1 Research and Development, Novo Nordisk, DK-2880 Bagsvaerd, Denmark; and 2 Garvan Institute of Medical Research, Darlinghurst, Sydney 2010, Australia
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ABSTRACT |
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Improvement of insulin
sensitivity and lipid and glucose metabolism by coactivation of both
nuclear peroxisome proliferator-activated receptor (PPAR) and
PPAR
potentially provides beneficial effects over existing PPAR
and
preferential drugs, respectively, in treatment of type 2 diabetes. We examined the effects of the dual PPAR
/
agonist
ragaglitazar on hyperglycemia and whole body insulin sensitivity in
early and late diabetes stages in Zucker diabetic fatty (ZDF) rats and
compared them with treatment with the PPAR
preferential agonist
rosiglitazone. Despite normalization of hyperglycemia and Hb
A1c and reduction of plasma triglycerides by both compounds in both prevention and early intervention studies, ragaglitazar treatment resulted in overall reduced circulating insulin and improved
insulin sensitivity to a greater extent than after treatment with
rosiglitazone. In late-intervention therapy, ragaglitazar reduced Hb
A1c by 2.3% compared with 1.1% by rosiglitazone.
Improvement of insulin sensitivity caused by the dual PPAR
/
agonist ragaglitazar seemed to have beneficial impact over that of the
PPAR
-preferential activator rosiglitazone on glycemic control in
frankly diabetic ZDF rats.
euglycemic clamp; peroxisome proliferator-activated receptor; ragaglitazar; rosiglitazone; type 2 diabetes; Zucker diabetic fatty
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INTRODUCTION |
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TYPE 2 DIABETES is a heterogeneous, progressive disorder initially
characterized by impaired glucose tolerance and compensatory hyperinsulinemia and, in the later stages, by severe insulin resistance and impaired -cell function. The syndrome is characterized by an
imbalanced interplay between endocrine pancreatic function, insulin
sensitivity of liver, muscle, and adipose tissues, and neural activity.
This is associated with hyperglycemia, dyslipidemia, hypertension, and
obesity and, in the long term, micro- and macrovascular complications,
leading to impaired life quality and increased mortality (10, 12,
13, 17, 28, 37, 48). Unfortunately, none of the available drugs
for clinical use has proved sufficiently efficacious in restoring
normal glucose metabolism alone or in combination therapy as the
disease progresses (35).
Previously, two classes of compounds, the thiazolidinediones (TZDs) and the fibrates, were empirically discovered by their ability to improve insulin sensitivity and lipidemia, respectively, in rodents. In type 2 diabetic patients, the TZDs reduce both hyperglycemia and the compensatory hyperinsulinemia but exert only marginal effects on plasma lipid parameters (3). In contrast, the fibrates are effective at lowering plasma triglycerides (TG) and free fatty acids (FFA) and increasing favorable high-density lipoprotein cholesterol via increased clearance and decreased synthesis of very low density lipoprotein (VLDL; see Ref. 45). In addition, they have been shown also to improve glycemic control in type 2 diabetic patients (23, 26).
The recent discovery that the nuclear peroxisome proliferator-activated
receptor (PPAR) and PPAR
are the primary targets for the TZDs and
fibrates, respectively (7, 51), has provided the
opportunity for application of target-directed approaches in the
optimization of drug candidates for the treatment of type 2 diabetes.
Consequently, agonists with dual PPAR
and -
activity would
potentially have beneficial effects superior to those obtained with
drugs activating either of the PPAR subtypes alone.
Here we report on the effects on carbohydrate metabolism invoked by
ragaglitazar, which is a non-TZD compound that was recently identified
as a combined PPAR and -
agonist with a good pharmacokinetic profile and full PPAR
and -
agonistic property both in vitro and
in vivo (28, 54). We have investigated the effects of ragaglitazar treatment on glycemic control and insulin sensitivity in
prediabetic and moderately diabetic Zucker diabetic fatty (ZDF) rats in
comparison with the preferential PPAR
agonist rosiglitazone. TZDs
have previously been shown capable of preventing or delaying the onset
of diabetes (44, 47) but unable to reverse fully established diabetes in the ZDF rat (41). Therefore, we
also conducted a late-intervention dose-response study with
ragaglitazar and rosiglitazone in severely diabetic ZDF rats to explore
the extent to which reversal of frank diabetes is feasible by dual activation of PPAR
and -
.
