitriol. Main Outcome: The main outcomes of the study were novel mutations leading to a severe loss of CYP27B1 activities for metabolism of 25-hydroxyvitamin D3. Results: Mitochondrial cytochrome P450s require adrenodoxin (FDX1) and adrenodoxin reductase. We created models of CYP27B1-FDX1 complex, which revealed negative effects of mutations H441Y and R459L. Upon structural analysis, near-identical folds, protein contact areas, and orientations of heme/iron-sulfur cluster suggested that both mutations may destabilize the CYP27B1-FDX1 complex by negating directional interactions with adrenodoxin. This system is highly sensitive to small local changes modulating the binding/dissociation of adrenodoxin, and electron-transporting efficiency might change with mutations at the surface. Functional assays confirmed this hypothesis and showed severe loss of activity of CYP27B1 by both mutations. Conclusions: This is the first report of mutations in CYP27B1 causing VDDR-1 by affecting protein-protein interactions with FDX1 that results in reduced CYP27B1 activities. Detailed characterization of mutations in CYP27B1 is required for understanding the novel molecular mechanisms causing VDDR-1

Vitamin D-Dependent Rickets Type 1 Caused by Mutations in CYP27B1 Affecting Protein Interactions With Adrenodoxin.

CONTEXT CYP27B1 converts 25-hydroxyvitamin D3 to active 1,25-dihydroxyvitamin D3, playing a vital role in calcium homeostasis and bone growth. Vitamin D-dependent rickets type 1 (VDDR-1) is a rare autosomal recessive disorder caused by mutations in CYP27B1. OBJECTIVE The objective of the study was an enzymatic and structural analysis of mutations in a patient with calcipenic rickets. Design, Setting, Patient, and Intervention: Two siblings presented with calcipenic rickets and normal 1,25-dihydroxyvitamin D3 levels. CYP27B1 gene analysis showed compound heterozygous mutations confirming VDDR-1. We studied wild-type CYP27B1 and mutations H441Y and R459L by computational homology modeling, molecular dynamics simulations, and functional studies using a luciferase assay. The patients were successfully treated with calcitriol. MAIN OUTCOME The main outcomes of the study were novel mutations leading to a severe loss of CYP27B1 activities for metabolism of 25-hydroxyvitamin D3. RESULTS Mitochondrial cytochrome P450s require adrenodoxin (FDX1) and adrenodoxin reductase. We created models of CYP27B1-FDX1 complex, which revealed negative effects of mutations H441Y and R459L. Upon structural analysis, near-identical folds, protein contact areas, and orientations of heme/iron-sulfur cluster suggested that both mutations may destabilize the CYP27B1-FDX1 complex by negating directional interactions with adrenodoxin. This system is highly sensitive to small local changes modulating the binding/dissociation of adrenodoxin, and electron-transporting efficiency might change with mutations at the surface. Functional assays confirmed this hypothesis and showed severe loss of activity of CYP27B1 by both mutations. CONCLUSIONS This is the first report of mutations in CYP27B1 causing VDDR-1 by affecting protein-protein interactions with FDX1 that results in reduced CYP27B1 activities. Detailed characterization of mutations in CYP27B1 is required for understanding the novel molecular mechanisms causing VDDR-1

Vitamin D-Dependent Rickets Type 1 Caused by Mutations in CYP27B1 Affecting Protein Interactions With Adrenodoxin

CONTEXT CYP27B1 converts 25-hydroxyvitamin D3 to active 1,25-dihydroxyvitamin D3, playing a vital role in calcium homeostasis and bone growth. Vitamin D-dependent rickets type 1 (VDDR-1) is a rare autosomal recessive disorder caused by mutations in CYP27B1. OBJECTIVE The objective of the study was an enzymatic and structural analysis of mutations in a patient with calcipenic rickets. Design, Setting, Patient, and Intervention: Two siblings presented with calcipenic rickets and normal 1,25-dihydroxyvitamin D3 levels. CYP27B1 gene analysis showed compound heterozygous mutations confirming VDDR-1. We studied wild-type CYP27B1 and mutations H441Y and R459L by computational homology modeling, molecular dynamics simulations, and functional studies using a luciferase assay. The patients were successfully treated with calcitriol. MAIN OUTCOME The main outcomes of the study were novel mutations leading to a severe loss of CYP27B1 activities for metabolism of 25-hydroxyvitamin D3. RESULTS Mitochondrial cytochrome P450s require adrenodoxin (FDX1) and adrenodoxin reductase. We created models of CYP27B1-FDX1 complex, which revealed negative effects of mutations H441Y and R459L. Upon structural analysis, near-identical folds, protein contact areas, and orientations of heme/iron-sulfur cluster suggested that both mutations may destabilize the CYP27B1-FDX1 complex by negating directional interactions with adrenodoxin. This system is highly sensitive to small local changes modulating the binding/dissociation of adrenodoxin, and electron-transporting efficiency might change with mutations at the surface. Functional assays confirmed this hypothesis and showed severe loss of activity of CYP27B1 by both mutations. CONCLUSIONS This is the first report of mutations in CYP27B1 causing VDDR-1 by affecting protein-protein interactions with FDX1 that results in reduced CYP27B1 activities. Detailed characterization of mutations in CYP27B1 is required for understanding the novel molecular mechanisms causing VDDR-1

Direct protein-protein interaction of 11 beta-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase in the endoplasmic reticulum lumen

Hexose-6-phosphate dehydrogenase (H6PDH) has been shown to stimulate 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1)-dependent local regeneration of active glucocorticoids. Here, we show that coexpression with H6PDH results in a dramatic shift from 11 beta-HSD1 oxidase to reductase activity without affecting the activity of the endoplasmic reticular enzyme 17 beta-HSD2. Immunoprecipitation experiments revealed coprecipitation of H6PDH with 11 beta-HSD1 but not with the related enzymes 11 beta-HSD2 and 17 beta-HSD2, suggesting a specific interaction between H6PDH and 11 beta-HSD1. The use of the 11 beta-HSD1/11 beta-HSD2 chimera indicates that the N-terminal 39 residues of 11 beta-HSD1 are sufficient for interaction with H6PDH. An important role of the N-terminus was indicated further by the significantly stronger interaction of 11 beta-HSD1 mutant Y18-21A with H6PDH compared to wild-type 11 beta-HSD1. The protein-protein interaction and the involvement of the N-terminus of 11 beta-HSD1 were confirmed by Far-Western blotting. Finally, fluorescence resonance energy transfer (FRET) measurements of HEK-293 cells expressing fluorescently labeled proteins provided evidence for an interaction between 11 beta-HSD1 and H6PDH in intact cells. Thus, using three different methods, we provide strong evidence that the functional coupling between 11 beta-HSD1 and H6PDH involves a direct physical interaction of the two proteins. (C) 2008 Elsevier B.V. All rights reserved