Regulation of insulin-like growth factor binding protein-1 and lipoprotein lipase by the aryl hydrocarbon receptor

Abstract

The aryl hydrocarbon receptor (Ahr), a ligand-activated transcriptional factor, mediates the transcriptional activation of a battery of genes encoding drug metabolism enzymes. In the present study, we investigated the hepatic mRNA expression profile in Ahr-null (Ahr KO) mice compared to wild-type mice by microarray analysis to find new Ahr target genes. Pooled total RNA samples of liver extracted from 7- and 60-week-old Ahr KO or wild-type mice were studied by DNA microarray representing 19,867 genes. It was demonstrated that 23 genes were up-regulated and 20 genes were down-regulated over 2 fold in Ahr KO mice compared with wild-type mice commonly within the different age groups. We focused on insulin-like growth factor binding protein-1 (Igfbp-1) and lipoprotein lipase (Lpl) that were up-regulated in Ahr KO mice. The higher expression in Ahr KO mice compared to wild-type mice were confirmed by real-time RT-PCR analysis. In the wild-type mice but not in the Ahr KO mice, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treatment increased the Igfbp-1 and Lpl mRNA levels. The expression profile of Igfbp-1 protein was consistent with that of Igfbp-1 mRNA. Since Lpl is the primary enzyme responsible for hydrolysis of lipids in lipoproteins, the serum triglyceride levels were determined. Indeed, the serum triglyceride levels in Ahr KO mice was lower than that in wild-type mice in accordance with the Lpl mRNA levels. Contrary to our expectation, TCDD treatment significantly increased the serum triglyceride levels in wild-type, but did not in Ahr KO mice. These results suggest that serum triglyceride levels are not correlated with hepatic Lpl expression levels. In the present study, we found that Ahr paradoxically regulates Igfbp-1 and Lpl expressions in the liver.

INTRODUCTION

Aryl hydrocarbon receptor (Ahr) is a ligand-activated transcription factor and a member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of chemosensors and developmental regulators. Various kinds of environmental stimuli including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are well known as ligands of Ahr (Schmidt and Bradfield, 1996; Sogawa and Fujii-Kuriyama, 1997). Upon binding to the ligand, Ahr translocates to the nuclei, coincident with formation of a heterodimeric complex with Ahr nuclear translocator (Arnt). The ligand-activated Ahr mediates the transcriptional activation of a battery of genes encoding enzymes such as cytochrome P450 (CYP) 1 family, NAD(P)H: quinone oxidoreductase and glutathione S-transferase Ya subunit that function in the metabolism of xenobiotics and endobiotics (Bock, 1994; Rowlands and Gustafsson, 1997).

Gene knockout technology is a useful tool to estimate the roles of certain genes in vivo. Ahr-null (Ahr KO) mice on a C57BL/6 strain background were established by three research groups (Fernandez-Salguero et al., 1995; Schmidt et al., 1996; Mimura et al., 1997). The Ahr KO mice established by Fernandez-Salguero et al. (1995) exhibited 40-50% neonatal lethality, although survivors reached maturity and were fertile. The Ahr KO mice established by the latter two groups exhibited no neonatal lethality, but the growth rate was decreased in the first few weeks. In the Ahr KO mice, the size of the liver has been reported to be decreased compared with that in wild-type mice (Fernandez-Salguero et al., 1995; Schmidt et al., 1996). Hepatic portal fibrosis and hepatic vascular hypertrophy were observed in the Ahr KO mice (Fernandez-Salguero et al., 1995; Fernandez-Salguero et al., 1997). In addition, the accumulation of retinoid in the liver owing to reduced retinoic acid metabolism has also been documented (Andreola et al., 1997). The abnormality of retinoid homeostasis was considered to be the reason for the liver fibrosis in the Ahr KO mice (Andreola et al., 2004). Zaher et al. (1998) found that transforming growth factor β is overexpressed in the liver of Ahr KO mice, and this could be a causal factor of liver fibrosis. Thus, these findings suggest that Ahr expression in the liver is important for normal liver development. In the present study, we sought to determine the hepatic mRNA expression profile in Ahr KO mice compared with that in wild-type mice by microarray analysis to identify new targets of Ahr.

