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Nutrient Metabolism

Increased Lipogenesis and Fatty Acid Reesterification Contribute to Hepatic Triacylglycerol Stores in Hyperlipidemic Txnip^–/– Mice^1,2

Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN, 55108 and ^* Departments of Medicine and Human Genetics, University of California, Los Angeles, CA 90095

³To whom correspondence should be addressed. E-mail: eparks{at}umn.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The effect of decreased fatty acid oxidation on liver lipid metabolism in HcB-19 mice, a mouse model of hyperlipidemia (Txnip^–/–), was investigated using metabolic labeling. De novo cholesterol synthesis and de novo lipogenesis were quantified using 1-¹³C₁ acetic acid, and liver triacylglycerol (TAG) derived from dietary fatty acids was quantified using dietary glyceryl tri(hexandecanoate-d₃₁). Tissue samples were analyzed for TAG, free cholesterol (FC), and cholesterol ester (CE) content. Txnip^–/– mice had significantly elevated (P < 0.05) serum nonesterified fatty acids compared with wild-type (WT) littermates; their livers weighed more and contained more TAG and total cholesterol. Txnip^–/– liver also contained measurable CE; CE was not detectable in WT mice. Liver CE content was elevated despite lower cholesterol fractional synthesis rates (16 vs. 31%/d in Txnip^–/– and WT mice, respectively). FC absolute synthesis rate (ASR) in WT mice (0.28 ± 0.0 µmol/d) was similar to the combined synthesis rates of FC (0.13 ± 0.10 µmol/d) and CE (0.10 ± 0.00 µmol/d) in Txnip^–/– mice. Lipogenesis, as assessed by TAG-palmitate ASR, was significantly greater in Txnip^–/– mice (1.47 ± 0.08 vs. 0.49 ± 0.06 µmol/d) and liver fatty acid synthase activity was also higher (7.96 ± 2.53 vs. 4.83 ± 1.44 U/mg protein). Both elevated lipogenesis and increased fatty acid reesterification to glycerol and cholesterol contributed to fat in the livers of Txnip^–/– mice. These data support elevated fatty acid synthesis as the primary contributor to liver TAG in Txnip^–/– mice, although increased esterification of fatty acids also contributed to excess liver TAG. The absolute total cholesterol synthesis rate was not altered, but esterification of fatty acids to cholesterol provided an additional means to buffer physiologically the negative results of excess fatty acid availability.

KEY WORDS: • de novo lipogenesis • cholesterol synthesis • dietary triacylglycerol • mouse liver metabolism