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EXPERIMENTAL PROCEDURES |
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Animals
ZDF (ZDF-fa/fa; genetic model) rats were housed under controlled ambient conditions after a 12:12-h light-dark cycle with light on at 6:00 AM and were fed Purina 5008 diet ad libitum with free access to water. All procedures were approved by the Danish Animal Experiments Inspectorate.Test Compounds
Ragaglitazar [NNC 61-0029, (Anesthesia and Surgical Preparation of Rats
Catheters (Tygon S-50-HL, ID: 0.016 in., OD: 0.031 in.; Norton Performance Plastics) were inserted (halothane anesthesia) in the left carotid artery (blood sampling) and jugular vein (infusions) and exteriorized on the back of the neck, as described (34). Prophylactic antibiotic (Streptocilin; Boehringer Ingelheim) and analgesic treatments (Anorphin) were employed for 3 days after the surgery. Diabetic control ZDF rats were given insulin (2-5 U/kg sc; Actrapid; Novo Nordisk) on the day of surgery and the following day to improve their postsurgical recovery. Rats recovered for 5-7 days before the clamp study. Rats subjected to dual-energy X-ray absorptiometry (DEXA) were anesthetized using a mixture of fentanyl (0.05 mg/kg), fluanizone (2.5 mg/kg; Hypnorm; Janssen Pharma, Copenhagen, Denmark), and midazolam (1.25 mg/kg; Dormicum; Roche, Basel, Switzerland).Hyperinsulinemic Euglycemic Clamp Studies
After an overnight fast (18 h), catheters were connected to the infusion system, and the rats were placed in clamp cages and allowed to settle for 45-60 min. A primed (80 µCi) continuous (0.8 µCi · kgR'g was calculated as described (21). Values were not corrected by a "discrimination constant" for 2-DG in the glucose metabolic pathways; therefore, results represent the index of glucose utilization.
DEXA
Body composition was determined by DEXA (pDEXA Sabre, Stratec Medizintechnic; Norland Medical Systems, Pörzheim, Germany). The coefficient of variation, as assessed by 10 repeated measurements (repositioning of the rat between each measurement), was 3.73% for the fat tissue mass.In Vivo Expression of Various PPAR-Regulated Genes
RNA was isolated by TRIzol (Invitrogen) according to the manufacturer's instructions. Total RNA was DNase treated, and reverse transcription reactions were performed using Superscript II RT (GIBCO-BRL) following the manufacturer's instructions. mRNA expression levels were determined using real-time fluorescent detection in a Lightcycler instrument (Roche) and the following primer combinations: adipsin: forward, 5'-AACCCGGCACGCTCTGCGAC-3' and reverse, 5'-TGCAAGTGTCCCTGCGGTTG-3'; adipocyte lipid-binding protein (aP2): forward, 5'-ATGCCTTTGTGGGAACCTGG-3' and reverse, 5'-CCCAGTTTGAAGGAAATCTCGG-3'; apolipoprotein CIII (ApoCIII): forward, 5'-TCCAGGTACGTAGGTGCCATGCA-3' and reverse, 5'-TTCCATGTAGCCCTGCACAGAGC-3'; acyl-CoA oxidase (ACO): forward, 5'-TAAGTCTGTGTCTGTGGCATTCG-3' and reverse, 5'-GCTGTGTACTGTCAATCTTAAGGG-3'; enoyl-CoA hydratase-3-hydroxyacyl-CoA dehrydrogenase bifunctional enzyme (BIFEZ): forward, 5'-CAACATAGATGAGTAGCCCAAGG-3' and reverse, 5'-CTGGGGATTTAGCTCAGTGG-3'; lipoprotein lipase (LPL): forward, 5'-GAACACCTACACACAAGCAAAGCC-3' and reverse, 5'-CATAGACAGTACCAGGCTCGTTGC-3'. mRNA expression levels were determined two times in each first-strand synthesis reaction and normalized to the expression levels of 36B4 mRNA using the following primers: forward, 5'-TAAAGACTGGAGACAAGGTGGGAG-3' and reverse, 5'-AGAAAGCGAGAGTGCAGGGC-3'.Analytical Assays
Blood (BG) and plasma glucose concentrations were analyzed by the glucose oxidase method using either an EBIO Plus autoanalyzer (Eppendorf) or, during clamp studies, a YSI 2500 STAT (Yellow Spring Instruments).Plasma concentrations of FFA (Wako Chemicals) and TG (Roche, Hvidovre, Denmark), as well as glycolated hemoglobin A1c (Hb A1c; Roche), were measured using a COBAS MIRA Plus autoanalyzer (Roche Dianognostic Systems). Plasma insulin (PI) concentration was measured by ELISA, as previously described (22).
