plate-based-large-scale-cultivation-caenorhabditis-elegans-sample

Plate-based Large-scale Cultivation of <em>Caenorhabditis elegans</em>: Sample Preparation for the Study of Metabolic Alterations in Diabetes

Culturing Caenorhabditis elegans (C. elegans) in a large-scale manner on agar plates can be time-consuming and difficult. This protocol describes a simple ...

Heidelberg University

Glucose derived metabolism generates reactive metabolites affecting the neuronal system and lifespan in C. elegans. Here, the role of the insulin homologue ins-7 and its downstream effectors in the generation of high glucose induced neuronal damage and shortening of lifespan was studied.

Reduction in ins-7 gene expression in non-neuronal cells of high glucose exposed Caenorhabditis elegans protects from reactive metabolites, preserves neuronal structure and head motility, and prolongs lifespan.

The aim of this study was to evaluate damage to the neurovascular unit in a mouse model of hyperglycaemic memory.

Hyperglycaemic memory affects the neurovascular unit of the retina in a diabetic mouse model.

Diabetic retinopathy (DR) is induced by an accumulation of reactive metabolites such as ROS, RNS, and RCS species, which were reported to modulate the activity of cation channels of the TRPC family. In this study, we use Trpc1/4/5/6 compound knockout mice to analyze the contribution of these TRPC proteins to diabetic retinopathy.

TRPC proteins contribute to development of diabetic retinopathy and regulate glyoxalase 1 activity and methylglyoxal accumulation.

The enzyme AICAR-transformylase/IMP cyclohydrolase (ATIC) catalyzes the last two steps of purine  synthesis. It metabolizes 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which is an AMP analogue, leading to activation of AMP-activated kinase (AMPK). We investigated whether the AICAR-ATIC pathway plays a role in the high glucose (HG)-mediated DNA damage response and AICAR-mediated AMPK activation, explaining the detrimental effects of glucose on neuronal damage and shortening of the lifespan. HG up-regulated the expression and activity of the  homologue of ATIC, C55F2.1 (), and increased the levels of reactive oxygen species and methylglyoxal-derived advanced glycation end products. Overexpression of  decreased the lifespan and head motility and increased neuronal damage under both standard and HG conditions. Inhibition of  expression, by RNAi, under HG was associated with increased lifespan and head motility and reduced neuronal damage, reactive oxygen species, and methylglyoxal-derived advanced glycation end product accumulation. This effect was independent of an effect on DNA damage or antioxidant defense pathways, such as superoxide dismutase () or glyoxalase-1 (), but was dependent on AMPK and accumulation of AICAR. Through AMPK, AICAR treatment also reduced the negative effects of HG. The mitochondrial inhibitor rotenone abolished the AICAR/AMPK-induced amelioration of HG effects, pointing to mitochondria as a prime target of the glucotoxic effects in  We conclude that  is involved in glucotoxic effects under HG conditions, either by blocked  expression or via AICAR and AMPK induction.

High-glucose toxicity is mediated by AICAR-transformylase/IMP cyclohydrolase and mitigated by AMP-activated protein kinase in .

Glucose and glucose degradation products (GDPs), contained in peritoneal dialysis (PD) fluids, contribute to the formation of advanced glycation end-products (AGEs). Local damaging effects, resulting in functional impairment of the peritoneal membrane, are well studied. It is also supposed that detoxification of AGE precursors by glyoxalase-1 (GLO1) has beneficial effects on GDP-mediated toxicity. The aim of the current study was to analyze systemic detrimental effects of PD fluids and their prevention by glyoxlase-1. Wild-type and GLO1-overexpressing  () were cultivated in the presence of low- and high-GDP PD fluids containing 1.5 or 4% glucose. Lifespan, neuronal integrity and neuronal functions were subsequently studied. The higher concentrations of glucose and GDP content resulted in a decrease of maximum lifespan by 2 (P<0.01) and 9 days (P<0.001), respectively. Exposure to low- and high-GDP fluids caused reduction of neuronal integrity by 34 (P<0.05) and 41% (P<0.05). Cultivation of animals in the presence of low-GDP fluid containing 4% glucose caused significant impairment of neuronal function, reducing relative and absolute head motility by 58.5 (P<0.01) and 56.7% (P<0.01), respectively; and relative and absolute tail motility by 55.1 (P<0.05) and 55.0% (P<0.05), respectively. Taken together, GLO1 overexpression protected from glucose-induced lifespan reduction, neurostructural damage and neurofunctional damage under low-GDP-conditions. In conclusion, both glucose and GDP content in PD fluids have systemic impact on the lifespan and neuronal integrity of . Detoxification of reactive metabolites by GLO1 overexpression was sufficient to protect lifespan, neuronal integrity and neuronal function in a low-GDP environment. These data emphasize the relevance of the GLO1 detoxifying pathway as a potential therapeutic target in the treatment of reactive metabolite-mediated pathologies.

Article	Year
Reduction in ins-7 gene expression in non-neuronal cells of high glucose exposed Caenorhabditis elegans protects from reactive metabolites, preserves neuronal structure and head motility, an... Journal of diabetes and its complications\| PubMed ID: 27776915	2017
Hyperglycaemic memory affects the neurovascular unit of the retina in a diabetic mouse model. Diabetologia\| PubMed ID: 28321468	2017
TRPC proteins contribute to development of diabetic retinopathy and regulate glyoxalase 1 activity and methylglyoxal accumulation. Molecular metabolism\| PubMed ID: 29373286	2018
High-glucose toxicity is mediated by AICAR-transformylase/IMP cyclohydrolase and mitigated by AMP-activated protein kinase in . The Journal of biological chemistry\| PubMed ID: 29414769	2018
Neuronal damage and shortening of lifespan in by peritoneal dialysis fluid: Protection by glyoxalase-1. Biomedical reports\| PubMed ID: 29805788	2018

Andrea Schlotterer

Biography

JoVE Journal Publications

Other Publications