A PDF file should load here. If you do not see its contents
the file may be temporarily unavailable at the journal website
or you do not have a PDF plug-in installed and enabled in your browser.
Alternatively, you can download the file locally and open with any standalone PDF reader:
https://link.springer.com/content/pdf/10.1007%2Fs00125-007-0916-5.pdf
Epigenetic regulation of PPARGC1A in human type 2 diabetic islets and effect on insulin secretion
C. Ling
S. Del Guerra
R. Lupi
T. Rnn
C. Granhall
H. Luthman
P. Masiello
P. Marchetti
L. Groop
S. Del Prato
Aims/hypothesis Insulin secretion in pancreatic islets is dependent upon mitochondrial function and production of ATP. The transcriptional coactivator peroxisome proliferator activated receptor gamma coactivator-1 alpha (protein PGC1; gene PPARGC1A) is a master regulator of mitochondrial genes and its expression is decreased and related to impaired oxidative phosphorylation in muscle from patients with type 2 diabetes. Whether it plays a similar role in human pancreatic islets is not known. We therefore investigated if PPARGC1A expression is altered in islets from patients with type 2 diabetes and whether this expression is influenced by genetic (PPARGC1A Gly482Ser polymorphism) and epigenetic (DNA methylation) factors. We also tested if experimental downregulation of PPARGC1A expression in human islets influenced insulin secretion. Methods The PPARGC1A Gly482Ser polymorphism was genotyped in human pancreatic islets from 48 non-diabetic and 12 type 2 diabetic multi-organ donors and related to PPARGC1A mRNA expression. DNA methylation of the PPARGC1A promoter was analysed in pancreatic islets from ten type 2 diabetic and nine control donors. Isolated human islets were transfected with PPARGC1A silencing RNA (siRNA).
-
Type 2 diabetes is characterised by chronic hyperglycaemia
as a result of impaired pancreatic beta cell function and
insulin resistance in peripheral tissues, i.e. skeletal muscle,
adipose tissue and liver. Although each of these
pathogenic defects could be accounted for by specific
mechanisms, impaired ATP production as a consequence of
reduced oxidative phosphorylation might provide an
intriguing common pathogenic pathway for all these defects.
The transcriptional coactivator peroxisome proliferator
activated receptor gamma coactivator-1 alpha (protein
PGC-1; gene PPARGC1A) is an important factor
regulating the expression of genes for oxidative phosphorylation
and ATP production in target tissues through coactivation
of nuclear receptors [1]. We have previously shown that the
expression of PPARGC1A and a set of genes involved in
oxidative phosphorylation is reduced in skeletal muscle
from patients with type 2 diabetes [2]. Furthermore, a
common polymorphism, Gly482Ser, in the PPARGC1A
gene has been associated with increased risk of type 2
diabetes and an age-related reduction in muscle PPARGC1A
expression [35]. In addition, genetic variation in the
PPARGC1A gene was associated with indices of beta cell
function [6]. Despite the central role of ATP for insulin
secretion, the function of PGC-1 in human pancreatic
islets and beta cells is less well established [7].
Obesity, reduced physical activity and ageing are well
known risk factors for type 2 diabetes. However, all
individuals exposed to an affluent environment do not
develop the disease. One likely reason is that genetic
variation modifies individual susceptibility to the
environment. However, the environment could also modify genetic
risk factors by influencing expression of a gene by DNA
methylation or histone modifications. Cytosine residues
occurring in CG dinucleotides are targets for DNA
methylation and gene expression is usually reduced when DNA
methylation takes place at a promoter. Whether DNA
methylation influences gene expression in target tissues
for type 2 diabetes and thereby the pathogenesis of the
disease remains to be demonstrated.
The present study investigated: (1) whether expression
of PPARGC1A is altered in human islets from patients with
type 2 diabetes; (2) if this expression is influenced by
genetic (the PPARGC1A Gly482Ser polymorphism) and
epigenetic (DNA methylation) factors; and (3) if expression
of PPARGC1A influences insulin secretion.
Multi-organ donors The characteristics of the 48
nondiabetic and 12 type 2 diabetic multi-organ donors,
whose pancreases were processed for islet preparation,
are presented in Table 1. Pancreases were obtained and
processed with the approval of the regional Ethics
Committee.
Data are expressed as meanSEM
a Donors used for DNA methylation analysis
b PPARGC1A Gly482Ser polymorphism
Human pancreatic islets and experimental plan Isolated
pancreatic islets were prepared by collagenase digestion and
density gradient purification [8, 9]. After isolation, islets
were cultured free floating in M199 culture medium
(SigmaAldrich, St Louis, MO, USA) at 5.5 mmol/l glucose
concentration and studied within 3 days from isolation. Cell
viability, measured by Trypan Blue exclusion, was higher
than 90% in control and diabetic islets after 3 days in culture.
Insulin secretion study Insulin secretion studies were
performed as previously described [8, 9]. Following a
45 min pre-incubation period at 3.3 mmol/l glucose, groups
of 30 islets of comparable size were kept at 37C for 45 min
in KRB, 0.5% (wt/wt) albumin, pH 7.4, containing
3.3 mmol/l glucose. At the end of this period, the medium
(...truncated)