Modulation of Metabolism Through
Transcriptional Control has created New Treatment Opportunities for Type 2
Diabetes
Esper
Boel*,
Tatjana Albrektsen, Jan Fleckner, and Johan Selmer
Health Care Discovery, Novo Nordisk
A/S, DK-2880 Bagsvćrd, Denmark
*Address correspondence to this author at the Molecular Genetics, Novo
Nordisk A/S, Novo Allé 6A1.027, DK-2880 Bagsvćrd, Denmark; Tel:
+45-4442-2872; FAX: +45-4442-1242; E-mail: Boel@novo.dk
Abstract: The discovery of the important
metabolic and physiological role played by a family of transcription factors,
the peroxisome proliferator activated receptors (PPAR), has opened up for a new
understanding of the mode of action for the lipid lowering drugs known as
fibrates and for the new glucose lowering compounds described as insulin
sensitizers.
Both of these classes of compounds have demonstrated significant
efficacy in both animal models of the metabolic derangements characteristic for
type 2 diabetes and in human clinical studies. The recognition of the role of
these drugs as ligands for PPAR transcription factors and the development of
new molecular and cellular tools to select and characterise new PPAR selective
compounds will open up for the development of even better new drug candidates
for the treatment of metabolic disorders associated with type 2 diabetes. With
the combined strength of new transcriptional mapping technologies developed in
the field of molecular biology, such as differential mRNA display and DNA
microarray hybridisations, it will be possible to perform a detailed molecular
characterisation of the transcriptional events involved in drug actions in
cellular and tissue systems, and information gathered from such types of
analysis will lead to an enormous amount of data, from which detailed knowledge
of drug actions at the gene regulatory level will emerge.
METABOLIC
DISTURBANCES IN TYPE 2 DIABETIC PATIENTS
Although the etiology of type 2 diabetes is
not known, several analyses suggest that the disease is the result of a
combination of genetic susceptibility and external factors of which an increase
in calorie consumption, especially fat, is by far the most important element.
The impact of excessive caloric consumption on the development of the type 2 diabetes
forms the basis for the almost epidemic increase in disease prevalence, which
is seen throughout most of the world.
The hyperglycemia of type 2 diabetes is
caused by a combination of impaired glucose uptake in muscle and fat tissue [1,2], increased hepatic glucose production [3] and finally a defect in glucose mediated insulin release [4,5]. The elevated level of plasma glucose is,
however, only part of a more general dysregulation of fuel utilization"
such as metabolism of glucose and lipids in these patients [6,7]. The disturbances in lipid metabolism are
profound and include elevation of plasma free fatty acids (FFA). The increase
in plasma FFA is probably a reflection of the impaired anti-lipolytic activity
of insulin in fat tissue and of the increased amounts of fat tissue in type 2
diabetic patients [6]. Analyses suggest that elevated levels of plasma FFA can induce almost
all of the changes in glucose metabolism seen in the patients. First, increased
concentration of plasma FFA induces a state of insulin resistance in muscle [8] and other tissues. Second, clinical studies suggest that it is serum
FFA and not insulin that primarily determines the glucose production from the
liver [9].
In addition to having effects on glucose
metabolism, the increased supply of FFA and glucose to the liver contributes to
the overproduction of plasma triglyceride rich VLDL particles [10]. Adding to this overproduction of lipoproteins from the liver is a
decreased activity of lipoprotein lipase [11], resulting in a prolonged circulation time of triglyceride rich
lipoproteins. This forms the basis for the development of the diabetic
dyslipidemic lipoprotein profile, characterized by elevated concentration of
small, dense LDL particles, increased concentration of small, cholesterol-rich
remnant particles, and decreased concentration of HDL cholesterol [12].
