Hepatic Fatty Acid Synthesis Is Suppressed in Mice With Fatty Livers Due to Targeted Apolipoprotein B38.9 Mutation
Humans and genetically engineered mice with hypobetalipoproteinemia due to truncation-producing mutations of the apolipoprotein B (apoB) gene frequently have fatty livers, because the apoB defect impairs the capacity of livers to export triglycerides (TGs). We assessed the adaptation of hepatic lipid metabolism in our apoB-38.9-bearing mice. Hepatic TG contents were 2- and 4-fold higher in heterozygous and homozygous mice, respectively, compared with wild-type mice. Respective in vivo hepatic fatty acid synthetic rates were reduced to 40% and 15% of the wild-type rate. Hepatic mRNAs for sterol regulatory element-binding protein (SREBP)-1c, fatty acid synthase (FAS), and stearoyl coenzyme A desaturase-1 were coordinately decreased. FAS and SREBP-1c mRNA levels were strongly and positively correlated with each other and inversely correlated with hepatic TGs, suggesting that impaired TG export is a potent inhibitor of fatty acid synthesis. In contrast, levels of plasma β-hydroxybutyrate and of hepatic carnitine palmitoyl transferase and peroxisome proliferator-activated receptor-α mRNAs were not altered, implying that β-oxidation was not affected. Fasting followed by refeeding increased hepatic fatty acid synthesis 56-fold over fasting in normal and heterozygous mice but only 24-fold in homozygous mice. Parallel changes occurred in FAS and SREBP-1c mRNAs. Thus, impairment of very low density lipoprotein export downregulates hepatic fatty acid synthesis, but the adaptation is incomplete, resulting in fatty livers. The signals mediating suppression of FAS and SREBP-1c levels remain to be identified.
- apolipoprotein B
- fatty acid synthesis
- fatty liver
- familial hypobetalipoproteinemia
- sterol regulatory element-binding protein 1c
In general, the accumulation of triglycerides in the liver (termed fatty liver) is due to an imbalance between the availability of hepatic triglycerides for export and the exporting capacity of the liver via VLDLs. In obesity, in diabetes mellitus, and during the feeding of diets high in carbohydrates,1,2⇓ the amounts of hepatic triglycerides available for export are increased because of the enhanced rates of uptake of unesterified fatty acids from plasma and/or because of increased de novo fatty acid synthesis. The fatty acids are then incorporated into triglycerides. The combined processes of fatty acid and triglyceride synthesis have been called lipogenesis. In response to increased needs for triglyceride export, the VLDL-exporting system adapts by packing more triglyceride molecules into each VLDL particle and also by producing increased numbers of particles.3,4⇓ This adaptation can result in hypertriglyceridemia.5 Still, despite the increased export, triglycerides accumulate in the livers of diabetic and obese subjects,6 suggesting that the VLDL export system contains a reserve capacity that is insufficient to handle the increased need for triglyceride export.
Recent reports document the presence of fatty livers in humans with familial hypobetalipoproteinemia (FHBL).7–9⇓⇓ However, in contrast to diabetes and obesity, this subset of fatty livers occurs not as a result of increased needs to export triglycerides but as a result of the reduced capacity of the hepatic VLDL-exporting system. ApoB is a crucial structural component of VLDLs. A variety of premature stop codon-producing mutations of the apoB gene (Apob) specifying abnormal apoB truncations are present in humans with FHBL.10,11⇓ In such cases, normal apoB-100-containing lipoproteins and apoB truncation-containing particles circulate. However, hepatic production rates of both apoB forms are reduced.12–14⇓⇓ Furthermore, the triglyceride-transporting capacities of short truncated forms of apoB are impaired.15,16⇓ Thus, the overall capacity of the VLDL export system is reduced.
