Differential effects of pharmacological liver X receptor activation on hepatic and peripheral insulin sensitivity in lean and ob/ob mice

Aldo Grefhorst,1 Theo H. van Dijk,1 Anke Hammer,1 Fjodor H. van der Sluijs,1 Rick Havinga,1 Louis M. Havekes,2 Johannes A. Romijn,3 Pieter H. Groot,4 Dirk-Jan Reijngoud,1 and Folkert Kuipers1

1Center for Liver, Digestive, and Metabolic Diseases, Laboratory of Pediatrics, University Medical Center Groningen, Groningen; 2TNO Prevention and Health and Departments of General Internal Medicine and Cardiology and of 3Endocrinology and Diabetes, Leiden University Medical Center, Leiden, The Netherlands and the 4Atherosclerosis Department, GlaxoSmithKline Pharmaceuticals, Stevenage, United Kingdom

Submitted 14 April 2005 ; accepted in final form 27 May 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURE
 REFERENCES
 
Liver X receptor (LXR) agonists have been proposed to act as anti-diabetic drugs. However, pharmacological LXR activation leads to severe hepatic steatosis, a condition usually associated with insulin resistance and type 2 diabetes mellitus. To address this apparent contradiction, lean and ob/ob mice were treated with the LXR agonist GW-3965 for 10 days. Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp studies. Hepatic glucose production (HGP) and metabolic clearance rate (MCR) of glucose were determined with stable isotope techniques. Blood glucose and hepatic and whole body insulin sensitivity remained unaffected upon treatment in lean mice, despite increased hepatic triglyceride contents (61.7 ± 7.2 vs. 12.1 ± 2.0 nmol/mg liver, P < 0.05). In ob/ob mice, LXR activation resulted in lower blood glucose levels and significantly improved whole body insulin sensitivity. GW-3965 treatment did not affect HGP under normo- and hyperinsulinemic conditions, despite increased hepatic triglyceride contents (221 ± 13 vs. 176 ± 19 nmol/mg liver, P < 0.05). Clamped MCR increased upon GW-3965 treatment (18.2 ± 1.0 vs. 14.3 ± 1.4 ml·kg–1·min–1, P = 0.05). LXR activation increased white adipose tissue mRNA levels of Glut4, Acc1 and Fasin ob/ob mice only. In conclusion, LXR-induced blood glucose lowering in ob/ob mice was attributable to increased peripheral glucose uptake and metabolism, physiologically reflected in a slightly improved insulin sensitivity. Remarkably, steatosis associated with LXR activation did not affect hepatic insulin sensitivity.

hepatic glucose production; hepatic steatosis; hyperinsulinemic euglycemic clamp; stable isotopes; triglycerides


NUCLEAR RECEPTORS act as cellular sensors of endogenous and exogenous compounds. When activated by their ligands, these receptors modulate transcription of their target genes to allow the cell to adapt adequately to changing conditions. The liver X receptor (LXR; NR1H3) has been identified as an oxysterol-activated nuclear receptor (26, 40, 47). After ligand binding, LXR forms a heterodimer with the retinoid X receptor (RXR; NR2B1). This complex binds to LXR response elements in promoter regions of genes, resulting in adaptation of gene transcription by attracting coactivator or corepressor complexes (13). LXR is well known to induce transcription of genes encoding proteins involved in reverse cholesterol transport, i.e., ATP-binding cassette transporter (ABC) A1 (10, 34, 36, 38, 44), ABCG1 (24, 45), ABCG5, ABCG8 (4, 35, 48), and Cyp7A1 (15, 26, 33). Pharmacological LXR activation increases high-density lipoprotein (HDL) cholesterol levels and stimulates fecal cholesterol excretion in mice (30, 34, 38, 44). Treatment with agonists such as T-0901317 or GW-3965 attenuated development of atherosclerosis in apolipoprotein E-deficient (Apoe–/–) and low-density lipoprotein (LDL) receptor-deficient (Ldlr–/–) mice (11, 41). Thus synthetic LXR agonists were considered as potential anti-atherosclerotic agents. Application of LXR agonists, however, was found to be associated with a number of undesirable side effects. LXR also controls expression of various genes involved in lipogenesis and triglyceride (TG) metabolism, and severe hepatic steatosis developed in mice upon LXR agonist treatment (14, 37). We have previously shown that pharmacological activation of LXR is associated with production of large, TG-rich very low density lipoprotein (VLDL) particles, leading to hypertriglyceridemia in a mouse model with a humanized lipid profile (14).

Hepatic steatosis is associated with hepatic insulin resistance and type 2 diabetes mellitus (2, 22). Counterintuitively, despite induction of hepatic steatosis, treatment with the LXR agonist GW-3965 improved the response to a glucose tolerance test in C57BL/6J mice on a high-fat diet (23) but not in chow-fed mice. Cao et al. (8) showed that LXR activation reduced blood glucose levels in diabetic rodents, which was associated with decreased hepatic expression of the gene encoding phosphoenolpyruvate carboxykinase (PEPCK), supposedly rate controlling in gluconeogenesis (GNG). Moreover, treatment of db/db mice with T-0901317 markedly lowered hepatic Pepck gene expression in combination with more severe hepatic TG accumulation (9), suggesting that suppressed hepatic Pepck expression might result in a shift from GNG toward lipogenesis.

