Disruption of hepatic C/EBPalpha results in impaired glucose tolerance and age-dependent hepatosteatosis.

C/EBPalpha is highly expressed in liver and regulates many genes that are preferentially expressed in liver. Because C/EBPalpha-null mice die soon after birth, it is impossible to analyze the function of C/EBPalpha in the adult with this model. To address the function of C/EBPalpha in adult hepatocytes, liver-specific C/EBPalpha-null mice were produced using a floxed C/EBPalpha allele and the albumin-Cre transgene. Unlike whole body C/EBPalpha-null mice, mice lacking hepatic C/EBPalpha expression did not exhibit hypoglycemia, nor did they show reduced hepatic glycogen in adult. Expression of liver glycogen synthase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase remained at normal levels. However, these mice exhibited impaired glucose tolerance due in part to reduced expression of hepatic glucokinase, and hyperammonemia from reduced expression of hepatic carbamoyl phosphate synthase-I. These mice also had reduced serum cholesterol and steatotic livers that was exacerbated with aging. This phenotype could be explained by increased expression of hepatic lipoprotein lipase and reduced expression of microsomal triglyceride transfer protein, apolipoproteins B100, and A-IV. These data demonstrate that hepatic C/EBPalpha is critical for ammonia detoxification and glucose and lipid homeostasis in adult mice.

Northern Blot Analysis-Northern blot analysis was carried out as described previously (17). All probes were amplified from a mouse cDNA library using gene-specific primers and cloned into a pCR II vector (Invitrogen). Sequences were verified using a CEQ 2000 Dye Terminator cycle sequencing kit (Beckman Coulter, Fullerton, CA) with a CEQ 2000XL DNA Analysis system (Beckman Coulter).
Blood and Serum Analysis-Blood from the tail vein was analyzed for glucose levels using an automatic glucometer (Bayer, Elkhart, IN). Mice were anesthetized with 2.5% avertin and decapitated, and the trunk blood was collected in a serum separator tube (BD Biosciences, Franklin Lakes, NJ). The serum was separated by centrifugation at 7000 ϫ g for 5 min and stored at Ϫ80°C prior to analysis for ammonia (Wako, Osaka, Japan) and insulin (RIA insulin kit, Linco Research, St. Charles, MO). Concentrations of total cholesterol and triglyceride (Sigma) as well as free cholesterol and phospholipid (Wako, Osaka, Japan) were measured from 12-l aliquots of serum using commercial kits and the Hitachi 911 automated chemistry analyzer (Roche Molecular Biochemicals, Indianapolis, IN).
Determination of Liver Glycogen Content-Liver glycogen content was determined using a slight modification of a previously described procedure (18). Liver pieces (50 mg) were digested in 250 l of 1 N KOH at 95°C for 30 min and neutralized with 250 l of 1 N HCl. 250 l of 0.3 M sodium acetate (pH 4.8) containing 10 mg/ml amyloglucosidase (Roche Molecular Biochemicals) was added into the 100 l of the homogenate and incubated at 30°C for 2 h. The resulting homogenate was centrifuged, and the produced glucose content was colorimetrically measured using a Glucose Trinder kit (Sigma).
Determination of Liver Lipid Content-Liver triglyceride, total cholesterol, and free cholesterol contents were determined using a modification of previously described procedures (19,20). Briefly, liver pieces (100 mg) were homogenized in 2.1 ml of chloroform/methanol (2:1) and incubated at 37°C for 40 min. Following incubation, 0.4 ml of water was added, and the mixture was stored at 4°C overnight. The lower phase was collected, and 0.83 volume of a 47:3:48 mixture of water/chloroform/ methanol was added and stored at 4°C overnight. The organic phase was collected and dried under nitrogen gas, followed by heating at 90°C for 10 min. The samples were dissolved in 250 l of 2-propanol and centrifuged at 10,000 ϫ g for 3 min. The resulting supernatant was assayed for total cholesterol and triglyceride (Sigma).
