Counteract of bone marrow of blotchy mice against the increases of plasma copper levels induced by high-fat diets in LDLR-/- mice

Abstract

Background: Bone marrow of blotchy mouse (blotchy marrow) reflects the function of transmembrane domain and relevant intramembrane sites of ATP7A in myeloid cells. By chronic infusion of angiotensin II, we previously found that blotchy marrow plays a minor role in regulating plasma copper. Moreover, the recipients of blotchy marrow presented a moderate reduction of plasma lipids and inflammatory mediator production. Little is known about whether these changes are a specific response to angiotensin II or reveal a more general role of ATP7A.

Objective and design: We investigated if blotchy marrow reduces plasma lipids and inflammatory mediators induced by high-fat diets. To test this hypothesis, blotchy and control marrows were reconstituted to the recipient mice (irradiated male LDLR-/- mice), followed by high-fat-diet feeding for 4 months. At the end points, plasma metals (copper, zinc and iron), lipid profiling (cholesterol, triglyceride, phospholipids and lipoprotein) and six inflammatory mediators (lymphotacin, MCP3, MCP5, TIMP1, VEGF-A and IP-10) were measured. Parallel experiments were performed using male LDLR-/- mice fed either high-fat diets or chow diets for 4 months.

Results: In addition to hyperlipidemia and low-grade inflammation, high-fat diets selectively increased plasma copper concentration compared to chow diets in LDLR-/- mice. After high-fat-diet feeding, the recipients with blotchy marrow showed a decrease in plasma copper (p < 0.01) and an increase in plasma iron (p < 0.05). The recipients with blotchy marrow also presented decreases in cholesterol (p < 0.01) and phospholipids (p < 0.05) in plasma. Surprisingly, plasma levels of MCP3 (p < 0.05), MCP5 (p < 0.05), TIMP1 (p < 0.01), VEGF-A (p < 0.01) and IP-10 (p < 0.01) were significantly increased in the recipients with blotchy marrow compared to controls; the increased levels of MCP3, MCP5 and TIMP1 were more than 50%.

Conclusion: Our studies showed that blotchy marrow counteracts the increased copper levels induced by high-fat diets, indicating that circulating myeloid cells can regulate blood copper levels via ATP7A. Moreover, transplantation of blotchy marrow followed by high-fat diets leads to a decrease in lipid profile and an increase in inflammatory mediator production. Overall, blotchy marrow mediates divergent responses to angiotensin II and high-fat diets in vivo.

Keywords: ATP7A; Bone marrow; Copper; Inflammation; Lipid metabolism.

Fig. 1

Concentrations of triglycerides, total cholesterol and phospholipids in the plasma of LDLR−/− mice reconstituted with control or blotchy marrow following by 4 mon of high-fat feeding. Fifteen microliters of plasma (nonfasting) was used to determine the concentrations of triglycerides, total cholesterol and phospholipids through specific colorimetric assays. Dots and squares represent values from individual mice, thin long lines are mean values of each group, and bars are SE. n = 4–5 for each group. *p < 0.05 and **p <0.01.

Fig. 2

Plasma lipoproteins in LDLR−/− mice reconstituted with control or blotchy marrow following by 4 mon of high-fat feeding. Pooled plasma from each group (nonfasting) was centrifuged at d = 1.215 g/mL, the lipoprotein fractions (d < 1.215 g/mL) were subjected to FPLC, and the total cholesterol and triglyceride content of each fraction was measured using an enzymatic assay kit.

Fig. 3

Concentrations of inflammatory mediators in the plasma of LDLR−/− mice reconstituted with control or blotchy marrow following 4 mon of high-fat feeding. Six inflammatory mediators were detected in plasma using a bead-based multiplexing immunoassay as previously described [33]. Dots and squares represent values from individual mice, thin long lines are mean values of each group, and bars are SE. n = 4–5 for each group. *p < 0.05 and **p <0.01.

Fig. 4

Concentration of copper, iron and zinc in plasma of LDLR−/− mice reconstituted with control or blotchy marrow following 4 mon of high-fat feeding. Fifty microliters of plasma were analyzed to determine biometal concentrations via inductively coupled plasma mass spectrometry. Three replicate measurements were performed for each sample, and the final reported concentration was the mean value of these three replicates. Dots and squares represent values from individual mice, thin long lines are mean values of each group, and bars are SE. n = 4–5 for each group. *p <0.05 and **p <0.01.