Reciprocal Metabolic Perturbations in the Adipose Tissue and Liver of GPIHBP1-deficient Mice

Materials and Methods

Gpihbp1^−/− mice³ were housed in a barrier facility and fed a chow diet containing 0.02% cholesterol (LabDiet #5001, Purina Mills) or a western diet containing 21.2% fat (anhydrous milk fat) and low (0.05%) amounts of cholesterol (TD #05311, Harlan Teklad, Madison, WI). In some experiments, mice were housed individually in sealed metabolic cages (Oxymax, Columbus Instruments, Columbus, Ohio). Mice were acclimated to the metabolic cages for 8 h before data collection (three 12-h light/dark cycles). Respiratory quotient data were compiled with OxyMax software and analyzed with Microsoft Excel by averaging readings taken every 20 min during each light/dark cycle.

Lipids were measured in the plasma and in three tissues (gonadal fat pad, liver, lung) of 14-week-old chow-fed Gpihbp1^−/− and littermate wild-type mice (n = 5/group). All mice were perfused extensively with PBS before harvesting tissues. We performed identical studies in Gpihbp1^−/− and littermate wild-type mice after 5 weeks on a high-fat diet. Lipids were extracted with chloroform and methanol in the presence of internal standards by the method of Folch et al.¹¹ For the separation of neutral lipids, thin layer chromatography (TLC) with a solvent system consisting of petroleum ether/diethyl ether/acetic acid (80/20/1, V/V/V) was employed. Individual phospholipids were separated by liquid chromatography (Agilent Technologies 1100 Series). Each lipid class was trans-esterified in 1% sulfuric acid in methanol in a sealed vial under a nitrogen atmosphere at 100°C for 45 min. The resulting fatty acid methyl esters were extracted from the mixture with hexane containing 0.05% butylated hydroxytoluene, and the hexane extracts were sealed under nitrogen until analysis by gas chromatography. Fatty acid methyl esters were separated and quantified by capillary gas chromatography (Agilent Technologies model 6890) equipped with a 30 m DB-88 capillary column (Agilent Technologies) and a flame-ionization detector. Absolute concentrations of each fatty acid in each lipid class were determined based on internal standards that were added to samples prior to the analysis. The resulting data were expressed in nanomoles/gram of tissue or plasma. The quantitative results were used to generate a normalized data set expressed as mol% fatty acid in each lipid class.

To quantify gene expression levels, RNA was prepared from tissues with TRI Reagent (Sigma). After treating the RNA with DNase I (Ambion), cDNA was prepared with a mixture of random primers, oligo(dT), and Superscript III reverse transcriptase (Invitrogen). Quantitative RT-PCR studies were performed in triplicate with SYBR Green PCR Master Mix (Quantace, UK) on a 7500 Fast Real-Time PCR System (Applied Biosystems). The primers for four lipid biosynthetic genes [acetyl–CoA carboxylase (Acc), fatty acid synthase (Fasn1), stearoyl–CoA desaturase (Scd1), and ATP citrate lyase (Acly)] and endothelial lipase (Lipg) are listed in the Supplementary Methods (Table I). We also measured expression of endothelial lipase (Lipg) in adipose tissue. Gene-expression levels were calculated with the comparative C_T method and normalized to levels of β2-microglobulin expression.

Results

The levels of triglycerides and cholesterol in the plasma of Gpihbp1^−/− mice were markedly elevated on both chow and high-fat diets, consistent with earlier studies.^{2, 3} The plasma triglyceride and cholesterol levels in Gpihbp1^−/− mice on the chow diet were 64.0 ± 19.1 and 12.5 ± 1.6 mmol/L, respectively [vs. 1.3 ± 0.4 and 3.2 ± 0.2, respectively in wild-type control mice (n = 6/group)]. On a high-fat diet, the triglyceride and cholesterol levels in Gpihbp1^−/− mice were 94.8 ± 37.2 and 26.22 ± 3. 9 mmol/L, respectively (vs. 1.0 ± 0.1 and 5.0 ± 0.3, respectively in wild-type control mice (n = 6/group). Earlier studies showed that Gpihbp1^−/− mice gained less weight after short-term feeding of a high-fat diet.¹² Here, we measured body weight and adiposity in littermate male Gpihbp1^−/− and wild-type mice that were maintained on chow or high-fat diets until they were 6 months of age. In wild-type mice, the body weights and adiposity were far greater on the high-fat diet, as expected, but with Gpihbp1^−/− mice, body weights and adiposity were nearly identical on chow and high-fat diets (Fig. 1).