Glucose Tolerance Tests-Mice were fasted overnight for 14 h followed by intraperitoneal glucose injection (2 g/kg body weight). Blood obtained from the tail vein at 0, 15, 30, 60, and 120 min after the injection was directly measured for glucose levels using an automatic glucometer. Similarly, blood obtained from the retro-orbital vein was collected, and the plasma was separated and stored at Ϫ80°C prior to analysis for insulin (Linco Research).
Fast Protein Liquid Chromatography Analysis-Plasma lipoproteins from pooled plasma samples (60 l; n ϭ 6) were analyzed by gel filtration on two Superose 6HR columns in series (fast-protein liquid chromatography (FPLC); Amersham Biosciences) at 0.3 ml/min in phosphate-buffered saline containing 0.1 mM EDTA and 0.02% sodium azide (21). Mouse apolipoproteins A-I, A-II, B, and E were identified by Western blotting of serum and FPLC fractions using a mixture of polyclonal rabbit anti-mouse immunoglobulin G antisera raised against purified apolipoproteins (Biodesign, Saco, ME).
Western Blot Analysis-Frozen livers, white adipose tissue, and brown adipose tissue were crushed on dry ice using a mortar and pestle, washed with cold phosphate-buffered saline, homogenized in a lysis buffer (9 M urea, 2% Triton X-100, 70 mM dithiothreitol, 1 g/ml aprotinin, 1 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride), and allowed to sit on ice for 30 min. The homogenate was centrifuged at 12,000 ϫ g for 30 min at 4°C, and the supernatants were used as whole cell lysates. Total protein (100 g), determined by the Bio-Rad assay, was diluted with 3ϫ Laemmli sample buffer, incubated at 65°C for 15 min, fractionated by 10% SDS-polyacrylamide gel electrophoresis, and blotted onto a polyvinylidene difluoride membrane (Amersham Biosciences). The membrane was incubated for 1 h with phosphate-buffered saline containing 0.1% Tween 20, 5% dry milk, 6% glycine, and 1% fetal bovine serum, and then for 1 h with a 1:1,000 -5,000 dilution of a primary antibody against C/EBP␣ (Santa Cruz Biotechnology, Santa Cruz, CA) and actin (Santa Cruz Biotechnology). After washing, the membrane was incubated for 1 h with 1:10,000-diluted horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), and the reaction product was visualized using an enhanced chemiluminescent system (ECL plus, Amersham Biosciences).
Histological and Immunohistochemical Analysis-Livers from 2-week-old and 2-, 6-, and 12-month-old liver-specific C/EBP␣-null and control mice were fixed in 10% neutral buffered formalin and embedded in paraffin, and sections cut at a thickness of 3 m were stained with hematoxylin and eosin (H&E). Immunohistochemical analysis was performed using antibody specifically against C/EBP␣ (Santa Cruz Biotechnology).

RESULTS
Generation of Liver-specific C/EBP␣-null Mice-A liver-specific C/EBP␣-null mouse line was generated to avoid the problems, including hepatotoxicity and transient recombination caused by recombinant adenovirus carrying the Cre gene (14). The albumin-Cre transgene (AlbCre) was introduced into mice carrying two C/EBP␣-floxed (flanked by loxP) alleles (C/ EBP␣ flox/flox ). The mice that were F 1 (C/EBP␣ flox/wt ; AlbCre ϩ ) were interbred with C/EBP␣ flox/flox littermates lacking AlbCre. All mice were genotyped by PCR, and C/EBP␣ flox/flox ; AlbCre ϩ (designated KO) and C/EBP␣ flox/flox ; AlbCre Ϫ (FLOX) mice were used for the following experiments. To assess loss of C/EBP␣ gene expression, Northern blot analysis was performed using total liver RNA from 4-and 7-week-old control (FLOX) and KO mice (Fig. 1A, the upper panel). Expression of C/EBP␣ mRNA was lost in the livers of KO mice, but expression in FLOX mice was unchanged. To confirm whether deletion of C/EBP␣ is a liver-specific event, Northern blot analysis was performed using white adipose tissue and brown adipose tissue where C/EBP␣ is highly expressed (4). There was no FIG. 1. Generation of liver-specific C/EBP␣-null mice. A, Northern blots of total RNA from liver, white adipose tissue (WAT), and brown adipose tissue (BAT). Tissue RNA was isolated from 4-and 7-week-old mice, and 10 g was separated on a 1% agarose gel, transferred to a nylon membrane, and hybridized with the 32 P-labeled cDNA probes for C/EBP␣ and GAPDH. B, Western blots of total proteins. Liver total protein (100 g) was separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Rabbit (C/EBP␣) or goat (actin) polyclonal antibodies were used to assess protein expression.
