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Molecular Endocrinology, doi:10.1210/me.2007-0156
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Molecular Endocrinology 21 (12): 3002-3012
Copyright © 2007 by The Endocrine Society

Dissection of the Insulin-Sensitizing Effect of Liver X Receptor Ligands

S. Renee Commerford, Leo Vargas, Suzanne E. Dorfman, Nico Mitro, Erik C. Rocheford, Puiying A. Mak, Xue Li, Patrick Kennedy, Tara L. Mullarkey and Enrique Saez

The Genomics Institute of the Novartis Research Foundation (L.V., N.M., P.A.M., P.K., E.S.), San Diego, California 92121; Novartis Institutes for BioMedical Research (S.R.C., S.E.D., E.C.R., X.L., T.L.M.), Cambridge, Massachusetts 02139; and Department of Cell Biology (N.M., E.S.), The Scripps Research Institute, La Jolla, California 92037

Address all correspondence and requests for reprints to: Enrique Saez, Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037. E-mail: esaez{at}scripps.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The liver X receptors (LXR{alpha} and β) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Treatment of insulin-resistant mice with synthetic LXR ligands enhances glucose tolerance, inducing changes in gene expression expected to decrease hepatic gluconeogenesis (via indirect suppression of gluconeogenic enzymes) and increase peripheral glucose disposal (via direct up-regulation of glut4 in fat). To evaluate the relative contribution of each of these effects on whole-body insulin sensitivity, we performed hyperinsulinemic-euglycemic clamps in high-fat-fed insulin-resistant rats treated with an LXR agonist or a peroxisome proliferator-activated receptor {gamma} ligand. Both groups showed significant improvement in insulin action. Interestingly, rats treated with LXR ligand had lower body weight and smaller fat cells than controls. Insulin-stimulated suppression of the rate of glucose appearance (Ra) was pronounced in LXR-treated rats, but treatment failed to enhance peripheral glucose uptake (R'g), despite increased expression of glut4 in epididymal fat. To ascertain whether LXR ligands suppress hepatic gluconeogenesis directly, mice lacking LXR{alpha} (the primary isotype in liver) were treated with LXR ligand, and gluconeogenic gene expression was assessed. LXR activation decreased expression of gluconeogenic genes in wild-type and LXRβ null mice, but failed to do so in animals lacking LXR{alpha}. Our observations indicate that despite inducing suggestive gene expression changes in adipose tissue in this model of diet-induced insulin resistance, the antidiabetic effect of LXR ligands is primarily due to effects in the liver that appear to require LXR{alpha}. These findings have important implications for clinical development of LXR agonists as insulin sensitizers.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
NUCLEAR RECEPTORS ARE ligand-activated transcription factors that modulate gene expression in response to a variety of endocrine and environmental signals. Members of the family that work as heterodimers with the retinoid X receptor act as sensors of dietary components, including lipids, fatty acids, retinoids, vitamins, cholesterol, bile acids, and xenobiotics (1). The liver X receptors (LXR{alpha} and β; NR1H3, NR1H2) are retinoid X receptor partners, the endogenous ligands of which include oxysterols (2, 3, 4). Expression of LXR{alpha} is restricted to liver, intestine, adipose tissue, and macrophages, whereas LXRβ is broadly expressed (5). Upon exposure to excessive accumulation of intracellular oxysterols, the LXRs activate a program of gene expression aimed at limiting pathogenic buildup of cholesterol (6). In the intestine, LXR activation decreases absorption of diet-derived cholesterol by promoting expression of efflux transporters of the ATP binding cassette (ABC) family, such as ABCA1, ABCG5, and ABCG8. In macrophages, the LXRs prompt increased expression of genes involved in high density lipoprotein formation and reverse cholesterol transport (e.g. ABCA1, apolipoprotein E), whereas in rodent liver they direct conversion of excess cholesterol into bile acids through regulation of the rate-limiting enzyme Cyp7a. Pharmacological activation of LXRs prevents formation of lesions in mice prone to atherosclerosis and can even induce regression of established plaques (7, 8, 9, 10). LXR ligands also have significant antiinflammatory activity, which suggests that the benefit of LXR activators in atherosclerosis may be due, not just to their effects on lipoprotein metabolism, but also to their ability to suppress proinflammatory gene expression (11, 12, 13, 14, 15).

