help button home button Endocrine Society Molecular Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Molecular Endocrinology, doi:10.1210/me.2005-0015
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
19/6/1593    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow NURSA Molecule Pages Link
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Reifel-Miller, A.
Right arrow Articles by Brozinick, J. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Reifel-Miller, A.
Right arrow Articles by Brozinick, J. T.
Right arrowPubmed/NCBI databases
*Compound via MeSH
*Substance via MeSH
Hazardous Substances DB
*CHOLESTEROL
*GLUCOSE
Medline Plus Health Information
*Diabetes
Molecular Endocrinology 19 (6): 1593-1605
Copyright © 2005 by The Endocrine Society

A Peroxisome Proliferator-Activated Receptor {alpha}/{gamma} Dual Agonist with a Unique in Vitro Profile and Potent Glucose and Lipid Effects in Rodent Models of Type 2 Diabetes and Dyslipidemia

Anne Reifel-Miller, Keith Otto, Eric Hawkins, Robert Barr, William R. Bensch, Chris Bull, Sharon Dana, Kay Klausing, Jose-Alfredo Martin, Ronit Rafaeloff-Phail, Chahrzad Rafizadeh-Montrose, Gary Rhodes, Roger Robey, Isabel Rojo, Deepa Rungta, David Snyder, Kelly Wilbur, Tony Zhang, Richard Zink, Alan Warshawsky and Joseph T. Brozinick

Endocrinology Division (A.R.-M., K.O., E.H., R.R.-P., K.W., J.T.B.), Lead Optimization Biology (R.B., C.R.-M., R.Z.), Cardiovascular Research (W.R.B., C.B., D.S.), and Medicinal and Developmental Chemistry (J.-A.M., G.R., R.R., I.R., T.Z., A.W.), Lilly Research Laboratories, Indianapolis, Indiana 46285; and Ligand Pharmaceuticals (S.D., K.K., D.R.), San Diego, California 92121

Address all correspondence and requests for reprints to: Anne Reifel-Miller, Ph.D., Building 98/C/2331, Endocrinology Division, Lilly Research Laboratories, Indianapolis, Indiana 46285. E-mail: a.r.miller{at}lilly.com.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LSN862 is a novel peroxisome proliferator-activated receptor (PPAR){alpha}/{gamma} dual agonist with a unique in vitro profile that shows improvements on glucose and lipid levels in rodent models of type 2 diabetes and dyslipidemia. Data from in vitro binding, cotransfection, and cofactor recruitment assays characterize LSN862 as a high-affinity PPAR{gamma} partial agonist with relatively less but significant PPAR{alpha} agonist activity. Using these same assays, rosiglitazone was characterized as a high-affinity PPAR{gamma} full agonist with no PPAR{alpha} activity. When administered to Zucker diabetic fatty rats, LSN862 displayed significant glucose and triglyceride lowering and a significantly greater increase in adiponectin levels compared with rosiglitazone. Expression of genes involved in metabolic pathways in the liver and in two fat depots from compound-treated Zucker diabetic fatty rats was evaluated. Only LSN862 significantly elevated mRNA levels of pyruvate dehydrogenase kinase isozyme 4 and bifunctional enzyme in the liver and lipoprotein lipase in both fat depots. In contrast, both LSN862 and rosiglitazone decreased phosphoenol pyruvate carboxykinase in the liver and increased malic enzyme mRNA levels in the fat. In addition, LSN862 was examined in a second rodent model of type 2 diabetes, db/db mice. In this study, LSN862 demonstrated statistically better antidiabetic efficacy compared with rosiglitazone with an equivalent side effect profile. LSN862, rosiglitazone, and fenofibrate were each evaluated in the humanized apoA1 transgenic mouse. At the highest dose administered, LSN862 and fenofibrate reduced very low-density lipoprotein cholesterol, whereas, rosiglitazone increased very low-density lipoprotein cholesterol. LSN862, fenofibrate, and rosiglitazone produced maximal increases in high-density lipoprotein cholesterol of 65, 54, and 30%, respectively. These findings show that PPAR{gamma} full agonist activity is not necessary to achieve potent and efficacious insulin-sensitizing benefits and demonstrate the therapeutic advantages of a PPAR{alpha}/{gamma} dual agonist.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
BY THE YEAR 2025, more than 300 million individuals worldwide will suffer from type 2 diabetes. This epidemic will be followed closely by a wave of cardiovascular disease, as diabetes is not just a disease characterized by elevated blood glucose levels but is also a serious vascular disease with poor prognosis. One important cardiovascular risk factor in type 2 diabetes is dyslipidemia, which is characterized by decreased high-density lipoprotein cholesterol (HDL-C), elevated very low-density lipoprotein cholesterol (VLDL-C), and an abundance of small, dense low-density lipoprotein cholesterol (LDL-C) (1). Unfortunately, glycemic control with diet, oral hypoglycemic agents, and insulin is often only partially effective in normalizing lipid levels in individuals with type 2 diabetes (2).

Two classes of compounds, the thiazolidinediones (TZDs) and fibrates, were empirically discovered by their abilities to improve insulin sensitivity and lipidemia, respectively, in rodent models. The TZDs reduce both hyperglycemia and the compensatory hyperinsulinemia but exert only marginal effects on plasma lipid parameters in patients with type 2 diabetes (3). In contrast, the fibrates are effective at lowering plasma triglycerides and free fatty acids and increasing favorable HDL-C via increased clearance and decreased synthesis of VLDL-C (4). In addition, fibrates have been shown to improve glycemic control in patients with type 2 diabetes (5, 6).

The recent discovery that peroxisome proliferator-activated receptor (PPAR){gamma} and PPAR{alpha} are the primary targets for TZDs and fibrates, respectively (7, 8), has provided chemists and biologists with the necessary information and tools to apply target-directed approaches to optimize drug candidates. Using this approach, compounds can be identified and studied that have both PPAR{alpha} and PPAR{gamma} agonist properties (PPAR{alpha}/{gamma} dual agonists), which combine the benefits of a TZD plus a fibrate in a single molecule. Further, compounds with partial agonist vs. full agonist activity can be designed and studied for their potential therapeutic benefits.

In the present study, a non-TZD PPAR ligand (Fig. 1Go) was fully characterized for its PPAR{gamma}, PPAR{alpha}, and PPAR{delta} binding and agonist activity in vitro and potential antidiabetic and lipid-altering properties in rodent models of type 2 diabetes and dyslipidemia, respectively.



