In patients with type 2 diabetes, a strong correlation between accumulation of intramuscular triclycerides (TGs) and insulin resistance has been found. The aim of the present study was to determine whether there is a causal relation between intramuscular TG accumulation and insulin sensitivity. Therefore, in mice with muscle-specific overexpression of human lipoprotein lipase (LPL) and control mice, muscle TG content was measured in combination with glucose uptake in vivo, under hyperinsulinemic-euglycemic conditions. Overexpression of LPL in muscle resulted in accumulation of TGs in skeletal muscle (85.5 ± 33.3 vs. 25.7 ± 23.1 μmol/g tissue in LPL and control mice, respectively; P < 0.05). During the hyperinsulinemic clamp study, there were no differences in plasma glucose, insulin, and FFA concentrations between the two groups. Moreover, whole-body, as well as skeletal muscle, insulin-mediated glucose uptake did not differ between LPL-overexpressing and wild-type mice. Surprisingly, whole-body glucose oxidation was decreased by ∼60% (P < 0.05), whereas nonoxidative glucose disposal was increased by ∼50% (P < 0.05) in LPL-overexpressing versus control mice. In conclusion, overexpression of human LPL in muscle increases intramuscular TG accumulation, but does not affect whole-body or muscle-specific insulin-mediated uptake, findings that argue against a simple causal relation between intramuscular TG content and insulin resistance.
It has previously been reported that mice lacking the VLDL receptor (VLDLR−/−) exhibit normal plasma lipid levels and a modest decrease in adipose tissue mass. In the present study, the effect of VLDLR deficiency on profound weight gain was studied in mice. Obesity was induced either by feeding of a high-fat, high-calorie (HFC) diet or by crossbreeding mice onto the genetically obese ob / ob background. After 17 weeks of HFC feeding, VLDLR−/− mice remained lean, whereas their wild-type littermates (VLDLR+/+) became obese. Similarly, the weight gain of ob / ob mice was less profound in the absence of the VLDLR. Moreover, VLDLR deficiency led to increased plasma triglycerides after HFC feeding. The protection from obesity in VLDLR−/− mice involved decreased peripheral uptake of fatty acids, because VLDLR−/− mice exhibited a significant reduction in whole-body free fatty acid uptake, with no clear differences in food intake and fat absorption. These observations were supported by a strong decrease in average adipocyte size in VLDLR−/− mice of both obesity models, implying reduced adipocyte triglyceride storage in the absence of the VLDLR. These results suggest that the VLDLR plays a role in the delivery of VLDL-derived fatty acids into adipose tissue.
Transgenic mice overexpressing human APOE* 3Leiden are highly susceptible to diet-induced hyperlipoproteinemia and atherosclerosis due to a defect in hepatic uptake of remnant lipoproteins. In addition to the human APOE *3Leiden gene, these mice carry the human APOC1 gene ( APOE *3Leiden- C1 ). To investigate the possible effect of simultaneous expression of the human APOC1 gene, we examined the phenotypic expression in these APOE *3Leiden- C1 mice in relation to transgenic mice expressing the APOE *3Leiden gene without the APOC1 gene ( APOE *3Leiden-HCR). APOE *3Leiden- C1 and APOE *3Leiden-HCR mice had comparable liver expression for the APOE *3Leiden transgene and high total cholesterol levels on a sucrose-based diet compared with control mice (4.3 and 4.3 versus 2.1 mmol/L). In addition, on this diet APOE *3Leiden- C1 mice displayed significantly higher serum triglyceride levels than APOE *3Leiden-HCR mice and control mice (4.4 versus 0.6 and 0.2 mmol/L). Elevated triglyceride and cholesterol levels were mainly in the VLDL-sized lipoproteins. In vivo turnover studies with endogenously triglyceride-labeled VLDL showed a reduced VLDL triglyceride fractional catabolic rate for APOE *3Leiden- C1 and APOE *3Leiden-HCR mice compared with control mice (3.5 and 11.0 versus 20.4 pools per hour). To study whether the difference in fractional catabolic rates between the two transgenic strains was due to an inhibiting effect of apoC1 on the extrahepatic lipolysis or hepatic-mediated uptake of VLDL, turnover experiments were performed in functionally hepatectomized mice. Strikingly, both APOE *3Leiden- C1 and APOE *3Leiden-HCR mice showed a decreased lipolytic rate of VLDL triglyceride in the extrahepatic circulation compared with control mice (1.5 and 1.8 versus 6.3 pools per hour). We conclude that next to an impaired hepatic uptake, overexpression of the APOE *3Leiden gene influences the extrahepatic lipolysis of VLDL triglycerides, whereas simultaneous overexpression of the APOC1 gene leads to a further decrease in hepatic clearance of VLDL.