So far, no quantitative data have been reported concerning the effects of LXR activation on hepatic and peripheral insulin sensitivity and on hepatic glucose metabolism. Moreover, the commonly used animal model for type 2 diabetes mellitus, the obese, leptin-deficient ob/ob mouse, has not been used to evaluate the anti-diabetic effects of LXR activation. Therefore, we quantified the effects of LXR activation by GW-3965 on whole body and hepatic insulin sensitivity in lean and obese (ob/ob) mice.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURE
 REFERENCES
 
Animals. Male lean and obese (ob/ob) C57BL/6J mice (Harlan, Horst, The Netherlands) were housed in a light- and temperature-controlled facility and were fed commercially available laboratory chow (RMH-B; Hope Farms, Woerden, The Netherlands) containing ~6.2% fat and ~0.01% cholesterol (wt/wt). For 10 days, the animals were fed the same diet with or without the synthetic LXR agonist GW-3965 (0.03% wt/wt; kindly provided by GlaxoSmithKline Pharmaceuticals, Stevenage, UK). On the 11th day, mice were subjected to one of the experiments described below. Experimental procedures were approved by the Ethics Committee for Animal Experiments of the State University Groningen.

Plasma and tissue sampling and analyses. Lean and ob/ob mice were killed under isoflurane anesthesia on the 11th day of treatment. A large blood sample was collected by cardiac puncture and centrifuged. Plasma was stored at –20°C until analyzed. The liver was quickly removed, weighed, and frozen in separate portions for RNA isolation and lipid analyses. Abdominal white adipose tissue (WAT) and backlimb muscle tissue were collected and frozen for RNA isolation.

Plasma TGs, phospholipids, nonesterified fatty acid (NEFA), HDL cholesterol, free cholesterol, and total cholesterol were determined using commercially available kits (Roche Diagnostics, Mannheim, Germany, and Wako Chemicals, Neuss, Germany). Plasma insulin concentrations were determined using RIA RI-13K (Linco Research, St. Charles, MO). Hepatic concentrations of TGs, free cholesterol, and total cholesterol were measured using commercial kits (Roche Diagnostics and Wako Chemicals) after lipid extraction according to Bligh and Dyer (6). Phospholipid content of the liver was determined according to Böttcher et al. (7) after lipid extraction. Protein concentrations in livers were determined according to Lowry et al. (28) using BSA (Pierce, Rockford, IL) as standard. Hepatic glycogen and glucose 6-phosphate (G-6-P) levels were determined as described before (17, 19). Fatty acid composition was determined by gas chromatography after methylation, as described previously (27).

Hyperinsulinemic euglycemic clamps. Lean and ob/ob mice were equipped with a permanent catheter in the right atrium via the jugular vein (21). The two-way entrance of the catheter was attached to the skull with acrylic glue. The mice were allowed a resting period of at least 5 days during which the treatment period was completed. Before the start of the experiment (9 h), food was withdrawn, but mice still had free access to water. They were kept in metabolic cages during the experiment, allowing frequent collection of blood spots from the mice without anesthesia (42).

The mice were infused for 6 h with two solutions. The first one (insulin solution) was a 1% BSA solution containing 40 µg/ml somatostatin (UCB, Breda, The Netherlands). This solution contained insulin (Actrapid; Novo Nordisk, Bagsvaerd, Denmark), leading to an insulin infusion rate of 10 mU·kg–1·min–1. To prevent high total infusion rates leading to possible higher morbidity, this solution contained 200 mg/ml glucose, from which 2% was [U-13C]glucose (99% 13C atom %excess; Cambridge Isotope Laboratories, Andover, MA). The solution was infused at a constant flow rate of 0.135 ml/h. The second infusate (glucose solution) was a 30% glucose solution, from which 3% was [U-13C]glucose. Its infusion rate was adjusted according to measured blood glucose levels to maintain euglycemic conditions. Just before the start of the experiment, a small blood sample was obtained by tail bleeding. Blood glucose levels were measured with a Lifescan EuroFlash glucose meter (Lifescan Benelux, Beerse, Belgium) in a small tail blood sample that was taken every 15 min. For gas chromatography/mass spectrometry (GC-MS) measurements, a blood spot was obtained by tail bleeding every hour. After the clamp, animals were killed by cardiac puncture under anesthesia. Blood samples were centrifuged, and the obtained plasma was stored at –20°C until analyzed.

Hepatic carbohydrate flux measurements. After 10 days of treatment, hepatic carbohydrate fluxes were determined using infusion of stable isotopes, as previously described by van Dijk et al. (42). The mice were allowed a resting period after surgery of at least 5 days during which the treatment period was completed. Before the start of infusion (9 h), food was withdrawn, but mice still had free access to water. They were infused at a rate of 0.3 ml/h (lean mice) or 0.6 ml/h (ob/ob mice) with a solution containing 13.9 µmol/ml [U-13C]glucose, 160 µmol/ml [2-13C]glycerol, 33 µmol/ml [1-2H]galactose, and 1.0 mg/ml paracetamol. Blood and urine spots were collected at hourly intervals on filter papers.

Measurement of mass isotopomer distribution by GC-MS. Analytical procedures for extraction of glucose from blood spots filter paper (hyperinsulinemic-euglycemic clamp and hepatic carbohydrate flux experiment) and paracetamol-glucuronide from urine filter paper (hepatic carbohydrate flux experiment), derivatization of the extracted compounds, and GC-MS measurements of derivatives were essentially, according to van Dijk and colleagues (42, 43).

Mass isotopomer distribution analysis for hepatic carbohydrate fluxes. The measured fractional isotopomer distribution by GC-MS (m0-m6) was corrected for the fractional distribution resulting from natural abundance of 13C by multiple linear regression as described by Lee et al. (25) to obtain the excess mole fraction of mass isotopomers M0-M6 due to incorporation of infused labeled compounds. For the determination of the hepatic carbohydrate fluxes, this distribution was used in mass isotopomer distribution analysis algorithms of isotope incorporation and dilution according to Hellerstein et al. (16) and as described by van Dijk and colleagues (42, 43).