View larger version (6K):
[in this window]
[in a new window]
  		Fig. 1. LSN862: A 2-Methoxydihydrocinnamic Acid-Based PPAR{alpha}/{gamma} Dual Agonist

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In Vitro Characterization of LSN862
Competitive binding assays were used to determine the affinity of LSN862 for PPAR{gamma}, PPAR{alpha}, and PPAR{delta}, and standard cotransfection (CTF) assays were used to determine the potency and efficacy of agonist activity. As shown in Table 1Go, LSN862 is a high-affinity [dissociation constant (Ki) = 7.0 nM], potent (EC50 = 239 nM) PPAR{gamma} agonist (77% efficacy) with relatively less, but significant affinity (Ki = 770 nM) and agonist activity (EC50 = 2622 nM; 35% efficacy) on PPAR{alpha}. This degree of PPAR{alpha} affinity is greater than that of two well-studied PPAR{alpha} agonists, fenofibrate (Ki > 10,000 nM) and WY14,643 (Ki = 9,570 nM) when examined in the same assays (data not shown in table). LSN862 displayed low affinity for PPAR{delta} (Ki = 4123 nM) with no agonist activity detected. The standard PPAR{gamma} CTF assay uses a trimeric PPAR response element (PPRE) from the acyl-coenzyme A (CoA) oxidase (AOX) gene. To further explore the PPAR{gamma} agonist potential of LSN862, additional CTF assays were performed using trimeric PPREs from promoters of the lipoprotein lipase (LPL) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (bifunctional enzyme, BIFEZ) genes. In addition, LSN862 was examined in a PPAR{gamma}, yeast galactosidase (GAL4) chimeric CTF assay. In all PAPR{gamma} CTF assays, rosiglitazone served as a standard with efficacy set at 100%. Interestingly, LSN862 showed less agonist activity on all PPREs compared with rosiglitazone (Table 2Go) with PPAR{gamma} partial agonist activity demonstrated regardless of the PPRE used in the CTF assay (Fig. 2Go). These data demonstrate that although LSN862 is a high-affinity ligand for PPAR{gamma}, it functions as a PPAR{gamma} partial agonist relative to a full agonist like rosiglitazone. LSN862 was also examined for activity on other nuclear hormone receptors. Results from these studies showed that LSN862 is selective for the PPARs with no activity on the retinoic acid receptor, retinoid X receptor, liver X receptor, farnesoid X receptor, and pregnane X receptor (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 1. PPAR Binding and Cotransfection Profiles for LSN862

 

View this table:
[in this window]
[in a new window]
  		Table 2. Efficacy Values from PPAR{gamma} CTF Assays Using Various PPREs

 


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 2. Representative Dose-Response Curves from CTF Assays

Rosiglitazone (squares) and LSN862 (triangles) were each examined in full log dilution from 0.1 nM to 10 µM. Dose-response curves from a BIFEZ (A) and AOX (B) CTF assay are shown.

 
To further characterize LSN862, PPAR{gamma} cofactor recruitment assays were performed using the following cofactors: cAMP response element-binding protein (CREB)-binding protein (CBP), peroxisome proliferator-activated receptor {gamma} coactivator-1 (PGC-1), activating signal cointegrator-2 (ASC-2), thyroid hormone receptor-activated protein complex (TRAP220), and the peptide C33. Rosiglitazone served as the standard in these assays, and recruitment of cofactors to PPAR{gamma} by rosiglitazone was set at 100%. As seen in Table 3Go, LSN862 showed a distinct pattern of cofactor recruitment compared with rosiglitazone with equivalent recruitment of ASC-2 and C33, and less recruitment of CBP, PGC-1, and TRAP220. Consistent with the distinct agonist activity of LSN862 seen in the CTF assays, LSN862 does not function as a full PPAR{gamma} agonist like rosiglitazone in cofactor recruitment.


View this table:
[in this window]
[in a new window]
  		Table 3. Efficacy Values for Recruitment of Cofactors to PPAR{gamma}

 
Antidiabetic Activity of LSN862
A study in Zucker diabetic fatty (ZDF) rats was done to investigate the antidiabetic properties of LSN862. Before the study described here, the maximally efficacious doses for glucose lowering of LSN862 and rosiglitazone in ZDF rats were determined to be 3.0 and 10.0 mg/kg per day, respectively. In the present study, maximally efficacious doses of LSN862 (3.0 mg/kg per day) and rosiglitazone (10 mg/kg per day) along with an equivalent dose of rosiglitazone (3.0 mg/kg per day) were each administered to ZDF rats for 7 d. Plasma glucose, triglycerides, and adiponectin levels were determined before the study began (d –1) and at the end of the study (d 7). Rosiglitazone decreased glucose and triglyceride levels at both doses examined, with LSN862 at 3.0 mg/kg per day achieving the same degree of efficacy as rosiglitazone at 10.0 mg/kg per day on both glucose and triglyceride lowering (Fig. 3Go, A and B). Adiponectin levels increased as expected and appear to reflect the glucose-lowering abilities of LSN862 and rosiglitazone; although, interestingly, the adiponectin elevation for LSN862 at 3.0 mg/kg per day was significantly greater than the equally efficacious dose of rosiglitazone (10 mg/kg per day, Fig. 3CGo).



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 3. Plasma Glucose, Triglyceride, and Adiponectin Levels in ZDF Rats after Administration of LSN862 or Rosiglitazone (RG)

LSN862 (3.0 mg/kg) or rosiglitazone (RG, 3.0 and 10.0 mg/kg) were administered to ZDF rats for 7 d. Blood samples were collected the day before the study started, d –1 ({square}) and on d 7 ({blacksquare}). Plasma samples were analyzed for glucose (A), triglyceride (B), or adiponectin (C) 1 h after the last dose. *, P < 0.05 from vehicle group; #, P < 0.05 from RG (10 mg/kg per day). NS, Not significantly different (NS ≥ 0.05).

 
Because mouse models of type 2 diabetes are often used to explore the side effect profile of PPAR ligands, LSN862 was also evaluated in db/db mice. LSN862 and rosiglitazone were each administered orally to db/db mice at 30 mg/kg per day for 7 d. LSN862 produced a statistically greater decrease in glucose levels (P = 0.003), a trend for better triglyceride lowering (P = 0.065), and equivalent body weight gain (P = 0.875) compared with rosiglitazone (Fig. 4Go, A–C). These data suggest that at equivalent glucose lowering, less weight gain would be seen after LSN862 administration.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 4. Changes in Plasma Glucose, Triglycerides, and Body Weight in db/db Mice after Administration of LSN862 or Rosiglitazone (RG)

LSN862 (30 mg/kg) or rosiglitazone (30 mg/kg) were each administered to db/db mice once daily for 7 d. Blood samples were collected the day before the study started, d –1, and 1 h after the last dose on d 7. Changes in plasma glucose (A) and triglyceride (B) levels from d –1 to d 7 were determined. Body weight gain (C) was determined by subtracting the weight of each mouse on d 1 from its weight on d 7. *, P < 0.05 from vehicle group; #, P < 0.05 from RG. NS, Not significantly different (NS ≥ 0.05).

