ABSTRACT
BACKGROUND AND PURPOSE: Heparanase is the only confirmed endoglycosidase that cleaves heparan sulfate (HS), a ubiquitous glycosaminoglycan with various essential roles in multiple pathological processes. Thus, the development of heparanase inhibitors has become an attractive strategy for drug discovery, especially in tumour therapy, in which HS mimetics are the most promising compounds. The various biological effects of heparanase also suggest a role for HS mimetics in many non-cancer indications, such as type 1 diabetes. However, the potential benefits of HS mimetics in obesity-related type 2 diabetes have not been elucidated. EXPERIMENTAL APPROACH: In this study, we investigated muparfostat (PI-88), a developed HS mimetic currently enrolled in Phase III clinical trials, in obese mouse models and in vitro cultured murine hepatocytes. KEY RESULTS: Daily administration of muparfostat for 4 weeks caused hyperlipidaemia and aggravated hepatic steatosis in obese mice models, but not in lean animals. In cultured hepatocytes, muparfostat did not alter lipid accumulation. Acute tests suggested that muparfostat binds to lipoprotein lipase in competition with HS on vascular endothelial cell surfaces, thereby reducing the degradation of circulating triglycerides by lipoprotein lipase and subsequent uptake of fatty acids into vascular endothelial cells and causing hyperlipidaemia. This hyperlipidaemia aggravates hepatic steatosis and causes liver injury in muparfostat-treated obese mice. CONCLUSIONS AND IMPLICATIONS: The binding activity of HS mimetics to lipoprotein lipase should be investigated as an additional pharmacological effect during heparanase inhibitor drug discovery. This study also provides novel evidence for an increased risk of drug-induced liver injury in obese individuals.
Subject(s)
Diabetes Mellitus, Type 2 , Fatty Liver , Animals , Mice , Endothelial Cells/metabolism , Heparitin Sulfate , Lipoprotein Lipase/metabolism , Mice, ObeseABSTRACT
Pancreatic islet ß cells secrete insulin in a biphasic manner when sensing high blood glucose level. This protocol describes the evaluation of different phases of insulin secretion, as well as basal, glucose-stimulated and total insulin secretion abilities, thereby enabling precise assessment of ß cell function both in vivo and ex vivo. The in vivo assay consists of intravenous tube imbedding surgery and hyperglycemic clamp. The ex vivo assay consists of islet isolation, dynamic perfusion and static immersion. For complete details on the use and execution of this protocol, please refer to Sun et al. (2021).
Subject(s)
Insulin Secretion/physiology , Islets of Langerhans/metabolism , Surgery, Veterinary/methods , Animals , Blood Glucose/metabolism , Glucose/metabolism , Hyperglycemia/metabolism , Insulin/blood , Insulin/metabolism , Insulin-Secreting Cells/metabolism , Islets of Langerhans/diagnostic imaging , Male , Mice , Mice, Inbred C57BLABSTRACT
The homeostatic balance of hepatic glucose uptake and production is exquisitely controlled by hormonal signals during feed-fast cycles. FoxO1, a transcription factor that functions in the regulation of glucose homeostasis, undergoes posttranslational modifications, such as acetylation, in response to hormonal signals, yet the mechanism remains poorly elucidated. Through expression profiling of 324 co-factors of CBP, a well-known acetyl-transferase of FoxO1, we identify Ets1 as a modulator of FoxO1 acetylation that is highly associated with feed-fast cycles. Mechanistic assays suggest that Ets1 enhances FoxO1 acetylation through the formation of a complex with CBP, which further promotes FoxO1 nuclear exclusion and inhibits its binding to gluconeogenic promoters. Functional studies further reveal that Ets1 inhibits gluconeogenesis under physiological and diabetes statuses, while the hyperinsulinemic-euglycemic clamp assay suggests hepatocyte Ets1 knockout mice have enhanced hepatic glucose production. Our study identifies Ets1 as an enhancer of FoxO1 acetylation and a repressor of hepatic gluconeogenesis in response to hormonal signals.
Subject(s)
Forkhead Box Protein O1/metabolism , Gluconeogenesis , Liver/metabolism , Proto-Oncogene Protein c-ets-1/metabolism , Acetylation , Animals , Cells, Cultured , HEK293 Cells , Humans , MAP Kinase Signaling System , Male , Mice , Mice, Inbred C57BL , Protein Binding , Protein Processing, Post-Translational , Proto-Oncogene Protein c-ets-1/geneticsABSTRACT
The recently popularized non-denaturing fluorescence in situ hybridization (ND-FISH) is a new technique that is both quick and efficient, in part because denaturing of both of the probes and the chromosomes is unnecessary. Synthetic simple sequence repeats (SSRs) labeled with fluorescein are used as probes to detect SSR-enriched chromosome regions and provide markers to identify the chromosomes. To date this method has not been applied to amphibians, even though the polymorphism of the distribution of SSRs may help to advance genetic polymorphism research. This paper also improved the double-colour FISH method by simultaneously using probes labelled with fluorescein and probes labelled with DIG to get double-color signals. This study found 5 SSRs markers that may be useful in the polymorphism research, and that the amphibian chromosomes must be denatured in ND-FISH.