MATERIALS AND METHODS

Chemicals

CodeLink^™ Expression Assay Reagent kit, Manual Prep and streptavidin-Cy5 were purchased from GE Healthcare Bio-Sciences (Piscataway, NJ, USA). QIAquick PCR Purification Kit and RNeasy Mini Kit were from Qiagen (Hilden, Germany). NEN Blocking Reagent and Biotin 11-UTP were from Perkin-Elmer Life Sciences (Boston, MA, USA). ReverTra Ace (Moloney Murine Leukemia Vims Reverse Transcriptase RNase H Minus) was from Toyobo (Osaka, Japan). SYBR Premix Ex Taq (Perfect Real Time) was from Takara (Shiga, Japan). Goat anti-mouse insulin-like growth factor binding protein 1 (Igfbp-1) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Triglyceride E Test Wako was from Wako Pure Chemical Industries (Osaka, Japan). TCDD was from Cambridge Isotope Laboratories (Cambridge, MA, USA). All primers were commercially synthesized at Hokkaido System Sciences (Sapporo, Japan). Other chemicals were of the highest grade commercially available.

Animals and treatment

Ahr KO mice generated by Fernandez-Salguero et al. (1995) were used. Animals were housed in the institutional animal facility in a controlled environment (temperature 25 ± 1°C, humidity 50 ± 10% and 12 hr light/12 hr dark cycle) with access to food and water ad libitum. Animal maintenance and treatment were conducted in accordance with the National Institutes of Health Guide for Animal Welfare of Japan, as approved by the Institutional Animal Care and Use Committee of Kanazawa University. Genotyping of animals was carried out by polymerase chain reactions (PCRs) described previously (Takemoto et al., 2004). For the DNA microarray experiment, 7- and 60-week-old Ahr KO and wild-type mice were used. For the TCDD treatments, TCDD in corn oil (40 μg/kg body weight per day) was intraperitoneally administered to 35-week-old Ahr KO mice and 14-week-old wild-type mice for four days. Corn oil (2 ml/kg body weight) was administered as a control.

Total RNA preparation

Mice were sacrificed and the livers were collected and immediately frozen in liquid nitrogen and stored at −80°C until use. Total RNA from liver was isolated using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. Equal amounts of total RNA from 5 - 7 mice were pooled.

Microarray analysis

Microarray analysis was performed using a CodeLink^™ Bioarray Perfect System according to the manufacturer’s protocol (GE Healthcare Bio-Sciences). A Codelink^™ UniSet Mouse 20K I Bioarray (GE Healthcare Bio-Sciences) consisting of 19,867 genes including expression sequence tags (ESTs) was used. Processed slides were scanned with an Agilent G2565BA Microarray Scanner using Agilent Scan Control Software (Agilent Technologies, Palo Alto, CA, USA) with the laser set to red (633 nm) and the photomultiplier tube value to 70%. The scanned images for each slide were analyzed using CodeLink^™ Expression Analysis Software (GE Healthcare Bio-Sciences). The microarray data quality control was as follows: present, no flags (neither marginal nor absent); marginal, low quality spots judged by analysis software; absent, low signal density spots. Microarray data management was performed with GeneSpring software (Agilent Technologies). Comparison of the present genes, expression filtering and experiment normalization were performed. The individual gene expression for each array was normalized to their respective median value. Expression filters included the requirement that the genes be present in over 200% of controls for up-regulated genes and below 50% of controls for down-regulated genes.

Real-time RT-PCR

Total RNA (4 μg) was reverse transcribed using ReverTra Ace according to the manufacturer’s instructions and the resulting cDNA was amplified by PCR. Real-time PCR was performed using the Smart Cycler (Cepheid, Sunnyvale, CA, USA). PCR reactions were carried out as follows: A 1 μl portion of the reverse transcribed mixture was added to a PCR mixture containing 0.4 μM of each primer and SYBR Premix Ex Taq solution in a final volume of 25 μl. The primers used for PCR are shown in Table 1. The PCR condition for Igfbp-1, Lpl, Cyp17a1, and GAPDH was as follows: after an initial denaturation at 95°C for 30 sec, the amplification was performed by denaturation at 94°C for 4 sec, annealing and extension at 64°C for 20 sec for 45 cycles. The amplified products were monitored directly by measuring the increase of the dye intensity of the SYBR Green I. To normalize RNA loading and PCR variations, the signals of targets were corrected with the signals of GAPDH mRNA as the internal standard.

Table 1.