3H and 14C counts in neutralized supernatants of deproteinized plasma samples and 2-[14C]DG and phosphorylated 2-[14C]DG (2-[14C]DG-P) counts in digested tissue samples before and after the Somogy extraction procedure (42) were measured in a scintillation counter. The Somogyi extraction procedure employed on the tissue samples removes both free intracellular 2-DG-P and any 2-DG incorporated in glycogen, and the value thus provides an estimate of the total R'g (33). For determination of [3H]- and [14C]glucose specific activities, glucose concentrations in subfractions of the deproteinized plasma supernatants were measured spectrophotometrically using a glucose oxidase-based colorimetric method (Boehringer Mannheim).
Glycogen content in muscle tissues was measured by a hexokinase method (19). Liver TG were extracted and quantified by a Peridochrom Triglyceride CPO-PAP kit (Boehringer Mannheim; see Ref. 6). Liver glycogen was measured (Bio-Rad, Copenhagen, Denmark) as previously described (1). Liver glycogen phosphorylase (GPa + GPb) activity and total protein content (Bio-Rad) was assayed in supernatants of homogenates of tissue samples, and glycogen phosphorylase activity was measured in the direction of glycogen breakdown from the photometric determination of the rate of NADPH formation in an assay coupled to phosphoglucomutase and glucose-6-phosphate dehydrogenase (29).
Immunohistochemistry
Immunohistochemical reagents were from Dako except guinea pig anti-insulin (ICN), goat anti-glucokinase (Santa Cruz), VectaStain Elite Kit and Vector Nova Red (Vector Laboratories), and monoclonal mouse anti-glucagon and anti-GLUT2 antibodies (Novo Nordisk). Sections were washed two times with Tris-buffered saline (TBS) plus Triton X-100 (TBS-T) and then one time with TBS if not mentioned otherwise. Antisera were diluted in 7% goat plus 3% rat serum in TBS-T. For the double staining ofStereological procedures.
Volume fractions for the -cell and non-
-cell were analyzed on an
Olympus BX-50 microscope (Olympus, Copenhagen, Denmark) with a video
camera and monitor, a PC-controlled motorized stage, and the CAST-GRID
software (Olympus). The
-cell proliferation rate was estimated by
the proportion of BrDU-positive
-cells from a total of
1,000-1,500 cells counted in two to three sections cut 250 µm
apart (38). The volume fractions of
- or non-
-cells were estimated by point counting at a total on-screen magnification of
×960 by random systematic scanning of the tissue sections controlled by the CAST-GRID software (27, 38). The sections were
examined with the observer blinded to the origin of the sections.
Statistics
Data are presented as means ± SE. One-way ANOVA with (time course) or without (single time points) repeated measures and with Tukey's post hoc test for pairwise group comparisons were employed. The total area under the curve was calculated using the trapezoidal rule. Differences between groups with P values <0.05 were considered significant.Protocol 1: Prevention and Early Intervention Studies
In two independent studies, initiated in 7-wk-old prediabetic (prevention study) and in 11-wk-old moderately diabetic (early-intervention study) ZDF male rats, animals were given either vehicle, ragaglitazar (3 mg · kg
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Protocol 2: Late-Intervention Dose-Response Study
Overtly diabetic, 16-wk-old male ZDF rats were used for a dose-response study with ragaglitazar (0.75, 1.50, and 3.00 mg · kg ![]() |
RESULTS |
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Prevention and Early Intervention of Diabetes (Protocol 1)
Body weight and food intake. Body weight and food and water intake data are shown in Table 1. Ragaglitazar and rosiglitazone increased body weight and 24-h communal food intake to the same extent as with vehicle. Water intake increased gradually during the treatment period in the vehicle-treated rats (data not shown), and, during the last week of the treatments, 24-h communal water intake was ~50% higher in control rats compared with both drug-treated groups.
Glycemic control.
BG and PI levels during the treatments are shown in Fig.
2. In the prevention study, pretreatment
BG levels were ~6 mmol/l in all groups. This level of BG remained
unchanged in both the ragaglitazar- and rosiglitazone-treated rats in
contrast to a gradual increase observed in the vehicle-treated rats, in
which BG reached 16 mmol/l at the end of the study (Fig.
