Kidney and liver are the main organs of expression of a key metabolic enzyme alanine:glyoxylate aminotransferase 2 in humans.

BACKGROUND: The metabolic syndrome is a cluster of cardiovascular risk factors and is highly predictive for development of cardiovascular diseases. An association between elevated plasma levels of the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) and risk of cardiovascular diseases has been demonstrated in numerous epidemiological studies. ADMA can be catabolized by dimethylarginine dimethylaminohydrolase (DDAH) or metabolized through a much less understood alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2) with the formation of α-keto-δ-(N,N-dimethylguanidino)valeric acid (ADGV). Previous RT-PCR and Western Blot studies suggested that Agxt2 is expressed in the mouse kidney and liver at comparable levels, while Northern Blot and in-situ RNA-hybridisation experiments demonstrated that the kidney is the main organ of Agxt2 expression in rats. Given this discrepancy, the goal of the current study was to analyse the expression of AGXT2 in human tissues. MATERIAL AND METHODS: We analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. RESULTS: We saw the strongest expression of AGXT2 in the kidney and liver and confirmed this results on protein level. By IHC staining we were able to show that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed mitochondrial localization of AGXT2. CONCLUSIONS: Our current data suggest that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans. We also demonstrated the mitochondrial localization of human AGXT2 enzyme.

Role of alanine:glyoxylate aminotransferase 2 in metabolism of asymmetric dimethylarginine in the settings of asymmetric dimethylarginine overload and bilateral nephrectomy.

BACKGROUND: Asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict complications and mortality in cardiovascular and renal diseases. Alanine:glyoxylate aminotransferase 2 (AGXT2) can metabolize both ADMA and SDMA; however, this metabolic pathway is still poorly understood. The goal of our study was to test the hypothesis that AGXT2 is compensatory upregulated in the settings of ADMA overload and bilateral nephrectomy. METHODS: ADMA was infused for 3 days using osmotic minipumps in mice. Half of the mice underwent bilateral nephrectomy 24 h before the end of the infusion. RESULTS: Infusion of ADMA caused a 3- to 4-fold increase in plasma and urine ADMA levels and a 2- to 3-fold increase in plasma and urine levels of the ADMA-specific metabolite of AGXT2 α-keto-δ-(N,N-dimethylguanidino)valeric acid (DMGV). Bilateral nephrectomy led to an ∼4-fold increase of plasma SDMA levels, but did not change plasma ADMA levels. Interestingly, plasma levels of DMGV were elevated 32-fold in the mice, which underwent bilateral nephrectomy. Neither bilateral nephrectomy nor ADMA infusion caused upregulation of AGXT2 expression or activity. CONCLUSIONS: Our data demonstrate that short-term elevation of systemic levels of ADMA leads to a dramatic increase of DMGV formation without upregulation of AGXT2 expression or activity, which suggests that AGXT2-mediated pathway of ADMA metabolism is not saturated under normal conditions and may play a major role in the maintenance of ADMA homeostasis in the setting of local or systemic elevation of ADMA levels.

Agonist of growth hormone-releasing hormone as a potential effector for survival and proliferation of pancreatic islets.

Therapeutic strategies for transplantation of pancreatic islet cells are urgently needed to expand beta-cell mass by stimulating islet cell proliferation and/or prolonging islet cell survival. Control of the islets by different growth factors provides a potential venue for augmenting beta-cell mass. In the present study, we show the expression of the biologically active splice variant-1 (SV-1) of growth hormone-releasing hormone (GHRH) receptor in rat insulinoma (INS-1) cells as well as in rat and human pancreatic islets. In studies in vitro of INS-1 cells, the GHRH agonist JI-36 caused a significant increase in cell proliferation and a reduction of cell apoptosis. JI-36 increased islet size and glucose-stimulated insulin secretion in isolated rat islets after 48-72 h. At the ultrastructural level, INS-1 cells treated with agonist JI-36 revealed a metabolic active stimulation state with increased cytoplasm. Coincubation with the GHRH antagonist MIA-602 reversed the actions of the agonist JI-36, indicating the specificity of this agonist. In vivo, the function of pancreatic islets was assessed by transplantation of rat islets under the kidney capsule of streptozotocin-induced diabetic non-obese diabetic-severe combined immunodeficiency (NOD-SCID) mice. Islets treated with GHRH agonist JI-36 were able to achieve normoglycemia earlier and more consistently than untreated islets. Furthermore, in contrast to diabetic animals transplanted with untreated islets, insulin response to an i.p. glucose tolerance test (IPGTT) in animals receiving islets treated with agonist Jl-36 was comparable to that of normal healthy mice. In conclusion, our study provides evidence that agonists of GHRH represent a promising pharmacological therapy aimed at promoting islet graft growth and proliferation in diabetic patients.

The vascular basement membrane: a niche for insulin gene expression and Beta cell proliferation.

Endocrine pancreatic beta cells require endothelial signals for their differentiation and function. However, the molecular basis for such signals remains unknown. Here, we show that beta cells, in contrast to the exocrine pancreatic cells, do not form a basement membrane. Instead, by using VEGF-A, they attract endothelial cells, which form capillaries with a vascular basement membrane next to the beta cells. We have identified laminins, among other vascular basement membrane proteins, as endothelial signals, which promote insulin gene expression and proliferation in beta cells. We further demonstrate that beta1-integrin is required for the beta cell response to the laminins. The proposed mechanism explains why beta cells must interact with endothelial cells, and it may apply to other cellular processes in which endothelial signals are required.

Nkx6.1 controls migration and axon pathfinding of cranial branchio-motoneurons.

As many studies have focused on the mechanisms of motoneuron specification, little is known about the factors that control the subsequent development of postmitotic motoneurons. Previously, we showed that the transcription factor Nkx6.1 is required for the early specification of somatic motoneuron progenitors in the spinal cord. Our present analysis of hindbrain motoneuron development in Nkx6.1-deficient mouse embryos reveals that the early specification of branchio-motoneurons is independent of Nkx6.1 function, but that it is required for their subsequent development. In Nkx6.1 mutant mice, we observed defects in the migration, as well as in the axon projections of branchio-motoneurons. A detailed analysis of the migratory defect in facial branchio-motoneurons reveals ectopic expression of the cell surface receptors Ret and Unc5h3 in premigratory neurons, but no changes in the rhombomeric environment. Taken together, our findings demonstrate a requirement for Nkx6.1 in the development of postmitotic motoneurons, and suggest a cell-autonomous function in the control of branchio-motoneuron migration.