New emerging role of protein-tyrosine phosphatase 1B in the regulation of glycogen metabolism in basal and TNF-α-induced insulin-resistant conditions in an immortalised muscle cell line isolated from mice

Abstract

Aims/hypothesis Protein-tyrosine phosphatase 1B (PTP1B) negatively regulates insulin action, promoting attenuation of the insulin signalling pathway. The production of this phosphatase is enhanced in insulin-resistant states, such as obesity and type 2 diabetes, where high levels of proinflammatory cytokines (TNF-α, IL-6) are found. In these metabolic conditions, insulin action on glycogen metabolism in skeletal muscle is greatly impaired. We addressed the role of PTP1B on glycogen metabolism in basal and insulin-resistant conditions promoted by TNF-α. Methods We studied the effect of TNF-α in the presence and absence of insulin on glycogen content and synthesis, glycogen synthase (GS) and glycogen phosphorylase (GP) activities and on glycogen synthesis and degradation signalling pathways. For this purpose we used immortalised cell lines isolated from skeletal muscle from mice lacking PTP1B. Results Absence of PTP1B caused activation of GS and GP with a net glycogenolytic effect, reflected in lower amounts of glycogen and activation of the glycogenolytic signalling pathway, with higher rates of phosphorylation of cyclic adenosine monophosphate-dependent kinase (PKA), phosphorylase kinase (PhK) and GP phosphorylation. Nevertheless, insulin action was strongly enhanced in Ptp1b (also known as Ptpn1)−/− cells in terms of glycogen content, synthesis, GS activation rates and GS Ser641 dephosphorylation. Treatment with TNF-α augmented the activity ratios of both GS and GP, and impaired insulin stimulation of glycogen synthesis in wild-type myocytes, whereas Ptp1b −/− myocytes restored this inhibitory effect. We report a glycogenolytic effect of TNF-α, as demonstrated by greater activation of the degradation signalling cascade PKA/PhK/GP. In our model, this effect is mediated by the activation of PKA. Conclusions/interpretation We provide new data about the role of PTP1B in glycogen metabolism and confirm the beneficial effect that absence of the phosphatase confers against an insulin-resistant condition.

Inducible liver-specific knockdown of protein tyrosine phosphatase 1B improves glucose and lipid homeostasis in adult mice

“…At the moment, it is unclear how hepatic PTP1B inhibition affects GS phosphorylation independently of its effects on the IR; however, PTP1B has been shown to regulate protein phosphatase 2 (PP2A) activation [36] as well as regulate hepatic Srebp1 gene expression through the PP2A axis [37], which may then affect the GS hepatic phosphorylation state [38]. This is currently under investigation in our laboratory, but is consistent with data from Ptp1b −/− immortalised cells treated with insulin, which were also shown to exhibit enhanced dephosphorylation of the S641 site on GS [39]. Liver Ptp1b deletion has previously been shown to decrease serum triacylglycerol with lower expression of lipogenic genes [17].…”

Prevalent role of the insulin receptor isoform A in the regulation of hepatic glycogen metabolism in hepatocytes and in mice

“…Glycogen was precipitated when the homogenates were transferred onto Whatman 3MM (Sigma-Aldrich) paper with 66% ethanol (vol./vol.) at −20°C, as previously described [20]. Precipitated glycogen was released in the form of glucose monomers via incubation with 1.6 mg/ml of α-amyloglucosidase (Sigma-Aldrich) for 4 h at 37°C, in 0.4 mol/l acetate buffer (pH 4.8).…”

“…At 70-80% confluence, cells were starved for 6 h in serum-free DMEM (5 mmol/l glucose) with 0.2% BSA (wt/vol.). At the moment of stimulation with 10 or 100 nmol/l insulin, the medium was changed to DMEM (25 mmol/l glucose) with 10 mmol/l of [U-14 C]glucose (11.1 GBq/μmol; PerkinElmer, Waltham, MA, USA), as previously described [20]. After glycogen precipitation using ice-cold 66% ethanol (vol./vol.…”