2A). After 10 days of treatment, a marked compensatory
hyperinsulinemia was observed in the vehicle-treated control group.
After an additional 4 wk of vehicle treatment, -cells were
exhausted, as evidenced by reduced hyperinsulinemia despite a marked
hyperglycemia. Hyperinsulinemia seemed less pronounced in the
ragaglitazar-treated animals compared with the rosiglitazone treatment
during the entire course of the study (Fig. 2B).
Plasma lipids. Results are given in Table 3. In the prevention study, ragaglitazar reduced FFA by 20% compared with vehicle treatment, whereas rosiglitazone had no such effect. Treatment with either compound reduced plasma TG levels by 80% compared with vehicle. In the early-intervention study, rosiglitazone increased FFA by ~35% compared with vehicle, whereas both drugs reduced TG by ~70% compared with vehicle.
Insulin sensitivity. Results from the clamp studies are presented in Fig. 4 and Table 4. In both studies, the GIR required to maintain matching glycemic levels during the clamp were higher in the ragaglitazar-treated rats compared with both vehicle and rosiglitazone-treated rats. Basal EGP was reduced significantly by both drugs compared with vehicle (Table 4). In both studies, the insulin-mediated suppression of EGP during the clamp tended to be more pronounced in ragaglitazar- than in rosiglitazone-treated rats, although this did not reach statistical significance.
In the prevention study, both ragaglitazar and rosiglitazone treatment increased the insulin-stimulated R'g 2.9- and 2.3-fold, respectively (P < 0.05 vs. vehicle), in the "fast-twitch white fiber" muscle tissue (white gastrocnemius). This effect was only inflicted by ragaglitazar in the early-intervention study (4.7-fold, P < 0.05 vs. vehicle). R'g in the muscles of the fast (red gastrocnemius)- and slow (soleus)-twitch red fiber types were overall unaffected by either of the two drug treatments. In both studies, treatment with ragaglitazar increased R'g in the white adipose tissue ~2.5-fold compared with vehicle treatment (P < 0.05) and 1.5-fold compared with rosiglitazone treatment (P < 0.05; data not shown).Biochemical parameters in tissues. Results are shown in Table 5. Both compounds significantly decreased hepatic glycogen content in the clamped rats compared with vehicle, with ragaglitazar being the more efficacious. Liver glycogen phosphorylase activity of both clamped (prevention study) and nonclamped (early intervention study) rats was reduced by the ragaglitazar treatment compared with vehicle and rosiglitazone treatment. In both studies, rosiglitazone treatment increased liver TG content in clamped rats compared with vehicle treatment, whereas ragaglitazar either had no such effect (early-intervention study) or even significantly reduced (prevention study) the liver TG content. The drugs did not exert significant changes in muscle (extensor digitorum longus and soleus) glycogen content obtained from the clamped and nonclamped rats compared with vehicle. Likewise, there was no effect of either rosiglitazone or ragaglitazar treatment on hepatic glycogen content in the nonclamped rats compared with vehicle.
Histology.
-Cell proliferation was measured by BrDU incorporation. Figure
5A depicts representative
fields of BrDU incorporation in insulin double-stained sections from
rats included in the prevention study. In both studies, however, the
labeling index of
-cells was low, and relatively more non-
-cells
and exocrine cells contained BrDU. There were no statistically
significant differences among the three groups, either in the
prevention or in the early-intervention study. The staining intensity
for insulin in
-cells was higher and less variable in ragaglitazar-
and rosiglitazone-treated rats, and the islets were more regular with
less ragged rims than in the vehicle-treated rats. The same trend was
found in the early-intervention study (data not shown). Separate
sections were stained for
- and non-
-cells, as shown in Fig.
5B (prevention study). The volume fractions of
- and
non-
-cells in ragaglitazar- and rosiglitazone-treated rats were not
different from the vehicle-treated rats. The appearance of the islets
showed a more regular distribution of non-
-cells in or near the rim
of the islets in the ragaglitazar- and rosiglitazone-treated rats. In
the intervention study, the overall morphology of the islets was also
more regular and rounded, although minor streaks of fibrosis revealed
that the rats had developed diabetes before treatment was commenced
(data not shown). GLUT2 staining intensity in
-cells from the
ragaglitazar- and rosiglitazone-treated rats in the prevention study
was higher than that of the vehicle-treated rats and more widely
distributed over the plasma membrane (Fig. 5C). In both
studies, GLUT2 staining intensity in hepatocytes from the rats treated
with ragaglitazar and rosiglitazone was higher compared with
vehicle-treated rats (data not shown). Improved glucokinase staining
patterns in
-cells were found in ragaglitazar- but not in
rosiglitazone-treated rats (data not shown). In both studies, the
staining intensity of glucokinase in liver was equally low, and the
distribution in liver cells was quite similar in all three groups of
ZDF rats (data not shown).