The regulation of fatty acid and cholesterol synthesis in the liver has been a field of intense interest given the influence of liver lipid metabolism and the development of chronic diseases. Genetic strains of mice have provided powerful models with which to investigate the regulation of lipid synthesis (1,2) and, as the molecular details of the genes affecting cholesterol synthesis have unfolded, additional strategies have been required for the study of whole-body cholesterol flux (3) and the coordinate regulation of triacylglycerol (TAG)⁴ and cholesterol flux at the organ level. Recent technical developments have supported investigation of the responses to genetic modifications to better define the metabolic phenotypes of genetically altered mice (4). These developments included surgical techniques to place jugular-vein catheters in mice and infusion of metabolites such as glucose and insulin (5), lipoprotein-particle turnover studies (6,7), and stable isotope administration in rodents to measure fatty acid (8) and bile acid synthesis (9). The method of having rodents consume stable isotopes was recently expanded by mixing deuterated water into the drinking water of animals to quantitate adipose TAG synthesis (10). Advances in isotope tracer methodology allowed for the quantitative determination of lipid synthesis in vivo (8–11). The first goal of the present investigation was to adapt tracer methodology using 2 isotopes in mice to determine the contribution of dietary vs. newly made fatty acids to hepatic TAG pools. Second, using this methodology, we sought to determine the rates of lipogenesis in a mouse model of hyperlipidemia (the Txnip^–/– mouse). The effect of excess fatty acid availability on cholesterol synthesis was also of interest, given the potential for coregulation between the pathways of lipogenesis and cholesterol synthesis (12). We hypothesized that fatty acids made through the de novo lipogenesis pathway would contribute significantly to the excess fat found in the livers of Txnip^–/– mice.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animal husbandry and overall study design. The breeding and genetic background of the C57BL/6J.HcB-19 (HcB-19 x C57BL/6J) N5 congenic mice homozygous (Txnip^–/–) or C57BL/6J (WT) for the Txnip nonsense mutation were published previously (13,14). For the present study, age-matched (2–6 mo) C57BL/6 and C57BL/6J.HcB-19 female mice were maintained on a 12-h light:dark cycle (dark from 0500 to 1700h) and meal-fed a cholesterol-free, nonpurified diet consisting of 60 g fat, 18 g protein, and 54 g carbohydrate/kg (Diet 7013, Teklad Research Diets). Mice were housed in pairs in metabolic cages and trained to meal-feed (1100 to 1700 h daily) for 1 wk before the start of the study. They initially lost 1 g of body weight that was regained within 3 d of the initiation of training. Mice were weighed to the nearest 0.1 gram throughout the study to verify that body weight was maintained during meal feeding. The daily food intake was determined by weighing the food cup and its contents to the nearest 0.01 g both before and after feeding. To determine the contributions of sources of lipids in liver lipid pools and the rates of de novo cholesterol synthesis and de novo lipogenesis, food was labeled with stable isotopes (see below). Groups of 3 mice were killed on d 1, 2, 4, 6, 8, 11, and 15 after the addition of isotopes to the food.

    Labeling of food. For the purpose of identifying sources of fatty acid and cholesterol in liver, 2 stable isotopes were added to the powdered food. ¹³C₁-Sodium acetate (60 g) and glyceryl tri(hexandecanoate-d₃₁) (6 g, Isotec) were dissolved in 6 L of 99% ethyl alcohol. The alcohol:isotope mixture was added to 3 kg of powdered mouse food, and mixed thoroughly, followed by complete alcohol evaporation. The food was mixed during the evaporation process to ensure homogenous distribution of the labels in the diet. The fatty acid composition of the fat in the diet was used to estimate the quantity of unlabeled palmitate present, and the amount of label added was designed to give a labeled to unlabeled enrichment of 20%. The final dietary palmitate d₃₁-enrichment, verified by GC-MS, was 32.8%. The amount of acetate added to the diet was determined from previous experiments to give an intracellular acetyl-CoA enrichment of >5% (8,15).

    Serum and tissue collection. Because mice were acclimated to the meal-feeding protocol, they would be presumed to not expect food to be available until 1100 h each day. On each study day, mice were killed at 0900 h, which means they were food deprived for a 16-h period. Mice were anesthetized using an i.m. injection of ketamine (1 mg/g body weight, Phoenix Pharmaceutical). Blood was obtained through intra-aortic puncture and the liver removed. Portions of the tissue samples were removed for lipid extraction and analysis. Serum samples were divided into aliquots for quantification of metabolites (e.g., TAG, cholesterol, ketone bodies), and the remaining serum samples from the 3 mice at each time point were pooled and used for serum lipid extraction.

    Lipid extraction and analysis. Total lipids were extracted from homogenized tissue and serum samples via the extraction method of Folch et al. (16). Free cholesterol (FC), TAG, and cholesterol esters (CE) were separated by TLC and transesterified as described previously (17). In the present text, the liberated cholesterol (which originally existed as CE in tissue) is referred to as cholesterol-ester cholesterol (denoted CEC) to distinguish it from the FC also present in tissue. Fatty acid composition of the TAG fraction in liver and in mouse food was determined by GC with flame-ionization detection using a 6890 gas chromatograph (Hewlett-Packard) fitted with a 7683 automatic split injection system and a flame ionization detector using a fused silica capillary column 30 m in length, with an i.d. of 0.25mm (DB-225, J&W). GC-MS of TAG-palmitate, FC, and CEC was performed on an HP 6890 with a Mass Selective Detector HP 5973 (Hewlett-Packard) as previously described (18,19). Enzymatic kits were used to measure serum and tissue TAG concentrations (kit #336, Sigma Diagnostics), and serum ß-hydroxybutyrate concentrations (kit #310-3, Sigma Diagnostics). Tissue total cholesterol, FC, and serum nonesterified fatty acids (NEFA) were also measured in duplicate by enzymatic kit (kits #276-64909, 274-47109 E and 994-75409 E, respectively, Wako Chemicals). Tissue CE was determined by the calculated difference between total cholesterol and FC. Liver fatty acid synthase (FAS; EC 2.3.1.85) enzyme activity was assayed kinetically from the rate of ß-NADPH oxidation (20,21).