Primers used in the present study.

Primer	Sequence
Cyp17a1 S	5’ -GTA TTC AGC ACC TTT TCC CT-3’
Cyp17a1 AS	5’ -AAT ATG TCC ACC AGA TCG CT-3’
IGFBP-1 S	5’ -CAA ACT GCA ACA AGA ATG G-3’
IGFBP-1 AS	5’ -TGT ATC AAG CAG TAT GTG G-3’
LPL S	5’ -AGA AGC AGC AAG ATG TAC CT-3’
LPL AS	5’ - GAA ACT TTC TCC CTA GCA CA-3’
GAPDH S	5’ -AAA TGG GGT GAG GCC GGT-3’
GAPDH AS	5’ -ATT GCT GAC AAT CTT GAG TGA-3’

Western blot analysis of Igfbp-1

Ahr KO and wild-type mice were sacrificed 24 hr after the last treatment with TCDD. The livers were homogenized with buffer (0.1 M Tris-HCl (pH 7.4), 0.1 M KCl, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF) and liver homogenates (100 μg protein) subjected to SDS-polyacrylamide gel electrophoresis with 10% polyacrylamide gels followed by Western blotting using a PVDF membrane (Immobilon-P, Millipore, Billerica, MA, USA). The membrane was incubated with goat anti-Igfbp-1 antibody at a dilution of 1:200. Biotinylated anti-goat IgG and a VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA) were used for diaminobenzidine staining. The quantitative analysis of protein expression was performed using ImageQuant TL software (GE Healthcare Bio-Sciences).

Serum triglycerides concentration

Blood samples were collected from the postcaval vein 24 hr after the last treatment with TCDD. The serum triglyceride concentration was measured using Triglyceride E Test Wako.

Statistical analysis

Statistical significance was determined by analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons.

RESULTS

Comparison of gene expression profiles in Ahr KO and wild-type mice

Among 19,867 genes, 11,509 (58%) genes were categorized into 15 groups (Table 2). Among these, 7,255 (37%) genes showed sufficient spot density. In 7-week-old Ahr KO mice, the expression levels of 133 genes were elevated, whereas those of 95 genes were suppressed compared with age-matched wild-type mice. In 60-week-old Ahr KO mice, the expression levels of 76 genes were elevated, whereas those of 136 genes were suppressed compared with age-matched wild-type mice. In both 7- and 60-week-old Ahr KO mice, 23 genes were commonly elevated and 20 genes were commonly suppressed. The 43 common genes are shown in Table 3. Cyp1a2 and Ugt1a6, which are known to be highly regulated by Ahr, were down-regulated in Ahr KO mice. In addition, we found that Slc22a7 (organic anion transporter 2, Oat2) and Slc2a2 (facilitated glucose transporter 2) were also down-regulated in Ahr KO mice. These results suggest that these genes might be targets of Ahr regulation.

Table 2.

Number of genes of which expression were significantly changed in Ahr KO mice.

			Up-regulated (> 2 fold)			Down-regulated (< 2 fold)
Category	Total 1)	Present 2)	7 w	60 w	7 & 60 w	7 w	60 w	7 & 60 w
Apoptosis regulator	265	198	5	3	0	0	5	0
Cancer	168	109	2	2	2	1	2	0
Cell cycle	386	252	5	3	1	0	0	0
Chaperone	429	276	4	3	0	4	9	0
Enzyme	3,829	2,615	59	28	12	54	59	11
Immunity	143	93	1	1	0	2	2	1
Microtubular	63	38	0	0	0	0	0	0
Motor	40	24	2	1	1	0	0	0
Nucleic acid binding	1,812	1,183	18	6	0	9	17	0
RNA	14	12	0	0	0	0	0	0
Signal transducer	839	387	6	6	2	7	6	1
Storage	11	5	0	0	0	0	0	0
Stractural protein	1,341	700	17	11	3	7	11	2
Transport	1,465	955	11	9	2	10	20	5
Others	704	408	3	3	1	1	5	0
	11,509	7,255	133	76	23	95	136	20

Up-regulated and down-regulated genes showed more than 200% expression and less than 50% expression, respectively, compared with those in wild type mice.

¹⁾Number of genes of which categories were defined.

²⁾Number of genes showing enough spot density.

Table 3.

Up- or down-regulated genes in Ahr KO mice.