The low levels of adiposity in Gpihbp1^−/− on the high-fat diet heightened our suspicion that we might find perturbed lipid metabolism in adipose tissue. To address this issue, we examined the composition of triglycerides in the adipose tissue in Gpihbp1^−/− and wild-type mice on a chow diet containing 1.2% palmitoleic acid (16:1), 44.3% linoleic acid (18:2), and 5.0% α-linolenic acid (18:3). In adipose tissue, the ratio of 18:2 and 18:3 fatty acids to 16:1 fatty acids was lower in Gpihbp1^−/− mice than in wild-type mice (Fig. 2A). Also, the absolute levels of 18:2 and 18:3 fatty acids in adipose tissue triglycerides were lower in Gpihbp1^−/− mice than in wild-type mice, while levels of 16:1 fatty acids were higher (Fig. 2B). Interestingly, the composition of triglycerides in the liver exhibited reciprocal changes: the 18:2,18:3/16:1 fatty acid ratio in the liver triglycerides was actually higher in Gpihbp1^−/− mice than in wild-type mice (Fig. 2C). Also, the amounts of 18:2 fatty acids in liver triglycerides tended to be higher while 16:1 fatty acids tended to be lower (Fig. 2D). The amounts of triglycerides in adipose tissue and liver, per gram of tissue, were similar in Gpihbp1^−/− and wild-type mice (Fig. I).

We also observed reciprocal differences in the composition of free fatty acids in adipose tissue and liver. The ratio of 18:2 and 18:3 free fatty acids to 16:1 free fatty acids in adipose tissue was lower in Gpihbp1^−/− mice than in wild-type mice (Fig. 3A, B), while in the liver, the ratio was higher in Gpihbp1^−/− mice than in wild-type mice (Fig. 3C, D).

The 18:2,18:3/16:1 fatty acid ratio in the plasma triglycerides was higher in Gpihbp1^−/− mice than in wild-type mice (Fig. 3E), mirroring the compositional changes in liver triglycerides (Fig. 2C). In a recent study, we reported that the lung has very high levels of GPIHBP1 expression, facilitating LPL’s role in lipid metabolism in that tissue.¹³ Interestingly, the 18:2,18:3/16:1 fatty acid ratio in triglycerides of the lung was lower in Gpihbp1^−/− mice than in wild-type mice (Fig. 3F), similar to results with adipose tissue triglycerides (Fig. 2A).

We also identified reciprocal changes in phospholipid composition in adipose tissue and liver. The phospholipids in the adipose tissue of Gpihbp1^−/− mice contained a “lower than wild-type” 18:2,18:3/16:1 fatty acid ratio (Fig. 3G), while the phospholipids in the liver contained a “higher than wild-type” 18:2,18:3/16:1 fatty acid ratio (Fig. 3H).