Myocardinal infarction, ischemic heart
disease, and stroke are some of the macrovascular complications responsible for
80% of the mortality in patients with type 2 diabetes. Type 2 diabetics have a
3 to 5 fold increase in morbidity and mortality of coronary heart disease (CHD)
compared to the normal population [13], and macrovascular complications have a much greater impact on patient
mortality than microvascular complications. Recent clinical data suggests that
small, dense LDL and low HDL cholesterol are very important factors in the
development of macrovascular diseases. In fact, the risk factor posed upon the
patients by the special diabetic dyslipidemic phenotype, far exceeds more
traditional risk factors like elevated LDL cholesterol [14,15].
TRANSCRIPTION
FACTOR LIGANDS AS MODULATORS OF LIPID METABOLISM AND PLASMA LIPOPROTEINS
Gene
Regulation of Lipid and Fatty Acid Oxidation
Lipid
lowering fibrates, of which many have been used in the clinic, are classified
as peroxisome proliferators because they cause proliferation of peroxisomes.
This proliferation is associated with hepatomegaly and hepatocellular
carcinomas in rodents, phenomenons that so far have not been reported in man.
At the physiological level, an increased number of peroxisomes in the liver
increase the capacity of this organ to perform oxidation of fatty acids, and
fibrates thus cause a general reduction in the load of lipids and fatty acids
in the body.
At
the molecular level, the fibrates along with other groups of compounds such as
certain leukotrienes, eicosanoids, and long chain fatty acids have been
identified as ligands [16-18] for a recently discovered class of
transcription factors described as peroxisome proliferator activated receptors
(PPARs) of the a
subclass. PPARa
is primarily expressed in liver, kidney and heart, where it through
heterodimerisation with another family of nuclear transcription factors, the
retinoid X receptors, RXRs, induces the transcription of mRNAs encoding enzymes
like, e.g. acyl-CoA oxidase [19],
bifunctional enzyme (enoyl-CoA hydratase/3-hydroxy-acyl-CoA dehydrogenase [20],
lipoprotein lipase [21],
malic enzyme [22]
and medium chain acyl-CoA dehydrogenase [23],
which are involved in the metabolism and oxidation of lipids and fatty acids.
Importantly, disruption of the ligand-binding domain of PPARa by homologous recombination in
mice resulted in animals that did not display the peroxisome proliferator
pleiotropic response when exposed to these compounds. After treatment of PPARa knock-out mice with peroxisome
proliferators no hepatomegaly, peroxisome proliferation, and transcriptional
activation of target genes were observed [24].
Fibrate mediated activation of PPARa
also results in transcriptional induction of the major HDL apolipoproteins,
apoA-I and apoA-II, which causes an increased HDL cholesterol. PPARa
activation can decrease plasma triglycerides through a decreased hepatic
apoC-III production and through an increase in LPL-mediated lipolysis. Thus,
fibrates stimulate cellular fatty acid uptake, conversion to acyl-CoA
derivatives, and catabolism by the beta-oxidation pathways, which combined with
a reduction in fatty acid and triglyceride synthesis, results in a decrease in
VLDL production [25]. In addition to inhibition of the VLDL production, inhibition of
cholesterol transfer from HDL to VLDL results in normalization of the
transformation of VLDL precursors to receptor-active LDL, thereby reducing the
atherogenic small dense LDL particles [26-28].
The
accelerated atherosclerosis, that is the major morbidity factor for type 2
patients, is associated with a lipid profile characterised by low HDL
cholesterol, increased plasma triglycerides, and increased level of small,
dense LDL. It is, therefore, tempting to speculate that fibrates may be
valuable in preventing cardiovascular diseases in these patients. Most
fibrates, in use as treatment for dyslipidemia, preferentially activate PPARa
and recently, a positive correlation between PPARa EC50 in transient
transactivation assays and in vivo
MED40-60 (producing 40 60 % lowering of serum VLDL+LDL
cholesterol) was reported [29].
In
the Helsinki Heart Study [30]
the effect of gemfibrozil on risk of coronary heart disease was evaluated in
patients with elevated levels of non-HDL cholesterol. The study has not had
major clinical impact, in that a 34% reduction of cardiac events for some
reason did not translate into a reduction of mortality. Subsequent subgroup
analysis has demonstrated, however, that almost all of the beneficial effect
was seen in the population with a combination of low HDL-cholesterol and high
triglycerides. Obese patients with this lipid profile had a cardiac risk
reduction of 75 % [31].