    Calculations and statistics. Liver de novo lipogenesis and cholesterol synthesis were calculated using the Mass Isotopomer Distribution Analysis method (MIDA) (19,22,23). Dietary enrichment of fatty acids in liver-TAG was calculated using a 5-point d₃₁ standard curve. Fractional synthesis rate (FSR) of FC, CEC, and palmitate from de novo pathways, as well as palmitate from dietary TAG, were calculated by modeling the rise toward plateau enrichment of the isotope in liver tissue (24). The data were fit to the equation y = A x [1–e^–ks(t)], where y is the liver tissue enrichment, A is the plateau or asymptote value of the isotope in liver tissue, and t is the time in days. Half-lives were calculated by dividing 0.693 by the FSR. Absolute synthesis (or accumulation) rate (ASR) was determined by multiplying the FSR determined for a lipid fraction in a single group of mice (d 11) by the corresponding pool size of lipid in each mouse in that group. Significant differences between Txnip^–/– mice and wild-type (WT) littermates were determined by unpaired Student’s t-tests; a paired t test was used to assess differences between 2 points on a turnover curve. Statistical analyses were performed using Statview (version 5.0.1., SAS Institute). Differences with P-values < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Txnip^–/– and WT mice did not differ in age, body weight, food intake, or serum total cholesterol (Table 1). Txnip^–/– mice had elevated serum ß-hydroxybutyrate concentrations, as was observed previously in this mouse strain (13). Serum NEFA concentrations were 70% higher in Txnip^–/– mice and TAG concentrations were 56% higher. These values were lower than previously observed by Castellani et al. (14), a possible result of the difference in age between our mice and those investigated previously (6 mo). Txnip^–/– livers weighed 13% more than those of WT mice and contained greater amounts of TAG, total cholesterol, and CE (Table 1). Liver CE concentrations in WT mice were below the detection limit of the assay. Liver FAS activity was significantly elevated in Txnip^–/– compared with WT mice (Table 1). Analysis of liver TAG-fatty acid composition revealed that Txnip^–/– mice had a lower percentage of palmitic acid (25.4 ± 2.6% vs. 27.7 ± 2.5%, for Txnip^–/– and WT mice, respectively, P = 0.004) and a higher percentage of oleic acid (38.6 ± 2.3% vs. 35.6 ± 2.8%, P = 0.0006). One other monounsaturated fatty acid, gadoleic [20:1(n-9)], also tended to be higher in Txnip^–/– mice (0.9 ± 0.6% vs. 0.7 ± 0.2%, P = 0.06).

Acetyl-CoA enrichments for each of the lipids were determined by analysis of liver TAG-palmitate (Table 2) and liver-FC and -CEC (Table 3) by MIDA. Only the TAG-palmitate acetyl-CoA differed between the groups, with Txnip^–/– mice having a lower enrichment (Table 2). The shape of liver TAG-fatty acid curves for lipogenesis (Fig. 1A) and dietary enrichment (Fig. 1B) supported the concept of complete liver-TAG turnover by 11 d in both Txnip^–/– and WT mice. Within each group’s curve, the mean percentage of enrichment of TAG-fatty acids (de novo or dietary) at d 8 was not different from the mean at d 11. No differences were observed between steady-state percentage of enrichments in liver-TAG derived from the de novo pathway (Table 2) in Txnip^–/– and WT mice. The steady-state percentage of dietary enrichment in liver-TAG also did not differ between the groups. However, because the absolute quantity of liver-TAG was different between the groups, the absolute pool size of liver TAG derived from the de novo pathway in Txnip^–/– mice was twice that in WT mice (Table 2). The same was true for the absolute content of TAG from diet in Txnip^–/– mouse liver (0.61 ± 0.29 µmol/liver), which was 2-fold higher than that in WT mice (0.21 ± 0.11 µmol/liver).