In addition to regulating cholesterol homeostasis in multiple tissues, the LXRs are also intimately involved in the control of hepatic lipid metabolism. They are essential for expression of sterol-regulatory element-binding protein (SREBP)-1c and carbohydrate response element-binding protein, the central regulators of lipogenesis, and direct modulators of lipid-synthesizing enzymes such as fatty-acid synthase (16, 17, 18, 19). Because physiological regulation of lipid and carbohydrate metabolism is tightly associated, we previously evaluated the role of LXRs in glucose homeostasis (20). Our studies showed that LXR agonists enhance glucose tolerance in a mouse model of diet-induced insulin resistance. Treatment with synthetic LXR ligands altered the expression of genes in liver and adipose tissue in a manner expected to reduce hepatic glucose output and increase peripheral glucose uptake. In the liver, LXR ligands indirectly suppressed expression of gluconeogenic enzymes [e.g. phosphoenol pyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase)], whereas in fat, LXR regulated expression of the insulin-sensitive glucose transporter glut4 by directly binding to its promoter. Moreover, we have recently reported the surprising observation that glucose itself can bind and activate LXR (21). Additional studies have suggested that the LXRs, in particular LXRβ, play an important role in pancreatic insulin secretion, and that LXR activators promote insulin secretion (22, 23). LXR ligands have also proven effective in other models of insulin resistance and type 2 diabetes, such as db/db and ob/ob mice, and fa/fa and Zucker diabetic fatty rats, highlighting the potential of LXR agonists as insulin sensitizers (24, 25, 26).

To better understand the mechanism of antidiabetic action of LXR ligands, we have measured the relative effects of LXR activation on hepatic and peripheral insulin sensitivity in obese insulin-resistant rats. Our results indicate that LXR activation enhances glucose tolerance primarily through direct effects in the liver that appear to require LXR{alpha}. These findings have important implications for clinical development of LXR ligands as insulin sensitizers.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LXR Ligands Diminish Weight Gain Due to High-Fat Feeding without Affecting Energy Expenditure
To explore the antidiabetic mechanism of LXR ligands, wild-type rats were fed a high-fat diet for 5 wk to induce insulin resistance and subsequently treated for 3 wk with the dual LXR{alpha}/β agonist GW3965 (20 mg/kg·d), or the peroxisome proliferator-activated receptor-{gamma} (PPAR) ligand pioglitazone (PIO, 20 mg/kg·d) used as a positive control for insulin sensitization. This model was favored because LXR ligands are known to have physiological effects in fat, so we thought it best to use animals with intact adipose tissue (e.g. unimpaired leptin signaling). In contrast to animals treated with PIO, which gained considerably more weight than controls, rats treated with GW3965 put on weight at a significantly slower rate than controls (Fig. 1Go). Although food intake over the 2-wk period was not considerably different between the GW3965 group and the vehicle group, the GW3965 group consumed significantly less than the PIO group over this time (wk 1: vehicle = 107 ± 4 g; GW3965 = 96 ± 3 g; PIO = 118 ± 4 g; week 2: vehicle = 161 ± 13 g; GW3965 = 147 ± 7 g; PIO = 186 ± 10 g). Food intake over the first 2 wk of dosing explained a significant proportion of the associated body weight gain among the groups during that time (Pearson’s correlation = 0.90, P < 0.01). Interestingly, although there was no difference among groups in epididymal fat pad weight (vehicle, 10.7 ± 0.9 g; GW3965, 10.0 ± 0.4 g, PIO, 9.1 ± 1.0 g;), fat cell size (Fig. 2Go) was significantly smaller in the GW3965 group (1262 ± 50 µm2, P < 0.01) relative to both vehicle (1615 ± 29 µm2) and PIO groups (1517 ± 81 µm2).


Figure 1
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  		Fig. 1. LXR Ligand Treatment Ameliorates Weight Gain in High-Fat-Fed Rats

A, Body weight evolution in rats fed a high-fat diet for 5 wk and treated orally with the LXR{alpha}/β agonist GW3965 (20 mg/kg·d), PIO (20 mg/kg·d), or dosing vehicle for 14 d. B, Body weight change in the same rats. Values are means ± SEM (n = 6/group). *, All groups differ significantly from one another beginning on d 6.

 

Figure 2
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  		Fig. 2. LXR Ligand Treatment Decreases Fat Cell Size

Representative pictures of hematoxylin eosin-stained fat cells from epididymal fat taken from high-fat-fed rats dosed for 21–23 d with vehicle (A), the LXR {alpha}/β agonist GW3965 (B), or PIO (C). Fat cells were significantly smaller in the GW3965 group relative to the other two groups (P < 0.05, n = 6/group). Magnification, x20.