 
In Vivo Molecular Activity of LSN862
To gain an understanding of the antidiabetic mechanism of LSN862, expression of candidate genes involved in metabolic pathways in the liver and two fat depots (visceral and epididymal) were investigated. The tissue samples used for these studies were procured from the ZDF rat study described above. Administration of equally efficacious doses of LSN862 (3.0 mg/kg per day) and rosiglitazone (10.0 mg/kg per day) led to a similar decrease in expression of phosphoenol pyruvate carboxykinase (PEPCK) in the liver (Fig. 5AGo), the enzyme responsible for the rate-limiting step in the gluconeogenesis pathway. In contrast, regulation of pyruvate dehydrogenase kinase isozyme 4 (PDK4) and BIFEZ in the liver by LSN862 and rosiglitazone was very different compared with that seen for PEPCK. A statistically significant decrease in PDK4 expression was produced with rosiglitazone, whereas a large increase in expression of PDK4 was seen with LSN862 (Fig. 5BGo). A similar pattern was seen for regulated expression of BIFEZ in the liver (Fig. 5CGo).



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 5. Regulation of Liver PEPCK (A), PDK4 (B), and BIFEZ (C) Gene Expression

Liver samples from ZDF rats administered an equally efficacious dose of LSN862 (3.0 mg/kg per day) or rosiglitazone (RG, 10.0 mg/kg per day) were used for analysis of PDK4, PEPCK, and BIFEZ mRNA levels. mRNA was extracted from the liver samples, cDNA was synthesized, and RT-PCR was performed as described in Materials and Methods. *, P < 0.05 from vehicle group; #, P < 0.05 from RG (10 mg/kg per day).

 
Administration of LSN862 led to a significant increase in BIFEZ expression. A trend toward lowered BIFEZ expression was seen with rosiglitazone administration, although this finding was not statistically significant. The differences in LSN862- vs. rosiglitazone-regulated expression of PDK4 and BIFEZ are most likely due to the PPAR{alpha} activity of LSN862.

Changes in expression of adiponectin, malic enzyme, uncoupling protein 1 (UCP-1), glycerol kinase, and LPL were investigated in visceral and epididymal fat. As seen in Fig. 6AGo, administration of LSN862 (3.0 mg/kg per day) or rosiglitazone (10 mg/kg per day) for 7 d led to a large increase in both malic enzyme and UCP-1 expression and a smaller, although still statistically significant, increase in adiponectin and glycerol kinase expression in visceral fat. Only LSN862 administration led to a statistically significant increase in LPL expression in visceral fat. Gene expression results from the epididymal fat depot were similar to those seen in visceral fat after LSN862 administration, whereas rosiglitazone produced a statistically significant increase in the expression of only malic enzyme (Fig. 6BGo). Although UCP-1 expression was increased in epididymal fat after rosiglitazone and LSN862 administration, a fold-induction could not be calculated due to the extremely low levels of expression in the vehicle control samples. Interestingly, transcription of only glycerol kinase was statistically different between LSN862 vs. rosiglitazone administration.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 6. Regulation of Adiponectin, Malic Enzyme, UCP-1, Glycerol Kinase, and LPL Gene Expression in Visceral (A) and Epididymal (B) Fat

Fat samples from ZDF rats administered an equally efficacious dose of LSN862 (3.0 mg/kg per day) or rosiglitazone (RG, 10.0 mg/kg per day) were used for analysis of adiponectin (ACRP30, {square}), malic enzyme (ME, light gray square, UCP-1 (UCP1, medium gray square), glycerol kinase (GK, dark gray square) and LPL ({blacksquare}) mRNA levels. mRNA was extracted from the fat samples, cDNA was synthesized, and RT-PCR was performed as described in Materials and Methods. *, P < 0.05 from vehicle group; #, P < 0.05 from RG (10 mg/kg per day).

 
Effect of LSN862 on Lipid Levels
To determine the effect, if any, of LSN862 on modulating lipid levels, a study in humanized apolipoprotein A1 (hApoA1) transgenic mice was performed. LSN862 (0.3, 1.0, 3.0, 10, 30, and 100 mg/kg per day), rosiglitazone (0.3, 1.0, 3.0, 10, 30, and 100 mg/kg per day), and fenofibrate (100 mg/kg per day) were each administered to hApoA1 mice for 7 d. VLDL-C and HDL-C were determined using blood samples collected 3 h after the last dose. As shown in Table 4Go, administration of LSN862 led to a decrease in VLDL-C and a dose-dependent increase in HDL-C reaching a 65% increase at the 100 mg/kg per day dose. In contrast, fenofibrate administration produced a 5% decrease in VLDL-C and a 54% increase in HDL-C. Rosiglitazone administration led to variable VLDL-C levels and modest increases in HDL-C with the greatest increase produced at the 3.0 mg/kg per day dose. These data demonstrate that LSN862 administration led to improvements in the lipid profile of a rodent model of dyslipidemia, which likely reflect the PPAR{alpha} activity of this compound.


View this table:
[in this window]
[in a new window]
  		Table 4. Summary of VLDL-C and HDL-C Values from hApoA1 Mice

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the current report, we describe a new non-TZD PPAR ligand, LSN862, and show that it has a unique in vitro profile and potent antidiabetic and lipid-altering effects when tested in vivo. LSN862 is not a traditional PPAR{gamma} agonist with a TZD structure.

Results from our in vitro studies show that LSN862 has a unique profile compared with the well-characterized PPAR{gamma} agonist rosiglitazone. LSN862 binds to PPAR{gamma} with high affinity and to PPAR{alpha} with lower affinity. Using standard CTF assays, LSN862 demonstrated PPAR{gamma} agonist activity with weaker but significant PPAR{alpha} agonist activity. Very low affinity was shown for PPAR{delta}, and no PPAR{delta} agonist activity was detected. By examining LSN862 in PPAR{gamma} CTF assays using a variety of PPREs and in cofactor recruitment assays with PPAR{gamma} and five different cofactors, a broader, more detailed in vitro profile was revealed. Results from these studies show that LSN862 has a distinct pattern of activity compared with rosiglitazone functioning as a PPAR{gamma} partial agonist in CTF assays with differential agonist activity (partial and full agonist activity) displayed in cofactor recruitment assays. Although it would be easy to speculate that the CTF and cofactor data are related, i.e. lower agonist activity on the BIFEZ PPRE may be due to the inability to recruit CBP, the data have not been compiled to make this type of correlation. The PPAR{gamma} partial agonist activity of LSN862 may become a distinct advantage for this compound because a number of studies have shown that PPAR{gamma} partial agonists including selective PPAR modulators have improved side effect profiles compared with full agonists (9, 10, 11, 12, 13, 14). These reports are consistent with our findings in db/db mice, which demonstrate that LSN862 has better antidiabetic efficacy with the same weight gain and suggest that at equivalent glucose-lowering doses, LSN862 administration would lead to less weight gain compared with rosiglitazone. Although others have reported on the development of a PPAR{gamma} partial agonist for the treatment of type 2 diabetes (12, 14), this is the first report to describe a compound with PPAR{gamma} partial agonist plus PPAR{alpha} activities.

Although the PPAR{alpha} affinity and agonist activity of LSN862 appears weak compared with its PPAR{gamma} activity, the affinity of LSN862 for PPAR{alpha} is greater than that of the well-characterized PPAR{alpha} agonists, fenofibrate and WY14,643. In addition, beneficial in vivo effects were seen, which are most likely due to the PPAR{alpha} activity of LSN862. These PPAR{alpha}-mediated contributions will be discussed below.