significant difference in C/EBP␣ mRNA between these tissues in FLOX and KO mice (Fig. 1A, the middle and lower panel). Next, Western blot analysis was performed using liver total protein from 4-and 7-week-old KO and FLOX mice (Fig. 1B). Hepatic C/EBP␣ proteins (42 and 30 kDa) were detected in FLOX mice, but almost no expression was observed in KO mice, indicating that expression of C/EBP␣ mRNA and protein were extinguished in KO mice in a liver-specific manner.
Phenotypes of 2-Week-Old and 2-Month-Old Liver-specific C/EBP␣-null Mice-To determine the effects of C/EBP␣ deletion in adult liver, body weights, liver weights, and histology were investigated. In 2-month-old mice, there was no significant difference in body weights between KO and FLOX mice, but the livers in KO mice were enlarged relative to those in FLOX mice (Table I). Furthermore, no significant histological difference in livers of KO mice was observed as compared with FLOX mice in both 2-week-old and 2-month-old mice (Fig. 2, A-D). Immunohistochemistry with C/EBP␣ antibody confirmed that C/EBP␣ was expressed in the nuclei of FLOX mice but not in KO mice (Fig. 2, E and F). Whole body C/EBP␣-null mice showed a marked reduction in liver glycogen as revealed by periodic acid-Schiff staining (9), but liver-specific C/EBP␣-null mice did not exhibit reduced hepatic glycogen levels (FLOX, 9.8 Ϯ 2.0 mg/g of liver; KO, 10.8 Ϯ 2.4 mg/g of liver). There was no significant difference in fat-positive cells determined with oil red O staining (data not shown).
Liver-specific 2-Month-Old C/EBP␣-null Mice Exhibit Normal Glucose and Serum Lipid Levels-To further characterize liver-specific C/EBP␣-null mice at the age of 2 months, serum chemistry was analyzed. The levels of triglyceride, total, free, and esterified cholesterol, and phospholipids in KO mice were indistinguishable from those in FLOX mice, but the levels of free fatty acid were significantly reduced in KO mice (Table I). Furthermore, there was no significant difference in glucose levels between KO and FLOX mice.

Altered Expression of Genes Involved in Glucose Metabolism, Except Glucokinase, Was Not Observed in 2-Week-Old and 2-Month-Old Liver-specific C/EBP␣-null Mice-Because
C/EBP␣ is known to regulate genes involved in glucose metabolism such as phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), and liver glycogen synthase (Gys2) (9) at birth, Northern blot analysis was performed to determine whether expression of these genes is reduced in liver-specific C/EBP␣-null mice. It was also reported that the PEPCK and Gys2 expression was reduced in the adult livers of conditional C/EBP␣-null mice generated using the adenovirus-Cre gene delivery system (14), but no significant differences of PEPCK and Gys2 were observed in the livers of both 2-weekold and 2-month-old KO mice (Fig. 3, A and B). Expression of G6Pase and glucose-6-phosphatase transporter (G6Pase-T)   was increased in 2-week-old KO mice, but unchanged in 2-month-old KO mice as compared with FLOX mice (Fig. 3, A  and B). Expression of other enzymes involved in glucose homeostasis such as glucose transporter 2 (GLUT2), liver pyruvate kinase, pyruvate carboxylase (PC), fructose-1,6-bisphosphatase, and glycogen phosphorylase was also unchanged (Fig.  3, A and B). Interestingly, expression of glucokinase (GK) was reduced in both 2-week-old and 2-month-old KO mice compared with FLOX mice (Fig. 3, A and B). Expression of C/EBP␤, a closely related member of the C/EBP family and predominantly expressed in liver (22), was unchanged (Fig. 3, A and B). Liver-specific C/EBP␣-null Mice Have Hyperammonemia due to Down-regulation of Carbamoyl Phosphate Synthase-I-The urea cycle is composed of five enzymes; carbamoyl phosphate synthase-I (CPS-I), ornithine transcarbamylase (OTC), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), and arginase-I. Hyperammonemia was caused by reduced expression of four genes except OTC in whole body C/EBP␣-null mice (10). To investigate whether the same results are obtained in adult liver-specific C/EBP␣-null mice, serum ammonia levels were measured and Northern blot analysis of genes involved in the urea cycle was performed. The ammonia levels in both 2-week-old and 2-month-old KO mice were significantly higher as compared with FLOX mice (Fig. 4A). Northern blot analysis revealed that expression of CPS-I was significantly decreased in both KO mice, but expression of the other four genes was unchanged (Fig. 4B), indicating that reduced expression of CPS-I caused hyperammonemia in liver-specific C/EBP␣-null mice. Aged Liver-specific C/EBP␣-null Mice Exhibit Steatotic Livers and Reduced Serum Cholesterol-To investigate whether older liver-specific C/EBP␣-null mice exhibit a phenotype, further analysis was performed using 6-and 12-month-old mice. In 6-month-old mice, body and liver weights in KO mice were significantly increased as compared with those in FLOX mice (Table I). In 12-month-old mice, body and liver weights in KO mice appeared to be increased as compared with those in FLOX mice, but only the liver weight in KO mice was significantly increased (Table I). These KO mice exhibited steatotic livers that were not observed in 2-week-old and 2-month-old KO mice (Fig. 2, G-J). Lipid accumulation in these KO mice was confirmed by oil red O staining (data not shown). Based on H&E staining, 83% (n ϭ 20/24) and 80% (n ϭ 8/10) of livers of KO mice were steatotic in 6-and 12-month-old mice, respectively. However, control FLOX mice did not exhibit steatotic livers (6-month-old; n ϭ 0/13, 12-month-old; n ϭ 0/6). To quantitate hepatic lipid contents in these mice, hepatic triglyceride and cholesterol levels were determined (Fig. 5). There was no difference of both levels between 2-month-old FLOX and KO mice. However, their levels were significantly increased in 6-month and 12-month-old KO mice as compared with those FLOX mice, indicating that these results are consistent with the histological results shown in Fig. 2. Serum triglyceride levels in these aged mice were unchanged and similar to 2-month-old mice, but serum total and esterified cholesterol and phospholipid levels in both 6-and 12-month-old KO mice were dramatically reduced as compared with FLOX mice (Table I). As mentioned above, this reduction was not observed in 2-month-old KO mice (Table I).
To further characterize the reduction of serum lipids, serum from 6-month-old FLOX and KO mice was subjected to FPLC analysis. The levels of very low density lipoprotein (VLDL)-, LDL-, and HDL-cholesterol were decreased in KO mice compared with FLOX mice (Fig. 6). Western blot analysis of lipoproteins indicated that KO mice exhibited reduced apoB100 in LDL fraction as well as decreased apoA-II in HDL fraction, whereas apoA-I, E, and B48 were not significantly affected as compared with FLOX mice (Fig. 6, inset).
Liver-specific C/EBP␣-null Mice Exhibit Impaired Glucose Tolerance-To further characterize the influence of disruption of hepatic C/EBP␣ in aged mice, blood glucose levels were investigated. The levels were unchanged in 6-month-old mice, but significantly increased in 12-month-old KO mice (Table I). Hepatic glycogen content in 6-month-old mice was not significantly different between FLOX and KO mice (8.9 Ϯ 3.6 and 6.5 Ϯ 1.7 mg/g liver, respectively), and similar results were found in 2-month-old mice. A glucose tolerance test was performed to investigate whether KO mice exhibit impaired glucose tolerance. As shown in Fig. 7 (A and B), both 2-month and 6-month-old KO mice significantly exhibited impaired glucose tolerance as compared with FLOX mice. However, there was no significant difference in the insulin levels in both 2-month and 6-month-old KO mice as compared with FLOX mice during the glucose tolerance test (Fig. 7, C and D). Furthermore, the basal levels of serum insulin in 2-, 6-, and 12-month old KO mice were unchanged from their FLOX counterparts (Table I).

