(A) Total RNA (15 μg) extracted from 3T3-L1 adipocytes cultured with or without glucose for 36 hours was subjected to Northern blot analysis with a probe specific for mouse MCP-1 mRNA. The region of the ethidium bromide–stained gel containing 28S rRNA is also shown. (B) The culture supernatants of 3T3-L1 adipocytes cultured with or without glucose for 36 hours were assayed for MCP-1. Data are mean ± SEM (n = 18). *P < 0.01 versus culture with glucose.

(A) Total RNA was extracted from the indicated tissues of 8-week-old db/db or db/+m mice and subjected to Northern blot analysis with a probe specific for mouse MCP-1 mRNA. WAT, white adipose tissue; BAT, brown adipose tissue. (B) Plasma concentration of MCP-1 in 11-week-old db/+m and db/db mice. Data are mean ± SEM (db/+m, n = 8; db/db, n = 11). *P < 0.05 versus db/+m. (C) Total RNA, extracted from the indicated tissues of 18-week-old C57BL/6J mice fed either a high-fat diet (HFD) or normal chow for 12 weeks, was subjected to Northern blot analysis with a probe specific for MCP-1 mRNA. (D) Plasma concentration of MCP-1 in 18-week-old C57BL/6J mice fed normal chow or the high-fat diet for 12 weeks. Data are mean ± SEM (normal chow, n = 11; high-fat diet, n = 9). *P < 0.05 versus normal chow. (E) Total RNA (15 μg), extracted from the SVF and adipocyte fraction (Adipo) of epididymal fat tissue from 11-week-old db/db mice or 18-week-old C57BL/6J mice fed a high-fat diet for 12 weeks, was subjected to Northern blot analysis with a probe specific for MCP-1 mRNA. The intensity of the band corresponding to MCP-1 mRNA in each fraction was quantitated and expressed relative to the value for the SVF of mice fed a high-fat diet. Data are mean ± SEM of values from 3 independent experiments (n = 3).

(A) Northern blot analysis with an MCP-1 probe of total RNA isolated from various tissues of 11-week-old MCP-1 Tg-B mice. (B) Plasma concentration of MCP-1 in 13-week-old MCP-1 Tg-B and WT mice. Data are mean ± SEM (n = 7). (C) Immunohistochemical detection of Mac3 in epididymal adipose tissue of 11-week-old MCP-1 Tg-B and WT mice. Macrophages are stained brown. Magnification, ×200 and ×400, as indicated. Scale bars: 50 μm. (D) Macrophage infiltration into epididymal fat tissue was quantitated in MCP-1 Tg-B (n = 7) and WT mice (n = 9) as the ratio of Mac3-positive cells to total cells. Data are mean ± SEM. (E) Quantitation by flow cytometry of the proportion of CD11b⁺CD45⁺ cells (macrophages) in the SVF of epididymal fat tissue from 14-week-old MCP-1 Tg-B (n = 7) and WT mice (n = 6). Data are mean ± SEM. (F) Quantitative RT-PCR analysis of total RNA isolated from epididymal fat tissue of 11-week-old MCP-1 Tg-B and WT mice for TNF-α, CD68, and F4/80 mRNAs. Data (mean ± SEM; n = 4) were normalized by the amount of 36B4 mRNA and expressed relative to the corresponding WT value. *P < 0.05, **P < 0.01 versus WT.

(A) Metabolic parameters of 11-week-old MCP-1 Tg-B and WT mice. Data are means ± SEM (body weight, n = 5; food intake, n = 3 [WT], 4 [Tg-B]; plasma glucose, n = 9 [WT], 11 [Tg-B]; serum FFA, n = 9 [WT], 11 [Tg-B]; plasma insulin, n = 5; serum triglyceride, n = 4 [WT], 6 [Tg-B]; serum total cholesterol, n = 9 [WT], 11 [Tg-B]; serum adiponectin, n = 5). (B) Plasma glucose level during insulin (upper panel) and glucose (lower panel) tolerance tests in WT and MCP-1 Tg-B mice at 12 to 13 weeks of age. Data are mean ± SEM (n = 5). (C) Hyperinsulinemic-euglycemic clamp analysis in MCP-1 Tg-B (n = 5) and WT (n = 7) mice. Data are mean ± SEM. GIR, glucose infusion rate; Rd, rate of glucose disappearance; BHGP, basal hepatic glucose production; CHGP, hepatic glucose production during clamp. (D) Hepatic triglyceride content of 11-week-old MCP-1 Tg-B and WT mice. Data are mean ± SEM (n = 5). (E) Quantitation by Northern blot of the abundance of PEPCK, G6Pase, and SREBP-1c mRNAs in the liver of MCP-1 Tg-B and WT mice after hyperinsulinemic-euglycemic clamp analysis. Data (mean ± SEM; PEPCK and SREBP-1c, n = 5 [WT], 4 [Tg-B]; G6Pase, n = 5.) are expressed relative to the corresponding value for WT mice. *P < 0.05, **P < 0.01 versus WT.