LSN862 functions as a potent and efficacious antidiabetic agent. In ZDF rats and db/db mice, rodent models of type 2 diabetes, LSN862 normalized glucose and triglyceride levels. Some of the antidiabetic activity of LSN862 may be due to its PPAR{alpha} activity. Recent findings have shown that activation of PPAR{alpha} leads to improved whole-body and muscle insulin resistance and hyperinsulinemia in various animal models of type 2 diabetes and insulin resistance (15, 16, 17, 18, 19). In addition, other investigators have shown that treatment with a PPAR{alpha}/{gamma} dual agonist results in reduced circulating insulin and improved insulin sensitivity to a greater extent than treatment with rosiglitazone (20), although, the underlying mechanisms responsible for this enhanced antidiabetic activity remain unclear.

Adiponectin levels were elevated after both LSN862 and rosiglitazone administration, although the levels were statistically higher with LSN862 administration at an equally efficacious dose. The greater adiponectin response is not due to the additional PPAR{alpha} activity of LSN862, as we have examined the adiponectin promoter in CTF assays and have shown that it is not activated by PPAR{alpha} agonists (Gillespie G., unpublished observations). It is possible that the additional adiponectin effect is due to the PPAR{gamma} partial agonist activity of LSN862; however, very little is known at this time regarding the mechanism for PPAR{gamma}-induced adiponectin expression. Regardless, the substantial increase in adiponectin could give LSN862 unique advantages according to recent reports describing adiponectin’s antiinflammatory (21) and antidiabetic effects without increasing body weight (22, 23, 24).

Expression of candidate genes in liver and fat were investigated to gain a better understanding of the molecular mechanisms underlying the therapeutic activity of LSN862. In the liver, LSN862 and rosiglitazone both reduced expression of PEPCK, the enzyme responsible for the rate-limiting step in gluconeogenesis. These findings are consistent with those of others who have reported that PPAR{gamma} agonists decrease PEPCK mRNA levels in streptozotocin-treated rats (25) and rodent models of type 2 diabetes (20, 26). The decrease in PEPCK expression suggests a molecular explanation for the findings that PPAR{gamma} agonists reduce hepatic glucose output (27, 28). The decrease in PEPCK expression may not be a direct effect of LSN862 but rather a secondary effect due to the increase in insulin sensitivity or elevated adiponectin levels (22).

Interestingly, LSN862 increased expression of PDK4 approximately 4-fold in the liver, whereas rosiglitazone decreased the expression of this enzyme. PDK (of which there are four isozymes) and pyruvate dehydrogenase phosphatase (of which there are two isozymes) control the flux of pyruvate through the pyruvate dehydrogenase complex (29, 30, 31, 32, 33). Up-regulation of PDK4 inactivates the pyruvate dehydrogenase complex, which blocks pyruvate oxidation and conserves lactate and alanine for gluconeogenesis; in contrast, a down-regulation of PDK4 allows complete oxidative decarboxylation of pyruvate to reduced nicotinamide adenine dinucleotide, acetyl-CoA, and CO2 in oxidative tissue or to the synthesis of lipids in lipogenic tissues. PPAR{alpha} agonists have been shown to increase the expression of PDK4 in heart, kidney, skeletal muscle, and liver (34). Thus, elevation of PDK4 expression in the liver by LSN862 is most likely due to the PPAR{alpha} agonist activity of this compound. Treatment with LSN862 also increased the expression of BIFEZ, the enzyme that catalyzes the second step in the ß-oxidation pathway of fatty acid metabolism (35), whereas rosiglitazone administration did not statistically affect expression of this enzyme. These data indicate that LSN862, like the PPAR{alpha} activating fibrates, stimulates peroxisomal fatty acid oxidation.

Because PPAR{gamma} is expressed at high levels in most fat depots, expression of genes was evaluated in visceral and epididymal fat from the ZDF rat study described above. Malic enzyme and UCP-1 mRNA levels were increased by LSN862 and rosiglitazone in epididymal and visceral fat depots. Due to the extremely low levels of UCP-1 expression in epididymal fat from the vehicle-treated animals, the fold-induction of this gene could not be accurately calculated for either compound. UCPs are small intramembranous mitochondrial proteins that are expressed in a tissue-selective manner and play key roles in thermogenesis (36, 37). UCP-1 is present primarily in brown adipose tissue (38, 39), whereas UCP-3 is expressed in brown fat and skeletal muscle (40, 41). UCP-2 is found in most tissues (42, 43). The thermogenic role of UCP-1 has been shown definitively by many gain- and loss-of-function experiments (44, 45, 46). Interestingly, overexpression of PGC-1 in cultured white fat cells, 3T3-F422A, has been shown to increase UCP-1 mRNA levels (47), suggesting that LSN862 and rosiglitazone might increase the expression of UCP-1 in visceral and epididymal fat depots, at least in part, through recruitment of PGC-1.

Consistent with our findings are those by Way et al. (26), who show that administration of GW1929, a non-TZD PPAR{gamma}-selective agonist, to ZDF rats for 7 d increased malic enzyme mRNA levels in white and brown adipose tissues. Because malic enzyme is required for fatty acid synthesis and storage, our data are consistent with the hypothesis mentioned above that at least some of the efficacy of PPAR{gamma} ligands on insulin sensitization is via increased disposal of free fatty acids.

LSN862, but not rosiglitazone, administration led to a statistically significant increase in glycerol kinase expression in epididymal fat, whereas both compounds increased expression of glycerol kinase in visceral fat. Importantly, the increase in transcription due to LSN862 administration is statistically greater than that after rosiglitazone administration in both fat depots. Glycerol kinase stimulates glycerol incorporation into triglycerides and thus reduces free fatty acid secretion from adipocytes. Elevated free fatty acids in the circulation are known to be associated with insulin resistance (48, 49), and thus reducing levels of circulating free fatty acids would lead to greater insulin sensitivity. Therefore, the greater elevation of glycerol kinase expression due to LSN862 compared with rosiglitazone administration could play a role in the trend toward better glucose- and triglyceride-lowering by LSN862 in the ZDF rat study. Guan et al. (50) write eloquently about TZDs stimulating a "futile" fuel cycle resulting from enhanced expression of glycerol kinase in adipocytes. In their study, ZDF rats were administered rosiglitazone at 4.0 mg/kg per day for 10 d, and a statistical increase of approximately 2.5-fold was seen in glycerol kinase mRNA from epididymal fat analyzed by Northern blotting. The discrepancy in the results presented here and those reported by Guan et al., could be due to the administered dose of rosiglitazone, duration of the study, and/or methods used to quantitative mRNA levels.

Increased expression of LPL in adipose tissue may explain some of the triglyceride-lowering activities of LSN862 and rosiglitazone, as LPL is a key player in triglyceride catabolism (51). Only LSN862 significantly increased LPL mRNA in both visceral and epididymal fat; however, a trend for enhanced LPL expression was seen with rosiglitazone in both fat depots. Although not statistically significant, LSN862-treated rats displayed a greater reduction in triglyceride levels compared with rosiglitazone-treated animals. This enhanced efficacy may reflect both the greater LPL expression and the additional PPAR{alpha} activity of LSN862, as PPAR{alpha} agonists are known to decrease liver apolipoprotein C-III transcription (52).