The composition of triglycerides in the adipose tissue (Fig. 2A) and plasma (Fig. 3E) of Gpihbp1^−/− mice was very different, while the composition of triglycerides in the liver (Fig. 2C) and plasma (Fig. 3E) of Gpihbp1^−/− mice was quite similar. These findings led us to predict that the composition of adipose tissue triglycerides in Gpihbp1^−/− mice would change minimally, if at all, in mice fed a saturated fat–rich diet, whereas the composition of liver triglycerides would change dramatically. Indeed, when we placed Gpihbp1^−/− mice on a high-fat diet (65.3% saturated fat and 4.5% polyunsaturated fat) for 5 weeks, the levels of saturated fatty acids and polyunsaturated fatty acids in the adipose tissue triglycerides were no different than those in chow-fed mice. In contrast, the composition of the adipose tissue in wild-type mice reflected the new diet, with higher levels of saturated fatty acids and lower levels of polyunsaturated fatty acids (Fig. 4A, B). Reciprocal changes were observed in the liver: the composition of triglycerides in the liver of Gpihbp1^−/− mice changed markedly on the high-fat diet, with increased levels of saturated fat and lower levels of polyunsaturated fat (Fig. 4C, D). The composition of the triglycerides in the liver of wild-type mice also changed, but the changes were smaller than those in Gpihbp1^−/− mice (Fig. 4C, D). In both wild-type and Gpihbp1^−/− mice, the plasma triglycerides contained higher levels of saturated fatty acids and lower levels of unsaturated fatty acids (Fig. 4E, F), but similar to the findings in the liver, those changes were more exaggerated in Gpihbp1^−/− mice.

The lower levels of essential fatty acids in adipose tissue triglycerides in Gpihbp1^−/− mice suggested that these mice would have higher levels of de novo lipogenesis⁷ and higher expression of lipid biosynthetic genes. Indeed, the expression of four key lipid biosynthetic genes was higher in adipose tissue of Gpihbp1^−/− mice than in wild-type mice, both on chow and high-fat diets (Fig. 5A). Reciprocal changes were observed in the liver: lipid biosynthetic gene expression in the liver was lower in Gpihbp1^−/− mice than in wild-type mice (Fig. 5B). Not surprisingly, the high-fat diet suppressed the expression of lipid biosynthetic genes in the liver in both wild-type and Gpihbp1^−/− mice (Fig. 5B). We also examined expression of endothelial lipase in adipose tissue, based on an earlier report that endothelial lipase expression is higher in adipose tissue of mice that express LPL exclusively in skeletal muscle.¹⁴ Interestingly, endothelial lipase expression in adipose tissue was significantly higher in Gpihbp1^−/− mice than in wild-type mice (Fig. II).

The reduced adiposity in Gpihbp1^−/− mice (Fig. 1), the striking differences in lipid composition in the adipose tissue of Gpihbp1^−/− mice (Figs. 2–4), and the increased expression of lipid biosynthetic genes in the adipose tissue of Gpihbp1^−/− mice (Fig. 5) suggested that Gpihbp1^−/− mice might exhibit significant defects in the utilization of dietary lipids and preferentially use carbohydrates. In line with this prediction, the respiratory quotient in Gpihbp1^−/− mice was significantly higher than in wild-type mice during the inactive period (a period of increased lipid utilization). These differences were particularly striking in mice fed the high-fat diet (Fig. 6).

Discussion

In the current study, we asked whether lipid composition and gene expression are perturbed in the adipose tissue of Gpihbp1^−/− mice—as they are in the setting of LPL deficiency^5-7—or whether the large stores of catalytically active LPL in the tissues of Gpihbp1^−/− mice might protect them from these changes. Our experiments provided a definitive answer. Like LPL-deficient adipose tissue,^5-7 the levels of essential fatty acids in the triglycerides of adipose tissue were far lower in Gpihbp1^−/− mice than in wild-type mice, and the expression of lipid biosynthetic genes in adipose tissue was higher. No one, as far as we are aware, has assessed the expression of lipid biosynthetic genes in the adipose tissue of LPL-deficient mice with chylomicronemia, but the expression of these genes is clearly increased in the adipose tissue of normolipidemic mice that express LPL exclusively in skeletal muscle.⁹ Thus, reduced amounts of catalytically active LPL in capillaries of adipose tissue, whether caused by a deficiency in LPL itself or a deficiency in GPIHBP1, perturbs adipose tissue composition and gene expression. The catalytically active LPL within the interstitium of adipose tissue in Gpihbp1^−/− mice^{2, 3} is incapable of preventing the perturbations in adipose tissue lipid composition or gene expression.

