Mechanisms by which Thiazolidinediones Enhance Insulin Action

REVIEWS
Mechanisms by which
Thiazolidinediones Enhance Insulin
Action
Mauricio J. Reginato and Mitchell A. Lazar
Thiazolidinediones (TZDs) are an exciting new class of insulinsensitizing drugs being used currently for the treatment of non-insulindependent diabetes mellitus. The molecular target of these compounds
is thought to be the nuclear hormone receptor, peroxisome proliferatoractivated receptor γ (PPARγ). PPARγ is expressed predominantly in
adipose tissue, yet a major site of TZD-responsive glucose disposal is
skeletal muscle. Potential explanations for this paradox are discussed
in this review.
Type 2 diabetes mellitus (DM) is a
common, chronic disease that is a
major cause of morbidity and mortality in industrialized societies. Type 2
DM has a strong genetic component
and is linked tightly to obesity. Although impaired insulin secretion contributes to type 2 DM, early in the
course of the disease insulin levels are
often increased. A major difference between type 2 DM and insulin-dependent diabetes is that type 2 DM is
characterized by peripheral insulin resistance1. The resistance occurs despite
qualitatively and quantitatively normal
insulin receptors, thus implicating one
or more defective steps in the insulin
signaling pathway downstream from
insulin binding to its receptor. Nevertheless, until recently the only available
M.J. Reginato is Research Fellow at the
Departments of Pharmacology and Medicine,
University of Pennsylvania Medical Center,
Philadelphia, PA 19104, USA. M.A. Lazar is
Chief of the Division of Endocrinology,
Diabetes, and Metabolism, Department of
Medicine, and Director of Penn Diabetes
Center, University of Pennsylvania Medical
Center, Philadelphia, PA 19104, USA.
TEM Vol. 10, No. 1, 1999
pharmacological treatments for type 2
DM were insulin or agents that increase insulin secretion. New pharmacological approaches to treating type 2
DM have been developed recently that
target other metabolic abnormalities2.
The thiazolidinediones (TZDs) are a
new class of orally active drugs that
are particularly exciting because they
decrease insulin resistance by enhancing the actions of insulin at a level distal to the insulin receptor3.
•
Effects of TZDs
TZDs, which include troglitazone, pioglitazone and rosaglitazone (Fig. 1),
are thought to sensitize target tissues
to the action of insulin. Indeed, they
are ineffective at lowering serum glucose levels in the absence of insulin3.
In animal models of type 2 DM, TZDs
reduce plasma glucose levels, and decrease insulin and triglycerides to near
normal levels4,5.
In human studies, about 75% of patients with type 2 DM responded to
troglitazone treatment6. In addition to
reduced plasma glucose levels, insulin
levels and/or dose requirements also
drop7. This reduction in insulin levels
is also associated with improved metabolic status of patients with the syndrome of insulin resistance and polycystic ovaries8,9. TZDs also significantly
reduce serum concentrations of
triglycerides and free fatty acids, with
a small rise in high-density lipoprotein
(HDL) cholesterol10,11. Recent hyperinsulinemic-euglycemic clamp studies
have suggested that troglitazone works
primarily by increasing the rate of peripheral glucose disposal in skeletal
muscle12.
In general, TZDs are well tolerated
by patients, although troglitazone
treatment has been associated with
hepatic dysfunction that has been fatal
in a few cases13–15. Thus, it is recommended that liver function tests be
monitored frequently, although the
causal relationship and mechanism
have not been established. Chronic
TZD treatment also leads to modest
weight gain in rodents and humans16.
•
Peroxisome Proliferator-activated
Receptor g: Molecular Target of
TZDs
TZDs were developed originally by
screening analogs of clofibric acid for
antilipidemic and antihyperglycemic
effects17. The antidiabetic effects of
these compounds were not understood,
but the discovery that TZDs enhanced
adipocyte differentiation18,19 was an
important clue to identifying their molecular target. Activators of a member
of the nuclear hormone receptor superfamily, peroxisome proliferator-activated receptor (PPAR), were also found
to induce adipogenesis20, and PPARγ
was shown to be expressed predominantly in adipose tissue21, and to function as a key transcription factor in
adipocyte differentiation22. Shortly
thereafter, TZDs were demonstrated to
be direct ligands for PPARγ (Ref. 23).
1043-2760/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S1043-2760(98)00110-6
9
O
S
O
O
NH
O
O
HO
Pioglitazone
O
S
NH
O
Troglitazone
Figure 1. Thiazolidinedione structures. The structures of pioglitazone and troglitazone are
shown.
in target genes25. The DNA sequences
recognized by the PPAR–RXR heterodimer are referred to as PPAR-response elements (PPREs). PPAR–RXR
heterodimers bind to PPREs in the absence of ligand, but ligand leads to a
conformational change that results in
activation of transcription of the target
gene. The active conformation recruits
a multiprotein coactivator complex
(reviewed in Ref. 26) that acetylates histones (leading to an open, more active
conformation of the nucleosome), as
well as interacting directly with the
basal transcription machinery (Fig. 2).
PPREs have been found in the regulatory regions of a number of genes involved in lipid metabolism and energy
balance27,28.