Calculation of endogenous glucose production and metabolic clearance rate under clamped conditions. Two solutions with [U-13C]glucose were infused with different rates. Therefore, the total rate of appearance of glucose into plasma [Ra(Glc;whole body)] was calculated as follows:

(1)
in which M6(Glc)glucose and M6(Glc)insulin are the excess mole fractions of infused [U-13C]glucose in the glucose and insulin solution, respectively, and infusion(Glc;M6)glucose and infusion(Glc;M6)insulin are the infusion rates of [U-13C]glucose of the glucose and insulin solution, respectively. M6(Glc)blood is the excess mole fraction of infused [U-13C]glucose in blood.

The rate of endogenous plasma glucose [Ra(Glc;endo)] was calculated as follows:

(2)
The metabolic clearance rate of glucose (MCR) was calculated according to:

(3)
where [Glc] is the blood glucose concentration (mM).

RNA isolation and measurement of mRNA levels by real-time PCR (Taqman). mRNA expression levels in liver, WAT, and skeletal muscle were measured by real-time PCR, as described previously (14). PCR results were normalized to {beta}-actin (hepatic tissue) or 18S (WAT, skeletal muscle) mRNA levels. The sequences of the primers and probes used are listed in Table 1.


View this table:
[in this window]
[in a new window]
 
Table 1. Primers and probes used for real-time PCR analysis

 
Statistics. All values represent means ± SE for the number of animals or experiments indicated. Statistical analysis of two groups was assessed by Mann-Whitney U-test (plasma and hepatic parameters) or ANOVA for repeated measurement (flux and clamp experiment). Level of significance was set at P < 0.05. Analyses were performed using SPSS for Windows software (SPSS, Chicago, IL).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURE
 REFERENCES
 
LXR activation increased hepatic TG content in both lean and ob/ob mice. Feeding the synthetic LXR agonist GW-3965 (0.03% wt/wt) for 10 days did not affect body weights of either lean or ob/ob mice (Table 2). Lean mice developed increased liver weights and a fivefold increase in hepatic TG content upon GW-3965 treatment (Table 2). Although ob/ob mice already showed severe hepatic steatosis on the control diet, LXR activation resulted in a further 25% increase in hepatic TG content. LXR activation resulted in a clearly altered hepatic fatty acid composition in lean mice (Fig. 1). GW-3965 treatment significantly reduced the relative amount of hepatic saturated fatty acids and polyunsaturated fatty acids from 37.8 ± 0.7 to 28.7 ± 0.6% and from 42.3 ± 2.8 to 27.5 ± 1.4%, respectively. In contrast, the relative amount of monounsaturated fatty acids significantly increased from 19.9 ± 3.4 to 43.8 ± 1.9% upon LXR activation. In ob/ob mice, LXR activation did not significantly affect hepatic fatty acid composition. Glycogen and G-6-P levels were higher in ob/ob mice than in lean mice. GW-3965 treatment did not affect glycogen and G-6-P levels of lean mice. In ob/obmice, in contrast, LXR activation reduced glycogen to levels comparable to those of lean mice, whereas G-6-P levels remained unchanged (Table 2).


View this table:
[in this window]
[in a new window]
 
Table 2. Hepatic and plasma parameters in lean and ob/ob mice fed a diet with or without the synthetic LXR agonist GW-3965 for 10 days

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Hepatic fatty acid composition in lean and ob/ob mice fed a diet with or without the synthetic liver X receptor (LXR) agonist GW-3965 for 10 days. Relative amounts of saturated (SAFA, open bars), monounsaturated (MUFA, gray bars), and polyunsaturated (PUFA, filled bars) fatty acids are shown; n = 6 mice. *P < 0.05, treated vs. untreated.

 
LXR activation resulted in elevated plasma cholesterol levels in lean mice (Table 2), mainly because of increased HDL cholesterol levels. Plasma TG and NEFA levels were not affected by the agonist in lean mice. Blood glucose levels were not affected by the treatment in lean mice (Fig. 2), but insulin levels were somewhat increased in the treated mice (Table 2). In ob/ob mice, LXR activation had no significant effect on plasma lipid levels, but the treatment resulted in reduced lower blood glucose (Fig. 2) and plasma insulin levels (Table 2).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2. Blood glucose levels (mM) in lean and ob/ob mice before and after 3 or 10 days diet with or without the synthetic LXR agonist GW-3965; n = 6 mice. P < 0.05, treated vs. untreated (*) and vs. before treatment ({dagger}).

 
Improved insulin sensitivity in ob/ob mice upon LXR activation. To test whether insulin sensitivity was influenced by LXR activation, we performed hyperinsulinemic-euglycemic clamp studies in conscious mice for 6 h. The total glucose infusion rate (GIR) was adjusted such that euglycemic conditions were maintained throughout the infusion period (Fig. 3A). In all four groups of mice, GIR reached constant values after ~3 h of infusion (Fig. 3B). In lean mice, GIR did not differ significantly between the treated and nontreated group. Calculated for the last 3 h of the experiment, GIR was 593 ± 13 and 564 ± 14 µmol·kg–1·min–1 for untreated and treated lean mice, respectively. In untreated ob/ob mice, GIR was markedly lower than in lean mice, i.e., 95 ± 5 µmol·kg–1·min–1. After the 10-day treatment period, insulin sensitivity was improved significantly, as is evident from the 50% increase in GIR to a value of 141 ± 5 µmol·kg–1·min–1.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3. Blood glucose level and glucose infusion rate during hyperinsulinemic-euglycemic clamps in lean and ob/ob mice fed a diet with (broken line) or without (solid line) the synthetic LXR agonist GW-3965 for 10 days. A: blood glucose levels (mM). B: glucose infusion rates (µmol·kg–1·min–1) during clamping; n = 6 untreated mice and n = 5 treated ob/ob mice. *P < 0.05, treated vs. untreated.