Late-Intervention Dose-Response Study (Protocol 2)
Glycemic control and body weight.
At the end of the 3-wk treatment period, ragaglitazar more
efficaciously improved the 24-h BG profile at all dose levels employed compared with rosiglitazone, which only partly had this effect at the
highest dose (Fig. 6A).
The improvement of glycemic control was paralleled
by a dose-related increase in body weight (Fig. 6B). PI
concentrations did not differ between the groups at any time point
during the treatment (data not shown). Hb A1c did not change in the control group, indicating that the diabetic state was
stabilized during the course of this study (data not shown). The
reduction in Hb A1c (Fig. 6C) caused by
rosiglitazone was ~1% at all three dose levels, suggesting that the
maximal response to this compound was reached. Ragaglitazar reduced Hb
A1c by ~0.7% at 0.75 mg · kg1 · day
1
and by 2.3% at 1.5 mg · kg
1 · day
1.
No further reduction in Hb A1c was observed at 3 mg · kg
1 · day
1,
indicating that the maximum response to the compound was reached at 1.5 mg · kg
1 · day
1.
The reduction of Hb A1c observed in the groups administered one time daily and two times daily with ragaglitazar at 3 mg · kg
1 · day
1
was identical. The reduction in Hb A1c correlated
positively with the increase in body weight in both ragaglitazar
(r = 0.747, P < 0.000005)- and
rosiglitazone (r = 0.772, P < 0.00001)-treated rats.
Body composition (DEXA) and correlations with Hb A1c.
The relative body fat mass was increased ~2- and 1.5-fold in the
ragaglitazar- and rosiglitazone-treated rats, respectively, compared
with vehicle treatment (Table 6). All
ragaglitazar-treated rats responded with a reduction in Hb
A1c by at least 1.75% and had a relative body fat mass of
at least 56%, whereas five out of eight rats treated with
rosiglitazone showed no significant reduction in Hb A1c
[0.49 ± 0.40 vs.
0.05 ± 0.18% for vehicle, not
significant (NS)] and correspondingly had a relative body fat mass
comparable to that of the vehicle-treated rats (36 ± 1 vs.
31 ± 2% for vehicle, NS). The three remaining
rosiglitazone-treated rats, however, all responded with a reduction in
Hb A1c comparable to that of the ragaglitazar-treated rats
(
2.08 ± 0.47 vs.
2.26 ± 0.23% for ragaglitazar, NS)
and correspondingly also had a relative body fat mass comparable to
that of the ragaglitazar-treated rats (60 ± 0.4 vs. 59 ± 1% for ragaglitazar, NS).
In vivo activation of PPAR and -
.
The PPAR
- responsive gene BIFEZ was induced in liver tissue up to
9.4-fold, whereas no response was detected in the animals treated with
rosiglitazone (Fig. 7, top).
Likewise, mRNA levels of ACO were induced to a maximum of 3.1-fold in
liver in a dose-dependent manner by ragaglitazar, and treatment with
rosiglitazone did not reveal any changes on this mRNA (data not shown).
Hepatic ApoCIII gene expression was reduced 1.7-fold at the higher
doses (1.5 mg/kg two times and 3 mg/kg one time daily) of ragaglitazar
and was not affected by rosiglitazone, lending further support to different actions of the two compounds in liver tissue. A similar pattern of differential action by the two compounds in the liver were,
although to lesser extent, noted on the levels of LPL mRNA, which was
slightly upregulated (1.5-fold) by ragaglitazar and not regulated by
rosiglitazone. In contrast, both ragaglitazar and rosiglitazone reduced
the mRNA levels of liver phosphoenolpyruvate carboxykinase
(PEPCK) approximately twofold at all doses. In the adipose tissue,
adipsin, aP2, and LPL levels were all dose-dependently increased by
both ragaglitazar and rosiglitazone. Adipsin mRNA levels were increased
a maximum of 6.3- and 5-fold upon treatment with ragaglitazar and
rosiglitazone, respectively (Fig. 7, bottom). aP2 gene
expression was induced up to 2.3-fold by ragaglitazar and 2-fold by
rosiglitazone, whereas the inductions for LPL reached 2.9- and
2.4-fold, respectively, for the two compounds. Adipocyte PEPCK mRNA was
elevated 3.2- and 1.8-fold by treatment with ragaglitazar and
rosiglitazone, respectively.