Emerging data suggest that visceral and sc adipose tissue have distinct physiological functions and contribute to obesity and type 2 diabetes to different extents (53). In the studies shown here, similar gene expression profiles were seen in epididymal and visceral fat depots from rats administered LSN862. In contrast, rosiglitazone administration led to a more robust response in visceral fat compared with epididymal fat. Interestingly, both compounds caused significant changes in the metabolic profiles of the treated animals, suggesting that visceral fat may play a greater role in contributing to the overall metabolic homeostasis of this rodent model of type 2 diabetes.

LSN862 was administered to hApoA1 mice to investigate the lipid-altering properties of this compound. These mice were selected for the study based on findings that human and mouse apolipoprotein A1 (ApoA1), the major protein constituent of HDL-C, are regulated in opposite directions by PPAR{alpha} agonists, with mouse ApoA1 decreased and human ApoA1 increased after administration of PPAR{alpha} agonists (54). Administration of LSN862 led to a modest decrease in VLDL-C and a dose-dependent increase in HDL-C, with neither response showing dose dependency. The effect of LSN862 on HDL-C was similar to that seen with an equivalent dose of fenofibrate. In contrast, rosiglitazone showed modest alterations in both VLDL-C and HDL-C. These data are consistent with the in vitro data, which showed that only LSN862 had significant affinity for PPAR{alpha}. Other investigators have also reported that a small amount of PPAR{alpha} activity measured by in vitro binding and CTF assays can have surprising effects on lipid levels in vivo (55). Therefore, the improved lipid profile of LSN862 is most likely due to its additional PPAR{alpha} agonist activity.

In conclusion, LSN862 is a new PPAR{alpha}/{gamma} dual agonist with a unique in vitro profile. Based on its potent antidiabetic activity, beneficial effects on lipid levels, potential for less body weight gain, and unique gene regulation profile, LSN862 may be an improved therapeutic agent for the treatment of type 2 diabetes and associated dyslipidemia. In addition, the data presented here demonstrate that PPAR{gamma} full agonist activity, as seen with rosiglitazone in vitro, is not necessary to achieve potent and efficacious antidiabetic benefits in vivo.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Competitive Displacement Binding Assays
Binding assays were performed using scintillation proximity assay technology, PPAR receptors, and corresponding radiolabeled ligands. PPAR{alpha}, PPAR{delta}, and PPAR{gamma} along with their heterodimeric partner, retinoid X receptor {alpha}, were each produced using a baculovirus expression system. Biotinylated oligonucleotides containing PPREs were used to couple the corresponding receptor dimers to yttrium silicate streptavidin-coated scintillation proximity assay beads. PPAR{gamma}- and PPAR{alpha}/{delta}-specific ligands were labeled with tritium and used in the appropriate corresponding assays. The Ki values for each competing compound were calculated after deduction of nonspecific binding (measured in the presence of 10 µM unlabeled ligand). Compounds were evaluated using an 11-point dose-response curve with concentrations ranging from 0.169 nM to 10 µM. Reported values represent means from three separate experiments.

CTF Assays
PPAR{gamma}, PPAR{alpha}, or PPAR{delta} were constitutively expressed using plasmids containing the cytomegalovirus promoter. Reporter plasmids for the PPAR{gamma} CTF assays contained PPREs from the following genes: AOX, LPL, or BIFEZ plus the thymidine kinase (TK) promoter upstream of the luciferase reporter cDNA. A PPAR{gamma} GAL4 chimeric system was also used. For PPAR{alpha} and PPAR{delta}, a GAL4 chimeric system was the standard CTF assay performed. All assays were done in CV-1 cells. Compounds were tested in full-log dilution, from 0.1 nM to 10 µM in duplicate. Percent efficacy was determined relative to reference molecules with the efficacy value reflecting the greatest amount of agonist activity achieved in the CTF assay for each compound. The reference compounds were rosiglitazone (PPAR{gamma} assays) and LG0070660 (PPAR{alpha} and PPAR{delta} assays). EC50 values were determined by computer fit to a concentration-response curve. An EC50 value was not calculated if the efficacy for the compound was less than 20%. Reported values represent means from three to 10 separate experiments. CTF assays for additional nuclear hormone receptors (retinoid X receptor, retinoic acid receptor, liver X receptor, farnesoid X receptor, pregnane X receptor) were performed as described above using appropriate nuclear receptors and corresponding reference ligands.

Cofactor Recruitment Assays
A mammalian two-hybrid assay system in CTF format was done in CV-1 cells. The following plasmids were used: a mammalian expression vector encoding a fusion of the GAL4 DNA-binding domain with the PPAR{gamma} ligand-binding domain; a mammalian expression vector encoding a fusion of the VP16 transactivation domain with the nuclear receptor interaction domain of the respective coactivators: CBP, PGC-1, ASC-2, TRAP220, and the peptide C33; and a reporter plasmid (multimerized GAL4 binding sites/minimal TK promoter driving a luciferase cDNA). Cells were transfected in batch format and treated with compound (full-log dilution from 0.1 nM to 10 µM) or vehicle for 24 h. Subsequently, the cells were lysed and luciferase activity was measured. Luciferase activity serves as the endpoint for interaction between coactivator and receptor. The data are presented as percent efficacy relative to rosiglitazone. Reported values represent means from three separate experiments.

RNA Quantitation
Liver mRNA was isolated using a FastTrack 2.0 kit from Invitrogen (Carlsbad, CA), and adipose mRNA was isolated using guanidine isothiocyanate/phenol/chloroform extraction. Isolated mRNA was first treated with DNase using a DNA-free kit from Ambion, Inc. (Austin, TX) and then 2.5 µg of DNase-treated mRNA was used for cDNA synthesis. Primer and probe sets for each gene of interest were designed using Primer Express 1.5 from Applied Biosystems (Foster City, CA). Each cDNA sample was analyzed in triplicate per gene in a 96-well plate using an ABI Prism 7700 Sequence Detector (Applied Biosystems). Results were averaged and normalized to 36B4 expression. Samples from each group of animals (n = 5) were averaged and compared with the vehicle group. Data are presented as mean standard error of the mean for each group. After taking the base 10 logarithm of the normalized expression results, group differences were assessed by ANOVA with pairwise contrasts examined using Fisher’s protected least significant difference, where the significance level for the overall ANOVA was P < 0.05.