 
Hyperinsulinemia reduced hepatic glucose production irrespective of GW-3965 treatment. LXR agonist treatment (10 days) did not affect any of the measurable fluxes in lean mice, but resulted in a tendency toward increased GNG and significantly increased fluxes through glycogen phosphorylase (GP), glucose-6-phosphatase (G-6-Pase), and glucokinase (GK) in ob/ob mice (Table 3). The ob/ob mice showed slightly higher glucose cycling rates upon LXR activation (Table 3), confirming recent findings from our laboratory (3). Most importantly, endogenous glucose production [Ra(Glc;endo), hepatic glucose production (HGP)] did not differ between untreated and treated lean and ob/obmice (Table 3).


View this table:
[in this window]
[in a new window]
 
Table 3. Hepatic carbohydrate fluxes in lean and ob/ob mice fed a diet with or without the synthetic LXR agonist GW-3965 for 10 days

 
In lean mice, steady-state HGP during the last 3 h of the clamp was strongly reduced and not affected by administration of the agonist: 11 ± 18 vs. 23 ± 15 µmol·kg–1·min–1, untreated vs. treated (Fig. 3A). Thus HGP was almost completely inhibited in both groups, i.e., by 94 and 86%, respectively. Steady-state HGP under clamped conditions was higher in ob/ob mice compared with lean mice, indicating hepatic insulin resistance, without significant differences between untreated and treated ob/ob mice, i.e., 79 ± 7 vs. 64 ± 16 µmol·kg–1·min–1, respectively (Fig. 4A). The insulin-mediated suppression of HGP was similar in untreated and GW-3965-treated ob/ob mice, i.e., 48 and 61% respectively.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4. Hepatic glucose production and metabolic clearance rate under basal (stable isotope infusion) and clamped conditions in lean and ob/ob mice fed a diet with or without the synthetic LXR agonist GW-3965 for 10 days. A: hepatic glucose production (µmol·kg–1·min–1). B: metabolic clearance rate (ml·kg–1·min–1); n = 6 untreated mice and n = 5 clamped treated ob/ob and basal lean mice. P < 0.05, ob/ob vs. lean untreated (*) and clamped vs. basal, same mice and treatment ({dagger}).

 
Slightly improved metabolic clearance of glucose under clamped conditions in GW-3965-treated ob/ob mice. From the data available, it is possible to calculate the MCR under both basal and clamped conditions (Fig. 4B). In lean mice, basal MCR was not affected by LXR activation. During the clamp, values of MCR increased 5.5-fold and 5.7-fold in untreated and treated C57BL/6J mice, respectively. In contrast, MCR did not change upon hyperinsulinemia in untreated ob/ob mice, but clamping increased MCR by 80% in treated ob/ob mice. Thus clamped MCR values were 18.2 ± 1.0 and 14.3 ± 1.4 ml·kg–1·min–1 (P = 0.05) in treated and untreated ob/ob mice, respectively.

Effects of LXR activation on hepatic gene expression in lean and ob/ob mice. As expected, hepatic mRNA levels of genes encoding sterol-regulatory element-binding protein-1c (SREBP-1c) and fatty acid synthetase (FAS) increased upon LXR activation in the lean mice (Table 4). Expression of Fas was higher in ob/ob mice than in lean mice but was not affected by LXR activation. Expression of Lxr{alpha}, the major LXR isoform, was not affected by LXR activation.


View this table:
[in this window]
[in a new window]
 
Table 4. Hepatic gene expression in lean and ob/ob mice fed a diet with or without the synthetic LXR agonist GW-3965 for 10 days

 
From the genes encoding relevant enzymes in hepatic carbohydrate metabolism, only expression of Gk, encoding for glucokinase, was significantly increased by 54% upon GW-3965 treatment in the lean mice. (Table 4). Data suggest a tendency toward lower expression of Pepck and higher expression of the pyruvate kinase gene (Pk) in treated lean mice. Compared with lean mice, ob/ob mice showed significantly higher expression of Gk and G6pt. The latter gene encodes for G-6-P translocase, which is part of the G-6-Pase complex that controls the flux of G-6-P toward glucose. Upon LXR activation, expression of the genes encoding for both subunits of the G-6-Pase complex, G6pt and G6ph, was markedly reduced in ob/ob mice.

Normalization of Glut4, Acc1, and Fas WAT mRNA levels in ob/ob mice upon LXR activation. Because previous studies reported effects of LXR agonists on adipose and muscle mRNA expression profiles (23, 39), we determined WAT in skeletal muscle mRNA levels of several genes encoding proteins involved in glucose or lipogenesis (Fig. 5). LXR activation resulted in a threefold increase of Srebp-1c in adipose tissue of the lean mice, but Fas and Acc1 (encoding for acetyl-CoA carboxylase-1) mRNA levels were not affected. Expression of these lipogenic genes was lower in untreated ob/ob mice compared with untreated lean mice. Yet, LXR activation resulted in a fourfold increased expression of Srebp-1c, whereas Fas and Acc1 expression were increased 2.6- and 2.3-fold, respectively. Untreated ob/ob mice showed lower adipose mRNA levels of Glut4than untreated lean mice, but Glut4 expression normalized upon LXR activation in ob/ob mice. Expression of the genes encoding for hexokinase-1 (Hk1) and hexokinase-2 (Hk2) was not different between the two strains and not affected by LXR activation. In muscle tissue, LXR activation led a 8.6- and 3.5-fold increase of Srebp-1cmRNA levels in lean and ob/ob mice, respectively. Neither Fas, Acc2(the isoform of ACC predominantly expressed in muscle; see Ref. 1), nor Glut4 muscle mRNA levels in lean and ob/ob mice were affected upon LXR activation. In ob/ob mice only, LXR activation slightly reduced muscle mRNA levels of Hk1 and Hk2. WAT and muscle Lxr{alpha}expression was not affected upon GW-3965 activation in either lean or ob/ob mice.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5. White adipose tissue (WAT) and skeletal muscle gene expression in mice fed a diet with (filled bars) or without (open bars) the synthetic LXR agonist GW-3965 for 10 days. A: WAT mRNA levels. B: skeletal muscle mRNA levels. results normalized to 18S mRNA levels; data from untreated lean mice defined as 1; n = 3 (untreated), n = 5 (treated). P < 0.05 treated vs. untreated (*) and ob/ob vs. lean untreated ({dagger}). Srebp-1c, sterol-regulatory element-binding protein-1c; Fas, fatty acid synthetase; Acc1, acetyl-CoA carboxylase-1; Glut4, glucose transporter-4; Hk, hexokinase; Lxr{alpha}, liver X receptor-{alpha}.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURE
 REFERENCES
 
