Omega-3 Fatty Acids Reduce Adipose Tissue Macrophages
The effects of drug or placebo treatment on standard clinical outcomes are shown in Table 2. Although the subjects in this study demonstrated modestly elevated baseline triglycerides, there was a small but significant decrease in triglycerides in the FO treatment group and no change with placebo, and there were no other significant changes in serum lipids between the groups. The oral glucose tolerance test was performed before and after treatment, and 23 subjects were either impaired glucose tolerance or impaired fasting glucose before randomization. After FO or placebo treatment, no changes were observed in fasting or 2-h glucose, insulin sensitivity (SI), or first-phase insulin secretion (AIRglu) (measured using the frequently sampled intravenous glucose tolerance test) in either group.
Previous studies have demonstrated that erythrocyte ω-3 PUFA content is altered by FO administration, but fewer studies have examined adipose tissue or muscle levels of ω-3 fatty acids in response to treatment. To characterize adipose tissue and muscle lipid content and determine the impact of the treatment groups, tissue total lipids were analyzed using mass spectrometry, as described in research design and methods. The distribution of total adipose and muscle lipids is shown in Supplementary Table 2. The vast majority of adipose tissue lipid was oleate (18:1, n-9), palmitate (16:0), and linoleate (18:2, n-6); the ω-3 PUFA linolenic acid, eicosapenanoic acid (EPA), and DHA constituted 1.1, 0.26, and 0.40% (by weight) of adipose lipids, respectively. In muscle, the relative abundance of lipids was somewhat different, with proportionally more of the saturated lipids palmitate and stearate and very low levels of EPA and DHA (Supplementary Table 2). Figure 1 shows the response of adipose and muscle EPA and DHA to FO and placebo treatments. All subjects treated with FO demonstrated an increase in tissue EPA and DHA, whereas there was no change in the placebo-treated subjects. Further, the FO-treated subjects demonstrated no changes in any other fatty acid. Although the placebo-treated subjects were receiving 4 g/day of corn oil, which is composed predominantly of linoleate, oleate, and palmitate, there was no significant change in any lipid component in the adipose tissue or muscle in these subjects.
(Enlarge Image)
Figure 1.
FO treatment increased adipose and muscle EPA and DHA. Adipose (A) and muscle (B) lipids were analyzed in biopsy samples before and after treatment with FO or placebo. There were no significant changes in the placebo-treated subjects, and data are expressed in relation to oleate, which is abundant in both tissues. The changes in EPA and DHA were significant (P < 0.05) compared with baseline and with placebo-treated subjects. Data are expressed as mean ± SEM.
Table 3 illustrates the measurements of a number of different blood adipokine levels. Previous studies have demonstrated that FO can invoke a PPARγ effect in vitro, and when given to animals. However, in these subjects, no changes in adiponectin were observed. In addition, no changes in the levels of IL-6, IL-10, IL-12, TNF-α, resistin, PAI-1, or leptin were noted. However, a significant decrease in the blood MCP-1 level was observed.
To further examine changes in inflammation, fibrosis, and vascularity, adipose tissue and muscle from the FO- and placebo-treated subjects were examined using histochemistry and immunohistochemistry. As shown in Fig. 2, there was a significant decrease in macrophage number in adipose after FO treatment and no change with placebo. Crown-like structures are clusters of macrophages, often involving giant cells, surrounding a necrotic adipocyte (large arrow in Fig. 2). After FO treatment, there was a significant decrease in crown-like structures, but there was no change in placebo-treated subjects. Macrophage number was also assessed in muscle, and no significant changes were observed in either group (data not shown).
(Enlarge Image)
Figure 2.
Effects of FO treatment on adipose macrophages and capillaries. Before and after treatment with FO, adipose tissue from biopsies was analyzed histochemically. A: CD68 staining. A representative image showing macrophages (small arrows) and crown-like structures (large arrow). Data are expressed as mean ± SEM. B: Effects of FO and placebo on macrophage number (*P < 0.05 vs. pretreatment). C: Effects of treatment on the number of crown-like structures (CLS) (*P < 0.05 vs. pretreatment). D: Capillary and large vessels were identified by staining with lectin and α-smooth muscle actin, and representative images are shown. E: Effects of placebo and FO on the number of capillaries in adipose tissue.
Previous studies have found that the adipose from obese, insulin-resistant subjects contained fewer capillaries and more large vessels than lean, insulin-sensitive subjects. To determine whether the treatment of subjects with FO altered vascularity, adipose tissue capillaries and larger vessels were quantified. As shown in Fig. 2, there was a small but consistent and significant increase in adipose capillaries, with no change in placebo-treated subjects. The number of large blood vessels was unchanged in both groups. There were no significant changes in muscle capillaries after FO treatment (data not shown).