View larger version (12K): [in this window] [in a new window]   FIGURE 1 Comparison of kinetics of liver TAG-palmitate derived from de novo lipogenesis (DNL) (A) and from dietary TAG-palmitate (B) in Txnip–/– and WT mice fed a nonpurified diet for up to 15d. One curve was generated for each strain of mice and, on a single curve, each time point represents mean ± SE from at least 3 mice. For WT mice, the enrichment of dietary fatty acids at d 8 and 11 did not differ (3.9 ± 0.1 and 4.3 ± 0.5%, respectively). The d 8 value (4.1 ± 0.4%) was also not different from d 15. This was also the case for both de novo and dietary fatty acids in liver-TAG of Txnip–/– mice and de novo fatty acids in liver-TAG of WT mice

View larger version (19K): [in this window] [in a new window]   FIGURE 2 Comparison of absolute pool sizes of TAG derived from the diet and de novo lipogenesis (DNL) (A) and contribution of newly made cholesterol to liver cholesterol pools (B) in Txnip–/– and WT mice. Data are means from 3 mice (d 11) in each group. The liver-TAG pool sizes were multiplied by the d 11 FSR for DNL and dietary fatty acids (A), whereas liver-FC and -CEC pool sizes were multiplied by their respective FSR (B). "Unaccounted for" denotes liver TAG, FC, and CEC pools that remained unlabeled at d 11.

The kinetics of newly synthesized cholesterol moving into liver FC and CEC pools are depicted in Figures 3A and B, and the data are presented in Table 3. Comparison of the final FC enrichment in Txnip^–/– mice to the asymptote predicted by modeling suggested that the liver-FC pool had not reached steady state because the asymptote was higher. The values for WT mice were more similar (29.14% measured vs. 30.17% predicted). Day 11 Txnip^–/– and WT liver-FC did not differ in the percentages of newly synthesized molecules (Table 3, Fig. 3A). The FSR of FC appeared lower for Txnip^–/– mice vs. WT mice (0.16 vs 0.31/d) and the ASR was significantly lower (Table 3).

View larger version (14K): [in this window] [in a new window]   FIGURE 3 Comparison of kinetics of liver FC (A) and liver CE-cholesterol molecules (B) derived from de novo synthesis in Txnip–/–and WT mice. One curve was generated for each strain of mice and, on a single curve, each time point represents the mean ± SE from at least 3 mice.

Finally, the absolute quantities of newly-made cholesterol at d 11 were calculated by multiplying the percentage of FC or CEC derived from the liver de novo cholesterol synthesis pathway by the pool size in these mice. The FC contents of the livers from the 2 mouse strains were similar (Fig. 2B, Table 3) and the excess cholesterol in Txnip^–/– liver was in the CEC fraction. In Txnip^–/– liver, the same proportion of FC and CEC became labeled (Fig. 3). These data support the concept that the FC and CEC pools were in equilibrium. Of the liver cholesterol accounted for in Txnip^–/– mice, approximately equal amounts were present in the FC and CEC pools. The sum of the total cholesterol ASR for Txnip^–/– mice (FC, 0.13 µmol/d + CEC, 0.10 µmol/d) was similar to the ASR of FC alone (0.28 µmol/d) in WT mice.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The present experiment had 2 goals. The first was to establish a methodology that used stable isotopes to determine the contribution of various fatty acid sources to hepatic TAG pools. The second goal was to apply this methodology to investigate rates of lipid turnover in a mouse model of hyperlipidemia. Presented below are our conclusions regarding the use of this technique, followed by a discussion of the metabolic consequences of a mutation in the Txnip gene.