These data and the advent of more efficacious fibrates gives hope to ongoing
intervention trials like the Diabetes Athero-sclerosis Intervention Study [32]
and the Fenofibrate Intervention and Event Lowering in Diabetes Study [33],
in which dyslipidemic type 2 patients are treated with micronized fenofibrate.
Gene
Regulation of Fat Storage and Adipocyte Differentiation
Chemical
synthesis of fibrate derivatives lead the way to the establishment of yet
another new group of pharmacologically versatile compounds, the
thiazolidinediones, which in animal models of type 2 diabetes and in the clinic
have shown beneficial effects primarily as hypoglycemic agents and therefore
have become known as insulin sensitizers. Early members of this class include
Pioglitazone [34,35],
Troglitazone [36,37] and Rosiglitazone [38-40],
which are now available in or entering the diabetes markets in many countries
including the USA and Europe.
In 1995 Lehmann et al. discovered that potent insulin sensitizers acted as high
affinity ligands for PPARg [41]. This finding naturally led to the idea that the hypoglycaemic
properties of the insulin sensitizers were mediated by this orphan nuclear
receptor. This has subsequently been substantiated by the finding of a positive
correlation between potency of PPARg activation
in transient trans-activation assays (EC50, see below) and the
minimum effective dose needed to attain 25 % maximum hypoglycaemic effect (MED25)
in ob/ob mice [35].
In
contrast to PPARa,
PPARg
is primarily responsible for the transcriptional regulation of genes involved
in adipocyte differentiation and at the metabolic level in FFA and lipid
anabolism and storage. Examples of such target genes include acyl-CoA synthase [42],
adipocyte lipid binding protein (aP2) [43],
phosphoenolpyruvate carboxykinase [44],
brown adipocyte uncoupling protein [45]
and stearoyl-CoA desaturase 1 [46].
The
PPARg
mediated reduction of hyperglycemia observed in type 2 diabetes is speculated
to be secondary to adipocyte differentiation [47]
or activation [42,48].
Metabolically active adipocytes will presumably store more lipids and thus
decrease circulating levels of free fatty acids, and upon treatment with
insulin sensitizers the body can gradually base more of its energy consumption
on the oxidation of glucose in insulin responsive tissues such as muscle. While
one might expect an increased body weight from such treatment, this has
apparently so far not been a major problem, but future long term treatment of
patients will answer this question.
Characterisation
of New PPAR Ligands in in-vitro
Assays
Over the past decade, a number of
technologies have been applied to describe the interaction between ligands and
PPAR receptors. These interactions have been characterized essentially at three
levels: First, the interaction has been described in terms of direct binding
using classical and novel type binding assays yielding Kd values as
measure of the interaction. Second, interactions have also been characterized
by the use of functional assays in which the transcriptional activity of the
ligand stimulated receptors have been determined either on native promoters
containing peroxisome proliferator response elements (PPREs) or from
heterogeneous promoters as e.g. Gal4 fusion proteins. This type of assay yields
both a measure of the efficacy and the potency of a compound. Third, drug
interactions with PPARg and PPARd have also been investigated
in spatial terms through the co-crystallization of PPARs with their ligand.
The first step in the identification of a
new ligand is often the use of a ligand-binding assay to directly detect
binding to the receptor in question [18,49-52]. Classically, this involves the use of a
radio labeled ligand and recombinant PPAR protein typically expressed in E. coli. Displacement of the receptor
bound radio labelled ligand allows determination of Ki for the
compound in question. This technology can be formatted for high throughput
screening in the form of an SPA (scintillation proximity assay) based assay [53]. In addition, a number of alternative techniques are also available to
detect and quantify direct binding. These include the protease sensitivity
assay, ligand-induced complex formation assay (LIC) and co-activator-dependent
receptor ligand assay (CARLA) [54]. These assays take advantage of the conformational change induced in
the receptors upon binding to ligand. The protease sensitivity assay is based
on the observation that when the receptor is exposed to protease a protected
fragment that is not observed with receptor alone emerges from the
ligand-receptor complex. The LIC assay was developed using band shift assay to
detect PPAR-receptor-RXR interaction [16], whereas the CARLA assay detects ligand dependent interaction of the
receptor with a co-activator protein or a fragment hereof [55].
