Table 1
Sequences of primer pairs used to amplify each PCR product
| Gene | Sequence | Predicted Size (bp) | GenBank Accession No. |
| Acc1 | |||
| Sense | 5'-GCACTCCCGATTCATAATTG-3' | 141 | NM_133360 (34–185) |
| Antisense | 5'-CCCAAATCAGAAAGTGTATC-3' | ||
| Aco | |||
| Sense | 5'-ATCTATGACCAGGTTCAGTCGGGG-3' | 237 | NM_015729 (1312–1548) |
| Antisense | 5'-CCACGCCACTTCCTTGCTCTTC-3' | ||
| β-actin | |||
| Sense | 5'-TGACAGGATGCAGAAGGAGA-3' | 131 | AK075973 (1009–1139) |
| Antisense | 5'-GCTGGAAGGTGGACAGTGAG-3' | ||
| Dbp | |||
| Sense | 5'-CCGTGGAGGTGCTAATGACCT-3' | 105 | NM_016974 (984–1087) |
| Antisense | 5'-CCTCTGAGAAGCGGTGTCT-3' | ||
| Mtp | |||
| Sense | 5'-GCCCTAGTCAGGAAGCTGTG-3' | 127 | NM_008642 (1300–1426) |
| Antisense | 5'-CCAGCAGGTACATTGTGGTG-3' | ||
| Per2 | |||
| Sense | 5'-TGTGTGCTTACACGGGTGTCCTA-3' | 142 | AF036893 (5563–5704) |
| Antisense | 5'-ACGTTTGGTTTGCGCATGAA-3' | ||
| Ppar α | |||
| Sense | 5'-TCTTCACGATGCTGTCCTCCT-3' | 142 | NM_011144 (1395–1475) |
| Antisense | 5'-GGAACTCGCGTGTGATAAAGC-3' |
Figure 1
Chronic ethanol exposure increases liver weight in Clock-mutant mice. Eight- to ten-week-old wild-type (WT, n = 16) and Clock-mutant (Ck, n = 16) mice were given 15% ethanol in drinking water for 8 weeks. At the end of this treatment, liver and body weights were measured. Clock-mutant mice exhibited a significant (ANOVA, Fisher PLSD, P < 0.05) increase in liver/body weight (A) as well as % change in liver/body weight (B).
Table 2
Impact of ethanol exposure on wild-type and Clock-mutant mice
| Wild-type | Clock-mutant | |||
| Control | Ethanol | Control | Ethanol | |
| Ethanol Intake (g/kg/day) | na | 30.5 ± 2.0 | na | 27.2 ± 1.6 |
| Food Intake (g/kg/day) | 154 ± 11 | 136 ± 13 | 154 ± 14 | 136 ± 10 |
| Body Weight (g) | 32.3 ± 0.6 | 32.3 ± 0.6 | 33.0 ± 0.5 | 31.9 ± 0.5 |
| Serum Ethanol (mM) | na | 9.0 ± 2.3 | na | 9.4 ± 1.8 |
| Serum TG (mg/dl) | 68 ± 6.4 | 84 ± 8.5 | 62 ± 4.8 | 71 ± 4.9 |
| Serum CH (mg/dl) | 64.2 ± 4.7 | 60.2 ± 4.8 | 64.5 ± 3.0 | 62.2 ± 2.9 |
| Serum FFA (mEq/l) | 0.50 ± 0.03 | 0.53 ± 0.03 | 0.52 ± 0.03 | 0.46 ± 0.02 |
| Serum ALT (Karmen) | 28.0 ± 0.7 | 26.1 ± 0.5 | 30.1 ± 1.3 | 27.2 ± 0.3 |
| Serum AST (Karmen) | 75.0 ± 6.5 | 61.8 ± 2.9 | 62.8 ± 4.2 | 59.6 ± 2.3 |
TG, triglycerides; CH, cholesterol; FFA, free fatty acids; ALT, alanine aminotransferase; AST, aspartate aminotransferase, na; not available
Figure 2
Chronic ethanol increases liver triglycerides (TG) content in Clock-mutant mice. (A) Ethanol exposure significantly increased the TG levels in both WT (n = 16) and Ck (n = 16) mice. Further analysis by two-way ANOVA indicates that the ethanol-induced increase was greater in the mutant mice (P < 0.01). (B) In WT mice, ethanol exposure significantly increased liver TG at ZT 0, 6, and 18 (n = 4 for each time point). (C) In Ck mice, ethanol exposure significantly increased liver TG at ZT 12 and 18 (n = 4 for each time point). Values are means ± SEM. Significance was initially determined using the one-way ANOVA followed by Fischer PLSD (*P < 0.05, **P < 0.01).
Figure 3
Chronic ethanol increases liver lipid content in Clock-mutant mice. The intensity of staining of liver tissue with oil red O provides a qualitative measure of the lipid content. We found that ethanol exposure increases the intensity of the oil red O staining. Representative oil red O staining of liver tissue in WT and Ck mice on control and ethanol (A-D). Images from the stained liver were magnified ×400 and scale bar is shown.
Figure 4
Chronic ethanol alters the expression of genes involved in lipid metabolism in Clock-mutant mice. We investigated the impact of ethanol treatment on 4 genes that are critically involved in lipid metabolism. Expression of Acc1 (A), Aco (B), Mtp (C), and Pparα (D) were measured from the liver (n = 16) with RT-PCR and product normalized to that of β-actin. Significance was determined using the one-way ANOVA followed by Fischer PLSD (*P < 0.05, **P < 0.01).
Figure 5
Chronic ethanol does not alter the expression of circadian clock genes. After exposure to ethanol, mouse livers were collected every 6 hours (n = 4 per time point). Expression of Per2 (A, B) and Dbp (C, D) was measured from the liver with RT-PCR and product normalized to that of β-actin. Values are means ± SEM and normalized as average levels of water-received wild-type mice become 1. Significance was determined using the one-way ANOVA followed by Fischer PLSD.