590s BiochemicalSocietyTransactions( 1 995)23
rffrct o f the thirzolidirudionr BRL49653 and g o n r t i c
a k s i t y on hmpatic gmnr q r e s s i o n i n the Gudrer rat
#
T
JAMES E. SIDAWAY, ALAN J. DICKSON, STEPHEN A. SMITH*
and DAVID S.W. BOAM
School of Biological Sciences, University of
Manchester, Manchester M13 9PT, U.K.
*Department of Vascular Biology, SmithKline Beecham
Pharmaceuticals, The Frythe, Welwyn, Herts AL6 9AR,
U.K.
Both obesity and non-insulin-dependent diabetes
mellitus (NIDDM) are associated with insulin
resistance in the liver and peripheral tissues [l].
The thiazolidinediones are a class of novel
antidiabetic compounds which decrease hyperglycaemia
and hyperlipidaemia in rodent models of obesity and
NIDDM by enhancing insulin action in target tissues
12-41. Recently BRL49653, a potent and selective
thiazolidinedione [5,61, and fatty acid derivatives
have both been shown to enhance gene expression in
pre-adipocytes via a mechanism that involves a novel
member of the peroxisome proliferator-activated
receptor (PPAR) family [7].
Given the important role of the liver in glucose
and lipid homeostasis we have sought to determine
whether the compound influences hepatic gene
expression using northern analysis. Previously it has
been shown, using the euglycaemic clamp technique,
that
the
compound
enhances
insulin-mediated
suppression of hepatic glucose output (HGO) in obese
[El.
AS
insulin
resistant
Zucker
rats
phosphoenolpyruvatecarboxykinase (PEPCK,EC 4.1.1.32)
is a key enzyme in gluconeogenesis (and therefore HGO)
we chose to study the effect of BRL49653 on hepatic
PEPCK mRNA levels in Zucker rats.
RNA was extracted [91 from the livers of 10 week
old Zucker rats.
RNA (15 pg) was resolved by
electrophoresis on 1.2% agarosej 7.5% formaldehyde
gels and transferred to nylon membranes by capillary
blotting. Hybridisation was carried out with a "P
labelled CDNA probe for PEPCK [lo]. PEPCK mRNA levels
were quantified on a phosphorimager and normalized to
18s ribosomal RNA.
In obese Zucker rats, hepatic
PEPCK mRNA content (Fig.1.) was half that of lean
littermates. No significant effects of obesity were
observed on hepatic mRNA content of two other genes
studied, glucokinase and gene 33. Oral administration
of BRL49653 to obese animals for 21 days
(3pmol/kg.day-')restored PEPCK mRNA to control levels.
BRL49653 treatment had no effect on PEPCK mRNA content
in control rats.
The decreased PEPCK mRNA content in obese Zucker
rats may be related to altered effects of insulin on
HGO.
Oral administration of BRL49653 reduced the
hyper-insulinaemia associated with obese animals [el,
thus the restoration of PEPCK mRNA to control levels
in obese BRL49653-treated rats may be an indirect
effect.
We have therefore used the rat H4IIE hepatoma cell
line to investigate direct effects of the compound on
PEPCK gene expression. H4IIE cells (seeded at 4 x
10'lcm') were grown in alpha minimum essential media
(aMEM) containing 10% charcoal-stripped foetal calf
serum for 3 days under an atmosphere of 95% air / 5%
CO,. Cells were placed in serum-free aMEM and
challenged with 10 pM BRL49653 for periods of up to 15
hours.
At 4 hours PEPCK mRNA level in BRL49653
treated cells was decreased to 50% of control, whereas
by 15 hours the compound had produced a 250% increase.
As these effects occurred in the absence of serum, we
hypothesise that BRL49653 may be able to directly
influence gene expression in this hepatoma cell line.
PPARs bind to DR-1 type hormone response elements
The
as heterodimers with retinoid x receptors [ l l l .
thiazolidinedione pioglitazone induces binding of a
factor to a DR-I type element in adipocytes [121.
Interestingly, nucleotides -468 to -440 of the PEPCK
promoter also contain a DR-1 element. As an initial
step to determining the mechanism of BRL49653 action
in H4IIE cells we have used the gel retardation assay
Abbreviation used: NIDDM, non-insulin-dependent
diabetes mellitus; PPAR, peroxisome proliferatoractivated receptor; HGO, hepatic glucose output;
PEPCK, phosphoenolpyruvate carboxykinase EC 4.1.1.32;
aMEM, alpha minimum essential media;
"
1
Iwn
lean+BRL
Fa
Fa+BRL
Fig. 1. Effect of qenetic obesity and BRL49653
treatment in Zucker rats on hepatic PEPCK mRNA
content. lean, lean control; lean+BRL, ERL49653
treated lean control; Fa, obese; Fa+BRL, BRL49653
treated obese rats. RNA was extracted after a 5 hour
fast. Data show the meantSEM of combined results of
10 animals/group as percentage of lean control, *
represents P<O.Ol when compared to lean control by the
Student's t test, # represents PcO.05 when compared to
obese animals by the Student's t test.
to detect BRL49653 mediated changes in transcription
factor binding to this element.
Using an
oligonucleotide containing this element and nuclear
extracts made from control and BRL49653-treated H4IIE
cells we were able to detect the formation of several
complexes. However there was no difference in complex
formation between control and treated cells
The financial support of the BBSRC and SmithKline
Beecham Pharmaceuticals are gratefully acknowledged.
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