Ligands for the PPARs can also be
identified and characterized in cell based assays measuring the ligand
dependent transcriptional capacity of the receptors [41,29,52]. In these assays a reporter gene under
PPAR transcriptional control is measured after addition of compound to the
medium of transiently transfected cells. The reporter gene, may be controlled
by a native PPRE, in which case the full-length receptor is used in the assay.
Alternatively, a heterogeneous enhancer/ promoter such as the yeast GAL4
response element can control the reporter gene expression. In this setting the
ligand-binding domain (LBD) of the PPAR that contains the ligand binding pocket
and the ligand responsive activating function is fused to the DNA binding
domain (DBD) of the Gal4 protein thus allowing for PPAR controlled reporter
gene transcription. The full-length receptor approach most likely reflects the in vivo situation more closely than the
fusion protein approach. However, since these assays are frequently carried out
in cells from higher eukaryotes, the latter assay is likely to be less obscured
by activities of endogenous factors.
The crystal structures of the human PPARg
and d
LBDs in the presence or absence of ligand have been solved. The LBD consist of
13 a-helices
and a small b sheet forming a hydrophobic ligand-binding cavity. Although
the binding pockets of the two receptors are similar to that of other know
nuclear receptors two major differences have been observed. Firstly, the
binding pocket of the PPARs is about twice the size of that for other
receptors. Moreover, only 30 and 40 % of the volume in the cavity of PPARg
and d
binding pockets is occupied by rosiglitazone and eicosapentaenoic acid
respectively [56,57]. This is unusual, since for instance the thyroid hormone receptor
ligand occupies 90 % of the volume in the binding pocket. This raises the question
of whether other larger ligands remain to be identified. Secondly, the PPAR
LBDs contain an additional a-helix at the bottom of the ligand-binding
pocket that takes part in the entry into the pocket of the ligand.
Understanding the spatial features of the ligand binding pockets of these
receptors will be of great significance for the future development of novel
improved insulin sensitizers and / or lipid lowering agents.
Characterisation
of New PPAR Ligands in in vivo Animal
Models
Characterisation
of PPARg
ligands can be done in a variety of animal models. The db/db mouse is a
convenient model for evaluation of the potency of larger series of PPARg compounds that will lower plasma
glucose, insulin and serum triglycerides [58].
It is far more difficult to establish rodent animal models that in a
reproducible way reflect the lipid lowering properties of PPARa ligands. Cholesterol lowering in
cholesterol fed rats [59]
or triglyceride lowering in hamsters fed a high fat diet (in-house
observations) are some of the current alternative possibilities. Transgenic
animal models like the human apoA-1 gene transgenic mice [60]
or the apoE3-Leiden mice [61]
may prove to be very useful.
TRANSCRIPTIONAL
MAPPING OF EXPRESSED GENES AS A TOOL IN THE IDENTIFICATION OF NEW POTENTIAL
DRUG TARGETS
In
many diseases, it is still not known which genes or gene products are involved
in the pathobiology in question, but gene-technology now offers new approaches
in the search for new candidate genes encoding putative pharmaceutical targets
for drug discovery. In any differentiated cell type only a subset of all
available genes is active to specify the particular functions characteristic
for this cell type. However, disease conditions can modify the activity or
expression levels of many important genes leading in some cases to changes that
will have a direct causative relation to a particular disease state.