 
To assess the effect of the LXR agonist on energy expenditure, oxygen consumption rate (VO2) and nonprotein respiratory exchange ratio (npRER) were measured in treated rats. No differences were found in rates of oxygen consumption (VO2, ml/kg·min) or npRER during fasted-to-fed or fed-to-fasted transitions among the three groups (data not shown). Likewise, fasted VO2 and npRER (taken as the mean over the last 80 min of a 12-h period without food) were not different among groups (Table 1Go). There was also no difference in fed VO2 or npRER between groups [taken as the mean over the last 80 min of free access to food (Table 1Go)]. These results indicate that LXR ligands can ameliorate diet-induced obesity without an apparent increase in energy expenditure.


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  		Table 1. Oxygen Consumption and npRER after 2 wk of LXR Ligand Dosing

 
LXR Ligands Enhance Glucose Tolerance Primarily through Effects in Liver
Compared with rats maintained on a normal diet, high-fat-fed rats showed significant insulin resistance (data not shown). To dissect the effect of LXR activation on hepatic and peripheral insulin sensitivity, high-fat-fed rats were treated for an additional week with compounds and subjected to euglycemic-hyperinsulinemic clamp studies. Before clamp studies, overnight fasted plasma glucose, total cholesterol, and triglycerides were not significantly different among groups (Table 2Go). Preclamp plasma insulin was decreased in the PIO group (P < 0.05), and preclamp plasma free fatty acids were significantly lower in the GW3965 group (P < 0.05) compared with the vehicle group. Insulin suppression of plasma free fatty acids (t = 180 min) was more significant in the GW3965 and PIO groups.


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  		Table 2. Basal and Clamp Hormone and Substrate Concentrations at 3 wk of LXR Ligand Dosing

 
Glucose infusion rates (GIRs) were significantly higher in rats dosed with GW3965 and PIO compared with the vehicle-treated group (Fig. 3AGo; P < 0.05), confirming the beneficial effect of LXR ligands on whole-body insulin action. A significant increase in insulin-stimulated glucose disappearance (Rd) was observed in rats administered PIO, but not in those treated with LXR ligand (Fig. 3BGo; P < 0.05). In contrast, insulin suppression of glucose appearance (Ra) was significantly lower in LXR-ligand-treated rats (Fig. 3CGo; P = 0.05). These changes were accompanied by decreased expression of hepatic gluconeogenic enzymes, such as PEPCK and G6pase (Fig. 4AGo). PIO had no effect on glucose Ra. GIR for the GW3965 group correlated significantly with rates of glucose Ra (Pearson’s r = –0.56; P < 0.05), whereas for the PIO group, GIR related significantly to rates of glucose Rd (Pearson’s r = 0.869; P < 0.05), suggesting that GW3965 improved whole-body insulin action primarily through effects on insulin suppression of endogenous glucose Ra, whereas PIO’s actions were mediated largely through effects on rates of glucose disposal. Direct measurement using [U-14C]2-deoxyglucose of tissue-specific rates of glucose uptake (R'g) supports this notion. Glucose R'g into gastrocnemius (a muscle group of mixed fiber type that contributes significantly to total muscle mass) was significantly increased in the PIO group (P < 0.05), whereas GW3965 had no significant effect on R'g in any tissue (Table 3Go). A nonstatistically significant increase in R'g was noticed in sc fat of GW3965-treated rats. However, the contribution of sc fat to whole-body glucose disposal in this model is thought to be small. The inability of GW3965 to stimulate R'g in epididymal fat was surprising given our prior observations in obese insulin-resistant mice demonstrating that glut4 is directly up-regulated by LXR agonists in this tissue (20). To examine this matter further, we evaluated glut4 levels in epididymal white adipose tissue and gastrocnemius muscle of treated rats. GW3965 dramatically induced expression of glut4 in epididymal fat but not in muscle, in accordance with our findings in mice (Fig. 4BGo). These results indicate that additional posttranscriptional regulation is necessary for the LXR-agonist-mediated increase in glut4 levels to translate into functional glucose uptake in vivo.


Figure 3
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  		Fig. 3. Hyperinsulinemic-Euglycemic Clamp Glucose Kinetics in High-Fat-Fed Rats Treated with LXR Ligand

A, GIR in milligrams per kg/min calculated over the final 30 min of a 90-min hyperinsulinemic clamp period. B, Rate of glucose disappearance (Glucose Rd) in milligrams per kg/min calculated over the final 20 min of the pancreatic clamp period (Basal Rd, min 70–90) or the final 20 min of the hyperinsulinemic-euglycemic period (Clamp Rd, min 160–180). C, Rate of glucose appearance (Glucose Ra) in milligrams per kg/min calculated over the final 20 min of the pancreatic clamp period (Basal Ra) or the final 20 min of the hyperinsulinemic-euglycemic period (Endo Ra). Values are mean ± SEM (n = 7–12/group). *, Significantly different from vehicle (P < 0.05).