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DISCUSSION |
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The present study demonstrates that combined activation of PPAR
and -
markedly prevents and ameliorates insulin resistance in the
diabetic ZDF rat and with superior beneficial impact on glycemic
control over that achieved upon activation of PPAR
only. Recently, a
limited number of investigations have reported on the effects of dual
PPAR
/
agonism in animal models of dyslipidemia, insulin
resistance, and/or type 2 diabetes (5, 11, 14, 30, 40,
54). In those studies, the focus has been directed toward
glycemic control and diabetic late complications in the ZDF rat or on
insulin sensitivity and lipid metabolism in obese Zucker or
high-fat-fed rats. In contrast, the present investigation aimed to
explore the comparable effects of PPAR
and dual PPAR
/
agonism
on both glycemic control and insulin sensitivity during different
stages of diabetes in the ZDF rat, enabling an evaluation of the
linkage between the two parameters within the same animal model that we
consider relevant for the clinical situation.
Most importantly and in general for both the prevention and
early-intervention studies (see protocol 1) is the finding
that normalization of glycemic control was obtained with a generally reduced requirement for insulin in ragaglitazar-treated rats compared with that of rosiglitazone treatment. This suggests that dual PPAR/
activation by ragaglitazar more effectively improves
insulin sensitivity relative to that obtained upon PPAR
activation
by rosiglitazone. In both studies, this observation was further
substantiated by the fact that significantly increased GIR were
required to maintain euglycemia in ragaglitazar-treated animals during
the clamp studies. The importance hereof is that maintenance of
euglycemia together with the improved insulin sensitivity brought about
by the ragaglitazar treatment result in a reduced burden on the
-cells, as evidenced by reduced PI levels, which, in the long term,
may delay or even prevent
-cell exhaustion and consequently the
development of frank diabetes. Although not accessed directly, it is
reasonable to assume that ragaglitazar treatment also more effectively
improved insulin sensitivity in the late-intervention study (see
protocol 2) and that this is the reason why further
improvement of glycemic control was obtained compared with
rosiglitazone treatment, since PI levels were indistinguishable between
the study groups. The reason why PI levels were unaffected by the drugs
is probably that the
-cells had irreversibly deteriorated already at
the time of initiation of the late-intervention treatment. Therefore, in an attempt to compensate for the hyperglycemia present to various degrees in all groups, maximal insulin secretion was required.
The physiological consequences of simultaneous activation of both
PPAR and PPAR
suggest that additional therapeutic benefit can be
achieved on glycemic control using a dual PPAR
/
agonist compared
with treatment with a preferential PPAR
agonist. This is supported
by the recent findings of improved whole body and muscle insulin
resistance and hyperinsulinemia in various models of insulin resistance
and type 2 diabetes upon activation of PPAR
(18, 31, 52,
53). The mechanisms underlying those findings are still unclear,
but apparently they differ from that known for PPAR
agonists, which
reduce hepatic glucose production and improve glucose disposal to
peripheral tissue such as muscle and adipose tissue (4,
49). It is known that activation of PPAR
mediates catabolism
of fatty acids mainly through hepatic fatty acid oxidation. Activation
of PPAR
, by contrast, predominantly causes direct effects on adipose
tissue, supposedly leading to secondary beneficial effects on muscle
and/or liver. The fact that PPAR
agonists lower circulating fatty
acids, presumably via increased sequestering of fatty acids into
adipose tissue and decreased lipolysis, is consistent with this notion
(32). In any respect, activation of both PPAR
and
PPAR
affects fatty acid homeostasis, which, in turn, may result in
beneficial changes of glucose homeostasis as originally proposed by
Randle et al. (36).