Reduction of Expression of Hepatic Genes Involved in Lipid and Glucose Metabolism in Aged Liver-specific C/EBP␣-null
Mice-To determine the mechanism of accounting for the phenotypes of the aged KO mice, Northern blot analysis was performed using the livers of 6-month-old KO mice (Fig. 8). The expression of genes encoding apoB, A-IV, and C-III and serum amyloid A4 protein was reduced in KO mice as compared with that in FLOX mice, but apoA-II was unaffected, although serum apoA-II was slightly decreased in HDL-cholesterol fraction as shown in Fig. 6 (Fig. 8A). Furthermore, the expression of microsomal triglyceride transfer protein (MTP) was slightly reduced in KO mice (Fig. 8B). Conversely, the expression of lipoprotein lipase (LPL) that is normally silent in liver was markedly increased in KO mice (Fig. 8B). Peroxisome proliferator-activated receptor ␥ (PPAR␥) mRNA showed a slight increase, but the level was still very low, suggesting that the effect of PPAR␥ would be minimal (Fig. 8B). The expression of other genes, including liver X receptor ␣ (LXR␣), PPAR␣, scavenger receptor class B type I, LDL receptor, fatty acid transporter, hepatic lipase, liver-fatty acid-binding protein, and sterol response element binding protein-1c, was unchanged (Fig.  8B). The expression of 11␤-hydroxysteroid dehydrogenase type 1 (11␤-HSD1), a direct target for C/EBP␣ exhibiting dramatically reduced expression in whole body C/EBP␣-null mice (23), was also decreased in KO mice (Fig. 8B).
The expression of genes encoding proteins involved in glucose metabolism was then examined. Expression of GK was reduced in KO mice, but the expression of GLUT2, liver pyruvate kinase, PC, PEPCK, glucose-6-phosphatase transporter, Gys2, and glycogen phosphorylase was unchanged (Fig. 8C); these results were similar to those in 2-week-old and 2-monthold KO mice as shown in Fig. 3.
Expression of CPS-I was markedly reduced in 6-month-old KO mice (Fig. 8D), consistent with the results in 2-week-old and 2-month-old KO mice as shown in Fig. 4. Expression of ASL and arginase-I was slightly reduced in KO mice, but the expression of OTC and ASS was unchanged (Fig. 8C). Similarly, serum ammonia levels in these aged KO mice were significantly increased as well as those in 2-week-old and 2-month-old KO mice (data not shown). Thus, aged KO mice have altered expression of genes involved in lipid, glucose, and ammonia metabolism pathways, consistent with the histological and serological alternation. DISCUSSION C/EBP␣ is known to regulate the expression of liver-specific genes as revealed by in vitro and in vivo studies using C/EBP␣null mice. In this study, it was determined that C/EBP␣ is involved in glucose and lipid metabolism in adult liver.
C/EBP␣-null mice cannot survive beyond a day after birth due to impaired energy homeostasis, including decreased glycogen synthesis and gluconeogenesis in liver and adipose tissues (9). However, no significant difference in the expression of genes involved in glucose metabolism pathways such as Gys2 and PEPCK was observed in liver-specific C/EBP␣-null mice at any age, and no reduction of blood glucose levels and hepatic glycogen was observed. Thus, the function of C/EBP␣ appears to differ between newborn and adult mice or deletion of C/EBP␣ in other tissues, mainly in adipose tissues, might influence the impaired glucose metabolism in liver. On the other hand, liver-specific C/EBP␣-null mice produced using FIG. 7. Intraperitoneal glucose tolerance test in liver-specific C/EBP␣-null and control mice. Blood glucose and plasma insulin levels from 2-month-old (A and C) and 6-month-old (B and D) mice were determined at 0, 15, 30, 60, and 120 min after intraperitoneal (IP) glucose injection. Data are means Ϯ S.E. (FLOX, n ϭ 15 (A), 12 (B), 6 (C), and 6 (D); KO, n ϭ 