 

Figure 4
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  		Fig. 4. Changes in Gene Expression Induced by LXR Ligand Treatment

A, Hepatic gene expression in high-fat-fed rats treated with LXR ligand for 3 wk. Tissues were collected after an overnight fast. Expression of SREBP-1c (a known LXR target) was used to confirm ligand exposure. B, Gene expression in adipose tissue and gastrocnemius muscle. Expression of Abca1 was used to verify ligand exposure. Note that glut4 is highly induced by the LXR ligand in white adipose tissue but not in muscle. Values are means ± SEM (n = 6/group). *, Significantly different from vehicle (P < 0.05). GASTROC, Gastrocnemius; WAT, white adipose tissue.

 

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  		Table 3. Tissue-Specific Glucose Disposal (R'g, µg/g·min) during Euglycemic-Hyperinsulinemic Clamp

 
To evaluate potential differences in glucose fate, tissue glycogen and lipid content were measured in treated rats. Liver and gastrocnemius muscle glycogen content were not significantly different under basal and clamp conditions among groups; hyperinsulinemic conditions did not increase tissue glycogen content in any group (Table 4Go). All groups demonstrated a similar increase in net hepatic glycogenesis from basal to insulin-stimulated conditions. In contrast, the increase from basal to hyperinsulinemic conditions in net glycogen synthesis in gastrocnemius muscle was significantly greater in the PIO-treated group (Table 4Go; P < 0.05). Total hepatic lipid content was significantly lower (P < 0.05) in the GW3965- and PIO-treated groups relative to vehicle-treated animals after a 90-min basal period (pancreatic clamp conditions). Hepatic triglyceride concentrations, however, were not different among groups (Table 4Go). These results were confirmed by histopathology (data not shown). In gastrocnemius muscle, no differences in total lipid content or tissue triglycerides were found among treatment groups, although the group treated with LXR ligand tended to accumulate less triglyceride in this muscle (Table 4Go).


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  		Table 4. Tissue Glycogen, Net Glycogenesis, and Lipid Content under Basal and Clamp Conditions

 
LXR Ligands Act Directly on the Liver to Suppress Expression of Gluconeogenic Enzymes
To investigate whether pharmacological activation of LXR acts directly on the liver to decrease Ra, as opposed to inducing a factor in the periphery that signals the liver to decrease glucose production, mice lacking LXR{alpha} (the dominant LXR isotype expressed in liver) were treated acutely with GW3965. LXR{alpha} null mice have deficiencies in hepatic LXR target gene expression that are not compensated for by the small levels of LXRβ present in this tissue (27, 28, 29). Hence, we reasoned that if suppression of gluconeogenic genes was observed in LXR{alpha} null mice treated with GW3965, that would suggest that systemic LXR activation leads to the production of a signal in the periphery (e.g. in adipose tissue or brain) that modulates hepatic gluconeogenesis. In contrast to the pattern observed in wild-type or LXRβ null mice, GW3965 failed to suppress PEPCK, G6Pase, or PPAR{gamma} coactivator 1{alpha} (PGC1-{alpha}) expression in the liver of mice lacking LXR{alpha}, suggesting that the beneficial effects of LXR activation on hepatic insulin sensitivity require LXR{alpha} (Fig. 5Go).


Figure 5
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  		Fig. 5. LXR Ligand Fails to Suppress Liver Gluconeogenic Gene Expression in the Absence of LXR{alpha}

Mice of the indicated genotype (n = 5–6/group) were treated orally for 2 d with GW3965 and fasted overnight, and livers were collected on the third day 3 h after dosing. Gene expression was analyzed by quantitative RT-PCR. Values are mean ± SEM (n = 5–6/group). *, Significantly different from vehicle (P < 0.05). KO, Knockout; wt, wild type.

 
LXR Ligands Suppress Expression of Gluconeogenic Enzymes in a Cell-Autonomous Manner in Primary Human Hepatocytes
Because the ultimate aim of our work is to evaluate the potential of LXR ligands as human insulin sensitizers, we treated human primary hepatocytes with GW3965 to assess whether our findings in rodents translate to human cells. This is an important issue because there are described species differences in the response of hepatocytes to LXR ligands (30, 31, 32). As shown in Fig. 6Go, GW3965 efficiently suppressed expression of PEPCK, G6Pase, and PGC1-{alpha} in human hepatocytes under conditions of maximal induction of gluconeogenic genes, suggesting that our finding in rodents can be extrapolated to human cells.