This study documents that pharmacological LXR activation improves glucose metabolism in ob/ob mice by increased peripheral glucose uptake and slightly increased peripheral insulin sensitivity. Remarkably, HGP and hepatic insulin sensitivity remained unaffected although LXR activation increased hepatic TG content dramatically. These observations indicate tissue-specific effects of LXR activation on fat and glucose metabolism. Treatment with the LXR agonist resulted in reduced blood glucose concentrations in ob/ob mice but had no effect whatsoever in lean mice (Fig. 2), in accordance with previous studies (8, 9, 23). Laffitte et al. (23) showed that obese, but not lean, C57BL/6 mice had improved glucose tolerance after 1 wk of GW-3965 treatment. The LXR agonist T-0901317 lowered blood glucose in male db/db mice and male Zucker diabetic fatty rats but not in their nondiabetic controls (8, 9).

Previous studies (8, 9, 23) suggested that reduced GNG might, at least in part, account for the anti-diabetic effect of LXR agonists. However, this conclusion was based merely on the finding of reduced PepckmRNA levels. To assess the actual effects of LXR activation on GNG flux and HGP, we quantified hepatic carbohydrate fluxes using stable isotope techniques (42, 43). From these data (Table 3) it is evident that neither GNG nor HGP was affected upon LXR activation in lean or ob/ob mice. We found reduced blood glucose levels in ob/obmice upon GW-3965 treatment, but our data clearly indicate that this effect of LXR activation is not the result of reduced GNG. We found hepatic Pepck expression to be slightly reduced in both strains of mice upon GW-3965 treatment. Previous reports (8, 23) showed more drastic effects on Pepck expression. The type of agonist (T-0901317 vs. GW-3965), mode and dose of administration, and the diabetic animal model could account for the differences in this respect between these studies and our results. For Gk expression, we found a 54% increase in lean mice and no change in ob/ob mice upon LXR activation, whereas others found a more than threefold induction in 12-h fasted female C57BL/6J mice (23). Changes in Gk mRNA levels could, at least in part, be the result of Srebp-1c induction (20). Nonetheless, our study demonstrates that changes in hepatic gene expression do not fully translate into changes in the metabolic fluxes. The slight reduction of Pepck gene expression was not reflected in the GNG flux (Table 3). Recently, is was pointed out that only severe reductions (–90%) or drastic increases (+300%) of Pepckexpression are associated with changes in GNG in mice (29). Consequently, relatively small alterations in Pepck mRNA levels per se do not predict changes in gluconeogenic flux.

Cao et al. (8) suggested that LXR activation improves insulin sensitivity. In contrast, we found a slight increase of plasma insulin levels in lean mice upon LXR activation (Table 2), which might suggest worsening of insulin sensitivity. The best possible technique to determine insulin sensitivity in vivo is the hyperinsulinemic-euglycemic clamp. Hence, to determine whether insulin sensitivity was actually affected, we used this technique in conscious, unrestrained mice to mimic the normal, physiological situation. Insulin-mediated glucose uptake did not differ between treated and untreated lean mice (Fig. 4A), indicating that LXR activation did not affect whole body insulin sensitivity. In addition, insulin-mediated suppression of HGP in lean mice was not affected by treatment with the LXR agonist (Fig. 4B), indicative for unaffected hepatic insulin sensitivity. In ob/ob mice, LXR activation resulted in a 50% increase of the GIR required to maintain euglycemia (Fig. 3, A and B). HGP was not significantly affected (Fig. 4A), indicating that the agonist had no measurable effect on hepatic insulin sensitivity. Yet, MCR was slightly increased upon clamping (Fig. 4B), indicating a positive, albeit only marginal, effect of the agonist on peripheral insulin sensitivity. Overall, insulin sensitivity of the treated ob/ob mice was still poor, since the effects on GIR, insulin-mediated suppression of HGP, and stimulation of MCR by no means yielded values for these parameters observed in lean C57BL/6J mice.

LXR activation induced a considerable increase in hepatic TG content. Steatosis is usually associated with hepatic insulin resistance, which means that the liver is less sensitive to the suppressive effects of insulin on hepatic glucose and VLDL-TG production. There are multiple endocrine, metabolic, and transcriptionally active factors involved in the interaction between hepatic TG metabolism and hepatic insulin sensitivity. The hierarchy between these different factors in modulating hepatic insulin sensitivity is at present unclear (12). In the present study, however, we report the striking observation that LXR-induced hepatic steatosis lacks the association with insulin resistance, at least with respect to insulin-mediated suppression of HGP. This might be a result of potential counteracting antidiabetic effects of the LXR agonist. In previous reports, it was already noticed that beneficial effects of LXR agonists on carbohydrate metabolism were only present in diabetic animal models (8, 9, 23). In these animals, insulin resistance associated with hepatic steatosis probably failed to overrule the effects of LXR on glucose homeostasis. Changes in hepatic fatty acid profile upon LXR activation may contribute in this respect (Fig. 1). LXR activation enhances transcription of the gene encoding stearoyl-CoA desaturase-1, Scd1, an enzyme involved in the conversion of saturated fatty acid into monounsaturated fatty acids (31). An increase in dietary monounsaturated fatty acids resulted in improved insulin sensitivity in healthy men and women (46), but had no effect on insulin secretion. Therefore, hepatic steatosis in which TG contain relatively more monounsaturated fatty acids, as observed in GW-3965-treated lean mice, might be "healthier" than steatosis with predominantly saturated fatty acids containing TG.

