Expression of the human apoE2 isoform in adipocytes: altered cellular processing and impaired adipocyte lipogenesis

Abstract

Expression of apoE in adipocytes has been shown to have an important role in modulating adipocyte triglyceride (TG) metabolism and gene expression that is independent of circulating and extracellular apoE. The impact of adipocyte expression of common human apoE isoforms was evaluated using adipocytes harvested from human apoE2, -3, and -4 knock-in mice. Expression of the apoE2 isoform was associated with an increase in adipocyte apoE gene expression and apoE synthesis. Newly synthesized apoE2 was unstable in adipocytes and demonstrated increased degradation and decreased secretion. ApoE2-expressing mice were hyperlipidemic, and had increased size of gonadal fat pads and of adipocytes, compared with apoE3 mice. In isolated cells, however, expression of the apoE2 isoform produced defective lipogenesis and increased TG hydrolysis. Incubation of adipose tissue with apoE3-containing TG-rich lipoproteins resulted in a significant increase in TG in adipose tissue from apoE3 and -E4 mice, but not apoE2 mice. Reduced capacity to internalize FFA as lipogenic substrate contributed to defective lipogenesis. Newly synthesized apoE2 is unstable in adipocytes and results in decreased adipocyte TG synthesis and defective FA uptake. These changes recapitulate those observed in apoE knockout adipocytes and have implications for understanding metabolic disturbances in humans expressing the E2 isoform.

Fig. 1.

ApoE mRNA levels in adipocytes, macrophages, and liver of apoE isoform knock-in mice. RNA was isolated from (A) freshly isolated mature adipocytes, (B) adipocytes differentiated from preadipocytes and maintained in culture, (C) liver, or (D) peritoneal macrophages of apoE knock-in mice. ApoE mRNA levels were analyzed by quantitative RT-PCR as described in Materials and Methods. The results are from five mice per group, and each sample was analyzed in triplicate. **P < 0.01 compared with apoE3.

Fig. 2.

ApoE protein levels in freshly isolated mature and cultured adipocytes from apoE isoform knock-in mice. A: Freshly isolated mature adipocytes isolated from the perigonadal fat pad or (B) adipocytes cultured from preadipocytes isolated from the same depot and maintained in culture were utilized for Western blot for apoE as described in Materials and Methods. *P < 0.05, **P < 0.01 compared with apoE3.

Fig. 3.

ApoE synthesis and turnover in adipocytes from apoE isoform knock-in mice. A: Cultured adipocytes were pulse-labeled with 200 μCi/ml ³⁵[S]methionine for 45 min and immediately harvested to measure incorporation of label into newly synthesized cellular apoE. B: Cultured adipocytes were pulse-labeled as described above followed by a 60 min chase incubation in DMEM supplemented with 500 μM unlabeled methionine. The amount of apoE secreted and retained in cells was directly measured, and the amount degraded was calculated as described in Materials and Methods. *P < 0.05, **P < 0.01 for comparison to apoE3.

Fig. 4.

Adipocyte size in apoE isoform knock-in mice. Mature adipocytes were freshly isolated from the perigonadal fat pad of five apoE isoform mice of each genotype as described in Materials and Methods. A: Adipocyte diameter was measured as described in Materials and Methods. B: Mean adipocyte diameter. *P < 0.05 compared with apoE3

Fig. 5.

TG turnover in apoE isoform knock-in adipocytes. A: TG synthesis was measured in apoE2, -E3, and -E4 freshly isolated mature adipocytes in the absence or presence of 100 μg/ml human VLDL isolated from an apoE3/E3 donor, as described in Materials and Methods. B: TG hydrolysis was measured in freshly isolated mature adipocytes isolated from apoE isoform mice as described in Materials and Methods. *P < 0.05, **P < 0.01 for comparison to E3 under the same incubation conditions.

Fig. 6.

Effect of incubation with VLDL on TG mass in adipose tissue explants from apoE isoform knock-in mice. ApoE2, -E3, or -E4 perigonadal adipose tissue explants were prepared and incubated with human VLDL (100 μg/ml) harvested from an apoE3/E3 donor in 0.2% BSA for 24 h. After incubation, TG mass was measured. *P < 0.05 for the change in TG mass due to inclusion of VLDL.

Fig. 7.

LPL activity and FA internalization in apoE isoform knock-in adipocytes. A: Mature adipocytes were freshly isolated from perigonadal fat pads of apoE isoform mice, and TG synthesis was measured over 6 h during incubation with 100 μg/ml VLDL isolated from an apoE3/E3 donor with or without the LPL inhibitor THL. B: Total LPL activity was measured from mature apoE isoform knock-in adipocytes. The insert shows secreted LPL activity. C: FA uptake was measured in freshly isolated mature apoE isoform adipocytes as described in Materials and Methods. A representative measurement of uptake from triplicate samples is shown as a function of time. The inset shows total FA uptake mean ± SD over the monitoring period from two separate experiments, each performed in triplicate using freshly isolated mature adipocytes pooled from five mice. *P < 0.05, **P < 0.01 for comparison to E3 adipocytes under the same incubation conditions.