Figure 6
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  		Fig. 6. LXR Ligand Suppresses Gluconeogenic Gene Expression in Primary Human Hepatocytes

Cells were grown in 2 mM glucose, and gluconeogenic genes were induced using 8-bromo-cAMP and dexamethasone for 8 h before GW3965 treatment. Levels of SREBP-1c were measured under standard conditions (25 mM glucose, no gluconeogenic stimuli). *, Significantly different from vehicle (P < 0.05); **, P < 0.001. DMSO, Dimethylsulfoxide.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Synthetic LXR ligands have proven effective in multiple rodent models of type 2 diabetes, raising the possibility that LXR modulators may become useful insulin sensitizers. We have taken a pharmacological approach to discern how LXR activation enhances glucose tolerance and to quantitate the effect of LXR ligands on hepatic and peripheral glucose metabolism. By combining clamping experiments with studies involving mice genetically deficient for each LXR isotype, we have established that the beneficial effects of LXR activation in the insulin resistance model we have used are primarily due to suppression of hepatic gluconeogenesis mediated by LXR{alpha}, the dominant LXR in this tissue.

Our results also show that LXR ligands reduce weight gain associated with high-fat feeding, even though the molecular mechanism underlying this effect remains to be unraveled. Rats treated with the LXR ligand displayed no difference in oxygen consumption, suggesting that pharmacological activation of LXR does not result in altered energy balance. Although rats treated with GW3965 did not eat significantly less than those treated with vehicle, there was a strong trend toward decreased food intake that can explain a considerable proportion of the weight gain difference, and perhaps some of the insulin-sensitizing effects of GW3965. Because the LXRs are widely expressed in the intestine, it is also possible that LXR ligands influence the absorption of dietary components, thereby decreasing effective energy intake. Moreover, whereas we saw no difference in fat pad mass, we noticed a significant decrease in fat cell size in LXR ligand-treated rats, indicating that LXR activation results in alterations in adipocyte physiology or function that lead to remodeling and the emergence of a greater number of smaller cells in adipose tissue. These observations add to recent work with LXR null mice that demonstrates that LXRβ is important for diet- or age-induced adipocyte growth (23).

Our findings contrast with previous work in which ob/ob mice were treated with GW3965 before glucose clamping studies (26). In that model, LXR activation led to a significant increase in GIR, but no apparent effect on hepatic insulin sensitivity or glucose production was detected. The improvement on whole-body insulin sensitivity was ascribed to the effect of LXR ligands in the periphery, but peripheral glucose uptake was not measured directly, as we have done. This study also reported a dramatic increase in hepatic lipid content upon LXR ligand treatment, which we did not observe, perhaps a reflection of the different model systems used. Induction of hepatic steatosis by LXR ligands has been a common finding in studies using rodent models with impaired leptin signaling. Because hepatic steatosis has been traditionally associated with liver insulin resistance, it has been challenging to explain how LXR activation can significantly enhance hepatic insulin sensitivity in the face of a massive increase in liver lipid accumulation. One potential explanation is that some of these studies used a second LXR ligand, T0901317, which it is not specific for LXR. This compound is known to activate the pregnane X receptor and the farnesoid X receptor, nuclear receptors that are also involved in hepatic lipid and carbohydrate metabolism (16, 33, 34). Hence, it is difficult to discern the contribution of LXR activation to the beneficial or deleterious effects observed with this compound. However, increases in liver lipid levels have also been observed in studies using GW3965, but these findings appear restricted primarily to experiments with severely obese/diabetic animals (35). Consistent with our present work, we did not observe significant liver triglyceride accumulation in our original study of the effect of GW3965 in diet-induced mouse obesity. These results suggest that LXR ligands have differential effects in obese insulin-resistant animals vs. an overtly diabetic context. Alternatively, these findings hint that LXR ligands may have systemic effects that require intact adipose tissue signaling, perhaps by influencing the balance between fat storage and oxidation as has been suggested (36).