    The methodology. Two pilot studies were performed in C57Bl6 mice to establish the time course of the present study (unpublished data). Because each time point on the kinetic curve was generated by a separate group of mice, it is remarkable that the shape of the curves is representative of physiologic, biological turnover from a single mouse. The use of different mice at each time point negated the need for repeated blood drawings and tissue biopsies in the same mouse. Although the technique of adding a stable isotope of acetate to the mouse diet was published previously (8), the present scheme expands the technique by labeling dietary fatty acids and simultaneously analyzing various tissue lipid fractions. The data suggest that 20 g of ¹³C₁-acetate/kg food provided enough label consumed by a mouse per day to result in a 5% acetyl-CoA enrichment intracellularly, as was observed previously (8). The technique of MIDA allows for the calculation of the enrichment of the acetyl-CoA pool used for lipid synthesis. Comparison of these enrichments revealed a similarity between cholesterol and TAG pools except for the pool that contributed to TAG-palmitate in Txnip^–/– mice. This finding is consistent with the previously hypothesized reduction in flux of fatty acid carbons through the tricarboxylic acid (TCA) cycle described in this mouse (13,14). A block in the TCA cycle after isocitrate would result in a backup of acetyl-CoA and dilution of this pool. Alternatively, elevations in the production of acetyl-CoA through increased flow of glycolytic carbons may have also caused the lower acetyl-CoA enrichments.

    Mechanisms of liver TAG accrual. The second goal of this research was to use the methodology to investigate the sources of liver fat in a mouse model of hyperlipidemia. The Txnip^–/– mutation was mapped (13) to a region containing the gene for thioredoxin-interacting protein (Txnip). Txnip is a protein thought to limit the reducing activity of thioredoxin, a ubiquitous protein that regulates the redox state of the cell. The mutation in Txnip may allow thioredoxin’s reducing activity to go unrestrained, with the putative effects of elevating NADH/NAD⁺ ratios. Several regulatory enzymes in the TCA cycle would be affected by an increased NADH/NAD⁺ ratio, resulting in a decreased flux though this cycle. The data presented here provide in vivo support for reduced oxidation, but also suggest that fatty acid synthesis and reesterification were also upregulated. From the standpoint of the absolute contribution of fatty acids moving into liver-TAG stores, de novo fatty acids contributed 3 times as many fatty acids to Txnip^–/– liver than to WT liver, and dietary fatty acids contributed 1.7 times more fatty acids to liver stores.

If reduced fatty acid oxidation were the only hepatic defect present, de novo fatty acids should have been treated no differently from those of dietary origin and both would have amassed proportionally. Fatty acid synthesis was significantly elevated in Txnip^–/– mice and this may have supported greater elongation of palmitate to oleate. The elevation in oleic acid is consistent with previous observations made in these mice (25); it was attributed to greater activity of stearoyl CoA desaturase (SCD), an enzyme that introduces the first cis-double bond at the -9 position in fatty acids. In the class of oxidoreductases, SCD may also be susceptible to alterations in the activity of the Txnip protein. If excess oleic acid (and also another monounsaturated fatty acid, 20:1) were due solely to an upregulation of SCD, we would not have expected to see an excess of dietary fatty acids as well. Thus, in addition to increased fatty acid synthesis, esterification of fatty acids also contributed to liver lipid accumulation. A mechanism that could have led to reesterification is increased availability of 2-carbon units from glucose. How the availability of 2-carbon units increased TAG synthesis is unclear and will be important to delineate, as well as a potential role for increased insulin to glucagon ratios in this model. The shunting of excess fatty acids toward esterification to glycerol and cholesterol, as well as to ketone body synthesis, provides a compensatory mechanism for protecting cells from the potential toxicity of fatty acid overload.

