Adipose tissue depot-specific differences in adipocyte apolipoprotein E expression

Abstract

Important differences in gene expression have been documented in adipocytes derived from specific adipose tissue depots. We have previously documented an important role for adipocyte apolipoprotein E (apoE) in modulating adipocyte and adipose tissue triglyceride and lipoprotein metabolism. We now evaluate the endogenous expression of apoE in adipocytes isolated from unique adipose tissue depots in 4 different species. Adipocyte apoE expression is higher in subcutaneous fat compared with visceral fat in humans, mice, rats, and baboons. In baboons, evaluation of apoE expression in 5 adipose tissue depots (subcutaneous abdominal, subcutaneous gluteal, visceral, pericardial, epicardial) showed that, compared with subcutaneous abdominal adipocytes, the level of apoE expression is similar in subcutaneous gluteal, lower in visceral and pericardial, and higher in epicardial adipocytes. Consistent with previously demonstrated suppression of adipocyte apoE by adipose tissue inflammation, adipose tissue depots with lower apoE expression demonstrated greater infiltration of macrophages and an increased expression of tumor necrosis factor-α messenger RNA. Depot-specific differences in apoE expression were maintained after in vitro differentiation. Adipocytes isolated from depots with lower apoE expression manifested lower rates of triglyceride synthesis in the absence and presence of triglyceride-rich lipoproteins. Adenoviral-mediated increase of apoE expression in omental adipocytes increased triglyceride synthesis in these cells. Our results demonstrate significant heterogeneity in adipocyte apoE expression across adipose tissue depots in several species. Because of its role in modulating adipocyte triglyceride and lipoprotein metabolism, depot-specific differences in endogenous adipocyte apoE could have important implications for modulating the accumulation of lipid in these depots.

Figure 1. ApoE mRNA levels in human subcutaneous and visceral mature adipocytes

Mature adipocytes were isolated from matched sets of freshly harvested subcutaneous and visceral adipose tissue depots (n=17) as described in Methods. ApoE mRNA levels were measured by qRT-PCR. Results presented in the box plot graph are ΔCT number of subcutaneous and visceral samples as described in Methods. Each sample was run in duplicate. *P<0.05 for the difference in apoE mRNA level between subcutaneous and visceral adipocytes.

Figure 2. ApoE mRNA levels in rat subcutaneous and omental mature adipocytes

Mature adipocytes were isolated from freshly harvested subcutaneous or omental fat depots from Zucker fatty rats or lean littermates. ApoE mRNA levels were measured by qRT-PCR. Results shown are mean ± SD of 3 rats per group. ***P<0.001 for the difference in apoE mRNA level between subcutaneous and omental adipocytes.

Figure 3. ApoE mRNA and protein expression in mouse subcutaneous and intra-abdominal mature adipocytes

Mature adipocytes were isolated from freshly harvested subcutaneous and visceral fat depots from chow fed or high-fat fed (12 weeks) mice (n=6–8 mice per group). (A) ApoE mRNA levels were measured by qRT-PCR, and (B) ApoE protein expression was estimated by Western blot. ApoE and β-actin signals are shown from the Western blot of samples from 5 different mice. (C) Changes in apoE expression in visceral and subcutaneous adipocytes with HFD-induced obesity. *P<0.05, **P<0.01 for the indicated comparison.

Figure 3. ApoE mRNA and protein expression in mouse subcutaneous and intra-abdominal mature adipocytes

Mature adipocytes were isolated from freshly harvested subcutaneous and visceral fat depots from chow fed or high-fat fed (12 weeks) mice (n=6–8 mice per group). (A) ApoE mRNA levels were measured by qRT-PCR, and (B) ApoE protein expression was estimated by Western blot. ApoE and β-actin signals are shown from the Western blot of samples from 5 different mice. (C) Changes in apoE expression in visceral and subcutaneous adipocytes with HFD-induced obesity. *P<0.05, **P<0.01 for the indicated comparison.

Figure 4. Evaluation of apoE, CD68, and TNFα levels in five adipose tissue depots from the baboon

Subcutaneous abdominal, omental, subcutaneous gluteal, pericardial, and epicardial adipose tissue was harvested from 6 baboons. ApoE, CD68 and TNF mRNA levels were measured by qRT-PCR. Expression levels in omental, subcutaneous gluteal, pericardial, and epicardial adipose tissue depots are expressed in box plot format relative to expression in subcutaneous abdominal, which is represented by the dashed line at “1” in each panel. Results are presented as fold change. *P<0.05, **P<0.01, ***P<0.001 for the difference compared to the subcutaneous abdominal fat depot.

Figure 5. ApoE expression in freshly isolated mature adipocytes compared to cultured adipocytes

RNA was isolated from mature adipocytes isolated from the 5 adipose tissue depots noted in Figure 4. RNA was also isolated from adipocytes differentiated from preadipocytes isolated from the same 5 depots, and maintained in culture for 10–14 days. ApoE mRNA level was measured by qRT-PCR, and its level of expression in freshly isolated mature adipocytes in each depot was compared to that measured in freshly isolated mature adipocytes from the subcutaneous abdominal fat depot. The level of expression in cultured adipocytes from each depot was compared to that in cultured adipocytes from the subcutaneous abdominal fat depot. Results are presented in box plot format as fold changes with the dashed line at “1” representing level of expression in the mature adipocytes or cultured adipocytes from the subcutaneous abdominal depot. *P<0.05, **P<0.01, ***P<0.001 for the difference in expression level compared to mature adipocytes or cultured adipocytes isolated from the subcutaneous abdominal fat depot.

Figure 6. ApoE expression and TG synthesis/mass

A. Baboon adipocytes were prepared from preadipocytes isolated from omental and subcutaneous adipose tissues and maintained in culture for 14 days as described in Methods. To measure TG synthesis, adipocytes were incubated with no addition or with 100µg/ml VLDL in DMEM medium containing [¹⁴C]glucose and 0.1%BSA for 6h. TG synthesis over 6h was measured as described in Methods. Results presented are mean of experiments from 2 separate baboons, each run in triplicate. *P<0.05, **P<0.01 for the difference between subcutaneous and omental adipocytes. B. Rat or human cultured adipocytes from the omental adipose tissue depot were incubated with an apoE or LacZ adenovirus as described in Methods. After an additional four days incubation in lipoprotein-containing growth medium, cells were harvested for measurement of TG mass. Results shown are mean of 2 separate experiments, each ran in triplicate. *P<0.05, **P<0.01 for the indicated comparison.