	Genbank ID	Common name	Relative mRNA expression
Gene name			7 w	60 w
Up regulation (23 genes)
Cancer
Rab38, member of RAS oncogene family	NM_028238	Rab38	2.1	4.3
Vav2 oncogene	NM_009500	Vav2	2.1	2.2
Cell cycle regulator
Cyclin B2	NM_007630	Ccnb2	3.8	2.1
Enzyme
Aldo-keto reductase family 1, member B7	NM_009731	Akr1b7	2.0	2.6
Arylacetamide deacetylase (esterase)	NM_023383	Aadac	2.5	2.0
Asparagine synthetase	U38940	Asns	6.5	5.0
Cis-retinol/3alpha hydroxysterol short-chain dehydrogenase-like	BC018263	CRAD-L	3.2	3.1
Cytochrome c oxidase, subunit VIIa 1	NM_009944	Cox7a1	2.8	2.1
Cytochrome P450, family 4, subfamily a, polypeptide 14	NM_007822	Cyp4a14	3.6	4.1
Cytochrome P450, family 17, subfamily a, polypeptide 1	NM_007809	Cyp17a1	12.5	2.1
Glutaredoxin 2 (thioltransferase)	NM_023505	Glrx2	2.3	2.2
Hydroxysteroid (17-beta) dehydrogenase 9	NM_013786	Hsd17b9	3.0	3.5
Lipoprotein lipase	NM_008509	Lpl	14.0	5.5
Methylmalonyl-Coenzyme A mutase	NM_008650	Mut	23.6	9.8
RIKEN cDNA 2310016A09 gene	BC024580	RIKEN	4.7	2.7
Motor
Dynein, axonemal, intermediate chain 1	AK004387	Dnaic1	4.1	8.2
Signal transducer
Insulin-like growth factor binding protein 1	NM_008341	Igfbp1	3.2	4.8
Structural protein
CD59a antigen	NM_007652	Cd59a	8.4	3.7
Collectin sub-family member 11	AK003121	Colec11	2.2	2.3
Olfactory receptor 65 (Olfr65)	NM_013617	Olfr65	2.3	2.5
Transport
Fatty acid binding protein 5, epidermal	NM_010634	Fabp5	2.3	2.7
Solute carrier organic anion transporter family, member 1a4	NM_030687	Slco1a4	2.9	2.3
Others
Ubiquitin-associated protein 1	NM_023305	Ubap1	5.8	3.3
Down regulation (20 genes)
Enzyme
Betaine-homocysteine methyltransferase	NM_016668	Bhmt	0.48	0.49
Cytochrome P450, family 1, subfamily a, polypeptide 2	NM_009993	Cyp1a2	0.30	0.36
Dopachrome tautomerase	NM_010024	Dct	0.26	0.32
Glutathione peroxidase 6	NM_145451	Gpx6	0.45	0.37
Interferon gamma-induced GTPase	NM_018738	Igtp	0.28	0.27
Isovaleryl coenzyme A dehydrogenase	NM_019826	Ivd	0.16	0.18
NADH dehydrogenase (ubiquinone) Fe-S protein 5	NM_134104	Ndufs5	0.02	0.01
Sulfotransferase family 5A, member 1	NM_020564	Sult5a1	0.30	0.29
UDP glucuronosyltransferase 1 family, polypeptide A6	U16818	Ugt1a6	0.38	0.48
Expressed sequence AI586015	NM_019992	AI586015	0.26	0.42
RIKEN cDNA E430034L04 gene	NM_011816	RIKEN	0.43	0.42
Immunity
Plasminogen	NM_008877	Plg	0.49	0.40
Signal transducer
Transforming growth factor beta 1 induced transcript 4	NM_009366	Tgfb1i4	0.23	0.46
Structural protein
Growth arrest specific 5	NM_013525	Gas5	0.27	0.46
Prion protein	NM_011170	Prnp	0.48	0.50
Transport
Amiloride-sensitive cation channel 5, intestinal	NM_021370	Accn5	0.12	0.23
Aquaporin 8	NM_007474	Aqp8	0.46	0.39
Solute carrier family 2 (facilitated glucose transporter), member 2	NM_031197	Slc2a2	0.47	0.26
Solute carrier family 22 (organic anion transporter), member 7	NM_144856	Slc22a7	0.35	0.47
Sorting nexin 1	NM_019727	Snx1	0.42	0.45

Up-regulated and down-regulated genes showed more than 200% and less than 50% expressions, respectively, compared with those in wild-type mice at the same weeks old.