The mechanism whereby LXR activation increases liver insulin sensitivity and suppresses hepatic gluconeogenic gene expression remains an important lingering question. Some studies have suggested that the inhibitory effect of LXR on expression of these genes may be due to the ability of LXR activators to interfere with glucocorticoid receptor (GR) function (37). Treatment of db/db mice with T0901317 normalized the aberrant expression of GR and 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1, the enzyme responsible for generating active GR ligand in liver) in this model of type 2 diabetes. These findings are consistent with earlier reports that T0901317 down-regulates 11β-HSD1 activity in liver and brown adipose tissue (38, 39). However, we have been unable to document a decrease of GR or 11β-HSD1 mRNA in the livers of our GW3965-treated insulin-resistant rats (data not shown). This observation suggests that either additional mechanisms exist that allow LXR to down-regulate expression of gluconeogenic enzymes, or that the effects of T0901317 on glucocorticoid receptor function may not be due to LXR activation. Very recent work has suggested the molecular basis of one of these potential mechanisms: interaction of LXR with the cofactor receptor-interacting protein 140 (RIP 140) appears necessary for suppression of PEPCK by LXR ligands (40).

Our results have important implications for clinical development of LXR agonists as insulin sensitizers. We have shown that LXR ligands increase insulin responsiveness to the same degree as PPAR{gamma} ligands in this model of diet-induced obesity, for both showed a similar increase in GIR. Our work also suggests that modulation of hepatic LXR{alpha} activity appears required for LXR-mediated improvement of whole-body insulin action. Because activation of LXR{alpha} in the liver typically results in deleterious increases in lipogenesis, one approach to pharmaceutically target LXR has been to search for LXRβ-selective compounds (41). Due to the low expression of LXRβ in liver, it is thought that these molecules may retain efficacy by acting on peripheral tissues that universally express LXRβ and avoid the lipogenic side effects of LXR activation. Although this strategy may prove fruitful for indications such as atherosclerosis, our results imply that LXRβ-selective molecules may not be efficient insulin sensitizers. Treatment of type 2 diabetes with LXR agonists may require the development of more sophisticated gene- or tissue-selective modulators of the kind that already exist for other nuclear receptors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Rat Studies
Rat studies were approved by the Novartis Animal Care and Use Committee. Male Sprague Dawley rats weighing 100–125 g arrived from Charles River Laboratories (Kingston, NY) at 4 wk of age. They were allowed free access to a high-fat diet (60% fat by calories, Diet D12492i; Research Diets, New Brunswick, NJ) and maintained on a normal light cycle (lights on from 0800 h to 2000 h). At 5 wk of high-fat diet feeding (9 wk old), rats were assigned to one of three weight-matched groups and treated orally for 21–23 d with either vehicle (0.5% methyl cellulose, 1 ml/kg), GW3965 (20 mg/kg·d), or pioglitazone (PIO, 20 mg/kg·d). On d 17–d 19 of dosing, cannulas were implanted into the carotid artery and jugular vein for subsequent infusion studies (42). Two sets of rats were studied. Rats in the first group (n = 6/group) were assessed for whole-body fuel utilization and energy expenditure on d 14–16 of dosing, and tissues were collected on d 21–24 of dosing under basal conditions, 150 min after dosing. A second group of rats (n = 7–12/group) handled similarly with regards to diet and dosing regimens underwent euglycemic-hyperinsulinemic clamp studies on d 21–24 of dosing. Tissues were collected after a 90-min clamp (240 min after dosing).

Assessment of Whole-Body Fuel Utilization and Energy Expenditure
On d 14–16 of dosing, rats were housed for 60 h in calorimetry cages (Oxymax System; Columbus Instruments, Columbus, OH) to assess rates of oxygen consumption (VO2) and nonprotein respiratory exchange ratio (npRER), calculated as previously described (43). The first 12 h in the system (beginning d 14, 9 h after dosing) was considered an acclimatization period. Rats were allowed free access to food and water. On d 16 of dosing, food was removed after dosing for the entire light cycle period and returned at the start of the dark cycle 12 h later. The 12-h period of food deprivation was meant to assess whether drug treatment affected whole-body fuel selection during the fed-to-fasted or fasted-to-fed transition.