We wondered whether the impairment of VLDL transport would have any consequences for hepatic lipid metabolism. Because it is not possible to examine intrahepatic metabolism in humans, we used our apoB-38.9 mouse model.16 This mouse, produced by using the gene-targeted homologous recombination in embryonic stem cells and the Cre-loxp system,16 harbors a single base-pair deletion in the coding region of Apob, but there are no retained foreign targeting construct sequences in its genome. The livers of heterozygous and homozygous mice selectively accumulate triglycerides. We used these mice to study hepatic adaptive mechanisms. We measured hepatic fatty acid synthetic rates in vivo and hepatic mRNA levels of key lipogenic enzymes, including acetyl coenzyme A carboxylase-1 (ACC-1),17 fatty acid synthase (FAS),18 stearoyl coenzyme A desaturase-1 (SCD-1),19 glycerol-3-phosphate acyltransferase (GPAT),20 and acyl coenzyme A:diacylglycerol acyltransferase (DGAT),21 and the hepatic mRNA level of the microsomal triglyceride hydrolase (TGH),22 an enzyme that plays an important role in the mobilization of cytosolic triglycerides in the liver.22 We also quantified the mRNA levels of the sterol regulatory element-binding protein (SREBP)-1c23 (also known as adipocyte determination and differentiation-dependent factor 1 [ADD1]24), which is a key mediator in the nutritional and hormonal control of lipogenesis. It regulates the expression of lipogenic genes.25,26⇓ Fatty acid oxidation was evaluated by quantifying plasma β-hydroxybutyrate levels and hepatic mRNA levels of carnitine palmitoyl transferase (CPT-1), the key enzyme controlling the rate of fatty acid oxidation,27 and its transcriptional activator, peroxisome proliferator-activated receptor-α (PPARα).28 Finally, we determined the effect of fasting and refeeding on fatty acid synthesis and on the expression of the cognate genes.
Heterozygous mice (Apob+/38.9) and homozygous mice (Apob38.9/38.9)16 have a mixed genetic background of 50% 129/SVJ and 50% C57BL/6. Wild-type, heterozygous, and homozygous mice of various ages and both sexes were fed a standard chow diet and housed in a pathogen-free barrier facility with a 12-hour light and 12-hour dark cycle. For the fasting and refeeding experiments, 3 mice of each genotype were allocated to each treatment: (1) ad libitum feeding, (2) 24-hour fasting with water available, or (3) 24-hour fasting followed by 12 hours of refeeding with a fat-free high-carbohydrate diet (Purina). Experiments were timed so that mice were euthanized between 10:00 am and 12:00 pm.
In Vivo Rates of Fatty Acid Synthesis
Hepatic rates of de novo fatty acid synthesis were determined on the basis of the incorporation rates of [1-14C]acetate into fatty acids during a 1-hour period. Mice (≈30 g) were injected with 25 μCi of [1-14C]acetate (0.2 μCi/μL) via the orbital vein. One hour after injection, the animals were euthanized, and the livers were excised immediately. Slices (≈250 mg) of the liver were digested in a potassium hydroxide solution (30%) at 95°C for 30 minutes, followed by saponification in 30% KOH/50% alcohol at 95°C for 3 hours.29 After extraction of the nonsaponifiable lipids with petroleum ether, the sample solution containing the saponified lipids was acidified with sulfuric acid, and fatty acids were extracted with petroleum ether as described.29 The radioactivity in total fatty acids was determined by scintillation counting. The fatty acid synthesis rates are reported as disintegrations per minute per hour per milligram liver.
All cDNA probes except for mouse β-actin, which was purchased from Sigma Chemical Co, were generated by reverse transcriptase (RT)-polymerase chain reaction (PCR) amplification of mouse liver RNA by using a One-tube RT-PCR kit (Roche Biochemicals). Online Table I (please see http://atvb.ahajournals.org) lists the RT-PCR primers for all gene transcripts examined except for FAS,30 SREBP-1,30 and apoB.16 The resulting PCR products were purified and subcloned into a TA-cloning PCRII vector (Invitrogen), and their identity was verified by nucleotide sequencing on an ABI PRISM 377 DNA sequencer (Applied Biosystems). For Northern blot analysis, the cDNA inserts were excised from the vector and labeled with [α-32P]dCTP by using a random prime-labeling kit (Promega). The SREBP-1 cDNA probe was amplified from a region that is common for SREBP-1c and SREBP-1a isoforms and was expected to hybridize to both transcripts.30 However, because SREBP-1c mRNA is 9-fold more abundant than SREBP-1a mRNA in the mouse liver,31 the hybridization signals obtained with this probe mainly reflect the SREBP-1c mRNA abundance.