The LXR agonist increased expression of lipogenic genes Acc1 and Fas in WAT of ob/ob mice, but not in lean mice (Fig. 5A). A similar pattern was found for Glut4. Being aware of the discrepancy between mRNA expression and real enzyme activity and hence fluxes of substrates, it is tempting to speculate that LXR activation leads to increased uptake of glucose by adipocytes in ob/ob mice. The observation that GW-3965 treatment did not affect expression of Acc2, Fas, and Glut4 in muscle tissue of lean and ob/ob mice (Fig. 5B) suggests that LXR activation specifically improved glucose uptake by adipocytes and not of muscle tissue, suggestive for a fat-specific mechanism for improved peripheral insulin sensitivity. After uptake in adipocytes, glucose is metabolized into acetyl-CoA, which serves as substrate for lipogenesis. Juvet et al. (18) already reported that LXR agonists increased the lipid content of 3T3-L1 adipocytes in culture. Therefore, in the long turn, LXR activation may lead to a further increase in adipose tissue mass in the already obese ob/ob mice. The fact that GW-3965 treatment failed to increase Acc1, Fas, and Glut4 expression in WAT of lean mice might be attributable to circulating leptin. Orci et al. (32) reported that adenovirus-induced hyperleptinemia led to transformation of adipocytes to fat-oxidizing cells in wild-type Zucker diabetic fatty rats. Moreover, WAT lipogenic gene expression was decreased in these rats. In our study, normal plasma leptin levels in the lean mice were apparently still able to suppress lipogenic gene expression, but the ob/ob mice lack this capability. As a result, the WAT lipogenic mRNA levels were increased in ob/ob mice but not in lean mice.

This study unequivocally demonstrates that antidiabetic effects of LXR agonists in ob/ob mice are exclusively the result of increased glucose uptake by peripheral tissues, i.e., probably by WAT. Moreover, LXR activation had no effect whatsoever on hepatic carbohydrate metabolism in lean or ob/ob mice. In adipose tissue, glucose might be more rapidly used as substrate for de novo lipogenesis. Because effects of general LXR activation on (peripheral) insulin sensitivity are limited in ob/ob mice and coincide with undesirable side effects as hepatic steatosis and hypertriglyceridemia, potential application of LXR modulators in diabetes treatment will require the development of gene- and/or organ-specific compounds.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURE
 REFERENCES
 
This work was supported by Grant 903-39-291 from the Netherlands Organisation for Scientific Research.


    DISCLOSURE
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURE
 REFERENCES
 
P. H. Groot is a former employee of GlaxoSmithKline.


    ACKNOWLEDGMENTS
 
We thank Theo Boer, Klaas Bijsterveld, Maaike H. Oosterveer, and Renze Boverhof for skillful technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Grefhorst, Center for Liver, Digestive, and Metabolic Diseases, Laboratory of Pediatrics, Rm. Y2117, CMC IV, Univ. Medical Center Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands (e-mail: A.Grefhorst{at}med.umcg.nl)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURE
 REFERENCES
 