Euglycemic-Hyperinsulinemic Clamp
Rats were fasted overnight before assessing insulin action by euglycemic-hyperinsulinemic clamp. They were dosed as usual 1 h before initiation of the basal period of the clamp studies. Five minutes before initiating infusions (55 min after dosing), a basal blood sample was taken from the arterial line to assess basal hormone and substrate concentrations. At t = 0 min (60 min after dosing), a bolus of HPLC-purified [3-3H]glucose was given (7.5 µCi) over 1 min into the venous line followed by a constant venous infusion at 0.12 µCi/min. A pancreatic clamp was also initiated by infusion of somatostatin (1.2 µg/kg·min) to suppress endogenous insulin and glucagon production. Insulin (0.24 mU/kg·min) and glucagon (0.8 ng/kg·min) were infused at rates that maintained these hormones at basal concentrations. Arterial blood samples were taken at t = 70, 80, and 90 min to assess tracer steady state and determine basal rates of endogenous glucose appearance (Ra) and disappearance (Rd). At t = 90 min, a second bolus of [3-3H]glucose was given (3.75 µCi) over 1 min into the venous line, and the constant infusion rate of [3-3H]glucose was increased to 0.25 µCi/min for the remainder of the study to minimize changes in blood glucose-specific activity with the initiation of the clamp period of the study. A venous infusion of insulin at 3.0 mU/kg·min was also initiated along with a variable infusion of glucose (30% glucose into the jugular vein) while maintaining the pancreatic clamp. Small arterial blood samples (5 µl) were taken every 5–10 min to assess circulating blood glucose concentrations and determine appropriate changes in the rate of infusion of exogenous glucose necessary to maintain glucose at 120 mg/dl. To assess peripheral glucose uptake into adipose tissue and skeletal muscle, a venous bolus of [U-14C]-2-deoxyglucose (45 µCi in 100 µl 0.1% BSA/saline; American Radiolabeled Chemicals, Inc., St. Louis, MO) was administered at t = 135 min over a 60-sec period. Arterial blood samples were taken at t = 137.5, 140, 145, 150, 155, 160, 165, 170, 175, and 180 min for 14C-labeled plasma glucose specific activity. At t = 160, 170, and 180, larger blood samples were taken to assess 3H-labeled plasma glucose specific activity and insulin suppression of glucose Ra. At t = 180 min (240 min after dosing), rats were anesthetized with pentobarbital sodium (100 mg/kg, iv) and tissues were collected and placed in liquid nitrogen. Before freezing, the epididymal fat pad was weighed and a piece taken for histopathology. A small piece of liver was handled likewise. For some animals in each treatment group, tissues were collected at the end of the 90-min basal period (150 min after dosing).

Plasma Hormones and Substrates
Plasma glucose for the determination of glucose specific activity was assessed using a 2700 YSI Select (Yellow Springs International, Yellow Springs, OH). Insulin was measured by ELISA (Linco Research, Inc, St. Charles, MO). Free fatty acids were measured spectrophotometrically (Wako-C NEFA kit; Wako Pure Chemical Industries Ltd., Richmond, VA). Total plasma cholesterol and triglycerides were estimated fluorometrically using validated in-house enzymatic methods utilizing horseradish peroxidase oxidation of Amplex Red to resorufin. During clamp studies, glucose was estimated with a One-Touch Ultra glucometer (LifeScan, Milpitas, CA).

Plasma and Tissue Radioactivity
Plasma samples were deproteinized with 0.3 N Ba(OH)2 and 0.3 N ZnSO4 and 30 min later were centrifuged (44). One portion was dried to remove all 3H2O, reconstituted in distilled deionized water, and counted for 3H and 14C using dual-mode liquid scintillation counting (Beckman LS6000IC; Beckman Instruments, Fullerton, CA). Phosphorylated [14C]-2-deoxyglucose concentrations were determined in gastrocnemius muscle, superficial vastus lateralis muscle, soleus muscle, and epididymal fat as described previously (45). In brief, tissues (50–100 mg for muscle; 350 mg for epididymal fat) were homogenized in 0.5% perchloric acid at 4 C (13,400 x g, 20 min) and neutralized with KOH. An aliquot of this supernatant was counted, representing total 14C-labeled 2-deoxyglucose, 2-deoxyglucose phosphate, and cellular glycogen. To a second aliquot of the supernatant, Ba(OH)2 and ZnSO4 (0.3 M) were added to precipitate glycogen and 2-deoxyglucose phosphate, and the samples were vortexed, centrifuged, and counted with the difference in 14C counts representing 2-deoxyglucose phosphate and glycogen. Tissue-specific glucose uptake (R'g for epididymal fat, gastrocnemius, superficial vastus lateralis, and soleus muscle) was calculated using accumulation of phosphorylated 2-deoxyglucose and glycogen, which are metabolically trapped in tissues lacking glucose-6-phosphatase activity (e.g. liver or kidney), and the area under the curve for the decay of [14C]glucose specific activity in plasma over the last 45 min of the clamp. This approach makes the assumption that differences in metabolic handling of 2-deoxyglucose and glucose are similar across drug treatments.

Liver and Gastrocnemius Muscle Glycogen Content and Net Tissue Glycogen Synthesis
Liver and skeletal muscle glycogen content and net tissue glycogen synthesis were determined by methods previously described (46).