Total RNA was isolated from mouse livers by using TRIAzol (Life Technologies). Poly(A)+ RNA was purified by using the Oligotex mRNA Purification System (Qiagen). The mRNA steady-state levels of the genes tested were determined by Northern blotting on pooled RNA samples from each genotype in each experimental treatment. The pooled RNA [30 μg total RNA or the poly(A)+ RNA purified from 80 μg of total RNA] was separated by electrophoresis on 1.0% agarose-formaldehyde gels,16 transferred, and immobilized onto GeneScreen Plus nylon membranes (NEN Life Science Products). Northern hybridization was carried out by using Rapid-hyb buffer (Amersham Pharmacia Biotech) at 65°C for 2 hours after prehybridization for 1 hour at 65°C. After hybridization, the blots were subjected to washing in 2× SSC/0.1% SDS at room temperature, followed by high-stringent washing in 0.2× SSC/0.1% SDS at 67°C. Relative intensities of the hybridization signals were quantified by using the GS-525 PhosphorImager System (Bio-Rad). The blots were also exposed to Kodak X-Omat films to obtain images of the signal. Thereafter, blots were stripped and probed with the 32P-labeled mouse β-actin cDNA probe. The relative mRNA levels were expressed as ratios of the phospho-stimulated luminescence intensity of the target mRNA to that of β-actin.
We also measured the hepatic SREBP-1c and SREBP-1a and FAS mRNA levels of individual animals in the ad libitum feeding experiment by a ribonuclease protection assay (RPA). For the RPA, the cDNA fragments were amplified by RT-PCR (One-tube RT-PCR kit, Roche Biochemicals) with the use of mouse liver total RNA as a template and primers as follows: mouse FAS (132-bp), 5′ primer, 5′-AGGGGTCGACCTGGTCCTCA-3′, 3′ primer, 5′-GCCATGC- CCAGAGGGTGGTT-3′32; mouse SREBP-1 (264-bp), 5′-GCCGGCGCCATGGAACGAGCTGGCC-3′, 5′-CAGGAAGGCT- TCCAGAGAGGAGGC-3′33; and mouse β-actin (100-bp), 5′-TCCCTGGAGAAGAGCTATGA-3′, 5′-CGATAAAGGAAGGCT- GGAA-3′.34 All amplified cDNA fragments were subcloned into a TA-cloning PCRII vector (Invitrogen), and their identity was verified by nucleotide sequencing on an ABI PRISM 377 DNA sequencer (Applied Biosystems). [32P]UTP-labeled FAS, SREBP-1, and β-actin riboprobes were generated by using a MAXIscript in vitro transcription kit (Ambion). Total RNA (10 μg) was hybridized with the labeled FAS, SREBP-1, and β-actin riboprobes with the use of the RPA III kit (Ambion) according to the manufacturer’s protocol (Ambion). The protected mRNA fragments were separated on a 5% acrylamide/8 mol/L urea gel and quantified with a PhosphorImager (Bio-Rad).
Commercial kits were used to measure plasma concentrations of glucose (Sigma), free fatty acids (FFAs), cholesterol, and triglycerides (Wako Chemicals). Insulin concentration in plasma was analyzed by radioimmunoassay (Linco Inc). Cellular protein contents were determined as described.16
Data are expressed as mean±SE. ANOVA followed by the Tukey procedure, which was performed for comparison between treatments. The Pearson correlation and stepwise linear regression analyses were performed on results across genotypes by using SAS Proc CORR and Proc REG (SAS Institute). The normality test was performed by using SAS Proc UNIVARIATE (SAS/STAT version 8, 2001, SAS Institute). In the analysis, genotypes were coded as follows: wild-type mice=1, heterozygous mice=2, and homozygous mice=3.