ZDF Rat Studies
Male ZDF rats were obtained from Genetic Models, Inc., (Indianapolis, IN) at 6 wk of age. After a 2-wk acclimation period, rats were prebled and assigned to four groups (five animals per group; vehicle, LSN862 at 3.0 mg/kg per day; rosiglitazone at 3.0 or 10.0 mg/kg per day) based on starting plasma glucose levels and body weight (d –1). Rats were administered compound daily by oral gavage between 0830 and 0930 h for 7 d. The dosing vehicle was 1% (wt/vol) carboxymethylcellulose, 0.25% Tween 80. Blood samples were obtained 1 h postdose on d 7 from the tail vein of conscious animals by gentle massage after tail snip. Blood was collected in EDTA tubes and kept chilled on ice. After centrifugation of blood samples, plasma was used for measurements of glucose, adiponectin, and triglyceride levels. Statistical significance was determined by one-way ANOVA. When statistical significance was detected with this method, group differences were determined by Neuman-Keuls post hoc analyses. Samples of liver and fat (visceral and epididymal) were removed on d 7, 6 h after the final dose of compound. Principles of laboratory animal care (NIH publication no. 85–23, revised 1985) were followed, and the use of animals was in accordance with the local animal ethics committee at Lilly Research Laboratories.

Db/db Mouse Studies
db/db Mice (5 wk of age) were purchased from Jackson Laboratories (Bar Harbor, ME). After a 2-wk acclimation period, the mice were prebled and assigned to three groups (vehicle, rosiglitazone, and LSN862; five animals per group) based on starting plasma glucose and body weight. The dosing vehicle for all studies was 1% (wt/vol) carboxymethylcellulose, 0.25% Tween 80. Compound was administered once daily by oral gavage between 0830 and 0930 h at a dose of 30 mg/kg for 7 d. Plasma was collected 1 h after compound administration on the last day of the study for measurement of plasma glucose and triglyceride levels. Statistical significance was determined by one-way ANOVA. When statistical significance was detected with this method, group differences were determined by Neuman-Keuls post hoc analyses. Principles of laboratory animal care (NIH publication no. 85–23, revised 1985) were followed, and the use of animals was in accordance with the local animal ethics committee at Lilly Research Laboratories.

Homozygous Human ApoA-1 Transgenic Mouse Studies
hApoA1 transgenic mice (56) were purchased from Jackson Laboratories. After a 2-wk acclimation period, the mice were assigned (based on weight) to individual groups with five animals per group. The mice were administered compound daily by oral gavage between 0600 and 0700 h for 7 d. Fenofibrate was administered at 100 mg/kg per day, whereas LSN862 and rosiglitazone were each given at 0.3, 1.0, 3.0, 10, 30, and 100 mg/kg per day. The dosing vehicle was 1% (wt/vol) carboxymethylcellulose, 0.25% Tween-80 with control animals receiving dosing vehicle only. Blood was collected by heart draw for analysis 3 h after the final dose. Principles of laboratory animal care (NIH publication no. 85–23, revised 1985) were followed, and the use of animals was in accordance with the local animal ethics committee at Lilly Research Laboratories.

Determination of VLDL-C and HDL-C
Lipoproteins were separated by fast protein liquid chromatography, and cholesterol was quantitated with an in-line detection system based on that described by Kieft et al. (57). Briefly, 35-µl plasma samples/50-µl pooled sample was applied to a Superose 6 HR 10/30 size exclusion column (Amersham Pharmacia Biotech, Piscataway, NJ) and eluted with PBS, pH 7.4 (diluted 1:10), containing 5 mM EDTA, at 0.5 ml/min. Cholesterol reagent from Roche Diagnostics (Indianapolis, IN) at 0.16 ml/min was mixed with the column effluent through a T connection; the mixture was then passed through a 15 m x 0.5 mm knitted tubing reactor (Aura Industries, New York, NY) immersed in a 37 C water bath. The colored product produced in the presence of cholesterol was monitored in the flow stream at 505 nm, and the analog voltage from the monitor was converted to a digital signal for collection and analysis. The change in voltage corresponding to change in cholesterol concentration was plotted vs. time, and the area under the curve corresponding to the elution of VLDL-C and HDL-C was calculated using Turbochrome (version 4.12F12) software from PerkinElmer (Norwalk, CT).


   FOOTNOTES
 
No grants or fellowships supported the writing or work described in this paper.

First Published Online April 14, 2005

Abbreviations: AOX, acyl coA oxidase; ASC-2, activating signal cointegrator-2; BIFEZ, bifunctional enzyme; CBP, cAMP response element-binding protein (CREB)-binding protein; CoA, coenzyme A; CTF assay, cotransfection assay; hApoA1, humanized apolipoprotein A1; HDL-C, high-density lipoprotein cholesterol; LPL, lipoprotein lipase; PDK4, pyruvate dehydrogenase kinase isozyme 4; PEPCK, phosphoenol pyruvate carboxykinase; PGC-1, PPAR{gamma} coactivator-1; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; TRAP, thyroid hormone receptor-activated protein; TZD, thiazolidinedione; UCP, uncoupling protein; VLDL-C, very low-density lipoprotein cholesterol; ZDF rat, Zucker diabetic fatty rat.