The adipose tissue of obese, insulin-resistant subjects contains more collagen VI, along with other changes in the ECM. The overall degree of fibrosis in the adipose tissue of FO- and placebo-treated subjects was examined using histochemistry. No changes in overall fibrosis or collagen VI were noted (data not shown).
To analyze changes in gene expression in adipose tissue, RNA was extracted from the adipose tissue of FO- and placebo-treated subjects. As shown in Fig. 3, there were significant changes in MCP-1 and CD68 mRNA levels in the whole group of subjects. Although there was an overall decrease in adipose tissue macrophages after FO, there was considerable variation between subjects, which was strongly related to the degree of adipose tissue inflammation at baseline. As shown in Fig. 4A, the baseline adipose tissue macrophage number was strongly correlated with the change in macrophage number after FO treatment, suggesting that the subjects with the most inflammation benefitted the most from FO treatment.
(Enlarge Image)
Figure 3.
Changes in gene expression in adipose tissue after FO treatment. A: MCP-1. B: CD68. *P < 0.05 vs. pretreatment. Data are expressed as mean ± SEM. A.U., arbitrary units.
(Enlarge Image)
Figure 4.
Correlation between the number of macrophages in adipose at baseline, and the change after FO treatment. A: Change in macrophage number was associated with baseline macrophages. The expression of other genes was significantly associated with the change in macrophage number, and these include CTGF (B), IL-8 (C), and TIMP-2 (D). All correlations are significant at P < 0.05. A.U., arbitrary units.
To obtain a broader view of gene expression changes in response to FO, 116 genes (Supplementary Table 1) involving various aspects of inflammation, ECM, and vascularity were analyzed using NanoString in nine placebo and nine FO-treated subjects. Somewhat surprisingly, despite a significant decrease in macrophage number, and an increase in capillaries, there were no significant changes in gene expression, except for the previously described changes in MCP-1 and CD68. Specifically, there were no significant changes in the expression of the classic adipokines, including TNF-α, IL-1, IL-12, and IL-6. Although there were no net changes, the expression of several genes varied with the changes in macrophage number. For example, the change in expression of CTGF, IL-8, and TIMP-2 correlated significantly with the change in macrophage number with FO treatment (Fig. 4). CTGF and TIMP-2 decreased in parallel with a decrease in macrophage number, whereas IL-8 increased in subjects who demonstrated the greatest decrease in macrophages.
FO in adipose could directly affect macrophages or adipocytes, or could be secondary to other systemic effects. To examine a direct effect, THP-1 cells were polarized into M1, M2a, and M2c macrophages, as described in Research Design and Methods. The MCP-1 gene was highly expressed in M1 macrophages, with much lower expression by M2 macrophages (Fig. 5A). When DHA was added to cultures of M1 macrophages, there was a dose-dependent decrease in MCP-1 expression, even though there was no change in TNF-α (Fig. 5B). Further experiments were performed in M1 macrophages to determine the specificity of the effects of DHA. M1 macrophages were treated with different fatty acids: α-linolenic acid (ALA), DHA, EPA, and linoleic, oleic, and palmitic acids, all at 100 μmol/L, followed by the measurement of MCP-1 and TNF-α expression. As shown in Fig. 5C, all the ω-3 PUFAs induced a significant reduction in MCP-1 expression, and there was no effect of linoleic, oleic, or palmitic acids. In contrast, the ω-3 PUFAs had no significant effect on M1 macrophage TNF-α expression (Fig. 5D), whereas TNF-α expression was significantly increased by linoleic and palmitic acids. To examine the effects on adipocytes, ADHASC cells were induced to differentiate. Adipocytes secreted low levels of MCP-1 when cultured alone (Fig. 5A), but there was a six- to ninefold upregulation in expression in response to coculture with macrophages, and adipocyte expression of CTGF was upregulated twofold by macrophage coculture. The addition of DHA significantly diminished the expression of both MCP-1 (Fig. 5C) and CTGF (Fig. 5D) in adipocytes in coculture with each type of polarized macrophage.
(Enlarge Image)
Figure 5.
Effects of DHA on adipocytes and macrophages in tissue culture. A: MCP-1 expression by adipocytes and polarized macrophages. B: Effects of the addition of increasing concentrations of DHA to TNF-α and MCP-1 expression in M1 macrophages. *P < 0.05 vs. MCP-1. The following lipids were conjugated to BSA and added at a concentration of 100 μmol/L to M1 macrophages in culture: ALA, DHA, EPA, and linoleic, oleic, and palmitic acids. After 24 h, cells were harvested followed by the measurement of expression of MCP-1 (C) and TNF-α (D). *P < 0.05 vs. control. DHA (100 μmol/L) was added to the coculture of adipocytes with polarized macrophages followed by measurement of expression of MCP-1 (E) and CTGF (F). *P < 0.05 vs. control. Data are expressed as mean ± SEM. A.U., arbitrary units.
