The diacylglycerol and protein kinase C pathways are not involved in insulin signalling in primary rat hepatocytes

Abstract

Diacylglycerol (DAG) and protein kinase C (PKC) isoforms have been implicated in insulin signalling in muscle and fat cells. We evaluated the involvement of DAG and PKC in the action of insulin in adult rat hepatocytes cultured with dexamethasone, but in the absence of serum, for 48 h. Our results show that although insulin stimulated glycolysis and glycogen synthesis, it had no effect on DAG mass or molecular species composition. Epidermal growth factor showed the expected insulin-mimetic effect on glycolysis, whereas ATP and exogenous phospholipase C acted as antagonists and abolished the insulin signal. Similarly to insulin, epidermal growth factor had no effect on DAG mass or molecular species composition. In contrast, both ATP and phospholipase C induced a prominent increase in several DAG molecular species, including 18:0/20:4, 18:0/20:5, 18:0/22:5 and a decrease in 18:1/18:1. These changes were paralleled by an increase in phospholipase D activity, which was absent in insulin-treated cells. By immunoblotting or by measuring PKC activity, we found that neither insulin nor ATP translocated the PKCa, -d, -e or -f isoforms from the cytosol to the membrane in cells cultured for six or 48 h. Similarly, insulin had no effect on immunoprecipitable PKCf. Suppression of the glycogenic insulin signal by phorbol 12-myristate 13-acetate, but not by ATP, could be completely alleviated by bisindolylmaleimide. Finally, insulin showed no effect on DAG mass or translocation of PKC isoforms in the perfused liver, although it reduced the glucagon-stimulated glucose output by 75%. Together these results indicate that phospholipases C and D or multiple PKC isoforms are not involved in the hepatic insulin signal chain.Keywords: hepatocytes; insulin; ATP; diacylglycerol molecular species; protein kinase C.Among the three major insulin-sensitive organs, i.e. liver, muscle and fat tissue, the liver plays a key role in the regulation of blood glucose homeostasis by channelling excess glucose into glycogen after food uptake and by producing glucose through glycogenolysis and gluconeogenesis in the states of hunger and starvation. Insulin, the dominant hormone of the absorptive phase, acts via receptor-mediated tyrosine phosphorylation of insulin receptor substrates (IRSs). Two well established signalling cascades are initiated when adaptor proteins are recruited to the IRSs through their src homology 2 domains (a) the growth factor receptor binding protein activates the ras/ mitogen-activated protein kinase pathway and (b) phosphatidylinositol 3-kinase activates the protein kinase B/glycogen synthase kinase-3 cascade. Recent data suggest that a third signalling pathway, downstream of phosphatidylinositol 3-kinase, may also be involved: phospholipase D (PLD)-dependent generation of phosphatidic acid (PA) and diacylglycerol (DAG), with subsequent activation of DAG-insensitive atypical protein kinase C (PKC) isozymes such as f and k, as well as activation of DAG-sensitive PKC isozymes [1][2][3]. These studies, which were performed on m...

Glucagon and Cyclic AMP: Time to Turn the Page?

“…The remaining 40 -50% of the PK phosphorylation and further enhancement of glucose output is presumably dependent on PKA activation and further phosphorylation of PK by higher concentrations of the hormone. Although phorbol esters can mimic some of the hepatic actions of insulin [198], insulin itself does not appear to act via this pathway in producing its rapid metabolic effects on this tissue [199,200]. The collective results thus strongly suggest that activation of the high-affinity glucagon receptor by physiological concentrations substantially inhibits PK, and consequently promotes gluconeogenesis, by activating the PLC/IP3/DAG signal pathway and CaCAMK, without affecting AC or increasing the production of cAMP.…”

Maintaining hepatocyte differentiation in vitro through co-culture with hepatic stellate cells

“…Four dishes each of 3×10 6 HSCs or hepatocytes were cultured for 24 h and used for extraction exactly as described previously (Probst et al 2003). The chloroform phase was transferred into a sterile 50-ml glass medium flask and the solvent was evaporated under N 2 in the dark.…”

Specific protein kinase C isoforms as transducers and modulators of insulin signaling

“…On the one hand, in a recent report [113] it was concluded that PKCδ (as well as isoforms α, ε or ς) was not stimulated by insulin and apparently not involved in insulin effects on primary rat hepatocytes. In this study, the investigators relied on DAG release and translocation of PKC to the plasma membrane as indicators of PKC stimulation.…”