Categories of first occurrence in Table 2 are listed.

The expression levels of methylmalonyl-Coenzyme A mutase, lipoprotein lipase (Lpl), and Cyp17a1 were highly (over 10 fold in 7-week-old mice) up-regulated in Ahr KO mice. Among these, the spot density of Lpl was highest. We additionally found that Igfbp-1 was unexpectedly up-regulated in Ahr KO mice, because previous studies reported that Igfbp-1 mRNA was induced by TCDD via Ahr activation (Adachi et al., 2004, Marchand et al., 2005). In a subsequent study, we investigated in detail the expression of Igfbp-1 and Lpl in the liver.

Real-time RT-PCR analysis

To confirm the results of the DNA microarray analysis, real-time RT-PCR analysis was performed (Fig. 1). The hepatic Igfbp-1 mRNA levels in Ahr KO mice were 7 fold (7-week-old) and 24 fold (60-week-old) higher than those in age-matched wild-type mice. The hepatic Lpl mRNA levels in Ahr KO mice were 8 fold (7-week-old) and 3 fold (60-week-old) higher than those in age-matched wild-type mice. Thus, the differences in the expression levels detected by microarray analysis were reproducible.

Fig. 1.

Relative expression levels of (A) Igfbp-1 and (B) Lpl mRNA in the liver of Ahr KO and wild-type mice determined by real-time RT-PCR. Total RNA was extracted from 7- and 60-week-old Ahr KO or wild-type mice. Samples from 5 - 7 mice were pooled within each group. The expression levels of Igfbp-1 and Lpl mRNA were normalized with the expression level of GAPDH as a control. Data are expressed as the mean of duplicate experiments.

Effects of TCDD treatment on Igfbp-1 and Lpl mRNA expression in Ahr KO and wild-type mouse livers

We investigated the effect of TCDD treatment on Igfbp-1 and Lpl mRNA expressions. Real-time RT-PCR analyses revealed that Igfbp-1 mRNA was significantly (7 fold) increased by TCDD in wild-type mice, but not in Ahr KO mice, showing 8-fold higher Igfbp-1 mRNA levels than those in wild-type mice (Fig. 2A). Lpl mRNA was also significantly (4 fold) increased by TCDD in wild-type mice, but not in Ahr KO mice, showing 4-fold higher Lpl mRNA levels than those in wild-type mice (Fig. 2B).

Fig. 2.

Effects of TCDD treatment on the expression levels of (A) Igfbp-1 and (B) Lpl mRNA in the liver of Ahr KO and wild-type mice determined by real-time RT-PCR analysis. TCDD (40 μg/kg weight) or corn oil was intraperitoneally administered to Ahr KO (35-week-old) and wild-type (14-week-old) mice for 4 days. The expression levels of Igfbp-1 and Lpl mRNA were normalized with the expression level of GAPDH as a control. Data are expressed as mean ± S.E. from 5 or 6 mice. **P < 0.01 by ANOVA.

Igfbp-1 protein expression

Western blot analysis demonstrated that Igfbp-1 protein was significantly (5 fold) induced by TCDD in wild-type mice (Fig. 3), but not in Ahr KO mice, showing 6-fold higher Igfbp-1 protein levels.

Fig. 3.

Effects of TCDD treatment on the expression level of Igfbp-1 protein level in the liver of Ahr KO and wild-type mice. TCDD (40 μg/kg weight) or corn oil was intraperitoneally administered to Ahr KO (35-week-old) and wild-type (14-week-old) mice for 4 days. Data are expressed as mean ± S.E. from 5 or 6 mice. *P < 0.05 by ANOVA.

Triglycerides concentration measurement

Since an antibody against Lpl is not commercially available, we sought to determine the Lpl activity to evaluate changes in the hepatic Lpl expression level. Lpl is the primary enzyme responsible for the metabolism of triglycerides. We investigated whether the differences in the Lpl expression level in liver might be inversely correlated with the serum triglyceride levels. The serum triglyceride level was lower in Ahr KO mice than in wild-type mice, being inversely correlated with the Lpl expression level. However, the serum triglyceride level was significantly (1.3 fold) increased by TCDD treatment in wild-type mice, but not in Ahr KO mice (Fig. 4).