  1. Abu-Elheiga L, Matzuk MM, Abo-Hashema KAH, and Wakil SJ. Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-coa carboxylase 2. Science 291: 2613–2616, 2001.[Abstract/Free Full Text]
  2. Angulo P. Nonalcoholic fatty liver disease. N Engl J Med 346: 1221–1231, 2002.[Free Full Text]
  3. Bandsma RHJ, Grefhorst A, van Dijk TH, van der Sluijs FH, Hammer A, Reijngoud DJ, and Kuipers F. Enhanced glucose cycling and suppressed de novo synthesis of glucose-6-phosphate result in a net unchanged hepatic glucose output in ob/ob mice. Diabetologia 47: 2022–2031, 2004.[CrossRef][ISI][Medline]
  4. Berge KE, Tian H, Graf GA, Yu L, Grishin NV, Schultz J, Kwiterovich P, Shan B, Barnes R, and Hobbs HH. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters. Science 290: 1771–1775, 2000.[Abstract/Free Full Text]
  5. Bligh EG and Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911–917, 1959.[ISI][Medline]
  6. Böttcher CFJ, van Gent CM, and Pries C. A rapid and sensitive sub-micro-phosphorus determination. Anal Chim Acta 24: 203–204, 1961.[CrossRef][ISI]
  7. Cao G, Liang Y, Broderick CL, Oldham BA, Beyer TP, Schmidt RJ, Zhang Y, Stayrook KR, Suen C, Otto KA, Miller AR, Dai J, Foxworthy P, Gao H, Ryan TP, Jiang XC, Burris TP, Eacho PI, and Etgen GJ. Antidiabetic action of a liver X receptor agonist mediated by inhibition of hepatic gluconeogenesis. J Biol Chem 278: 1131–1136, 2003.[Abstract/Free Full Text]
  8. Chisholm JW, Hong J, Mills SA, and Lawn RM. The LXR ligand T0901317 induces severe lipogenesis in the db/db diabetic mouse. J Lipid Res 44: 2039–2048, 2003.[Abstract/Free Full Text]
  9. Costet P, Luo Y, Wang N, and Tall AR. Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor. J Biol Chem 275: 28240–28245, 2000.[Abstract/Free Full Text]
  10. Davis RA and Hui TY. 2000 George Lyman Duff Memorial Lecture. Atherosclerosis is a liver disease of the heart. Arterioscler Thromb Vasc Biol 21: 887–898, 2001.[Abstract/Free Full Text]
  11. Den Boer M, Voshol PJ, Kuipers F, Havekes LM, and Romijn JA. Hepatic steatosis: a mediator of the metabolic syndrome. Lessons from animal models. Arterioscler Thromb Vasc Biol 24: 644–649, 2004.[Abstract/Free Full Text]
  12. Glass CK. Differential recognition of target genes by nuclear receptor monomers, dimers, and heterodimers. Endocr Rev 15: 391–407, 1994.[CrossRef][ISI][Medline]
  13. Grefhorst A, Elzinga BM, Voshol PJ, Plösch T, Kok T, Bloks VW, van der Sluijs FH, Havekes LM, Romijn JA, Verkade HJ, and Kuipers F. Stimulation of lipogenesis by pharmacological activation of the liver X receptor leads to production of large, triglyceride-rich very low density lipopoprotein particles. J Biol Chem 277: 34182–34190, 2002.[Abstract/Free Full Text]
  14. Gupta S, Pandak WM, and Hylemon PB. LXRalpha is the dominant regulator of CYP7A1 transcription. Biochem Biophys Res Commun 293: 338–343, 2002.[CrossRef][ISI][Medline]
  15. Hellerstein MK, Neese RA, Linfoot P, Christiansen M, Turner S, and Letscher A. Hepatic gluconeogenic fluxes and glycogen turnover during fasting in humans. A stable isotope study. J Clin Invest 100: 1305–1319, 1997.[Abstract/Free Full Text]
  16. Hohorst HJ. D-Glucose-6-phosphat und D-fructose-6-phosphat. In: Methoden der Enzymatischen Analyse, edited by Bergmeyer HU. Weinheim, Germany: Verlag Chemie, 1970, p. 1200–1204.
  17. Juvet LK, Andresen SM, Schuster GU, Dalen KT, Tobin KAR, Hollung K, Haugen F, Jacinto S, Ulven SM, Bamberg K, Gustafsson J, and Nebb HI. On the role of liver X receptors in lipid accumulation in adipocytes. Mol Endocrinol 17: 172–182, 2003.[Abstract/Free Full Text]
  18. Keppler D and Decker K. Glykogen. Bestimmung mit amyloglucosidase. In: Methden der Enzymatischen Analyse, edited by Bergmeyer HU. Weinheim, Germany: Verlag Chemie, 1970, p. 1089–1094.
  19. Kim SY, Kim H, Kim TH, Im SS, Park SK, Lee IK, Kim KS, and Ahn YH. SREBP-1c mediates the insulin-dependent hepatic glucokinase expression. J Biol Chem 279: 30823–30829, 2004.[Abstract/Free Full Text]
  20. Kuipers F, Havinga R, Bosschieter H, Toorop GP, Hindriks FR, and Vonk RJ. Enterohepatic circulation in the rat. Gastroenterology 88: 403–411, 1985.[ISI][Medline]
  21. Kumar KS and Malet PF. Nonalcoholic steatohepatitis. Mayo Clin Proc 75: 733–739, 2000.[ISI][Medline]
  22. Laffitte BA, Chao LS, Li J, Walczak R, Hummasti S, Joseph SB, Castrillo A, Wilpitz DC, Mangelsdorf DJ, Collins JL, Saez E, and Tontonoz P. Activation of liver X receptor improves glucose tolerance through coordinate regulation of glucose metabolism in liver and adipose tissue. Proc Natl Acad Sci USA 100: 5419–5424, 2003.[Abstract/Free Full Text]
  23. Laffitte BA, Repa JJ, Joseph SB, Wilpitz DC, Kast HR, Mangelsdorf DJ, and Tontonoz P. LXRs control lipid-inducible expression of the apolipoprotein E gene in macrophages and adipocytes. Proc Natl Acad Sci USA 98: 507–512, 2001.[Abstract/Free Full Text]
  24. Lee WN, Byerley LO, Bergner EA, and Edmond J. Mass isotopomer analysis: theoretical and practical considerations. Biol Mass Spectrom 20: 451–458, 1991.[ISI][Medline]
  25. Lehmann JM, Kliewer SA, Moore LB, Smith-Oliver TA, Oliver BB, Su JL, Sundseth SS, Winegar DA, Blanchard DE, Spencer TA, and Willson TM. Activation of the nuclear receptor LXR by oxysterols defines a new hormone response pathway. J Biol Chem 272: 3137–3140, 1997.[Abstract/Free Full Text]