We used a total of 50 mice in the ad libitum feeding experiments (Table 1) and 27 mice in the fasting and fasting/refeeding studies. The numbers of animals used for various determinations and experiments are indicated in the legends of the tables and figures. Several litters were used. Whenever possible, littermates of the various genotypes were compared with each other. Because there were no significant differences by sex in the parameters presented in the present study, the data were pooled across sexes. Significant differences by genotype were noted for plasma cholesterol and triglycerides (Table 1), as expected.16 Lower levels of FFAs were seen in homozygous mice. Liver/body weight ratios of the homozygous mice were elevated (Table 1). Plasma insulin and glucose levels were similar among the Apob+/+, Apob+/38.9,and Apob38.9/38.9 mice under ad libitum feeding conditions, whereas the Apob38.9/38.9 mice appeared to have reduced fasting insulin and glucose levels (Table 1).
Triglyceride contents of the homozygous mice were increased ≈4-fold (Figure 1A). Liver triglycerides of heterozygous mice were about twice normal. Hepatic triglyceride contents were significantly correlated with genotype (Table 2). On regression analysis, liver triglycerides were predicted by genotype and by the ratio of liver weight to body weight (R2=0.60, P<0.0001).
Rates of Fatty Acid Synthesis
We had hypothesized that the impaired hepatic triglyceride export via VLDL could affect hepatic lipid metabolism. We tested the rates of incorporation of [14C]acetate into hepatic total fatty acids. We found a gene dose-dependent stepwise decrease from a mean value of 370 660 disintegrations per minute (dpm)/h per gram liver to 175 519 and 54 550 dpm/h per gram liver for wild-type, heterozygous, and homozygous mice, respectively (Figure 1B). Logarithmic fatty acid synthetic, ie, log(Lipo), rates were inversely correlated with liver triglyceride contents (Table 2). In contrast, plasma concentrations of β-hydroxybutyrate were not altered in apoB-38.9 mice (Table 1), suggesting that hepatic fatty acid β-oxidation and ketogenesis were not changed.
Hepatic mRNA Levels
SREBP-1c and FAS play crucial roles in the regulation of fatty acid synthesis. On Northern blot analysis of poly(A)+ mRNA isolated from pooled hepatic RNA samples, FAS mRNA levels were reduced by 41% and 58% in the heterozygous and homozygous mice, respectively (not shown). SREBP-1c mRNA levels were also lower, by 38% in heterozygous mice and by 66% in homozygous mice (not shown). To be able to correlate the levels of the mRNAs with hepatic triglyceride contents and fatty acid synthetic rates, the levels of FAS and SREBP-1c mRNA were also measured in each individual sample in an RPA with the use of the appropriate riboprobes (Figure 2). SREBP-1a mRNAs were also quantified, and β-actin mRNA was used as the endogenous comparator. SREBP-1a mRNA levels were constant across genotypes. FAS mRNA levels were decreased by 37% and 57% and SREBP-1c mRNA levels were decreased by 31% and 67% in heterozygous and homozygous mice, respectively. In analyses including all of the genotypes, the levels of the 2 mRNAs were strongly and positively correlated with each other and with log(Lipo) rates, and levels of both mRNAs were inversely correlated with liver triglyceride, or log(liver triglyceride), contents (Table 2). This confirms the ANOVA results (Figure 2). However, correlations between mRNAs and liver fat were lost when genotypes were analyzed separately (not shown), suggesting that hepatic triglyceride contents may not be the proximate signal for suppression of the mRNA levels.
Significant decrements were also seen in SCD-1 mRNA levels, by 60% in heterozygous mice and by 80% in homozygous mice (Figure 3). No differences by genotype were seen in ACC-1, GPAT, DGAT, or TGH hepatic mRNA levels (Figure 3). In contrast to the observed changes in levels of fatty acid synthesis-related mRNAs, the levels of CPT-1 and PPARα were unchanged whether they were measured on pooled (Figure 3) or individual (not shown) samples, consistent with the unaltered plasma β-hydroxybutyrate concentrations (Table 1).
As reported previously,16 the apoB-38.9 mutation caused significant drops in total apoB mRNA levels (Figure 3). However, the mRNA levels of apoA-I, apoE, or the microsomal triglyceride transfer protein genes35 were unchanged.
Responses to Fasting and Refeeding
To assess their responses to a lipogenic stimulus, mice were subjected to 24 hours of fasting, and then some were refed with a high-carbohydrate/fat-free diet for 12 hours. Compared with plasma levels during ad libitum feeding, plasma levels of FFAs rose during the fast and fell during refeeding, as expected. Changes in plasma glucose levels were opposite in direction (Figure 4).