    Turnover of liver lipid. The kinetic data support the complete turnover of a fast-turnover TAG pool in the liver, with steady state being reached in 8 d. At this time, our method could account for 45% of liver TAG-palmitate (40% from lipogenesis and 4% from the diet), with 55% remaining unlabeled. What was the origin of these unlabeled fatty acids? One possibility is that a pool exists in the liver that is compartmentalized and turns over so slowly that it is unequilibrated with the faster turnover pool. This is unlikely given the total liver TAG pool size and the flux of fatty acids into and out of the liver. A 65% greater VLDL-TAG secretion rate was previously demonstrated in Txnip^–/– mice (13). A more likely explanation for the presence of unlabeled fatty acids in liver TAG at d 11 is that they arose from the periphery and came to the liver via the plasma NEFA pool. To test this hypothesis, we analyzed inguinal and subcutaneous adipose TAG and found TAG-fatty acid FSR in both strains to be between 14 and 19%/d, with a T of 4 d. These data suggest that fatty acids potentially originating from the adipose TAG contributed at least 55% of the fatty acids present in liver-TAG.

Although higher lipogenesis was confirmed in our studies, the cholesterol synthesis rate was not elevated. The combined production rate of FC and CEC in Txnip^–/– mice was similar to the production rate of FC in WT mice; yet, the Txnip^–/– total cholesterol pool size was larger. Furthermore, because the enrichments of FC and CEC in Txnip^–/– liver were still rising at the end of the experiment, the pool would have continued to expand with time. This suggests that reduced efflux of cholesterol from the liver contributed to the excess of CE in these mice. The activity of the hepatic enzyme, acyl coenzyme A:cholesterol acyltransferase (ACAT-2 in mouse liver), is upregulated by the presence of both cholesterol (26) and fatty acids (27,28). ACAT-2 also utilizes reducing equivalents and it is possible that the Txnip^–/– mutation stimulated ACAT activity through some direct mechanism leading to the increased stores of CE. However, recent work by Xie and colleagues (6) demonstrated that compared with other fatty acids, oleic acid raised liver CE content by 6-fold and increased secretion of apolipoprotein B-containing lipoproteins in mice. Oleic acid stimulation of cholesterol esterification occurred in an environment of similar cholesterol flux, which echoes our finding here. Finally, in both strains of mice, 70% of liver cholesterol remained unlabeled at d 11. Just as unlabeled fatty acids could have originated in the periphery, it is also possible that the periphery was also the origin of the unlabeled liver cholesterol. This methodology should be explored further to determine how it might be used to quantitate reverse cholesterol transport.

In summary, using stable isotope methodology, we identified metabolic mechanisms that contributed to elevated liver fat accrual in Txnip^–/– mice. Clearly, elevated fatty acid synthesis played a major role, and both fatty acid sources (dietary and de novo) were stored as TAG. It is intriguing that esterified cholesterol stores were elevated as well, suggesting that this route may also be a means to physiologically buffer the potentially negative results of excess fatty acids. How these lipid pools are altered acutely with feeding will be important to determine.

	ACKNOWLEDGMENTS

 
The authors thank Anne Pylkas, Rasa Michniovaite, Johanna Rehorst, and Cindy Gallaher for their technical assistance, and Fred Fierholm and John Freeburg of Agilent Technologies, for providing support in trouble-shooting the mass spectrometry software.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 02, April 2002, New Orleans, LA [Donnelly, K. L., Sheth, S. S., Lusis, A. J. & Parks, E. J. (2002) Measurement of lipid turnover in a mouse model of hyperlipidemia. FASEB J. 16: A633 (abs.)].

² Supported by a grant from the American Heart Association.

⁴ Abbreviations used: ACAT, acyl coenzyme A:cholesterol acyltransferase; ASR, absolute synthesis rate; CEC, cholesterol ester cholesterol; FAS, fatty acid synthase; FC, free cholesterol; FSR, fractional synthesis rate; MIDA, Mass Isotopomer Distribution Analysis; NEFA, nonesterified fatty acids; TAG, triacylglycerols; TC, total cholesterol; TCA cycle, tricarboxylic acid cycle.

Manuscript received 22 January 2004. Initial review completed 3 March 2004. Revision accepted 16 March 2004.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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