Ragaglitazar treatment altered gene expression in a PPAR- and
-dependent manner, as evidenced by changes observed in the liver and
in adipose tissue. In contrast, administration of rosiglitazone altered
gene expression in a PPAR
-dependent manner, since changes in PPAR
target gene levels could be found solely in the adipose tissue. Whereas
treatment with ragaglitazar induced the expression of genes in the
peroxisomal
-oxidation pathway such as liver ACO and BIFEZ, these
genes were unaffected by rosiglitazone. This indicates that
ragaglitazar, like the PPAR
-activating fibrates, elicits augmented
peroxisomal fatty acid oxidation. Similarly, as for fibrates and other
PPAR
agonists that have been reported to reduce the liver expression
of ApoCIII, a major component of VLDL particles, it was observed that
ragaglitazar but not rosiglitazone elicited this change. Both compounds
reduced the expression of PEPCK in liver. This may, however, be a
secondary response to the increase of insulin sensitivity in the
animals, leading to a decrease in hepatic gluconeogenesis. In the
adipose tissue, treatment with either ragaglitazar or rosiglitazone led
to increased expression of known adipocyte PPAR target genes. For all
genes tested (adipsin, aP2, and LPL), the maximum degree of increase brought about by ragaglitazar was ~20% higher than that obtained with rosiglitazone, suggesting a quantitatively slightly stronger in
vivo effect of ragaglitazar. Adipose tissue expression of PEPCK was
also regulated by both compounds, which may offer a clue to the
mechanism whereby increased reesterification of FFA, and thus sequestration into fat, is attained (16). In concert, the
data clearly demonstrate that the two compounds exert qualitatively similar effects on the adipose tissue.
Recently, a study with the dual PPAR/
agonist LY-465608
(14) demonstrated that complete restoration of euglycemia
could be obtained in ZDF male rats during treatment in the early
diabetic period (8-12 wk of age). That effect was reported along
with a lack of augmentation of food consumption and an increase in body weight, as seen with rosiglitazone. Unfortunately, that study evaluated
the effects of LY-465608 on insulin sensitivity in obese, nondiabetic
Zucker rats only and not in the ZDF rat as well. Furthermore, the study
did not include a rosiglitazone-treated group for comparison in the
assessment of insulin sensitivity. Nevertheless, their first
observation stands in contrast to the results obtained in the present
study (protocol 1) in which ragaglitazar and rosiglitazone both maintained normal glycemic control equally well, but at the same
time both also augmented food consumption and increased body weight.
The discrepancy between the results obtained in the two studies may
well be explained by different binding and activation ratios between
PPAR
and -
brought about by ragaglitazar and LY-465608. For
example, we have observed similar beneficial effects to those reported
for LY-465608 on body weight, food consumption, and early glycemic
control in ZDF rats during the characterization of another dual
PPAR
/
agonist (NNC 61-4655), which, like LY-465608, has a higher
than
binding and activation potency compared with ragaglitazar.
However, when treatment with NNC 61-4655 was extended beyond the 12 wk
of age in the ZDF rat (from 11 to 17 wk of age), the effect on glycemic
control and insulin sensitivity ceased despite continued reduction in
food intake and less increase in body weight compared with ragaglitazar
(Brand CL, unpublished data). Therefore, it remains to be demonstrated
how well dual PPAR
/
agonists with higher
than
binding and
activation potencies such as LY-465608 are suited for treatment of
later stages of type 2 diabetes.
Independent of the type of PPAR agonist employed in the present study,
it appeared that an improvement of Hb A1c in the ZDF rat
occurred concomitantly with an increase in body weight, particularly in
total body fat mass. This is generally thought to be because of
increased lipid storage in fat tissue caused by increased clearance of
circulating lipids along with PPAR-mediated stimulation of adipogenesis, as indicated by an increase in aP2 gene expression in
adipose tissue compared with that in untreated rats. In addition, however, the increased fat mass could also be caused by preventing loss
of calories through glucosuria when glycemic control is improved. In
fact, pilot studies performed in our laboratory suggest that 20-30% of the calories consumed are lost via glucosuria in
severely diabetic ZDF rats (Brand CL, unpublished observations).
Therefore, unless food intake is decreased and/or energy expenditure is
increased to counterbalance the increased amount of retained calories
caused by preventing glucosuria, an increase in body weight would
obviously be expected. Hence, by restoring glycemic control with
ragaglitazar or rosiglitazone, this animal model seems to revert to its
original phenotype, which is the obese Zucker rat. A similar
association between body weight gain and treatment of diabetes has
previously also been demonstrated employing chronic insulin therapy in
the ZDF rat (43), which is in support of this notion.