14 (A), 20 (B), 6 (C), and 6 (D)). Significant differences compared with FLOX mice: *, p Ͻ 0.05; **, p Ͻ 0.01. acute adenovirus-Cre delivery system to the liver exhibited reduced expression of PEPCK and Gys2 in adult, and these phenotypes are very similar to those observed in newborn whole body C/EBP␣-null mice (14). Thus, the phenotypes of liver-specific C/EBP␣-null mice produced using albumin-cre transgenic mice in this study differ from the liver-specific C/EBP␣-null mice produced using the adenovirus-Cre delivery system. The reason for this difference is not clear. However, serum alanine aminotransferase and aspartate aminotransferase levels were markedly increased, hypoglycemia was noted, and hepatic cell division was stimulated by adenovirus infusion in both C/EBP␣-null and wild-type mice in adenovirus-Cre delivery system (14). The phenotypes of these C/EBP␣null mice might have been influenced by the effect of adenovirus infusion in combination with C/EBP␣ disruption. In addition, the effect of adenovirus infusion is transient, where disruption of hepatic C/EBP␣ expression using albumin-Cre transgenic mice was already detected in 2-week-old mice and maintained in 12-month-old mice. It cannot be excluded that the difference in disrupted period of C/EBP␣ expression might cause the phenotypic differences between these two lines. Expression of C/EBP␤ in liver-specific C/EBP␣-null mice was unchanged at any age, but DNA binding activity of C/EBP␤ or other transcription factors might increase to compensate for the loss of function of hepatic C/EBP␣ only under the condition of continuous absence of C/EBP␣.
Liver-specific C/EBP␣-null mice exhibited impaired glucose tolerance and blood glucose levels at 12 months of age. Reduced expression of GK was common to young and aged KO mice. Heterozygous mutations of the GK gene is known to cause maturity-onset diabetes of the young in humans (24,25) and GKdeficient mice die with severe hyperglycemia (26). Furthermore, ␤ cell-specific GK-null mice had severe diabetes, but liver-specific GK-null mice were only mildly hyperglycemic (27). However, because the expression of hepatic GK was unaffected in some KO mice as shown in Fig. 3, it is still unknown whether the impaired glucose tolerance in KO mice was primarily the result of de-creased expression of GK. Thus, reduction of GK mRNA might partly cause impaired glucose tolerance in liver-specific C/EBP␣null mice. Because the expression pattern of C/EBP␣ mRNA by in situ hybridization is virtually identical to that of GK mRNA in adult liver (28), C/EBP␣ might directly regulate the expression of GK via C/EBP␣ binding site(s) in its promoter region.
Further reduction in expression of genes involved in glucose metabolism was not observed in the KO mice at any age. Because C/EBP␣ and ␤ can transactivate the promoter activity of GLUT2 (29), C/EBP␤ might compensate for the loss of function of C/EBP␣ in the regulation of PEPCK, Gys2, and GLUT2.
It is known that whole body C/EBP␣-null mice exhibit hyperammonemia due to down-regulation of the expression of hepatic CPS-I, ASS, ASL, and arginase-I (10). A similar phenotype was observed in liver-specific C/EBP␣-null mice; expression of CPS-I was markedly reduced, and arginase-I was slightly reduced in these mice. The expression patterns of genes involved in glucose metabolism was different from whole body C/EBP␣-null mice, but the urea cycle phenotype was conserved between both C/EBP␣-null mouse models. Thus, CPS-I appears to be strongly regulated by C/EBP␣ at any age. Because the expression of arginase-I was diminished in whole body C/EBP␣-null mice (10), arginase-I might be partly regulated by C/EBP␣ or rescued by C/EBP␤. C/EBP binding sites have been identified in the promoter regions of CPS-I (30) and arginase-I (31,32).