Liver and Gastrocnemius Muscle Triglyceride Content
Total lipids were extracted from liver and gastrocnemius muscle (50 mg) as previously described (47). The resulting lipid extract was solubilized (Triton X-100) and suspended in normal saline (0.8% Triton in final assay). Triglycerides were assessed spectrophotometrically (Sigma kit 320A; Sigma, St Louis, MO).

White Adipose Tissue Cell Size
Epididymal fat was collected at the end of basal infusion studies from some rats (n = 6/group), fixed in normal buffered formalin (10% phosphate buffer; Fisher Scientific, Pittsburgh, PA), and embedded in paraffin. Sections (10 µm) were stained with hematoxylin and eosin and viewed at x20 magnification. Images (10 per rat with 50–75 cells per field), obtained with an Aperio T2 ScanScope digital camera (Aperio Technologies, Vista, CA), were converted into a binary format with Aperio Image Scope. Fat cell area was estimated by tracing the cell membrane with an in-house image analysis program. Minimum and maximum field limits were set before analysis to exclude preadipocytes or nonadipose cells. The final readout of the system (pixels) was converted to square micrometers and reported as fat cell cross-sectional area.

Calculations
Area under the curve was calculated using the trapezoidal rule. Glucose Ra and Rd were determined using non-steady-state equations as described previously (48). Tissue R'g was determined by correcting the concentration of tissue [14C]-2-deoxyglucose-6-phosphate concentration by the area under the curve for plasma [14C]glucose specific activity over the last 45 min of the clamp studies. Glucose infusion rates were determined as a time-weighted average of the rates of glucose infusion over the last 30 min of the clamp studies. Net liver glycogenesis was calculated using a precursor-product relationship, where [3H]glucose associated with tissue glycogen was corrected for plasma [3H]glucose specific activity maintained over 90 (basal studies) or 180 min (clamp studies).

Data Analysis
Data are reported as means ± SEM. Two-way repeated measures ANOVA assessed any time-dependent effects of drug treatment. One-way ANOVA assessed whether differences existed among treatment groups for variables that were not dependent on time or condition. Tukey’s multiple comparison test determined between-group differences. Pearson’s correlation assessed the degree to which glucose Ra and Rd within groups explained differences in whole-body insulin action. Significance was set at P < 0.05. Outliers were assessed using Dixon’s Q-test. Gene expression data were analyzed using a two-tailed Student’s t test.

Mouse Studies
Mouse studies were approved by the Genomics Institute of the Novartis Research Foundation’s Animal Care and Use Committee. LXR mutant animals were obtained from Deltagen (San Carlos, CA). Mice (9 wk old) maintained on standard chow were dosed for 2 d with GW3965 (50 mg/kg orally in a 4:1 mixture of PEG300/Tween 20), before being fasted overnight. On the morning of the third day, mice received a final dose of GW3965 or vehicle 3 h before tissue collection.

Gene Expression Analysis
RNA was isolated from frozen tissues and analyzed by TaqMan quantitative RT-PCR using the one-step Superscript III platinum reagent (Invitrogen, Carlsbad, CA). Samples were run in triplicate as multiplexed reactions with a normalizing internal control (36B4). Probe and primer sequences are available on request.

Human Primary Hepatocytes Studies
Fresh frozen primary human hepatocytes (In Vitro Technologies) were thawed and plated according to vendor instructions. The next day, cells were washed twice with PBS to remove unattached hepatocytes and starved for 4 h. They were then cultured in DMEM with 2 mM glucose with 5% Lipoprotein-deficient fetal bovine serum (Intracel, Royston, Herts, UK) under conditions of sterol depletion. To induce gluconeogenic genes, cells were treated for 8 h with 8-bromo-cAMP (1 mM) and dexamethasone (1 µM) before test compound addition. Cells were harvested for gene expression analysis 14 h after ligand addition.


   FOOTNOTES
 
This work was supported by The Novartis Research Foundation and Novartis Pharmaceuticals.

Disclosure Statement: The authors have nothing to disclose.

First Published Online August 23, 2007

Abbreviations: ABC, ATP binding cassette; GIR, glucose infusion rate; G6Pase, glucose-6-phosphatase; GR, glucocorticoid receptor; 11β-HSD1, 11β-hydroxysteroid dehydrogenase type 1; LXR, liver X receptor; npRER, nonprotein respiratory exchange ratio; PEPCK, phosphoenol pyruvate carboxykinase; PGC-1{alpha}, PPAR{gamma} coactivator 1{alpha}; PIO, pioglitazone; PPAR, peroxisomal proliferator-activated receptor; SREBP, sterol-regulatory element-binding protein.

Received for publication March 26, 2007. Accepted for publication August 9, 2007.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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