In
the case of type 2 diabetes a selected number of genetic defects such as
defects in the genes encoding the insulin receptor, glucokinase, and hepatic
nuclear transcription factors lead to
more narrowly defined subtypes of the disease. However, there are no strong
indications that the common adult-onset type of diabetes should have as its
primary cause a defect at one or a few specific genetic loci [62-65].
Nevertheless, a better understanding of the changes in the regulation of gene
transcriptional activity, which is associated with the development of metabolic
dysregulation, might lead the way to identification of new treatment
modalities.
Transcriptional
Mapping of Genes Regulated in Disease or upon Treatment with Drugs
With
our current stage of knowledge, where only a fraction of the human genes have
been identified and functionally described in detail, we do not always know all
the genes that are involved or modulated by disease. This is even the case in
many conditions, where there is a significant genetic component involved in the
etiology of the disease. However, we can now systematically investigate if
transcriptional up or down regulation of genes in afflicted cells and tissues
may be associated with the pathological situation, and we may even be able to
identify novel and important genes, which may form the basis for pharmaceutical
intervention.
This
methodology is called transcriptional mapping of gene regulation, and is
currently mostly based on work conducted on cell culture derived material or on
tissue material from appropriate animal models, which may serve as simplified
surrogates for the human diseases under study. However, there is in principle
no reasons why such studies could not be conducted on tissue material collected
from e.g., patients enrolled in clinical studies, and we will presumably soon
see various applications of transcriptional mapping used in extensive studies
in man.
In
transcriptional mapping, all genes, irrespective of whether their exact
identity or function is known or not, can be compared between tissues collected
from patients and from control individuals. Such comparisons can lead to
identification of defects in the gene regulatory machinery, which might be
causative for specific disease conditions. This knowledge will have the
potential to lead to new opportunities for pharmaceutical development.
A
detailed characterisation of the mode of action at the molecular level of
investigational compounds and drugs that act at the level of gene regulation
can also be obtained through transcriptional mapping. In such studies the
mapping is performed on cells or on tissue material from animals treated with
the compounds in question. By administering ligands for PPARa and PPARg to responsive cell cultures or to
animal model systems, it is thus possible to obtain a detailed picture of
overall gene regulation caused by the ligands for these transcription factors.
Differential
mRNA Display Analysis of Gene Regulation
Differential
mRNA display analysis, which is one well-established way of performing
transcriptional mapping, can be used to investigate the overall gene regulatory
patterns in a biological sample. Differential mRNA display analysis was devised
and refined by Liang and Pardee [66]
as a method to compare gene expression in two or more cell types and to clone
differentially expressed genes rapidly. The method is based upon a series of
steps; reverse transcription of mRNA from anchored primers, rounds of polymerase
chain reactions (PCR) using the same anchoring primer and a short arbitrary
primer as well as radioactive nucleotides, and finally labelled cDNAs produced
by PCR are separated on a DNA sequencing gel and visualized by autoradiography.
DNA
Array/Chip Analysis of Gene Regulation
As
an alternative to the overall analysis, in which no prior knowledge of the
identity or DNA sequence of the genes investigated is required, it is now also
possible to immobilise thousands of gene fragments on solid supports either as
cDNA fragments [67-69] or as oligonucleotide counterparts [70].
Such immobilised DNA probes can be hybridised with dye-labelled cDNA mixtures,
which have been reverse transcribed from mRNA populations isolated from cells
and tissues under investigation. The resulting hybridisation patterns will give
detailed information on the regulation at the transcriptional level of all of
the pre-selected genes included in the immobilised set of probes.
New
highly automated technologies in this field have required significant resource
investments, both in terms of development of the necessary hardware needed for
robotization of the handling of gene fragments to be analysed, and in the
development of the software for final analysis of accumulated data.
Equipped
with sufficient and appropriate access to databases with a rapidly accumulating
number of mammalian and human gene sequences combined with access to the
relevant biological samples from adipocyte, muscle and hepatocyte cell cultures
and from animal models of dyslipidemia, the power of these new analytical
technologies is now explored, and the defined aim is to obtain a better
understanding at the level of gene transcriptional regulation of the
development of metabolic dysregulation seen in type 2 diabetes.
