The diacylglycerol and protein kinase C pathways are not involved
in insulin signalling in primary rat hepatocytes
Irmelin Probst
1
, Ulrich Beuers
2
, Birgit Drabent
1
, Kirsten Unthan-Fechner
1
and Peter Bu¨ tikofer
3
1
Institut fu
¨
r Biochemie und Molekulare Zellbiologie, Georg-August – Universita
¨
tGo
¨
ttingen, Germany;
2
Medizinische Klinik II-
Großhadern, Ludwig-Maximilians-Universita
¨
tMu
¨
nchen, Germany;
3
Institut fu
¨
r Biochemie und Molekularbiologie,

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,aswellasactivationof
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]

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) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03853.x
The aim of the present work was to study the possible
involvement of lipid signalling and PKC during hepatic
insulin action in a differentiated model for the adult organ,
the primary adult rat hepatocyte cultured serum-free with
dexamethasone. This system shows high insulin sensitivity
and responsiveness towards a multitude of insulin-depend-
ent parameters [25–27]. The effects of insulin were compared
with those of epidermal growth factor (EGF), ATP and
exogenous phospholipase C (PLC).
Materials and methods
Materials
Enzymes, M199 medium, collagenase A and the transfec-
tion agent DOSPER were from Roche Molecular Biochem-
icals (Mannheim, Germany). Bovine insulin was from Serva
(Heidelberg, Germany). Bisindolylmaleimide I and protein
G–agarose were from Calbiochem (Bad Soden, Germany).
Phorbol 12-myristate 13-acetate (PMA), rottlerin, PLC
from Clostridium perfringens, IGEPAL and dexamethasone
were from Sigma (Taufkirchen, Germany). A stock solution
of PMA (10 m
M
) was made in dimethylsulfoxide; before
use it was diluted 1 : 100 in M199 medium containing
0.2% (w/v) bovine serum albumin.
D

ln, Germany).
Cell culture
Hepatocytes from fed male Wistar rats (of weight
180–250 g) were isolated by recirculating collagenase per-
fusion in situ, purified by centrifugation through Percoll and
culturedinM199mediumon6-cmplasticdishes[28].For
the first 3 h, medium contained 4% newborn calf serum,
1n
M
insulin and 0.1 l
M
dexamethasone. Serum was then
omitted and the cells were cultured for the next 4 or 43 h
with 1 n
M
insulin and 0.1 l
M
dexamethasone. Medium was
changed at 22 h. The gas atmosphere contained CO
2
/O
2
/N
2
(5 : 17 : 78).
Cell experiments
After 4 or 46 h of continuous culture, dishes were washed
twice and incubated in M199 (2.5 mL per dish). After 1 h
the medium was replaced with M199 containing 2 m
M

M
pyruvate; 95% O
2
/5% CO
2
;37°C;
constant flow without recirculation, 5.5–6 mLÆmin
)1
Æg
)1
of
liver). Experiments were performed between 09.00 h and
11.00 h; preperfusion lasted for 20 min before the onset of
sampling from the inferior vena cava. Liver samples were
taken from the front lobe at 35 min.
Lipid extraction and separation by TLC
Hepatocytes from one 6-cm dish were scraped into 2 mL
of methanol and transferred into a glass tube. Chloroform
(1 mL) was added and lipids were extracted for 10 min at
4 °C. Subsequently, 1 mL of chloroform and 1.7 mL of
1
M
NaCl were added under vigorous mixing. After 5 min,
samples were centrifuged (400 g for 5 min), the aqueous
phase was discarded and the organic phase dried under N
2
at room temperature. Lipids were concentrated in the
V-shaped tip of the tube by repetitive solvent evaporation
and resuspension using diminishing volumes of chloro-
form. Dried lipids were stored under N