PPARγ is a member of the nuclear
hormone receptor superfamily of transcription factors that are activated by
small, lipophilic, non-genomically encoded ligands24. There are two PPARγ
isoforms, γ1 and γ2, derived from alternative promoter usage. PPARγ2 contains an additional 31 amino acids at its
N-terminus, but the functional significance is unclear. Interestingly, PPARγ2
is found exclusively in adipocytes,
whereas PPARγ1 is expressed predominantly in adipocytes, but is also expressed in other tissues (see below).
PPARγ belongs to a subset of nuclear receptors that form heterodimers
with the retinoid X receptor (RXR),
greatly enhancing the ability of the receptor to bind specific DNA sequences
SRC1
CBP
P/CAF
Others
Coactivator complex:
TZD
PPARγ
LBD
RXR
LBD
9-cis
RA
HAT
PPARγ
DBD
RXR
DBD
AANTAGGTCANAGGTCA
PPRE
Basal transcription machinery
Others
H
F
TAF
Nucleosome
AA A
A
c c cA
c
A
c
cAA A A
cc
cc
TBP
RNA polymerase II
B
E
TATA
Ectopic expression of PPARγ in
preadipocytes, fibroblasts and myoblasts induces adipocyte differentiation in the presence of the TZD ligand22,29. The ability of TZDs acting via
PPARγ to induce adipocyte differentiation might explain the modest weight
increases observed in vivo. However, it
is not clear how to reconcile the fact
that excess fat cell mass is a major risk
factor for insulin resistance and type 2
DM with the antihyperglycemic effects
of TZDs.
•
Evidence that PPARg Mediates the
Antidiabetic Effects of TZDs
Nevertheless, there is strong evidence
that TZDs function via PPARγ. PPARγ
has been shown to bind to a number of
different ligands, including a number of
fatty acids, as well as prostaglandin J
derivatives, such as 15-deoxy-∆12, 14prostaglandin J2 (Refs 30–32). However, none of these compounds binds to
PPARγ with affinities in the nanomolar
range. By contrast, TZDs have been
shown to bind to PPARγ with an affinity in the range of 40–200 nM (Refs
30,31). Not only are TZDs activating ligands for PPARγ at nanomolar concentrations, but there is a remarkable correlation between TZD potencies for in
vivo plasma glucose lowering and their
order of potency for both PPARγ activation and direct binding to PPARγ
(Refs 33,34). RXR ligands can also activate the PPARγ–RXR heterodimer35,36,
and synthetic RXR agonists increase insulin sensitivity in obese mice and work
in combination with TZDs to enhance
antidiabetic activity37. This further suggests that the PPARγ–RXR heterodimer
complex is the molecular target for
treatment of insulin resistance in vivo.
The evidence supporting PPARγ as
the target of TZD is summarized in
Table 1.
PPARγ target gene
•
Figure 2. Mechanism of thiazolidinedione (TZD) activation of transcription by peroxisome
proliferator-activated receptor γ (PPARγ). PPARγ binds to specific DNA sequences in target
genes as a heterodimer with retinoid X receptor (RXR). TZDs [and/or an RXR ligand, indicated
as 9-cis retinoic acid (RA)] recruit coactivator complexes to the target gene, resulting in increased transcription through inherent histone acetylase (HAT) activity or via interactions with
the basal transcription machinery. CBP, CREB-binding protein; CREB, cyclic AMP response
element-binding protein; DBD, DNA-binding domain; LBD, ligand-binding domain; PPRE,
PPAR-response element; P/CAF, p300/CBP-associated factor; SRC1, steroid receptor coactivator
1; TAF, TBP-associated factor; TBP, TATA-binding protein.
10
The Paradox
There is general agreement that TZDs
are effective antidiabetic agents because they enhance insulin-responsive
glucose disposal in vivo. It is also clear
that TZDs are high-affinity, activating
ligands for PPARγ. However, the
mechanism by which PPARγ mediates
the antidiabetic actions of TZDs is
TEM Vol. 10, No. 1, 1999
controversial. The problem is that the
main site of TZD-enhanced glucose disposal occurs primarily in skeletal muscle, whereas the main site of PPARγ
expression is in adipose tissue. Although PPARγ is expressed predominantly in adipocytes, PPARγ expression
has been demonstrated in a variety of
extra-adipose tissues, including liver38,
colon39,40, breast41, type II pneumocytes
of the lung42 and macrophages43–45.
PPARγ expression in skeletal muscle
has also been reported46. By northern
analysis, the level of PPARγ mRNA is
>50-fold higher in adipose tissue than
in skeletal muscle21,47, although protein
levels have not been compared quantitatively. Therefore, one missing piece
of the puzzle is the exact tissue site at
which TZDs function to promote insulin action in muscle.
•
Mechanisms
It is possible that mechanisms other
than PPARγ activation explain the effects of TZDs on glucose disposal in
muscle. However, given the nanomolar
binding to PPARγ and the remarkable
correlation between PPARγ activation
and enhancement of insulin action, it
seems likely that PPARγ binding and
activation are related to the in vivo actions of TZDs. A number of potential
mechanisms could link the activation
of PPARγ to insulin action. These are
summarized in Table 2.