The most significant phenotype in these mice was steatotic livers and decreased serum cholesterol levels only in old mice, but not in 2-month-old mice. As shown in Fig. 7, the expression of MTP and apoB was reduced only in 6-month-old KO mice, but unchanged in 2-month-old mice (data not shown). Further evidence was provided by the fact that there was only reduced apoB100 protein derived mostly from liver, but not apoB48 protein derived mostly from intestine in serum of 6-month-old KO mice. Liver-specific MTP-null mice have a large reduction in VLDL/LDL and HDL-cholesterol (33, 34) and reduction of MTP and apoB secretion (35). Furthermore, MTP inhibitors FIG. 8. Northern blot analysis of 6-month-old liver-specific C/EBP␣-null and control mice. Pooled total liver RNA (n ϭ 7 for each genotype) was isolated, and 10 g was separated on a 1% agarose gel, transferred to a nylon membrane, and hybridized with the indicated 32 P-labeled cDNA probes. F, FLOX; K, KO. cause steatotic livers and reduced lipoprotein production (36,37), indicating that down-regulation of these genes might cause age-dependent steatotic livers and reduced serum cholesterol levels in KO mice. However, it is still unclear why these genes are down-regulated only in 6-month-old mice and whether C/EBP␣ directly regulates the expression of these genes.
It is noteworthy that apoA-IV-null mice have decreased levels of plasma triglycerides and VLDL-and HDL-cholesterol associated with hepatic expression of apoC-III (38). These results are very similar to those in liver-specific C/EBP␣-null mice, except that no decreased levels of triglycerides were observed. The expressions of apoA-IV and C-III were also decreased in 2-month-old mice (data not shown), and this lower level remained in 6-month-old KO mice, indicating that it might take a longer time to manifest the significant decrease in serum cholesterol in combination with decreased expression of MTP and apoB. Because the expression of apoA-IV is higher in small intestine than liver in the rat (39), intestinal apoA-IV may partly compensate for the decline in its synthesis in livers of KO mice.
Interestingly, LPL is not normally expressed in liver, but its expression was markedly increased in the livers of KO mice. Liver-specific LPL transgenic mice, which were produced on an LPL knockout background, have excess lipid droplets in the liver (40). Similarly, other liver-specific LPL transgenic mice, which normally express LPL in other tissues, also revealed an increased number and size of lipid droplets in their livers (41). The function of LPL in extrahepatic tissues is to uptake fatty acid from triglyceride-rich VLDL and chylomicrons (42). However, it was reported that hepatic LPL can mediate the selective uptake of HDL-cholesterol (43), consistent with the results that the levels of HDL-cholesterol were significantly reduced in liver-specific C/EBP␣-null mice. Thus, these results indicate that the increased expression of hepatic LPL might explain why liver-specific C/EBP␣-null mice had steatotic livers. Furthermore, mice overexpressing hepatic LPL also had liverspecific insulin resistance due to defects in insulin signaling in liver (41). Because expression of hepatic LPL was also increased in 2-month-old KO mice (data not shown), elevated expression of hepatic LPL in KO mice might be the main reason for the impaired glucose tolerance, and the combination with decreased expression of GK might cause this phenotype.
It still remains unknown why the expression of hepatic LPL is increased in liver-specific C/EBP␣-null mice. Hepatic LPL is a direct target of LXR␣ via LXR response element in the mouse promoter region (44). Expression of LXR␣ was unchanged, but endogenous oxysterols that are ligands for LXR␣ might accumulate in these mice due to the steatotic livers. Furthermore, ligands for PPAR␣ and PPAR␥ can induce LPL expression (45). In addition, expression of PPAR␥ that is increased in steatotic livers (46) was slightly elevated in KO mice, indicating that induced expression of LPL might be partly due to transactivation of these PPARs by endogenous ligands.
Liver-specific C/EBP␣ transgenic mice, that were generated on the C/EBP␣ knockout background, showed steatotic livers at 7 days of age (47). This result differs from our studies. Because liver-specific C/EBP␣-null mice show steatotic livers only in aged mice and it was reported that aging switches the C/EBP␣ pathway of growth arrest (48), a function of C/EBP␣ that might differ between young and old mice. Furthermore, this discrepancy could be explained by the effect of C/EBP␣ expressed in adipose tissues.
In conclusion, liver-specific C/EBP␣-null mice revealed a role for this transcription factor in impaired glucose tolerance and steatotic livers that was only observed upon aging. These phenotypes could be partly caused by decreased expression of GK and increased expression of LPL in liver. Furthermore, these mice might be useful models to understand the hepatic function of C/EBP␣ and establish a possible search for novel therapeutic targets.