Received for publication January 10, 2005. Accepted for publication April 5, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Taskinen MR 2001 Diabetic dyslipidemia. Atheroscler Suppl 3:47–51
  2. Garber AJ, Karlsson FO 2001 Treatment of dyslipidemia in diabetes. Endocrinol Metab Clin North Am 30:999–1010[Medline]
  3. Aronoff S, Rosenblatt S, Braithwaite S, Egan JW, Mathisen AL, Schneider RL 2000 Pioglitazone hydrochloride monotherapy improves glycemic control in the treatment of patients with type 2 diabetes: a 6-month randomized placebo-controlled dose-response study. Diabetes Care 23:1605–1611[Abstract/Free Full Text]
  4. Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart JC 1998 Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation 98:2088–2093[Abstract/Free Full Text]
  5. Jones IR, Swai A, Taylor R, Miller M, Laker MR, Algerti KG 1990 Lowering of plasma glucose concentrations with bezafibrate in patients with moderately controlled NIDDM. Diabetes Care 13:855–863[Abstract]
  6. Kobayashi M, Shigeta Y, Hirata Y, Omori Y, Sakamoto N, Nambu S, Baba S 1988 Improvement of glucose tolerance in NIDDM by clofibrate. Randomized double-blind study. Diabetes Care 11:495–499[Abstract]
  7. Issemann I, Green S 1990 Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators. Nature 347:645–650[CrossRef][Medline]
  8. Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Wilson TM, Kliewer SA 1995 An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}). J Biol Chem 270:12953–12956[Abstract/Free Full Text]
  9. Rocchi S, Picard F, Vamecq J, Gelman L, Potier N, Zeyer D, Dubuquoy L, Bac P, Champy M-F, Plunket KD, Leesnitzer LM, Blanchard SG, Desreumaux P, Moras D, Renaud J-P, Auwerx J 2001 A unique PPAR{gamma} ligand with potent insulin-sensitizing yet weak adipogenic activity. Mol Cell 8:737–747[CrossRef][Medline]
  10. Berger JP, Petro AE, Macnaul KL 2003 Distinct properties and advantages of a novel PPAR{gamma} selective modulator. Mol Endocrinol 17:662–676[Abstract/Free Full Text]
  11. Shimaya A, Kurosaki E, Nakano R, Hirayama R, Shibasaki M, Shikama H 2000 The novel hypoglycemic agent YM440 normalizes hyperglycemia without changing body fat weight in diabetic db/db mice. Metabolism 49:411–417[CrossRef][Medline]
  12. Chakrabarti R, Vikramadithyan R, Misra P, Suresh J, Rajagopalan R 2003 Balaglitazone, a quinazalone analogue of thiazolidinedione shows excellent antidiabetic and hypolipidemic potential with less adipogenic activity. Diabetes 52(Suppl 1):601P (Abstract)
  13. Kawai T, Takei I, Oguma Y 1999 Effects of troglitazone on fat distribution in the treatment of male type 2 diabetes. Metabolism 48:1102–1107[CrossRef][Medline]
  14. Wulff E, Pedersen K, Sauerberg P 2003 Balaglitazone a new partial PPAR{gamma} agonist has a better cardiovascular safety profile and glycemic control compared with the full PPAR{gamma} agonist rosiglitazone. Diabetes 52(Suppl 1):594P (Abstract)
  15. Guerre-Millo M, Gervois P, Raspe E 2000 Peroxisome proliferator-activated receptor {alpha} activators improve insulin sensitivity and reduce adiposity. J Biol Chem 275:16638–16642[Abstract/Free Full Text]
  16. Nagai Y, Nishio Y, Nakamura T, Maegawa H, Kikkawa R, Kashiwagi A 2002 Amelioration of high fructose-induced metabolic derangements by activation of PPAR{alpha}. Am J Physiol Endocrinol Metab 282:E1180–E1190
  17. Winegar DA, Brown PJ, Wilkison WO, Lewis MC, Ott, RJ, Tong WQ, Brown HR, Lehmann JM, Kliewer SA, Plunket KD, Way JM, Bodkin NL, Hansen BC 2001 Effects of fenofibrate on lipid parameters in obese rhesus monkeys. J Lipid Res 42:1543–1551[Abstract/Free Full Text]
  18. Ye J-M, Doyal PJ, Iglesias MA, Watson DG, Cooney GJ, Kraegen EW 2001 Peroxisome proliferator-activated receptor (PPAR)-{alpha} activation lowers muscle lipids and improves insulin sensitivity in high fat-fed rats: comparison with PPAR-{gamma} activation. Diabetes 50:411–417[Abstract/Free Full Text]
  19. Aasum E, Belke DD, Severson DL, Riemersma RA, Cooper M, Andressen M, Larsen TS 2002 Cardiac function and metabolism in type 2 diabetic mice after treatment with BM17.0744, a novel ppar-{alpha} activator. Am J Physiol Heart Circ Physiol 283:H949–H957
  20. Brand CL, Sturis J, Gotfredsen CF, Fleckner J, Fledelius C, Hansen BF, Andersen B, Ye J-M, Sauerberg P, Wassermann K 2003 Dual PPAR{alpha}/{gamma} activation provides enhanced improvement of insulin sensitivity and glycemic control in ZDF rats. Am J Physiol Endocrinol Metab 284:E841–E854
  21. Ouchi N, Kihara S, Arita Y, Okamoto Y, Maeda K, Kuriyama H, Hotta K, Nishida M, Takahashi M, Muraguchi M, Ohmoto Y, Nakamura T, Yamashita S, Funahashi T, Matsuzawa Y 2000 Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-{kappa}B signaling through a camp-dependent pathway. Circulation 102:1296–1301[Abstract/Free Full Text]
  22. Combs TP, Berg AH, Obici S, Scherer PE, Rossetti L 2001 Endogenous glucose production is inhibited by the adipose-derived protein Acrp30. J Clin Invest 108:1875–1881[CrossRef][Medline]
  23. Fruebis J, Tsao T-S, Javorschi S 2001 Proteolytic cleavage product of 30 kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice. Proc Natl Acad Sci USA 98:2005–2010[Abstract/Free Full Text]
  24. Yamauchi T, Kamon J, Waki H 2001 The fat derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity. Nat Med 7:941–946[CrossRef][Medline]
  25. Hofmann C, Lorenz K, Williams D, Palazuk BJ, Colca JR 1995 Insulin sensitization in diabetic rat liver by an antihyperglycemic agent. Metabolism 44:384–389[CrossRef][Medline]
  26. Way JM, Harrington WW, Brown KK, Gottschalk WK, Sundseth SS, Mansfield TA, Ramachandran RK, Willson TM, Kliewer SA 2001 Comprehensive messenger ribonucleic acid profiling reveals that peroxisome proliferator-activated receptor {gamma} activation has coordinate effects on gene expression in multiple insulin-sensitive tissues. Endocrinology 142:1269–1277[Abstract/Free Full Text]
  27. Day C 1999 Thiazolidinediones: a new class of antidiabetic drugs. Diabet Med 16:179–192[CrossRef][Medline]
  28. Reginato MJ, Lazar MA 1999 Mechanisms by which thiazolidinediones enhance insulin action. Trends Endocrinol Metab 10:9–13[CrossRef][Medline]
  29. Popov KM, Kedishvili NY, Zhao Y, Shimomura Y, Crabb DW, Harris RA 1993 Primary structure of pyruvate dehydrogenase kinase establishes a new family of eukaryotic protein kinases. J Biol Chem 268:26602–26606[Abstract/Free Full Text]
  30. Popov KM, Kedishvili NY, Zhao Y, Gudi R, Harris RA 1994 Molecular cloning of the p445 subunit of pyruvate dehydrogenase kinase. J Biol Chem 269:29720–29724[Abstract/Free Full Text]
  31. Rowles J, Scherer SW, Xi T 1996 Cloning and characterization of PDK4 on 7q21.3 encoding a fourth pyruvate dehydrogenase kinase isoenzyme in humans. J Biol Chem 271:22376–22398[Abstract/Free Full Text]
  32. Lawson JE, Niu XD, Browning KS, Trong HL, Yan J, Reed LJ 1993 Molecular cloning and expression of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase and sequence similarity with protein phosphatase 2C. Biochemistry 32:8987–8993[CrossRef][Medline]
  33. Huang B, Gudi R, Wu P, Harris RA, Hamilton J, Popov KM 1998 Isoenzymes of pyruvate dehydrogenase phosphatase. DNA-derived amino acid sequences, expression, and regulation. J Biol Chem 273:17680–17688[Abstract/Free Full Text]
  34. Wu P, Peters JM, Harris RA 2001 Adaptive increase in pyruvate dehydrogenase kinase 4 during starvation is mediated by peroxisome proliferator-activated receptor {alpha}. Biochem Biophys Res Comm 287:391–396[CrossRef][Medline]
  35. Agnihotri G, Liu H-W 2003 Enoyl-CoA hydratase: reaction, mechanism, and inhibition. Bioorg Med Chem 11:9–20[CrossRef][Medline]
  36. Flier JS, Lowell BB 1997 Obesity research springs a protein leak. Nat Genet 15:223–224[CrossRef][Medline]
  37. Ricquier D, Bouillaud, F 1997 The mitochondrial uncoupling protein: structural and genetic studies. Prog Nucleic Acid Res Mol Biol 56:83–108[Medline]
  38. Jacobsson A, Stadler U, Glotzer MA, Kozak LP 1985 Mitochondrial uncoupling protein from mouse brown fat. Molecular cloning and genetic mapping, and mRNA expression. J Biol Chem 260:16250–16254[Abstract/Free Full Text]
  39. Bouillaud F, Weissenbach J, Ricquier D 1986 Complete cDNA-derived amino acid sequence of rat brown fat uncoupling protein. J Biol Chem 261:1487–1490
  40. Boss O, Samec S, Paoloni-Giacobino A, Rossier C, Dulloo A, Seydoux J, Muzzin P, Giacobino J-P 1997 Uncoupling protein-3: a new member of the mitochondrial carrier family with tissue-specific expression. FEBS Lett 408:39–42[CrossRef][Medline]
  41. Vidal-Puig A, Solanes G, Grujic D, Flier JS, Lowell BB 1997 UCP3: an uncoupling protein homologue expressed preferentially and abundantly in skeletal muscle and brown adipose tissue. Biochem Biophys Res Commun 235:79–82[CrossRef][Medline]
  42. Fleury C, Neverova M, Collins S, Raimbault S, Champigny O, Levi-Meyrueis C, Bouillaud F, Seldin MF, Surwit RS, Ricquier D, Warden CH 1997 Uncoupling protein-2: a novel gene linked to obesity and hyperinsulinemia. Nat Genet 15:269–272[CrossRef][Medline]
  43. Gimeno RE, Dembski M, Weeng X 1997 Cloning and characterization of an uncoupling protein homolog: a potential molecular mediator of human thermogenesis. Diabetes 46:900–906[Abstract]
  44. Lowell BB, Susulic VS, Hamann A, Lawitts JA, Himms-Hagen J, Boyer BB, Kozak LP, Flier JS 1993 Development of obesity in transgenic mice after genetic ablation of brown adipose tissue. Nature 366:740–742[CrossRef][Medline]
  45. Kopecky J, Clarke G, Enerback S, Spiegelman B, Kozak LP 1995 Expression of the mitochondrial uncoupling protein gene from the aP2 gene promoter prevents genetic obesity. J Clin Invest 96:2914–2923
  46. Enerback S, Jacobsson A, Simpson EM, Guerra C, Yamashita H, Harper M-E, Kozak LP 1997 Mice lacking mitochondrial uncoupling protein are cold-sensitive but not obese. Nature 387:90–94[CrossRef][Medline]
  47. Wu Z, Puigserver P, Andersson U 1999 Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98:115–124[CrossRef][Medline]
  48. Boden G 2001 Pathogenesis of type 2 diabetes and insulin resistance. Endocrinol Metab Clin North Am 30:801–815[CrossRef][Medline]
  49. Roden M, Stingl H, Chandramouli V 2000 Effects of free fatty acid elevation on postabsorptive endogenous glucose production and gluconeogenesis in humans. Diabetes 49:701–707[Abstract]
  50. Guan H-P, Li Y, Jensen MV, Newgard CB, Steppan CM, Lazar MA 2002 A futile metabolic cycle activated in adipocytes by antidiabetic agents. Nat Med 8:1122–1126[CrossRef][Medline]
  51. Goldberg I 1996 Lipoprotein lipase and lipolysis: central roles in lipoprotein metabolism and atherogenesis. J Lipid Res 37:693–707[Abstract]
  52. Lefebvre AM, Peinado-Onsurbe J, Leitersdorf I, Briggs MR, Paterniti JR, Fruchart JC, Fievet C, Auwerx J, Staels B 1997 Regulation of lipoprotein metabolism by thiazolidinediones occurs through a distinct but complementary mechanism relative to fibrates. Arterioscler Thromb Vasc Biol 17:1756–1764[Abstract/Free Full Text]
  53. Wajchenberg GL, Biannella-Neto D, Da Silva ME, Santos RF 2002 Depot-specific hormonal characteristics of subcutaneous and visceral adipose tissue and their relation to the metabolic syndrome. Horm Metab Res 34:616–621[CrossRef][Medline]
  54. Berthou L, Duverger N, Emmanuel F, Langouet S, Auwerx J, Guillouzo A, Fruchart JC, Rubin E, Denefle P, Staels B, Branellec D 1996 Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice. J Clin Invest 97:2408–2416[Medline]
  55. Wilson GG, Abou-Donia M, Frith L, Patel J, Fiedorek FT 2000 Monotherapy with GI262570, a tyrosine-based non-thiazolidinedione PPAR{gamma} agonist significantly reduces triglyceride and increases HDL-C concentrations in patients with type 2 diabetes mellitus. Diabetes 49(Suppl 1):158OR (Abstract)
  56. Rubin EM, Ishida BY, Clift SM, Krauss RM 1991 Expression of human apolipoprotein A-I in transgenic mice results in reduced plasma levels of murine apolipoprotein A-I and the appearance of two new high density lipoprotein size subclasses. Proc Natl Acad Sci USA 88:434–438[Abstract/Free Full Text]
  57. Kieft KA, Bocan TMA, Krause BR 1991 Rapid on-line determination of cholesterol distribution among plasma lipoproteins after high-performance gel filtration chromatography. J Lipid Res 32:859–866[Abstract]

