Læknablaðið - 15.12.1984, Side 60

362
LÆKNABLAÐIÐ
EFFECTS OF INSULIN, GLUCAGON AND
SOMATOSTATIN ON HEPATIC LIPID
METABOLISM IN VITRO.
Ó. G. Bjömsson, C. R. Pullinger, and G. F.
Gibbons.
MRC Metabolism Unit, Hammersmith Hospital,
London, U.K.
There are contradictory reports on the direct effects
of insulin and glucagon on hepatic cholesterol
metabolism (J. Biol. Chem 254: 6644-49, 1979).
Direct effect of another pancreatic hormone, soma-
tostatin, has not yet been investigated. In order to
assess the in vitro effects of these polypeptideson
hepatic lipid metabolisme incubated rat hepatocytes
in Krebs- Henselit buffer containing varying conc.
of insulin (7.2 x 10—11 — 7.2 x 10—8 M), glucagon
(10-8—10-5 M) and somatostatin (3.0xl0-7 —
3.0 x 10—5 M) respectively, and measured the rate of
fatty acid synthesis (incorporation of 3H20) and
sterol (sesmosterol) synthesis and the activity og
HMG-CoA reductase, the rate limiting enzym for
hepatic cholesterol biosynthesis. Hepatocytes were
prepared by the collagenase perfusion technique
from rats on a controlled lighting and feeding
schedule in which food was available for the first 8 h
of the 12 h dark period. Insulin (>7.2xl0—10 M)
increased reductase activity (78.2±15.5, SEM, n = 6,
vs 59.1 ±8.8, P<0.01, pmol mevalonate/min per mg
protein), but no effect on sterol synthesis was
observed. Insulin increaed fatty acid synthesis.
Glucagon (> 10_7 M) suppressed reductase activity
(48.2 ±3.7 vs. 60.0 ±9.5, p<0.05), sterol synthesis
(0.82 ±0.11 vs. 0.93 ±0.13, P = 0.05, nmol/mg protein)
and fatty acid synthesis (4.2 ±0.3 vs. 18.1 ±4.5, P<
0.01, nmol 3H20 incorporated/mg protein). Somato-
statin (> 1,2 x 10-6 M) weakly stimulated reductase
activity in hepatocytes form rats fed ad libitum
(36.5 ±4.2, SEM, n = 5, vs. 24.6 ±4.2, P<0.05), but
was without an effect on sterol and fatty acid
synthesis. — Insulin and glucagon had opposite
effects on HMG-CoA reductase activity and fatty
acid synthesis. The effect of these hormones on
sterol synthesis was none (insulin) or weak (gluca-
gon), and not within the expected physiological
conc. range of these hormones. The effect of
somatostatin on HMG-CoA reductase activity was
probably limited to pharmacological conc.
VARIATIONS IN THE RELATIONSHIP
BETWEEN HEPATIC STEROL SYNTHESIS
AND THE INCORPORATION OF pH]
WATER: EFFECTS OF LIPOGENIC
PRECURSORS, DRUGS AND PEPTIDE
HORMONES.
Ó. G. Bjömsson, C. R. Pullinger, and G. F. Gibbons.
MRC Lipid Metabolism Unit, Hammersmith
Hospital, London, U.K.
The use of incorporation of [3H] water into chole-
sterol as an estimate of sterol biosynthesis has
obviously advantages over [14C] acetate or [14C]
acetyl-CoA, as water penetrates rapidly most cell
membranes and [3H] water is also not subject to
extensive metabolism prior to incorporation into
sterol. There is, however, no general agreement as
to the exact number of 3H atoms which aree
incorporated per molecule of cholesterol synthesi-
zed. We have used a gas liquid chromatographic
method to measure the weight of a newly synthesi-
zed cholesterol precursor, desmosterol, in hepatocy-
tes. Simultaneously we measured tritium incorpora-
tion into desmosterol and this allowed us a direct
measurement and calculation of ng-atoms of 3H
incorporated per ng-atom carbon of cholesterol
synthesized (the H/C ratio). Isolated rat hepatocytes
in suspension were incubated up to 3 h in Krebs-
Henseleit buffer under various experimental condi-
tions, where lipogenic precursors (pyruvate, lactate)
or pancreatic hormones (insulin, glucagon) or drugs
(compactin, hydroxycitrate, dexamethasons, triari-
mol) were added to the incubations. Time course
studies showed an increasee in H/C ratio with 'time
(0.42 ±0.07 (1 h), 0,55 ±0.04 (2 h), 0,74 ± 0.08 (3 h),
SEM, respectively, P<0.001). Incubations with in-
creasing conc. of pyruvate (6 MM, 10 mM, 25 mM)
increased the H/C ratio in a dose-responsive
manner while incubation with 50 mM pyruvate
decreased it. Hydroxycitrate reduced the ratio
(0.51 ±0.02 vs. 0.63±0.04, control, P<0.01) and
dexamethasone increased it marginally (0.68 ±0.04,
P = 0.05). Insulin, lactate, compactin or triarimol had
no effects. The fact that the number of tritium atoms
incorporated into newly synthesized cholesterol
varied under different incubation conditions, might
be explained by changes in the contribution of
unlabelled NADPH produced via the pentose phos-
phate pathway (J. Biol. Cheem. 252;4667-73, 1977).
These findings may help to explain the conflicting
results which has been published recently on the
effects of glucagon and some lipogenic precursors
on sterol synthesis in vitro.
ISOLATION, PURIFICATION AND
SHORT-TERM INCUBATION OF RAT
HEPATOCYTES IN OXYGEN/CARBON
DIOXIDE BUFFER MEDIA:
Ó.G. Bjömsson, C.R. Pullinger, and G.F. Gibbons.
MRC Lipid Metabolism Unit, Hammersmith
Hospital, London, United Kingdom.
Isolated hepatocytes in suspension of culture have
partly replaced whole liver perfusion preparations,
liver slices or in vivo studies as research tools for
studying hepatic metabolism. The obvious advanta-
ge of using isolated hepatocytes is that they allow
direct assessment of the effects of hormones and
metabolic precursors on the hepatic metabolism
without interference from other cells or organs. We
have modified tha technique of Berry and Friend (].
Cell Biol. 43: 506-20, 1969) and Seglen (Exp. Cell
Res. 82: 391-8, 1973) for isolating and purifying rat