New
Target Identification and Evaluation
It
is expected that this technology will provide the scientific community with a
better understanding of the genetic circuits involved in metabolic regulation
as well as create new possibilities for identification of molecular targets for
therapeutic intervention. Once such candidate gene targets have been discovered
by transcriptional mapping, the pharmaceutical industry still has a long path
to travel before the specific biology of the newly discovered genes is
unravelled and before new drug candidates acting on these genes and their
protein products can be identified.
Gene
target evaluation will involve at least over- and underexpression of the
selected full length genes initially in in
vitro systems and possibly also in tissue specific and in development-stage
specific in vivo systems such as
transgenic and gene knock-out mouse models followed by careful biological
assessment of the physiological and metabolic consequences of such changes.
In
this respect it is important to realise that any drug that affects
transcriptional regulation has the potential to modulate promoter activities at
genetic loci that may be involved in other unrelated cellular functions. Such
functions, if they are centrally involved in regulation of cell growth and
differentiation, may represent targets that it would be potentially hazardous
to modulate in a non-physiological direction.
Therefore,
since the PPAR transcription factors are centrally involved in cell
differentiation in several tissues, transcriptional mapping technologies also
hold the potential to identify and characterise the regulation of genetic
elements that not only regulate metabolic functions, but also those that are of
particular relevance for coordinated growth and differentiation of other cell
types.
Of
particular interest is the involvement of PPARg
in the development of the epithelium in colon. PPARg ligands have been
shown to have potent antineoplastic and antiproliferative activities both in vitro [71,72] and in
vivo as judged by growth inhibition of human colonic tumours transplanted
in to mice [73].
These findings, however, contrast results obtained in rodent models
characterised by spontaneous development of multiple intestinal tumours due to
a mutation in the adenomatous polyposis coli (APC) tumour suppressor gene.
Treatment with PPARg
ligands was found to promote development of colonic tumours in these animals,
whereas no effect could be observed in wild-type animals [74,75].
THERE WILL BE
MUCH MORE TO BE LEARNED
An
important interplay between differentiation biology and gene regulation has
been taken for granted for many years, but the new recognition of the strong
relation between transcriptional regulation, cell differentiation and metabolic
regulation in common diseases like diabetes has added an interesting twist to
the synergy between molecular biology and drug discovery and development. We
have only begun to acquire a very superficial view and initial basic
understanding of how this new field of research will help us in designing new
treatment modalities based on transcription factor ligands. This will in the
future allow us to investigate at the molecular level the associated efficacy
and safety aspects related to the development of such drugs.
In
addition to the already described classes of PPAR ligands new and interesting
compounds for treatment of type 2 diabetes will be discovered and developed in
the near future, and we feel that transcription biology offers a unique
opportunity for providing access to potentially important new targets in type 2
diabetes and the new technology will definitely add to our understanding of the
mode of action of new active molecules.
Abbreviations
FFA = Free
fatty acids
VLDL = Very
low density lipoprotein
LDL = Low
density lipoprotein
HDL = High
density lipoprotein
CDH = Coronary
heart disease
PPAR = Peroxisome
proilferator activated receptor
RXR = Retinoid
X receptor
LPL = Lipoprotein
lipase
apoA-I = Apolipoprotein
A-I
apoA-II = Apolipoprotein
A-II
apoC-III = Apolipoprotein
C-III
EC50 = Efficient
concentration at which 50% maximum effect is obtained
MED40-60 = Minimal
effective dose needed to obtain 40 60 % in
vivo effect
PPRE = Peroxisome
proliferator activated receptor response element
SPA = Scintillation
proximity assay
LIC = Ligand-induced
complex formation assay
CARLA = Co-activator-dependent
receptor ligand assay
LBD = Ligand
binding domain
DBD = DNA
binding domain
apoE3 = Apolipoprotein
E3
PCR = Polymerase
chain reaction
APC =