Liver fatty acid synthetic rates were reduced in all groups after fasting to levels that were similar among all 3 genotypes (Figure 4). Thus, the fasting-induced fractional reductions in heterozygous and homozygous mice were not as dramatic as in the wild-type control mice. Refeeding increased fatty acid synthetic rates dramatically in all mice, but the rates achieved by the homozygous mice were significantly lower than those reached by their heterozygous or wild-type littermates. Fasting reduced SREBP-1c and FAS mRNA levels in wild-type mice (both mRNAs by 60%, Figure 5). The respective reductions for these 2 mRNAs in the heterozygous mice were 58% and 29%. It is worth emphasizing that the mRNA levels of homozygous mice fed ad libitum were similar to the suppressed levels seen after 24 hours of fasting in control and heterozygous mice, and no additional decreases were seen in homozygous mice after 24 hours of fasting. For mice in the fasted state, mRNA levels were similar among the 3 genotypes (Figure 5). Refeeding caused dramatic increases in SREBP-1c and FAS mRNA levels in all 3 groups of mice (Figure 5). However, the mRNA levels of SREBP-1c and FAS reached in the homozygous mice after refeeding continued to be lower than those in the control or heterozygous mice.
ApoB truncation-specifying mutations in mice reduce the production of the products of the normal Apob allele and the products of the mutant Apob allele, with the truncated forms of apoB have impaired abilities to transport triglycerides. These defects cause the selective accumulation of triglycerides in hepatocytes.16,36⇓ Fatty livers have also been reported in human subjects with FHBL.7–9⇓⇓ The present study has demonstrated that the impaired hepatic triglyceride transport that is due to the apoB defect downregulates the hepatic fatty acid synthetic pathway.
Hepatic fatty acid synthetic rates, measured in vivo, were reduced in heterozygous and homozygous mice on the ad libitum regimen, and there were parallel decreases of mRNA levels of FAS and SREBP-1c. FAS was chosen for measurement because it is a critical enzyme in fatty acid synthesis, catalyzing all steps in the biosynthesis of palmitic acid from acetyl coenzyme A and malonyl coenzyme A.18 It is rate-limiting in the long-term regulation of fatty acid synthesis,37 and its activity is regulated mainly at the transcriptional level.38 SREBP-1c too is very important; it plays a crucial role in transcriptional regulation of the genes of lipogenic enzymes.39,40⇓ Overexpression of SREBP-1c in mouse livers selectively stimulates lipogenesis,39 whereas disruption of the SREBP-1 gene blunts the response of hepatic lipogenesis to fasting and fasting/refeeding.40 The decreases in the fatty acid synthetic rates and in SREBP-1c and FAS mRNA levels seen in the present study were identical in direction and similar in magnitude. Similar analyses were performed in mice heterozygous for a gene-targeted apoB-27.6 truncation-specifying allele (data not shown) and in compound heterozygous (Apob38.9/27.6) mice.41 These mice also have fatty livers because of the low apoB-100 and apoB-48 production rates and the greatly reduced ability of apoB-27.6 to transport triglycerides.41 Here too the rates of fatty acid synthesis and mRNA levels of SREBP-1c and FAS were decreased. In addition, the mRNA level of SCD-1, the enzyme catalyzing the critical step in fatty acid desaturation,19 was also dramatically reduced in the apoB-38.9 and apoB-27.6 mice, indicating that the entire fatty acid synthetic pathway was affected. Recent studies on SCD-1-deficient mice have demonstrated a stringent requirement of the de novo synthesized monounsaturated fatty acids for liver triglyceride synthesis and VLDL production.42,43⇓ The coexistence of fatty livers and downregulation of hepatic SCD-1 mRNA in our apoB-38.9 mice suggested that the reduced rates of fatty acid synthesis in the livers of these mice still exceeded their triglyceride-exporting capacity.
It is worth noting that in homozygous mice fed ad libitum, FAS and SREBP-1c mRNA levels were similar to those in the 24-hour-fasted control mice. This suggests that the levels of both mRNAs were maximally suppressed in homozygous mice, even on ad libitum feeding.