Results
The effects of drug or placebo treatment on standard clinical outcomes are shown in Table 2. Although the subjects in this study demonstrated modestly elevated baseline triglycerides, there was a small but significant decrease in triglycerides in the FO treatment group and no change with placebo, and there were no other significant changes in serum lipids between the groups. The oral glucose tolerance test was performed before and after treatment, and 23 subjects were either impaired glucose tolerance or impaired fasting glucose before randomization. After FO or placebo treatment, no changes were observed in fasting or 2-h glucose, insulin sensitivity (SI), or first-phase insulin secretion (AIRglu) (measured using the frequently sampled intravenous glucose tolerance test) in either group.
Previous studies have demonstrated that erythrocyte ω-3 PUFA content is altered by FO administration, but fewer studies have examined adipose tissue or muscle levels of ω-3 fatty acids in response to treatment. To characterize adipose tissue and muscle lipid content and determine the impact of the treatment groups, tissue total lipids were analyzed using mass spectrometry, as described in research design and methods. The distribution of total adipose and muscle lipids is shown in Supplementary Table 2. The vast majority of adipose tissue lipid was oleate (18:1, n-9), palmitate (16:0), and linoleate (18:2, n-6); the ω-3 PUFA linolenic acid, eicosapenanoic acid (EPA), and DHA constituted 1.1, 0.26, and 0.40% (by weight) of adipose lipids, respectively. In muscle, the relative abundance of lipids was somewhat different, with proportionally more of the saturated lipids palmitate and stearate and very low levels of EPA and DHA (Supplementary Table 2). Figure 1 shows the response of adipose and muscle EPA and DHA to FO and placebo treatments. All subjects treated with FO demonstrated an increase in tissue EPA and DHA, whereas there was no change in the placebo-treated subjects. Further, the FO-treated subjects demonstrated no changes in any other fatty acid. Although the placebo-treated subjects were receiving 4 g/day of corn oil, which is composed predominantly of linoleate, oleate, and palmitate, there was no significant change in any lipid component in the adipose tissue or muscle in these subjects.
(Enlarge Image)
Figure 1.
FO treatment increased adipose and muscle EPA and DHA. Adipose (A) and muscle (B) lipids were analyzed in biopsy samples before and after treatment with FO or placebo. There were no significant changes in the placebo-treated subjects, and data are expressed in relation to oleate, which is abundant in both tissues. The changes in EPA and DHA were significant (P < 0.05) compared with baseline and with placebo-treated subjects. Data are expressed as mean ± SEM.
Table 3 illustrates the measurements of a number of different blood adipokine levels. Previous studies have demonstrated that FO can invoke a PPARγ effect in vitro, and when given to animals. However, in these subjects, no changes in adiponectin were observed. In addition, no changes in the levels of IL-6, IL-10, IL-12, TNF-α, resistin, PAI-1, or leptin were noted. However, a significant decrease in the blood MCP-1 level was observed.
To further examine changes in inflammation, fibrosis, and vascularity, adipose tissue and muscle from the FO- and placebo-treated subjects were examined using histochemistry and immunohistochemistry. As shown in Fig. 2, there was a significant decrease in macrophage number in adipose after FO treatment and no change with placebo. Crown-like structures are clusters of macrophages, often involving giant cells, surrounding a necrotic adipocyte (large arrow in Fig. 2). After FO treatment, there was a significant decrease in crown-like structures, but there was no change in placebo-treated subjects. Macrophage number was also assessed in muscle, and no significant changes were observed in either group (data not shown).
(Enlarge Image)
Figure 2.
Effects of FO treatment on adipose macrophages and capillaries. Before and after treatment with FO, adipose tissue from biopsies was analyzed histochemically. A: CD68 staining. A representative image showing macrophages (small arrows) and crown-like structures (large arrow). Data are expressed as mean ± SEM. B: Effects of FO and placebo on macrophage number (*P < 0.05 vs. pretreatment). C: Effects of treatment on the number of crown-like structures (CLS) (*P < 0.05 vs. pretreatment). D: Capillary and large vessels were identified by staining with lectin and α-smooth muscle actin, and representative images are shown. E: Effects of placebo and FO on the number of capillaries in adipose tissue.
Previous studies have found that the adipose from obese, insulin-resistant subjects contained fewer capillaries and more large vessels than lean, insulin-sensitive subjects. To determine whether the treatment of subjects with FO altered vascularity, adipose tissue capillaries and larger vessels were quantified. As shown in Fig. 2, there was a small but consistent and significant increase in adipose capillaries, with no change in placebo-treated subjects. The number of large blood vessels was unchanged in both groups. There were no significant changes in muscle capillaries after FO treatment (data not shown).