  26. Lepage G and Roy CC. Direct transesterification of all classes of lipids in a one-step reaction. J Lipid Res 27: 114–120, 1986.[Abstract]
  27. Lowry OH, Rosenbrough NJ, Farr AL, and Randall RJ. Protein measurement with Folin-phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
  28. Magnuson MA, She P, and Shiota M. Gene-altered mice and metabolic flux control. J Biol Chem 278: 32485–32488, 2003.[Free Full Text]
  29. Millatt LJ, Bocher V, Fruchart JC, and Staels B. Liver X receptors and the control of cholesterol homeostasis: potential therapeutic targets for the treatment of atherosclerosis. Biochim Biophys Acta 1631: 107–118, 2003.[ISI][Medline]
  30. Miyazaki M and Ntambi JM. Role of stearoyl-coenzyme A desaturase in lipid metabolism. Prostaglandins Leukot Essent Fatty Acids 68: 113–121, 2003.[CrossRef][ISI][Medline]
  31. Orci L, Cook WS, Ravazzola M, Wang M, Park BH, Montesano R, and Unger RH. Rapid transformation of white adipocytes into fat-oxidizing machines. Proc Natl Acad Sci USA 101: 2058–2063, 2004.[Abstract/Free Full Text]
  32. Peet DJ, Turley SD, Ma W, Janowski BA, Lobaccaro JMA, Hammer RE, and Mangelsdorf DJ. Cholesterol and bile acid metabolism are impaired in mice lacking the nuclear oxysterol receptor LXR{alpha}. Cell 93: 693–704, 1998.[CrossRef][ISI][Medline]
  33. Plösch T, Kok T, Bloks VW, Smit MJ, Havinga R, Chimini G, Groen AK, and Kuipers F. Increased hepatobiliary and fecal cholesterol excretion upon activation of the liver X receptor is independent of ABCA1. J Biol Chem 277: 33870–33877, 2002.[Abstract/Free Full Text]
  34. Repa JJ, Berge KE, Pomajzl C, Richardson JA, Hobbs H, and Mangelsdorf DJ. Regulation of ATP-binding cassette sterol transporters, ABCG5 and ABCG8 by the liver X receptors alpha and beta. J Biol Chem 2002.
  35. Repa JJ, Turley SD, Lobaccaro JMA, Medina J, Li L, Lustig K, Shan B, Heyman RA, Dietschy JM, and Mangelsdorf DJ. Regulation of absorption and ABC1-mediated efflux of cholesterol by RXR heterodimers. Science 289: 1524–1529, 2000.[Abstract/Free Full Text]
  36. Schultz JR, Tu H, Luk A, Repa JJ, Medina JC, Li L, Schwendner S, Wang S, Thoolen M, Mangelsdorf DJ, Lustig KD, and Shan B. Role of LXR in control of lipogenesis. Genes Dev 14: 2831–2838, 2000.[Abstract/Free Full Text]
  37. Schwartz K, Lawn RM, and Wade DP. ABC1 gene expression and ApoA-I-mediated cholesterol efflux are regulated by LXR. Biochem Biophys Res Commun 274: 794–802, 2000.[CrossRef][ISI][Medline]
  38. Stulnig TM, Steffensen KR, Gao H, Reimers M, Dahlman-Wright K, Schuster GU, and Gustafsson J. Novel roles of liver X receptors exposed by gene expression profiling in liver and adipose tissue. Mol Pharmacol 62: 1299–1305, 2002.[Abstract/Free Full Text]
  39. Teboul M, Enmark E, Li Q, Wikström AC, Pelto-Huikko M, and Gustafsson J. OR-1, a member of the nuclear superfamily that interacts with the 9-cis-retinoic acid receptor. Proc Natl Acad Sci USA 92: 2096–2100, 1995.[Abstract/Free Full Text]
  40. Terasaka N, Hiroshima A, Koieyama T, Ubukata N, Morikawa Y, Nakai D, and Inaba T. T-0901317, a synthetic liver X receptor ligand, inhibits development of atherosclerosis in LDL receptor-deficient mice. FEBS Lett 536: 6–11, 2003.[CrossRef][ISI][Medline]
  41. Van Dijk TH, Boer TS, Havinga R, Stellaard F, Kuipers F, and Reijngoud DJ. Quantification of hepatic carbohydrate metabolism in conscious mice using serial blood and urine spots. Anal Biochem 322: 1–13, 2003.[CrossRef][ISI][Medline]
  42. Van Dijk TH, van der Sluijs FH, Wiegman CH, Baller JFW, Gustafson LA, Burger HJ, Herling AW, Kuipers F, Meijer AJ, and Reijngoud DJ. Acute inhibition of hepatic glucose-6-phosphate does not affect gluconeogenesis but directs gluconeogenic flux toward glycogen in fasted rats. J Biol Chem 276: 25727–25735, 2001.[Abstract/Free Full Text]
  43. Venkateswaran A, Laffitte BA, Joseph SB, Mak PA, Wilpitz DC, Edwards PA, and Tontonoz P. Control of cellular cholesterol efflux by the nuclear oxysterol receptor LXR{alpha}. Proc Natl Acad Sci USA 97: 12097–12102, 2000.[Abstract/Free Full Text]
  44. Venkateswaran A, Repa JJ, Lobaccaro JMA, Bronson A, Mangelsdorf DJ, and Edwards PA. Human white/murine ABC8 mRNA levels are highly induced in lipid-loaded macrophages. A transcriptional role for specific oxysterols. J Biol Chem 275: 14700–14707, 2000.[Abstract/Free Full Text]
  45. Vessby B, Uusitupa M, Hermansen K, Riccardi G, Rivellese AA, Tapsell LC, Nälsén C, Berglund L, Louheranta A, Rasmussen BM, Calvert GD, Maffetone A, Pedersen E, Gustafsson IB, and Storlien LH. Substituting dietary saturated for monounsaturated fat impairs insulin sensitivity in healthy men and women: the KANWU study. Diabetologia 44: 312–319, 2001.[CrossRef][ISI][Medline]
  46. Willy PJ, Umesono K, Ong ES, Evans RM, Heyman RA, and Mangelsdorf DJ. LXR, a nuclear receptor that defines a distinct retinoid response pathway. Genes Dev 9: 1033–1045, 1995.[Abstract]
  47. Yu L, York J, von Bergmann K, Lutjohann D, Cohen JC, and Hobbs HH. Stimulation of cholesterol excretion by LXR agonist requires ATP-binding cassette transporters G5 and G8. J Biol Chem 278: 15565–15570, 2003.[Abstract/Free Full Text]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
289/5/E829    most recent
00165.2005v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Google Scholar
Articles by Grefhorst, A.
Articles by Kuipers, F.
PubMed
PubMed Citation
Articles by Grefhorst, A.
Articles by Kuipers, F.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.