Previous studies with hypertriglyceridemic APOC3 transgenic mice have suggested that apolipoprotein C-III (apoC-III) may inhibit either the apoE-mediated hepatic uptake of TG-rich lipoproteins and/or the lipoprotein lipase (LPL)-mediated hydrolysis of TG. Accordingly, apoC3 knockout (apoC3−/−) mice are hypotriglyceridemic. In the present study, we attempted to elucidate the mechanism(s) underlying these phenomena by intercrossing apoC3−/− mice with apoE−/− mice to study the effects of apoC-III deficiency against a hyperlipidemic background. Similar to apoE+/+ apoC3−/− mice, apoE−/−apoC3−/− mice exhibited a marked reduction in VLDL cholesterol and TG, indicating that the mechanism(s) by which apoC-III deficiency exerts its lipid-lowering effect act independent of apoE. On both backgrounds, apoC3−/− mice showed normal intestinal lipid absorption and hepatic VLDL TG secretion. However, turnover studies showed that TG-labeled emulsion particles were cleared much more rapidly in apoC3−/− mice, whereas the clearance of VLDL apoB, as a marker for whole particle uptake by the liver, was not affected. Furthermore, it was shown that cholesteryl oleate-labeled particles were also cleared faster in apoC3−/− mice. Thus the mechanisms underlying the hypolipidemia in apoC3−/− mice involve both a more efficient hydrolysis of VLDL TG as well as an enhanced selective clearance of VLDL cholesteryl esters from plasma.In summary, our studies of apoC3−/− mice support the concept that apoC-III is an effective inhibitor of VLDL TG hydrolysis and reveal a potential regulating role for apoC-III with respect to the selective uptake of cholesteryl esters.
To accelerate the biological annotation of novel genes discovered in sequenced regions of mammalian genomes, we are creating large deletions in the mouse genome targeted to include clusters of such genes. Here we describe the targeted deletion of a 450-kb region on mouse chromosome 11, which, based on computational analysis of the deleted murine sequences and human 5q orthologous sequences, codes for nine putative genes. Mice homozygous for the deletion had a variety of abnormalities, including severe hypertriglyceridemia, hepatic and cardiac enlargement, growth retardation, and premature mortality. Analysis of triglyceride metabolism in these animals demonstrated a several-fold increase in hepatic very-low density lipoprotein triglyceride secretion, the most prevalent mechanism responsible for hypertriglyceridemia in humans. A series of mouse BAC and human YAC transgenes covering different intervals of the 450-kb deleted region were assessed for their ability to complement the deletion induced abnormalities. These studies revealed that OCTN2 , a gene recently shown to play a role in carnitine transport, was able to correct the triglyceride abnormalities. The discovery of this previously unappreciated relationship between OCTN2 , carnitine, and hepatic triglyceride production is of particular importance because of the clinical consequence of hypertriglyceridemia and the paucity of genes known to modulate triglyceride secretion.
