Tài liệu Báo cáo khoa học: The diacylglycerol and protein kinase C pathways are not involved in

The diacylglycerol and protein kinase C pathways are not involved

in insulin signalling in primary rat hepatocytes

Irmelin Probst1

, Ulrich Beuers2

, Birgit Drabent1

, Kirsten Unthan-Fechner1 and Peter Bu¨ tikofer3

1

Institut fu¨r Biochemie und Molekulare Zellbiologie, Georg-August – Universita¨t Go¨ttingen, Germany; 2

Medizinische Klinik II￾Großhadern, Ludwig-Maximilians-Universita¨t Mu¨nchen, Germany; 3

Institut fu¨r Biochemie und Molekularbiologie,

Universita¨t Bern, Switzerland

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 dexa￾methasone, 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 sev￾eral 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, insu￾lin showed no effect on DAG mass or translocation of PKC

isoforms in the perfused liver, although it reduced the glu￾cagon-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 mole￾cular 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 gluconeo￾genesis 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 kin￾ase B/glycogen synthase kinase-3 cascade. Recent data

suggest that a third signalling pathway, downstream of

phosphatidylinositol 3-kinase, may also be involved: phos￾pholipase 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 [1–3]. These studies, which

were performed on muscle and fat cells, showed insulin￾dependent increases in lipid mediator concentrations [4–7]

and translocation and activation of various PKC isoforms

[6–13], suggesting their probable involvement in insulin

action [8,10,14,15].

In contrast, the available data on hepatic systems are

scarce, controversial and have been obtained using primary

adult rat hepatocyte suspensions and cultures, and different

hepatoma cell lines, as model systems. In hepatocyte

suspensions, insulin provoked increases in DAG mass

[16,17], whereas activation of PLD was demonstrated by

two groups [17,18], but not by another group [19]. Similarly,

activation of PKC was demonstrated, in two reports, in

both cytosolic and membrane fractions of crude extracts

[16,20], but not in a third [21]. Furthermore, activation of

atypical PKCf was demonstrated in hepatocytes cultured

without glucocorticoid for 3 days [22], whereas two other

reports showed enhanced translocation of the d isoform in

different hepatoma cell lines [23,24].

Correspondence to I. Probst, Institut fu¨r Biochemie und Molekulare

Zellbiologie, Humboldtallee 23, 37073 Go¨ttingen, Germany.

Fax: + 49 551 395960, Tel.: + 49 551 395961,

E-mail: [email protected]

Abbreviations: DAG, diacylglycerol; EGF, epidermal growth factor;

IRSs, insulin receptor substrates; ODN, oligodesoxynucleotides;

PA, phosphatidic acid; PMA, phorbol 12-myristate 13-acetate;

PKC, protein kinase C; PLC, phospholipase C; PLD,

phospholipase D; TGF-a, transforming growth factor-a.

Enzymes: phospholipase C (EC 3.1.4.3); phospholipase D

(EC 3.1.4.4); protein kinase C (EC 2.7.1.37).

(Received 25 April 2003, revised 26 August 2003,

accepted 25 September 2003)

Eur. J. Biochem. 270, 4635–4646 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03853.x