The second issue that we addressed is whether compositional and metabolic changes in the adipose tissue of Gpihbp1^−/− mice would be accompanied by reciprocal metabolic changes in tissues lacking a prominent role in LPL processing, namely the liver. We reasoned that, at steady state, large amounts of triglycerides must enter the plasma of Gpihbp1^−/− mice, exactly as in wild-type mice, and that the “triglyceride entry” must be matched by equal amounts of triglyceride clearance. And we further suspected that evidence of reduced triglyceride uptake in the adipose tissue of Gpihbp1^−/− mice (as reflected by lipid compositional abnormalities and increased expression of lipid biosynthetic genes) would be accompanied by reciprocal metabolic perturbations in the liver, a tissue that has a negligible role in LPL-mediated lipoprotein processing but a large role in the uptake of remnant lipoproteins.¹⁵ Our studies provided strong support for the “reciprocal metabolic changes” concept. Gpihbp1^−/− mice did not have elevated hepatic triglyceride stores, but the levels of essential fatty acids in liver triglycerides were higher in Gpihbp1^−/− mice than in wild-type mice (changes opposite to those occurring in adipose tissue). Also, unlike the situation in adipose tissue, the composition of triglycerides in the liver changed dramatically when Gpihbp1^−/− mice were fed a high-fat diet, and the magnitude of those changes was greater in Gpihbp1^−/− mice than in wild-type mice. Finally, the expression of lipid biosynthetic genes in the liver was lower-than-normal in chow-fed Gpihbp1^−/− mice, while the expression levels of these same genes in adipose tissue was higher-than-normal. As far as we are aware, our studies are the first to propose and document the existence of reciprocal metabolic perturbations in liver and adipose tissue in the setting of defective lipolysis.

Our studies suggest that the liver is capable of removing triglyceride-rich lipoproteins from the plasma under conditions when lipolysis is absent in adipose tissue (e.g., GPIHBP1 deficiency), leading to higher-than-normal levels of essential fatty acids in the liver and lower-than-normal expression of lipid biosynthetic genes. The liver’s role in triglyceride metabolism under these circumstances is hardly efficient, given the strikingly elevated plasma triglyceride levels in Gpihbp1^−/− mice (and the complete absence of hepatic steatosis in these mice), but increased triglyceride uptake by the liver likely compensates, at least to a degree, for the loss of LPL-mediated triglyceride delivery to peripheral tissues.

The ability to investigate the “reciprocal metabolic changes” hypothesis was facilitated by the existence of Gpihbp1^−/− mice, which are healthy and can be bred in unlimited numbers. Similar studies in LPL-deficient human subjects would be next to impossible, given ethical hurdles in obtaining human liver samples for elective scientific experiments. However, we believe that the concept set forth in the current studies—reciprocal metabolic changes in adipose tissue and liver in the setting of defective lipolysis—should be of interest to lipidologists. The pathogenesis of many cases of severe hypertriglyceridemia is unclear. Most often, patients with chylomicronemia have no obvious mutations in LPL, GPIHBP1, or APOCII,¹⁶ and it is often unclear whether hypertriglyceridemic patients actually have bona fide defects in LPL-mediated processing of lipoproteins in capillaries or whether they actually have lipoprotein overproduction or defects in remnant uptake. In the future, it could be interesting to determine whether “reciprocal metabolic changes” in adipose tissue and liver, as delineated in this paper, represents a “metabolic signature” for severely defective LPL-mediated lipolysis along the capillary wall. Perhaps “reciprocal metabolic changes” similar to those found in Gpihbp1^−/− mice are present only in patients with chylomicronemia due to LPL, APOCII, or GPIHBP1 deficiency (or yet-to-be-defined defects in LPL action) and are largely absent in many “garden variety” cases of hypertriglyceridemia. In the future, it might be possible to examine this issue in human patients characterizing adipose tissue and liver lipid synthesis profiles with stable isotope studies^{17, 18} rather than with the tissue biopsy experiments used in our mouse studies.

Supplementary Material