To test the expression and secretion of APOA2 in vitro, we analyzed culture medium from HTB-13 cells infected with AdGFP or AdGFP-APOA-Ⅱ. Analysis of the culture medium by Western blotting confirmed the efficient APOA-Ⅱ secretion at 48 hours post-infection (Fig. 2A).
To confirm human APOA-Ⅱ expression in vivo, C57BL/6 mice were infected with AdGFP or AdGFP-APOA-Ⅱ and five days post-infection, liver and plasma samples were collected. Then total hepatic RNA was analyzed by Northern blotting and plasma by Western blotting for APOA-Ⅱ mRNA and APOA-Ⅱ protein respectively, confirmed the efficient expression of APOA-Ⅱ in vivo (Fig. 2B and C).
The human APOA-Ⅱ protein contains a free cysteine residue capable of forming inter-molecular S-S bonds resulting in APOA-Ⅱ dimers. When expressed in cell cultures, APOA-Ⅱ could be detected at both its monomeric and dimeric forms (Fig. 2A). However, the dimeric form was no longer evident when APOA-Ⅱ was expressed in mice (Fig. 2C).
Analysis of plasma lipid levels five days post-infection showed that expression of APOA-Ⅱ resulted in a significant increase of plasma total cholesterol, triglyceride and phospholipid levels, compared to the same mice prior to infection (day 0) or the AdGFP infected mice five days post-infection (Fig. 3A–C). This increase was associated with a marked increase in chylomicrons/VLDL, IDL and LDL cholesterol triglycerides and phospholipids and a shift of HDL cholesterol distribution towards smaller and more dense HDL particles (Fig. 3D–F). The relative lipid content of APOA-Ⅱ-HDL expressed as mg of total HDL protein or wt% content of total lipids in HDL is shown in Table 1.
Lipid content per mg of protein wt% content of total lipids AdGFP AdGFP-APOA-Ⅱ AdGFP AdGFP-APOA-Ⅱ Total cholesterol/mg of protein 0.253±0.007 0.208±0.004 34.9±0.94 33.7±0.63 Triglycerides 0.030±0.004 0.019±0.005 4.3±0.56 3.2±0.80 Phospholipids 0.439±0.014 0.390±0.015 60.8±1.85 63.1±2.47 Total lipids 0.722±0.025 0.618±0.006 100 100 Total protein (mg/L) 1 330±80 1 810±260 HDL: high-density lipoprotein.
Table 1. Lipid composition of HDL expressed as milligrams of lipid per milligram of total HDL protein isolated from AdGFP- or AdGFP-APOA-Ⅱ infected mice
Fractionation of plasma samples by density gradient ultracentrifugation followed by Western blotting analysis of lipoprotein fractions for human APOA-Ⅱ and murine APOA-Ⅰ, APOE, APOC-Ⅰ, APOC-Ⅱ and APOC-Ⅲ, showed that APOA-Ⅱ expression resulted in qualitative and quantitative changes in apolipoprotein composition of HDL and other lipoprotein classes five days post-infection (Fig. 4). APOA-Ⅱ was distributed among all HDL fractions and was also present in intermediate-density lipoprotein (IDL) and low-density lipoprotein (LDL) subclasses. No measurable levels of APOA-Ⅱ could be found in the chylomicrons/very low-density lipoprotein (CM/VLDL) fraction. Expression of APOA-Ⅱ resulted in substantial recruitment of APOE in HDL, while APOA-Ⅰ expression remained unchanged. Some lipid-free APOE was also visible. Interestingly, APOA-Ⅱ expression also increased the levels of APOCs (APOC-Ⅰ, APOC-Ⅱ and APOC-Ⅲ) though in different lipoprotein fractions. In contrast to the control group, where trace amounts of APOC-I were present and APOC-Ⅱ and APOC-Ⅲ were not detectable, a significant increase in APOC-Ⅱ content was observed in all lipoproteins, while APOC-Ⅰ was significantly augmented in VLDL and LDL. Measurable levels of APOC-Ⅲ were also present mainly in LDL with trace amounts found in HDL.
To study the effects of human APOA-Ⅱ on HDL particle geometry, we next isolated HDL from the plasma of infected mice and performed a qualitative negative staining TEM analysis. As expected, negative TEM staining confirmed the presence of spherical HDL particles in the HDL fractions of plasma from AdGFP infected mice (Fig. 5A). However, similar analysis revealed a significant presence of discoidal (indicated by white arrows) HDL particles in the plasma of mice expressing APOA-Ⅱ (Fig. 5B). Average APOA-Ⅱ-HDL particle diameter, expressed as median (Min to Max) was calculated to be (12.54±0.32) nm, and displayed no statistically significant difference from the average diameter of control HDL, calculated to be (12.42±0.27) nm (Fig. 5C).
Given the distinct structural differences between APOA-Ⅱ-HDL and control-HDL, we next sought to investigate how these differences may influence HDL functionality such as HDL antioxidant activity and total cholesterol efflux from RAW 264.7 macrophage cells in vitro.
As shown in Fig. 6A, APOA-Ⅱ-HDL demonstrated a significantly higher antioxidant function (i.e. greater inhibition of substrate oxidation) compared to control-HDL, when equal amounts of HDL cholesterol from each group were used in the dihydrorhodamine assay. Control-HDL also inhibited substrate oxidation as expected, though to a much lesser extent.