Fig. 4.

Effects of TCDD treatment on serum triglyceride level in Ahr KO and wild-type mice. TCDD (40 μg/kg weight) or corn oil was administered to Ahr KO (35-week-old) and wild-type (14-week-old) mice for 4 days. Data are expressed as mean ± S.E. from 5 or 6 mice. *P < 0.05, ***P < 0.001 by ANOVA.

DISCUSSION

DNA microarray technology has been extensively used as a powerful tool for predicting unknown signaling pathways. Using DNA microarray analysis, the changes in mRNA expression levels in smooth muscle cells in Ahr KO mice were investigated by Guo et al. (2004) who found that transforming growth factor-beta 3 (Tgfb3) expression was higher in Ahr KO mice than in wild-type mice, indicating that Ahr suppresses Tgfb3 gene expression. It is well known that Ahr regulates various drug-metabolizing enzymes in liver. In the present study, we sought to investigate the mRNA profiles in liver from Ahr KO mice using microarray to find new Ahr gene targets. The overall gene expression profiles vary during development and the aging process. Therefore, we compared the data in young (7-week-old) and older (60-week-old) mice to determine common changes in gene expression by Ahr KO. The decreases in Cyp1a2 and Ugt1a6 in Ahr KO mice were consistent with those previously reported (Fernandez-Salguero et al., 1995). In addition, the decrease of Slc22a7 and increase of Cyp17a1 in livers in Ahr KO mice were consistent with a recent report by Tijet et al. (2006). These results suggest that our study was sufficiently reliable. In the present study, we first found that the Igfbp-1 and Lpl expression levels were higher in Ahr KO mice.

Despite the fact that the expression levels of Igfbp-1 and Lpl in the liver were increased in the Ahr KO, they were induced by TCDD in an Ahr-dependent manner. The latter results suggested that Ahr positively regulates Igfbp-1 and Lpl in the presence of the ligands. This is supported by a previous report indicating that a xenobiotic responsive element (XRE) to which Ahr binds is located in the promoter region of the Igfbp-1 gene at −87 (Marchand et al., 2005). In addition, we found two XRE sequences in the promoter region of the Lpl gene at −332 and −443 by a computer-assisted homology search. Further study will be necessary to determine whether the binding of Ahr to XRE might be responsible for the induction of Lpl by TCDD. Based on the higher expression levels of Igfbp-1 and Lpl in Ahr KO mice compared to those in the wild-type mice, Ahr might suppress Igfbp-1 and Lpl expressions in the absence of exogenous ligands and TCDD may interfere with the suppression. Alternatively, Ahr might positively regulate some suppressor of Igfbp-1 and Lpl expression in the absence of exogenous ligands.

IGFBP-1, one of the six IGFBPs, capable of sequestering insulin growth factors (IGF)s from their receptor. It was reported that transgenic mice of human IGFBP-1 gene showed postnatal growth retardation and impaired fecundity (Schneider et al., 2000). The pathophysiological abnormalities in Ahr KO mice such as decreased body weight, impaired fecundity as well as decrease liver size (Fernandez-Salguero et al., 1995) might be, in part, associated with Igfbp-1 overexpression.

LPL is produced by adipose tissue and then is transported to the endothelial cell surface (Matsumura, 1995). It is also expressed in heart, lung, adipose tissue, kidney, intestine and liver in mice (Kirchgessner et al., 1987). Lpl hydrolyses triglycerides. In Ahr KO mice, Lpl expression in adipocytes might also be increased in addition to that in liver, because the serum triglyceride levels in Ahr KO mice were decreased. The serum triglyceride level was increased by TCDD treatment in wild-type mice, being consistent with a previous report showing that adipose Lpl activity was decreased by TCDD treatment (Matsumura, 1995). TCDD inhibits the differentiation of preadipocytes to adipocytes (Alexander et al., 1998). The differentiation increases peroxisome proliferator activated receptor (PPAR) γ expression, which is a major regulator of Lpl in adipocytes. Therefore, the increase of serum trigrycerides by TCDD in wild-type mice was, in part, due to the inhibition of adipogenesis.

In summary, we found that hepatic Igfbp-1 and Lpl were paradoxically up-regulated by the activation and knockout of Ahr. Ahr would be responsible for glucose homeostasis and lipid metabolism.