In contrast with reduced lipogenesis, no significant changes were observed in plasma β-hydroxybutyrate concentrations or mRNA levels of PPARα and CPT-1, suggesting that mitochondrial β-oxidation of fatty acids may not be altered in the apoB-38.9 mice.
Rates of hepatic triglyceride synthesis and/or turnover were probably not affected, inasmuch as mRNA levels of GPAT and DGAT, the 2 critical enzymes in triglyceride synthesis,20,21⇓ remained unaltered. Consistent with these unaltered mRNA levels, we have previously shown that the incorporation rates of [3H]glycerol into hepatic triglycerides were not altered in primary hepatocyte cultures taken from the apoB-38.9 mice.16 Likewise, the rates of hepatic triglyceride turnover may not be altered, inasmuch as hepatic mRNA levels for TGH, an enzyme responsible for the hydrolysis of cytosolic triglycerides in the liver,22 were unaltered in the apoB-38.9 mice. Together, these results imply that the triglycerides of the cytosolic lipid droplets in the livers of these mice continued to undergo hydrolysis and reesterification in the presence of an impaired hepatic triglyceride-exporting capacity and suppressed fatty acid synthesis rates.
It is not clear how the apoB-38.9 mutation induced a negative feedback on the hepatic lipogenic pathway. Although there was a strong negative correlation between liver triglyceride contents and hepatic fatty acid synthetic rates or FAS and SREB-1c mRNA levels on the data pooled from the mice of all 3 Apob genotypes, such correlations were not observed when the data of each genotype were analyzed separately. These results indicate that the accumulation of hepatic triglycerides, per se, may not be responsible for the feedback inhibition. It is possible that the defect in hepatic VLDL secretion resulting from apoB-38.9 mutation may directly alter the size and/or compositions of the regulatory fatty acid or fatty acyl coenzyme A pools in the liver by mechanisms that remain to be identified, leading to downregulation of SREBP-1c mRNA, which, in turn, downregulates the gene expression of the lipogenic enzymes.
Despite the suppressed lipogenic rates in the basal state, our mutant mice were able to respond to the lipogenic stimulus of fasting and refeeding with rises in lipogenic rates and FAS and SREBP-1c mRNA levels. However, the lipogenic stimulus appeared only partially able to override the inhibitory effect resulting from the triglyceride export defect in our homozygotes, suggesting that the apoB mutation-induced chronic and substantial suppression of triglyceride secretion provoked a robust adaptive diminution of fatty acid synthesis. Insulin plays a crucial role in mediating the nutritional regulation of hepatic lipogenic activities.44,45⇓ An increased FFA uptake could cause lipotoxicity and induce insulin resistance in pancreatic beta cells46,47⇓ and in the liver.48 Despite the presence of fatty livers, our apoB-38.9 mice did not appear to have a global insulin-resistant condition, inasmuch as they had similar or even lower fasting plasma glucose and insulin levels than did their apoB wild-type littermates. However, it is not known whether the impairment in the hepatic VLDL-triglyceride export system could specifically interfere with the stimulatory role of insulin in the lipogenic pathway.
In conclusion, the results of the present study demonstrate that the mouse liver adapts to triglyceride accumulation caused by a well-defined apoB defect, mainly by downregulating the fatty acid synthetic pathway. Further studies are required to determine the mechanism(s) by which steady-state levels of the mRNAs of SREBP-1c and its target genes become reduced. Such studies may provide further insight into the understanding of the pathophysiological consequences of fatty livers as well as the FHBL syndrome.
This work was supported by National Institutes of Health grants R37 HL-424460 and RO1 HL-59515 and by funds from the General Clinical Research Center (GCRC-5 MO1RR0036), Diabetes Research Training Center (5P60DK20579), Clinical Nutrition Research Unit (P30DK56341), Digestive Disease Center (1P30DK52574), and Siteman Cancer Center (1P30CA91842-01) at Washington University School of Medicine. We are grateful to Robin L. Fitzgerald for assistance in maintaining the mouse colony and for her help in the whole-animal studies.
Received December 5, 2001; revision accepted December 21, 2001.
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