The adipose tissue of obese, insulin-resistant subjects contains more collagen VI, along with other changes in the ECM. The overall degree of fibrosis in the adipose tissue of FO- and placebo-treated subjects was examined using histochemistry. No changes in overall fibrosis or collagen VI were noted (data not shown).
To analyze changes in gene expression in adipose tissue, RNA was extracted from the adipose tissue of FO- and placebo-treated subjects. As shown in Fig. 3, there were significant changes in MCP-1 and CD68 mRNA levels in the whole group of subjects. Although there was an overall decrease in adipose tissue macrophages after FO, there was considerable variation between subjects, which was strongly related to the degree of adipose tissue inflammation at baseline. As shown in Fig. 4A, the baseline adipose tissue macrophage number was strongly correlated with the change in macrophage number after FO treatment, suggesting that the subjects with the most inflammation benefitted the most from FO treatment.
(Enlarge Image)
Figure 3.
Changes in gene expression in adipose tissue after FO treatment. A: MCP-1. B: CD68. *P < 0.05 vs. pretreatment. Data are expressed as mean ± SEM. A.U., arbitrary units.
(Enlarge Image)
Figure 4.
Correlation between the number of macrophages in adipose at baseline, and the change after FO treatment. A: Change in macrophage number was associated with baseline macrophages. The expression of other genes was significantly associated with the change in macrophage number, and these include CTGF (B), IL-8 (C), and TIMP-2 (D). All correlations are significant at P < 0.05. A.U., arbitrary units.
To obtain a broader view of gene expression changes in response to FO, 116 genes (Supplementary Table 1) involving various aspects of inflammation, ECM, and vascularity were analyzed using NanoString in nine placebo and nine FO-treated subjects. Somewhat surprisingly, despite a significant decrease in macrophage number, and an increase in capillaries, there were no significant changes in gene expression, except for the previously described changes in MCP-1 and CD68. Specifically, there were no significant changes in the expression of the classic adipokines, including TNF-α, IL-1, IL-12, and IL-6. Although there were no net changes, the expression of several genes varied with the changes in macrophage number. For example, the change in expression of CTGF, IL-8, and TIMP-2 correlated significantly with the change in macrophage number with FO treatment (Fig. 4). CTGF and TIMP-2 decreased in parallel with a decrease in macrophage number, whereas IL-8 increased in subjects who demonstrated the greatest decrease in macrophages.
FO in adipose could directly affect macrophages or adipocytes, or could be secondary to other systemic effects. To examine a direct effect, THP-1 cells were polarized into M1, M2a, and M2c macrophages, as described in Research Design and Methods. The MCP-1 gene was highly expressed in M1 macrophages, with much lower expression by M2 macrophages (Fig. 5A). When DHA was added to cultures of M1 macrophages, there was a dose-dependent decrease in MCP-1 expression, even though there was no change in TNF-α (Fig. 5B). Further experiments were performed in M1 macrophages to determine the specificity of the effects of DHA. M1 macrophages were treated with different fatty acids: α-linolenic acid (ALA), DHA, EPA, and linoleic, oleic, and palmitic acids, all at 100 μmol/L, followed by the measurement of MCP-1 and TNF-α expression. As shown in Fig. 5C, all the ω-3 PUFAs induced a significant reduction in MCP-1 expression, and there was no effect of linoleic, oleic, or palmitic acids. In contrast, the ω-3 PUFAs had no significant effect on M1 macrophage TNF-α expression (Fig. 5D), whereas TNF-α expression was significantly increased by linoleic and palmitic acids. To examine the effects on adipocytes, ADHASC cells were induced to differentiate. Adipocytes secreted low levels of MCP-1 when cultured alone (Fig. 5A), but there was a six- to ninefold upregulation in expression in response to coculture with macrophages, and adipocyte expression of CTGF was upregulated twofold by macrophage coculture. The addition of DHA significantly diminished the expression of both MCP-1 (Fig. 5C) and CTGF (Fig. 5D) in adipocytes in coculture with each type of polarized macrophage.
(Enlarge Image)
Figure 5.
Effects of DHA on adipocytes and macrophages in tissue culture. A: MCP-1 expression by adipocytes and polarized macrophages. B: Effects of the addition of increasing concentrations of DHA to TNF-α and MCP-1 expression in M1 macrophages. *P < 0.05 vs. MCP-1. The following lipids were conjugated to BSA and added at a concentration of 100 μmol/L to M1 macrophages in culture: ALA, DHA, EPA, and linoleic, oleic, and palmitic acids. After 24 h, cells were harvested followed by the measurement of expression of MCP-1 (C) and TNF-α (D). *P < 0.05 vs. control. DHA (100 μmol/L) was added to the coculture of adipocytes with polarized macrophages followed by measurement of expression of MCP-1 (E) and CTGF (F). *P < 0.05 vs. control. Data are expressed as mean ± SEM. A.U., arbitrary units.