A Peroxisome Proliferator-Activated Receptor
/
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.
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ABSTRACT
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LSN862 is a novel peroxisome proliferator-activated receptor (PPAR)
/
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
partial agonist with relatively less but significant PPAR
agonist activity. Using these same assays, rosiglitazone was characterized as a high-affinity PPAR
full agonist with no PPAR
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
full agonist activity is not necessary to achieve potent and efficacious insulin-sensitizing benefits and demonstrate the therapeutic advantages of a PPAR
/
dual agonist.
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INTRODUCTION
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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)
and PPAR
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
and PPAR
agonist properties (PPAR
/
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. 1
) was fully characterized for its PPAR
, PPAR
, and PPAR
binding and agonist activity in vitro and potential antidiabetic and lipid-altering properties in rodent models of type 2 diabetes and dyslipidemia, respectively.
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RESULTS
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In Vitro Characterization of LSN862
Competitive binding assays were used to determine the affinity of LSN862 for PPAR
, PPAR
, and PPAR
, and standard cotransfection (CTF) assays were used to determine the potency and efficacy of agonist activity. As shown in Table 1
, LSN862 is a high-affinity [dissociation constant (Ki) = 7.0 nM], potent (EC50 = 239 nM) PPAR
agonist (77% efficacy) with relatively less, but significant affinity (Ki = 770 nM) and agonist activity (EC50 = 2622 nM; 35% efficacy) on PPAR
. This degree of PPAR
affinity is greater than that of two well-studied PPAR
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
(Ki = 4123 nM) with no agonist activity detected. The standard PPAR
CTF assay uses a trimeric PPAR response element (PPRE) from the acyl-coenzyme A (CoA) oxidase (AOX) gene. To further explore the PPAR
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
, yeast galactosidase (GAL4) chimeric CTF assay. In all PAPR
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 2
) with PPAR
partial agonist activity demonstrated regardless of the PPRE used in the CTF assay (Fig. 2
). These data demonstrate that although LSN862 is a high-affinity ligand for PPAR
, it functions as a PPAR
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).

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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.
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To further characterize LSN862, PPAR
cofactor recruitment assays were performed using the following cofactors: cAMP response element-binding protein (CREB)-binding protein (CBP), peroxisome proliferator-activated receptor
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
by rosiglitazone was set at 100%. As seen in Table 3
, 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
agonist like rosiglitazone in cofactor recruitment.
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. 3
, 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. 3C
).
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. 4
, AC). These data suggest that at equivalent glucose lowering, less weight gain would be seen after LSN862 administration.

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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).
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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. 5A
), 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. 5B
). A similar pattern was seen for regulated expression of BIFEZ in the liver (Fig. 5C
).

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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).
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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
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. 6A
, 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. 6B
). 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.

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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, ), malic enzyme (ME, light gray square, UCP-1 (UCP1, medium gray square), glycerol kinase (GK, dark gray square) and LPL ( ) 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).
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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 4
, 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
activity of this compound.
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DISCUSSION
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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
agonist with a TZD structure.
Results from our in vitro studies show that LSN862 has a unique profile compared with the well-characterized PPAR
agonist rosiglitazone. LSN862 binds to PPAR
with high affinity and to PPAR
with lower affinity. Using standard CTF assays, LSN862 demonstrated PPAR
agonist activity with weaker but significant PPAR
agonist activity. Very low affinity was shown for PPAR
, and no PPAR
agonist activity was detected. By examining LSN862 in PPAR
CTF assays using a variety of PPREs and in cofactor recruitment assays with PPAR
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
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
partial agonist activity of LSN862 may become a distinct advantage for this compound because a number of studies have shown that PPAR
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
partial agonist for the treatment of type 2 diabetes (12, 14), this is the first report to describe a compound with PPAR
partial agonist plus PPAR
activities.
Although the PPAR
affinity and agonist activity of LSN862 appears weak compared with its PPAR
activity, the affinity of LSN862 for PPAR
is greater than that of the well-characterized PPAR
agonists, fenofibrate and WY14,643. In addition, beneficial in vivo effects were seen, which are most likely due to the PPAR
activity of LSN862. These PPAR
-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
activity. Recent findings have shown that activation of PPAR
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
/
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
activity of LSN862, as we have examined the adiponectin promoter in CTF assays and have shown that it is not activated by PPAR
agonists (Gillespie G., unpublished observations). It is possible that the additional adiponectin effect is due to the PPAR
partial agonist activity of LSN862; however, very little is known at this time regarding the mechanism for PPAR
-induced adiponectin expression. Regardless, the substantial increase in adiponectin could give LSN862 unique advantages according to recent reports describing adiponectins 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
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
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
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
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
activating fibrates, stimulates peroxisomal fatty acid oxidation.
Because PPAR
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
-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
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
activity of LSN862, as PPAR
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
agonists, with mouse ApoA1 decreased and human ApoA1 increased after administration of PPAR
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
. Other investigators have also reported that a small amount of PPAR
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
agonist activity.
In conclusion, LSN862 is a new PPAR
/
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
full agonist activity, as seen with rosiglitazone in vitro, is not necessary to achieve potent and efficacious antidiabetic benefits in vivo.
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MATERIALS AND METHODS
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Competitive Displacement Binding Assays
Binding assays were performed using scintillation proximity assay technology, PPAR receptors, and corresponding radiolabeled ligands. PPAR
, PPAR
, and PPAR
along with their heterodimeric partner, retinoid X receptor
, 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
- and PPAR
/
-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
, PPAR
, or PPAR
were constitutively expressed using plasmids containing the cytomegalovirus promoter. Reporter plasmids for the PPAR
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
GAL4 chimeric system was also used. For PPAR
and PPAR
, 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
assays) and LG0070660 (PPAR
and PPAR
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
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 Fishers 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. 8523, 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. 8523, 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. 8523, 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).
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FOOTNOTES
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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
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.
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