P]ATP[cP], as described by Preiss et al.[31].Themanu-
facturer’s instructions for the commercially available DAG
test kit were followed.
32
P-labelled PA was purified using
chloroform/methanol/acetic acid (65 : 15 : 5, v/v/v) as a
solvent system and quantified with a Storm 860 phosphoi-
mager (Pharmacia, Freiburg, Germany).
Analysis of DAG molecular species
Hepatocytes from one 10-cm dish were extracted as outlined
above. After drying the lipid extract under nitrogen, DAGs
were extracted with ether and immediately benzoylated, as
described by Blank et al. [32]. Diradylglycerobenzoates were
separated into their subclasses (diacyl, alkylacyl, and alk-1-
enylacyl types) by TLC using benzene/hexane/ether
(50 : 45 : 4, v/v/v) as a solvent system, and the individual
molecular species were separated by HPLC using an
octadecyl reverse-phase column in acetonitrile/isopropanol
(80 : 20, v/v) as the mobile phase. Individual peaks were
quantified by measuring absorbance at 230 nm. To identify
individual molecular species, representative samples were
analysed by combined HPLC/MS [33] using the instrumen-
tation described in Bu
¨
tikofer et al. [34]. Briefly, after the UV
detector, methanol/0.2
M
aqueous ammonium acetate
(10 : 90; v/v) was added via a T-connector, and the total
flow was introduced through a thermospray interface into a

M
phenylmethanesulfonyl fluor-
ide, 20 m
M
2-mercaptoethanol) and centrifuged at
100 000 g for 30 min. Supernatant and membrane fraction
were diluted with lysis buffer (without sucrose and EGTA)
and 8–14 lg of protein from each fraction was assayed for
the ability to phosphorylate a synthetic EGF-receptor
peptide (RKRTLRRL). The Amersham assay contained
25 lL of sample, 25 m
M
Tris/HCl, pH 7.5, 34 lgÆmL
)1
phosphatidylserine, 2.7 lgÆmL
)1
PMA, 102 l
M
receptor
peptide, 3.4 m
M
dithiothreitol, 1.36 m
M
calcium acetate,
109 l
M
ATP, and 6.5 m
M
MgCl
2

isoforms run on each gel, (c) PMA-induced PKC
translocation from the cytosol to the membrane fraction
(except for the nonmobile f-isoform; samples of control
and PMA-treated cells were run on each gel for compar-
ison), and (d) comparison of bands after incubation of a
membrane blot with buffer in the presence or absence of
an antigen (PKC isoform) of the respective PKC
antibody. The bands on the immunoblots at about
80 000 molecular mass, representing PKC isoforms a, d
and f, and at 90 000 molecular mass, representing PKC
isoform e, were quantified by densitometry.
Immunoprecipitation and activity assay of PKCf
Hepatocytes from one 6 cm dish were homogenized in
500 lL of PKC lysis buffer (see above) supplemented with
0.5% IGEPAL (Nonidet P-40) and 1% Triton X-100. The
lysate was sonicated for 10 s and centrifuged for 20 min at
20 000 g after 30 min of incubation at 4 °C. Supernatants
(200 ll, 1 mg of protein) were incubated under mild
agitation for 4 h at 4 °Cwith5lgofanti-PKCf,which
had been coupled to protein G–agarose (30 lL of agarose in
NaCl/P
i
,1h,4°C). Immobilized immune complexes were
recovered by centrifugation, washed three times with
complete lysis buffer and twice in kinase buffer (50 m
M
Tris pH 7.5, 10 m
M
MgCl
2

cultured serum-free in the presence of 0.1 l
M
dexametha-
sone for 46 h. In each subsequent short-term experiment,
measurement of lipid mediators or PKC was always
paralleled by the determination of the physiological action
of insulin on glucose metabolism. ATP and exogenous PLC,
which both stimulate DAG formation [36–38], were used as
positive controls. In addition, EGF, an insulin-mimetic as
Ó FEBS 2003 Insulin signalling in rat hepatocytes (Eur. J. Biochem. 270) 4637
well as an insulin-antagonistic factor [39–41], was included
in some experiments.
Metabolic effects
We found that the addition of insulin to our primary rat
hepatocyte cultures stimulated glycolysis 4.5-fold, with a
50% effective dose (ED
50
)of 0.3 n
M
,whereasEGF
increased glycolysis twofold, with an ED
50
of  0.5 ngÆmL
)1
(Fig. 1B). Similar results have been reported before for
other hepatocyte culture systems [27,41]. Furthermore,
transforming growth factor-a (TGF-a) completely mim-
icked EGF action in the lower concentration range
(0.1–3 ngÆmL
)1