The abundance of PPARγ in
adipocytes suggests that this is the site
of action of TZDs. Consistent with
this, TZD treatment increases the
number of small adipocytes in diabetic
rats; these small adipocytes might
have altered properties that promote
insulin action either directly or indirectly48. One possibility is that TZD activation of PPARγ directly induces
genes involved in glucose metabolism
in adipocytes. Indeed, TZDs increase
GLUT4 mRNA levels and glucose uptake in cultured adipocytes49,50. However, the increases in GLUT4 levels following TZD treatment are modest (twoto threefold). In addition, glucose disposal into adipocytes is unlikely to be
of sufficient quantitative impact to
explain the dramatic effects of TZDs
(Ref. 51).
TEM Vol. 10, No. 1, 1999
Table 1. Evidence that the mechanism of glucose lowering by thiazolidinediones (TZDs) in vivo involves peroxisome proliferator-activated
receptor g (PPARg)
• TZDs bind to PPARγ with affinities in the nanomolar range
• The rank order potency of TZDs for blood glucose lowering in vivo correlates
strongly with TZD binding and activation of PPARγ in vitro
• Activating ligands for retinoid X receptor (RXR), which forms PPARγ–RXR
heterodimers also have antidiabetic activity in vivo
Another possibility is that TZDdependent activation of PPARγ induces
adipocytes to send an endocrine signal
to muscle that enhances insulin action. This signal could be the decrease
in a factor that promotes insulin resistance, or the increase in a factor that
enhances insulin action. One potential
factor is adipocyte-derived tumor
necrosis factor α (TNF-α), which has
been shown to be associated with insulin resistance52–54. TZDs can block the
inhibitory effects of TNF-α on insulin
action55,56 and reduce TNF-α levels57.
Another peptide hormone secreted by
adipocytes is leptin58. The concentration of leptin is proportional to fat
cell mass, which is itself correlated
directly with type 2 DM (Ref. 59). This
raises the possibility of a link between
leptin and type 2 DM (Ref. 60), and
TZDs do reduce leptin gene expression
in vitro and in vivo61–63. Increased free
fatty acid (FFA) levels have also been
implicated in the pathogenesis of insulin resistance64,65. TZDs lower plasma
FFA levels, both by increasing β-oxidation in the liver and by increasing
adipocyte FFA uptake66. Other, as yet
undiscovered adipocyte factors, whose
gene expression and/or secretion is altered by TZDs, could also lead to insulin action in muscle.
It is also possible that the effects
of TZDs are independent of adipose
tissue. Lipodystrophic mice with little
or no adipose tissue develop insulin resistance and diabetes that responds
well to TZD treatment67. In this
model, TZDs might act upon hepatic
cells; these mice develop massive, fatty
livers that express PPARγ. It is also
possible that, despite its low abundance, PPARγ in skeletal muscle is the
target of TZDs. PPARγ mRNA was undetectable in skeletal muscle of the fatablated mice67. Nevertheless, activation of PPARγ in muscle, the main site
of glucose disposal, would provide a
direct mechanism to explain TZD action and, indeed, TZDs reportedly
stimulate glucose uptake and enhance
Table 2. Potential mechanisms by which thiazolidinediones (TZDs)
enhance insulin action
Mechanisms involving peroxisome proliferator-activated receptor g (PPARg)
Via PPARγ in adipocytes
• Direct stimulation of increased glucose disposal in adipocytes
• Stimulation of increased glucose disposal in skeletal muscle
• Reduced tumor necrosis factor α
• Reduced leptin
• Reduced free fatty acids
• Alteration of other adipocyte factors
Via extra-adipocytic PPARγ
• Direct stimulation of increased glucose disposal in skeletal muscle
• Action on other target tissue (such as liver) leading to increased glucose disposal
in skeletal muscle
Other mechanisms not involving PPARγ
11
GLUT4 mRNA levels in cultured
muscle cells68.
•
Future Directions
TZDs represent a breakthrough in the
treatment of type 2 DM. New insights
into the mechanism of TZD action in
type 2 DM are likely to result from
basic research in a variety of directions.
The discovery of a physiological ligand
for PPARγ might provide clues to the
site and substrates of the normal hormonal or metabolic pathways regulating insulin action. Tissue-specific
knockouts of PPARγ will not only test
the hypothesis that PPARγ is the molecular target of TZDs, but will support or
eliminate various cell types as candidate sites of TZD action. The discovery
of new TZD-dependent PPARγ target
genes will also contribute to a conceptual bridge between TZD activation of
PPARγ and insulin action. Finally, better understanding of the mechanistic
relationship between TZD binding to
PPARγ and enhanced insulin action in
vivo might lead to the development of
additional treatments directed at this
TZD receptor. For example, phosphorylation of PPARγ negatively regulates its
function69–72, suggesting that therapies
aimed at increasing the dephosphorylated state might synergize with TZDs in
potentiating insulin action.
•
Acknowledgements
Our work was supported by NIH grants
DK49780 and DK49210. We thank Dalei
Shao for valuable discussions.
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