AMPK differentially alters sulphated glycosaminoglycans under normal and high glucose milieu in proximal tubular cells.

Glycosaminoglycans (GAGs) and AMP-activated protein kinase (AMPK) are two critical molecular players involved in cellular homeostasis. Both of them are altered due to hyperglycaemia in the kidney, leading to the pathogenesis of diabetic nephropathy. Here, we have looked into the effect of AMPK modulation on sulphated GAG (sGAG) levels of tubular cells of proximal and distal origin to understand the mechanism of hyperglycaemia-mediated pathogenesis of the diabetic nephropathy. In MDCK cells (distal tubular cell) and NRK-52E (proximal tubular cell), AMPK inhibition resulted in increased sGAG levels under normal glucose conditions characteristically of heparan sulphate class, whereas AMPK activation did not have any effect. High glucose (HG) condition did not alter sGAG levels in MDCK cell despite a decrease in AMPK phosphorylation. Subjecting NRK-52E cells to HG milieu significantly decreased sGAG levels more so of chondroitin/dermatan sulphate, which is significantly prevented when HG is co-treated with AMPK activator. Interestingly, knockdown of AMPK by AMPKα1/α2 siRNA showed increased sGAG levels in NRK-52E. Our results suggest that changes in sGAG level, in particular, as a result of AMPK modulation is differentially regulated and is dependent on cell type as well as its physiological status. Furthermore, activation of AMPK is beneficial in preventing the HG-mediated decrease in sGAGs in proximal tubular cells.

AMPK in microvascular complications of diabetes and the beneficial effects of AMPK activators from plants.

BACKGROUND: Diabetes mellitus is a multifactorial disorder with the risk of micro- and macro-vascular complications. High glucose-induced derangements in metabolic pathways are primarily associated with the initiation and progression of secondary complications namely, diabetic nephropathy, neuropathy, and retinopathy. Adenosine monophosphate-activated protein kinase (AMPK) has emerged as an attractive therapeutic target to treat various metabolic disorders including diabetes mellitus. It is a master metabolic regulator that helps in maintaining cellular energy homeostasis by promoting ATP-generating catabolic pathways and inhibiting ATP-consuming anabolic pathways. Numerous pharmacological and plant-derived bioactive compounds that increase AMP-activated protein kinase activation has shown beneficial effects by mitigating secondary complications namely retinopathy, nephropathy, and neuropathy. PURPOSE: The purpose of this review is to highlight current knowledge on the role of AMPK and its activators from plant origin in diabetic microvascular complications. METHODS: Search engines such as Google Scholar, PubMed, Science Direct and Web of Science are used to extract papers using relevant key words. Papers mainly focusing on the role of AMPK and AMPK activators from plant origin in diabetic nephropathy, retinopathy, and neuropathy was chosen to be highlighted. RESULTS: According to results, decrease in AMPK activation during diabetes play a causative role in the pathogenesis of diabetic microvascular complications. Some of the plant-derived bioactive compounds were beneficial in restoring AMPK activity and ameliorating diabetic microvascular complications. CONCLUSION: AMPK activators from plant origin are beneficial in mitigating diabetic microvascular complications. These pieces of evidence will be helpful in the development of AMPK-centric therapies to mitigate diabetic microvascular complications.

One-pot analysis of sulfated glycosaminoglycans.

Routine isolation, estimation, and characterization of glycosaminoglycans (GAGs) is quite challenging. This is compounded by the fact that the analysis is technique-intensive and more often there will be a limitation on the quantity of GAGs available for various structural, functional and biological studies. In such a scenario, the sample which can be made available for estimation and elucidation of disaccharide composition and species composition as well remains a challenge. In the present study, we have determined the feasibility where isolated sulfated GAGs (sGAG) that is estimated by metachromasia is recovered for further analysis. sGAG-DMMB complex formed after estimation of sGAG by DMMB dye-binding assay was decomplexed and sGAGs were recovered. Recovered sGAGs were analysed by cellulose acetate membrane electrophoresis and taken up for disaccharide composition analysis by HPLC after fluorescent labelling. Good recovery of sGAGs after metachromasia was observed in all samples of varying levels of purity by this protocol. Further analysis using cellulose acetate membrane electrophoresis showed good separation between species of sGAGs namely chondroitin/dermatan sulfate and heparan sulfate, with comparatively lesser interference from hyaluronic acid, a non-sulfated GAG. Analysis of recovered sGAGs, specifically heparan sulfate by HPLC showed characteristic disaccharide composition akin to that of GAG obtained by the conventional protocol. Thus, in the present paper, we show that sGAG can be recovered in comparatively purer form after routine estimation and can be used for further analysis thus saving up on the precious sample.