Determination of DAG mass and DAG molecular species
Increases in DAG and PA, through insulin-dependent
activation of PLC and PLD, have been reported previously
for rat hepatocytes [16–18]. In contrast to these studies, we
found no increase in DAG mass when the cells were
stimulated with 1–100 n
M
insulin, either in 6-h cultures
(data not shown) or in 48 h cultures (Fig. 3). Similarly, the
addition of 10 ngÆmL
)1
EGF or 10 ngÆmL
)1
TGF-a also
showed no effect. As shown previously [36,37], ATP and
PLC are capable of rapidly elevating the level of DAG. In
agreement with these reports, we found that the addition of
100 l
M
ATP doubled DAG mass within 5 min; interest-
ingly, the presence of PLC increased DAG mass at both
insulin-mimetic (5 mUÆmL
)1
) and insulin-antagonistic
(100 mUÆmL
)1
) concentrations (Fig. 3).
It has been previously shown that the addition of tritium-
labelled fatty acids to hepatocytes results in the incorpor-
ation of label into the phospholipid fraction [17,18];

M
insulin and 0.1 l
M
dexamethasone. Subsequently, they were washed
free of hormones and incubated for 30 min in M199 medium con-
taining 0.1 l
M
dexamethasone and 2 m
M
lactate before the agonists
were added. [
14
C]Lactate production from 5 m
M
[
14
C]glucose was
measured for 2 h. Data represent mean values ± SD from three dif-
ferent hepatocyte preparations.
4638 I. Probst et al. (Eur. J. Biochem. 270) Ó FEBS 2003
contained almost exclusively diacyl-type molecular species
(> 98% of total species). The HPLC profile, and thus the
composition of DAG species, was not altered when cultures
were treated with insulin (100 n
M
), EGF (10 ngÆmL
)1
)
or TGF-a (10 ngÆmL
)1

shown). However, when we studied the metabolic insulin
responsiveness of the cells cultured under these steroid-free
conditions, we found that the activation of glycogen
Fig. 2. Modulation of basal and insulin-stimulated glycogen synthesis by epidermal growth factor (EGF), transforming growth factor a (TGF-a), ATP
and phospholipase C (PLC). Hepatocytes were cultured as described in the legend to Fig. 1. Incorporation of [
14
C]glucose into glycogen was
measured for 2 h. Data represent mean values ± SD from four to seven different hepatocyte preparations.
Fig. 3. Total cellular diacylglycerol (DAG) mass of hepatocytes after
treatment with ATP, phospholipase C (PLC), insulin and epidermal
growth factor (EGF)/transforming growth factor a (TGF-a). Hepato-
cytes were cultured as described in the legend to Fig. 1. The DAG
content was quantified in lipid extracts using the DAG kinase assay.
Data represent mean values ± SD from three to five different
hepatocyte preparations.
Ó FEBS 2003 Insulin signalling in rat hepatocytes (Eur. J. Biochem. 270) 4639
synthesis was severely reduced by 90% compared to cells
cultured with dexamethasone (Fig. 2).
Measurement of PLD activity
A possible involvement of PLD in insulin signalling was
investigated in cells using our serum-free culture condi-
tions, in the presence of dexamethasone, by determining
transphosphatidylation activity with 0.3% butanol as the
acceptor [17]. We found that cell exposure to insulin in
the presence of butanol did not increase the formation
of phosphatidylbutanol. As reported previously [19],
transphosphatidylation was, however, five- to 10-fold
enhanced in the presence of ATP (positive control, data
not shown).
Translocation of PKC