Figure 6. Functional properties of HDL isolated from AdGFP or AdGFP-APOA-Ⅱ infected mice and Lp-PLA2 activity.
As shown in Fig. 6B, when equal amounts of HDL cholesterol from each group were used in the cholesterol efflux assay, no differences between the capacities of APOA-Ⅱ-HDL and control-HDL to accept [14C]-cholesterol from [14C]-cholesterol-charged RAW 264.7 cells were observed.
To compare the effects of APOA-Ⅱ-HDL on inflammation, we employed the pertinent model of LPS-induced inflammation in RAW 264.7 macrophages, as described previously[18,27]. In the absence of LPS, addition of 10 μg/mL HDL from each tested sample (i.e. APOA-Ⅱ-HDL and control-HDL) did not result in any significant production of TNFα confirming that samples were properly prepared free of LPS. Stimulation of cells with LPS in the absence of HDL resulted in a significant production of TNFα in culture medium. When LPS stimulation was performed in the presence of APOA-Ⅱ-HDL, a significant inhibition of TNFα production was observed compared to control-HDL, which also showed a smaller yet measurable anti-inflammatory effect (Fig. 6C).
As shown in Fig. 6D, expression of APOA-Ⅱ in mice infected with AdGFP-APOA-Ⅱ resulted in a significantly higher plasma Lp-PLA2 activity.
In an attempt to study the effects of APOA-Ⅱ expression on adipose tissue metabolic activity, in the next set of experiments, mitochondria were isolated from BAT and WAT of mice infected with AdGFP-APOA-Ⅱ or AdGFP. In WAT mitochondrial fractions, we found that expression of APOA-Ⅱ stimulated a significant induction of mitochondrial CYTC levels (corrected for COX4 expression), suggesting elevated oxidative phosphorylation (Fig. 7A–C). Moreover, it appeared that oxidative phosphorylation was coupled with respiration towards ATP production, due to an apparent relative decrease of UCP1 when corrected for mitochondrial COX4 (Fig. 7A–C and G).
Figure 7. Representative Western blotting analysis and semiquantitative determination of CYTC and UCP1 relative to COX4 in mitochondrial extracts.
Similar analysis of BAT mitochondrial fractions showed that APOA-Ⅱ expression did not have any substantial impact on mitochondrial CYTC and UCP1 levels further indicating no effects on oxidative phosphorylation and non-shivering thermogenesis in this tissue (Fig. 7D–F).
To determine the effects of APOA-Ⅱ expression on plasma glucose homeostasis, GTT and IST tests were performed in mice infected with AdGFP-APOA-Ⅱ or AdGFP five days post-infection.
On day 0 (immediately prior to infection), all mouse groups displayed comparable glucose tolerance when GTT was performed (Fig. 8A and C). Similarly, no significant differences in insulin sensitivity between groups were detected when IST was performed (Fig. 8D and F). Moreover, both groups had normal fasting glucose levels between 60–70 mg/dL (Fig. 8G).
On day five post-infection, glucose tolerance of APOA-Ⅱ-expressing mice appeared significantly improved compared to the control group (Fig. 8B and C). Nevertheless, these mice displayed comparable insulin sensitivity to that of mice infected with AdGFP (Fig. 8E and F), indicating an unaffected physiological response to intraperitoneally administered exogenous insulin. Both the control and APOA-Ⅱ-expressing mouse groups showed a tendency towards higher fasting plasma glucose levels, between 80–90 mg/dL, though the increase did not reach statistical significance (Fig. 8G).
Pleiotropic effects of apolipoprotein A-Ⅱ on high-density lipoprotein functionality, adipose tissue metabolic activity and plasma glucose homeostasis
- Received Date: 2019-03-29
- Accepted Date: 2019-05-14
Abstract: Apolipoprotein A-Ⅱ (APOA-Ⅱ) is the second most abundant apolipoprotein of high-density lipoprotein (HDL) synthesized mainly by the liver and to a much lesser extent by the intestine. Transgenic mice overexpressing human APOA-Ⅱ present abnormal lipoprotein composition and are prone to atherosclerosis, though in humans the role for APOA-Ⅱ in coronary heart disease remains controversial. Here, we investigated the effects of overexpressed APOA-Ⅱ on HDL structure and function, adipose tissue metabolic activity, glucose tolerance and insulin sensitivity. C57BL/6 mice were infected with an adenovirus expressing human APOA-Ⅱ or a control adenovirus AdGFP, and five days post-infection blood and tissue samples were isolated. APOA-Ⅱ expression resulted in distinct changes in HDL apoproteome that correlated with increased antioxidant and anti-inflammatory activities. No effects on cholesterol efflux from RAW 264.7 macrophages were observed. Molecular analyses in white adipose tissue (WAT) indicated a stimulation of oxidative phosphorylation coupled with respiration for ATP production in mice overexpressing APOA-Ⅱ. Finally, overexpressed APOA-Ⅱ improved glucose tolerance of mice but had no effect on the response to exogenously administered insulin. In summary, expression of APOA-Ⅱ in C57BL/6 mice results in pleiotropic effects with respect to HDL functionality, adipose tissue metabolism and glucose utilization, many of which are beneficial to health.