Abstract —The apolipoprotein E2 (Lys146→Gln) variant is associated with a dominant form of familial dysbetalipoproteinemia. Heterozygous carriers of this variant have elevated levels of plasma triglycerides, cholesterol, and apolipoprotein E (apoE). It was hypothesized that the high amounts of triglycerides in the very low density lipoprotein (VLDL) fraction are due to a disturbed lipolysis of VLDL. To test this hypothesis, apoE knockout mice were injected with an adenovirus containing the human APOE*2 (Lys146→Gln) gene, Ad-E2(146), under the control of the cytomegalovirus promoter. ApoE knockout mice injected with an adenovirus vector encoding human apoE3 (Ad-E3) were used as controls. Five days after adenovirus injection, plasma cholesterol levels of mice injected with a high dose of Ad-E2(146) (2×10 9 plaque-forming units) were not changed compared with preinjection levels, whereas in the group who received a low dose of Ad-E2(146) (5×10 8 plaque-forming units) and in the groups injected with a low or a high dose of Ad-E3, plasma cholesterol levels were decreased 5-, 6-, and 12-fold, respectively. Plasma triglycerides were not affected in mice injected with Ad-E3. In contrast, a 7-fold increase in plasma triglycerides was observed in mice injected with the low dose of Ad-E2(146) compared with mice injected with Ad-E3. Injection with the high dose of Ad-E2(146) resulted in a dramatic increase of plasma triglycerides (50-fold compared with Ad-E3 injection). In vitro lipolysis experiments showed that the lipolysis rate of VLDLs containing normal amounts of apoE2 (Lys146→Gln) was decreased by 54% compared with that of VLDLs containing comparable amounts of apoE3. The in vivo VLDL-triglyceride production rate of Ad-E2(146)–injected mice was not significantly different from that of Ad-E3–injected mice. These results demonstrate that expression of apoE2 (Lys146→Gln) causes hypertriglyceridemia due to an apoE variant–specific inhibition of the hydrolysis of VLDL-triglycerides.
Abstract —The present study examined the relative contributions of the different pathways by which oxidatively modified VLDL (oxVLDL) promotes the uptake and intracellular accumulation of lipids in J774 macrophages. VLDL was oxidized for a maximum of 4 hours, resulting in an increase in thiobarbituric acid–reactive substances and an increased electrophoretic mobility on agarose gel. The lipid composition of the relatively moderately oxidized VLDL samples did not differ significantly from that of nonoxidized VLDL samples. The uptake of 125 I-labeled VLDL by the J774 cells increased with oxidation time and was completely blocked on coincubation with polyinosinic acid (PolyI), indicating that oxVLDL is taken up by the cells via the scavenger receptor only. Despite the 2-fold increased uptake of oxVLDL protein, the cell association of triglyceride (TG)-derived fatty acids by the J774 macrophages after incubation with oxVLDL was only 50% of that with native VLDL. In line with these observations, the induction of de novo synthesis of TG by J774 cells was ≈3-fold less efficient after incubation with oxVLDL than after incubation with native VLDL. The induction of de novo synthesis of TG with oxVLDL was even further decreased on simultaneous incubation with PolyI, whereas PolyI did not affect the native VLDL-induced TG synthesis. These results indicate that oxVLDL induces endogenous TG synthesis predominantly through particle uptake via the scavenger receptor and much less via the extracellular lipoprotein lipase (LPL)–mediated hydrolysis of TG, as is the case for native VLDL. In line with these observations, we showed that the suitability of VLDL as a substrate for LPL decreases with oxidation time. Addition of oxVLDL to the LPL assay did not interfere with the lipolysis of native VLDL. However, enrichment of the oxidized lipoprotein particle with native apoC2 was able to fully restore the impaired lipolysis. Thus, from these studies it can be concluded that on oxidation, VLDL becomes less efficient in inducing TG accumulation in J774 cells as a consequence of a defect in apoC2 as an activator for the LPL-mediated extracellular lipolysis.