indolylmaleimide I, which predominantly inhibits conven-
tional and novel isoforms, i.e. the a-, d-ande-isoforms
(Fig. 6). Owing to its isoform specificity, the inhibitor
completely alleviated the insulin-antagonistic effect of
PMA, which is mediated via DAG-dependent PKC
isoforms. In contrast, bisindolylmaleimide I was unable to
revert the ATP-mediated blockade of the insulin signal
(Fig. 6). Selective inhibition of PKCd by the inhibitor
rottlerin (5–10 l
M
) was also without effect on insulin
signalling (data not shown).
Finally, we tried to inhibit insulin signalling by transfect-
ing hepatocytes with antisense oligodesoxynucleotides
(ODN) targeted against PKCf. We found that cells
transfected with 2.5 lgÆmL
)1
cytofectin and 0.125 n
M
fluorescent ODN, or with 2–10 lgÆmL
)1
DOSPER and
0.5–2.5 l
M
fluorescent ODN, showed up to 80% fluores-
cent nuclei, and the amount of PKCf was reduced slightly
(< 30%) after 3 days of culture when PKCf antisense
ODN was added. It should be noted, however, that both cell
vitality (measured by the release of lactate dehydrogenase)
and insulin signalling (measured as glycogen synthesis) were

a
+ 14:0, 16:1
a
4.4 ± 0.2
11 16:1, 22:4
+ 18:0, 20:5
a
2.4 ± 0.2
12 16:0, 20:4 3.8 ± 0.2
13 18:1, 18:2
+ 18:0, 22:6
a
+ 16:1, 18:1
a
12.5 ± 0.3
14 16:0, 18:2
+ 18:0, 18:3
a
8.8 ± 0.5
15 16:0, 16:1 0.7 ± 0.1
16 < 0.5
17 18:0, 22:5 4.1 ± 0.6
18 18:0, 20:4 4.3 ± 0.9
19 17:0, 18:2
+ 16:1, 17:0
a
3.6 ± 0.7
20 18:1, 18:1 13.6 ± 0.8
21 16:0, 18:1
+ 18:0, 18:2

M
PMA (positive
control for PKC translocation), a second bolus of 50 p
M
glucagon or a staggered infusion of 10 n
M
insulin
(25–35 min) and 50 p
M
glucagon (Fig. 7B,C). The anti-
glucagon effect of insulin was demonstrated by a 75%
reduction of the glucagon-stimulated glucose output. PMA
alone stimulated glucose production (data not shown) [45].
Of all agonists used, only PMA translocated PKC isoforms
a, d and e (Fig. 7A). Differences in DAG mass (lgÆmg
)1
of
protein) were not observed between control liver (8.3) and
livers treated with glucagon (7.9), insulin/glucagon (8.2), or
PMA (8.2, n ¼ 3 for all treatments).
Discussion
In muscle and fat tissue, lipid messengers such as DAG
and PA, as well as DAG-dependent and -independent
PKC isoforms, have recently been proposed to play a role
in the insulin signal leading to activation of glucose
uptake [1–3].
Fig. 4. HPLC profile of diacylglycerol (DAG) molecular species of control and ATP-stimulated hepatocytes. Hepatocytes cultured for 2 days were
incubated for 5 min with M199 as vehicle (A) or 100 l
M
ATP (B), and the molecular species of DAG were analysed as described in the Materials