Insulin-stimulated (clamp) R'g and glycogen content were both measured only in the soleus muscle, which, on the other hand, seemed unaffected by the drug treatments. However, both drugs increased R'g in white gastrocnemius muscle, which represents ~70% of the hindlimb musculature, whereas only ragaglitazar improved R'g in white adipose tissue (2). The differential effects of the two drugs on glucose uptake by muscle and fat tissues could therefore partly account for the extrahepatic effects observed.
Somewhat surprisingly, the liver glycogen content in all groups of nonfasted rats was near maximal, which is considered normal in healthy rats. However, despite an impaired glucose homeostasis in vehicle-treated rats, non-insulin-dependent (GLUT2) hepatic glucose uptake was probably markedly increased because of the mass action of glucose (BG >18 mmol/l). The consequent increase in intracellular glucose 6-phosphate concentrations would then activate glycogen synthase, which in turn will lead to the high glycogen content observed in the vehicle-treated rats.
Compared with vehicle-treated rats, postclamp liver glycogen stores
were reduced significantly after an overnight fast in drug-treated
animals from both studies, which is considered normal in fasted,
healthy rats, since hepatic glycogen content is not expected to
increase during a clamp (9). The reduced liver glycogen
phosphorylase activity found in some but not all ragaglitazar-treated groups of rats would expectedly decrease hepatic glycogen breakdown and
thereby reduce hepatic glucose production. This is also supported by
the finding that insulin-mediated suppression of EGP seemed more
pronounced by ragaglitazar than by rosiglitazone treatment. Whether
this is a direct PPAR-mediated effect by the ragaglitazar treatment
or caused indirectly via an increase in hepatic insulin sensitivity
resulting from, e.g., marked reduction in hepatic TG levels, is not
clear (54).
Histological analyses of the pancreas were only carried out on a small
subset of rats (4 rats/group). We observed a partial restoration of
both GLUT2 and glucokinase in -cells from the drug-treated ZDF rats,
but both were still far from being normal. Troglitazone has previously
been shown to partially normalize the reduced GLUT2 in
-cells from
ZDF rats (20). PPAR
is expressed in
-cells, and
peroxisome proliferator response element is found in the GLUT2
(24) and in the glucokinase promoter region
(25). We have at present no explanation for the
lack of change for the GLUT2 and glucokinase expression in the liver,
except that the indirect effect of the reduced insulin resistance and
improved glucose metabolism in liver is less important than in the
-cells. In vehicle-treated ZDF rats, the islets showed substantial
fibrosis, whereas there was little fibrosis in the ragaglitazar- and
rosiglitazone-treated ZDF rats. In the early-intervention study, the
general picture from the immunostainings of
-cell markers and of
the BrDU incorporation was quite similar to that of the prevention
study. Because the animals were diabetic at the start of the
experiment, there was, however, some fibrosis present in a number of
the big islets.
In summary, effects of dual PPAR/
(ragaglitazar) and only PPAR
(rosiglitazone) agonism on glycemic control and insulin sensitivity
were studied during different stages of diabetes in the ZDF rat. In
prevention and early-intervention settings, both drugs normalized
glycemic control. Yet, ragaglitazar-treated rats generally required
less insulin compared with rosiglitazone treatment, suggesting that
dual PPAR
/
activation more effectively improves insulin
sensitivity compared with activation of PPAR
alone. This was
substantiated by the hyperinsulinemic euglycemic clamp technique for
assessment of insulin sensitivity. During treatment of frank diabetes,
ragaglitazar improved glycemic control more effectively than
rosiglitazone without affecting circulating insulin, suggesting that
improvement of insulin sensitivity by treatment with ragaglitazar, over
the long term, may result in a reduced burden on the
-cells and
consequently increase their likelihood of survival. Dual PPAR
/
activation by ragaglitazar may therefore represent a superior therapy
over that of PPAR
activation alone by, e.g., rosiglitazone in early,
intermediate, and late stages of type 2 diabetes.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank S. Gronemann, B. S. Hansen, M. B. Jappe, W. Listov-Saabye, S. Primdahl, P. Rothe, A. Seneca, and A. Vinterby for excellent technical assistance, and the staff at the animal unit for daily care, monitoring, and dosing of animals.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: C. L. Brand, Pharmacological Research 1, Novo Nordisk A/S, Novo Allé, DK-2880 Bagsvaerd, Denmark (E-mail: clbr{at}novonordisk.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 10, 2002;10.1152/ajpendo.00348.2002
Received 7 August 2002; accepted in final form 8 December 2002.
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