(A) Weight of various white and brown adipose tissues from the KO and WT mice fed a high-fat diet from 12 to 24 weeks of age. Data are mean ± SEM (n = 5). (B) Size distribution of adipocytes in epididymal fat tissue of mice fed a high-fat diet from 12 to 24 weeks of age. Data are means from analysis of 5 sections from each of 5 mice. (C) Immunohistochemical detection of Mac3 in epididymal adipose tissue of mice fed a high-fat diet from 12 to 24 weeks of age. Magnification, ×200 and ×400, as indicated. Scale bars: 50 μm. (D) Macrophage infiltration into epididymal fat tissue. Data are mean ± SEM (WT, n = 8; KO, n = 6). (E) Quantitation by flow cytometry of the proportion of macrophages in the SVF of epididymal fat tissue from mice fed a high-fat diet from 12 to 28 weeks of age. Data are mean ± SEM (WT, n = 4; KO, n = 6). (F) Abundance of macrophage-related protein mRNAs in epididymal fat tissue of mice fed a high-fat diet from 12 to 24 weeks of age. Data (mean ± SEM; n = 4) were normalized by the amount of 36B4 mRNA and expressed relative to the corresponding WT value. *P < 0.05, **P < 0.01 versus WT.

(A) Metabolic parameters of mice fed a high-fat diet from 12 to 24 weeks of age. Data are means ± SEM (body weight, n = 5 [WT], 9 [KO]; food intake, n = 4; plasma glucose, n = 5; serum FFA, n = 6 [WT], 5 [KO]; plasma insulin, n = 7 [WT], 6 [KO]; serum triglyceride, n = 7 [WT], 6 [KO]; serum total cholesterol, n = 6 [WT], 5 [KO]; serum adiponectin, n = 7 [WT], 5 [KO]). (B) Plasma glucose level during insulin (upper panel) and glucose (lower panel) tolerance tests in mice fed a high-fat diet from 12 to 24 weeks of age. Data are mean ± SEM (n = 6). (C) Hyperinsulinemic-euglycemic clamp analysis in mice fed a high-fat diet from 12 to 28 weeks of age. Data are mean ± SEM (WT, n = 6; KO, n = 5). (D) Hepatic triglyceride content of mice fed a high-fat diet from 12 to 28 weeks of age. Data are mean ± SEM (n = 5). (E) Quantitation by Northern blot of the hepatic abundance of PEPCK, G6Pase, and SREBP-1c mRNAs in mice after hyperinsulinemic-euglycemic clamp analysis. Data (mean ± SEM; n = 5) were expressed relative to the corresponding WT value. *P < 0.05, **P < 0.01 versus WT.

(A) Insulin tolerance test in db/db mice determined insulin sensitivity 21 days after transfection of 8-week-old animals with the 7ND vector (n = 10) or the corresponding empty plasmid as a control (Con, n = 6). (B) Insulin (left) and glucose (right) tolerance tests performed 21 days after transfection of 8-week-old db/+m mice with the 7ND vector (n = 8) or the corresponding empty plasmid (n = 7). (C) Insulin (left) and glucose (right) tolerance tests performed 21 days after transfection of 30-week-old C57BL/6J mice fed a high-fat diet since 6 weeks of age with either the 7ND expression vector or the corresponding empty plasmid. Data are mean ± SEM (insulin tolerance test, n = 4; glucose tolerance test, n = 5 [7ND], 7 [empty vector]). (D) Hyperinsulinemic-euglycemic clamp analysis of db/db mice performed 21 days after transfection of 8-week-old animals with the 7ND vector (n = 4) or the corresponding empty plasmid (n = 5). Data are mean ± SEM. (E) Appearance of the liver (left) and hepatic triglyceride content (n = 4, right) in db/db mice 21 days after injection of 8-week-old animals with the 7ND expression plasmid or the corresponding empty vector. *P < 0.05, **P < 0.01 versus mice injected with the empty vector.