preparations.
Agonist Culture
Percentage of membrane-bound PKC
PKCa PKCd PKCe PKCf
Control 6 h 19.5 ± 7.7 51.5 ± 11.1 41.5 ± 5.8 41.2 ± 7.7
48 h 13.3 ± 11.4 37.7 ± 5.9 40.1 ± 4.0 41.7 ± 11.7
PMA 6 h 39.7 ± 11.0* 77.3 ± 10.0* 66.5 ± 12.2* 43.5 ± 7.0
48 h 44.0 ± 12.3* 67.9 ± 9.7* 54.7 ± 11.1* 37.5 ± 16.6
Insulin 6 h 20.0 ± 8.0 52.8 ± 9.6 41.6 ± 12.1 38.8 ± 2.3
48 h 20.7 ± 15.3 39.2 ± 13.3 35.6 ± 7.4 36.7 ± 10.1
ATP 6 h 27.8 ± 4.9 41.2 ± 9.6 58.8 ± 8.7 39.0 ± 9.9
48 h 19.0 ± 7.3 37.1 ± 7.4 42.7 ± 10.4 36.7 ± 7.9
* P < 0.05 vs. control.
4642 I. Probst et al. (Eur. J. Biochem. 270) Ó FEBS 2003
DAG molecular species composition. Second, an
involvement of PLD could not be demonstrated as
insulin-stimulated hepatocytes showed no evidence for
transphosphatidylation activity. Third, we found no
evidence of translocation of PKC isoforms from the cytosol
to the membrane fraction after stimulation of hepatocytes
with insulin. Fourth, insulin-stimulated cells showed no
increase in membrane-bound PKC activity and did not
increase the activity of immunoprecipitated PKCf.Fifth,
the action of insulin on glycogen synthesis was not abolished
by the specific PKC inhibitor, bisindolylmaleimide, whereas
it completely reversed the insulin-antagonistic effect of
PMA. Sixth, insulin did not alter DAG mass and PKC
isozyme distribution in the perfused liver.
Our results are in good agreement with two previous
reports showing a lack of PLD [19] and PKC [21] activation

Table 3. Determination of protein kinase C (PKC) activity in crude extracts (pmolÆmin
-1
Æmg
-1
of protein) and PKCf immunoprecipitates
(pmolÆmin
-1
Æmg
-1
of lysate protein). Hepatocytes cultured for 6 h and 48 h were exposed to vehicle, 0.1 l
M
phorbol 12-myristate 13-acetate (PMA)
or 10 n
M
insulin for 10 min. Data represent mean values ± SD from three different hepatocyte preparations.
Treatment Culture
Protein kinase C activity
Crude extracts
PKCf-immunoprecipitateCytosol Membrane
Control 6 h 121.3 ± 4 95.1 ± 10 ND
48 h 243.2 ± 16 148 ± 34 ND
PMA
a
6 h 58.0 ± 8
d
218.5 ± 19
d
ND
48 h 141.5 ± 33
c

Hepatocytes cultured for 48 h were incubated with the agonists and
the inhibitor for 2 h. Data represent the mean values ± SD from three
different hepatocyte preparations.
Ó FEBS 2003 Insulin signalling in rat hepatocytes (Eur. J. Biochem. 270) 4643
increases in DAG, PA, inositoltrisphosphate and cytosolic
calcium, were detected to various degrees when hepatocyte
suspensions or glucocorticoid-free hepatocyte cultures were
used [36,39,41,46,47]. In contrast, in our dexamethasone-
treated cultures, EGF had no effect on DAG levels, which is
in agreement with the results of Dajani et al.[38]whoused
similar culture conditions. Working with hepatocyte sus-
pensions and cultured cells, Nojiri & Hoek [47] pointed out
that EGF-induced inositoltrisphosphate formation was
effectively reduced by actin rearrangement, which occurs
during the transition of the cells from the suspended to the
cultured state. As dexamethasone is known to retain
cuboidal hepatocyte morphology in cultures and to
influence actin polymerization [48], the differences in insulin
signalling (and also in EGF signalling) observed between
steroid-treated and untreated cultures might well reflect the
differences in cytoskeletal cell architecture and thus point to
a major regulatory role of actin fibers in the propagation of
hormone and growth factor signals. The recent finding that
focal adhesion kinase regulates protein kinase B, glycogen
synthase kinase-3 and glycogen synthase, in an insulin-
dependent manner [49], supports the hypothesis of cross-
talk between insulin and integrin-signalling pathways.
Thelackofaninsulin-elicitedincreaseinDAG,shown
here for dexamethasone-treated hepatocytes and for the
perfused liver, excludes the involvement of conventional and

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