NURSA Molecule Pages Link:

Nuclear Receptors:   PPARα  |  PPARδ  |  PPARγ
Coregulators:   TRAP220  |  PGC-1  |  CBP  |  ASC-2
Ligands:   Rosiglitazone



This article has been cited by other articles:


Home page
 DiabetesHome page
D. R. Cha, X. Zhang, Y. Zhang, J. Wu, D. Su, J. Y. Han, X. Fang, B. Yu, M. D. Breyer, and Y. Guan
Peroxisome Proliferator Activated Receptor {alpha}/{gamma} Dual Agonist Tesaglitazar Attenuates Diabetic Nephropathy in db/db Mice
Diabetes, August 1, 2007; 56(8): 2036 - 2045.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
Y. Liu, M. D. Michael, S. Kash, W. R. Bensch, B. P. Monia, S. F. Murray, K. A. Otto, S. K. Syed, S. Bhanot, K. W. Sloop, et al.
Deficiency of Adiponectin Receptor 2 Reduces Diet-Induced Insulin Resistance but Promotes Type 2 Diabetes
Endocrinology, February 1, 2007; 148(2): 683 - 692.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
A. Tsuchida, T. Yamauchi, S. Takekawa, Y. Hada, Y. Ito, T. Maki, and T. Kadowaki
Peroxisome Proliferator-Activated Receptor (PPAR){alpha} Activation Increases Adiponectin Receptors and Reduces Obesity-Related Inflammation in Adipose Tissue: Comparison of Activation of PPAR{alpha}, PPAR{gamma}, and Their Combination
Diabetes, December 1, 2005; 54(12): 3358 - 3370.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow