Sexual Differentiation, Pregnancy, Calorie Restriction, and Aging Affect the Adipocyte-Specific Secretory Protein Adiponectin

Increased insulin sensitivity in female FVB mice. Plasma glucose (A) and plasma insulin levels (B) were measured in young male and female FVB mice. C: Insulin tolerance test in young male and female FVB mice. Recombinant insulin at equal amounts per body weight were injected (0.5 units/kg body wt) and glucose was measured at the designated time points. Two-way ANOVA using sex, glucose, and time as independent variables identified significant (P < 0.05) effects of sex. *Significant (P < 0.05) effects of sex.

A: Acrp30 mRNA levels in adipose tissue in adult male ([cjs2108]; n = 3) and female (▪; n = 3) FVB mice. Total RNA was extracted from dorsal subcutaneous white adipose tissue (WAT), interscapular brown adipose tissue (BAT), and epididymal or perimetrial/ovarian white adipose tissue and analyzed for Acrp30 mRNA by Northern blot. Two-way ANOVA using fat depot and sex as independent variables identified a significant (P < 0.05) effect of fat depot in males and females but no effect of sex. Representative 1.2-kb Northern bands are shown below the bar graph. Subcut., subcutaneous. *Significant (P < 0.05) effect of fat depot. B: Intracellular Acrp30 levels in adipose tissue from adult ([cjs2108]; n = 3) and female (▪; n = 3) FVB mice. Equal amounts of protein (50 μg) from various fat depots were analyzed for Acrp30 by Western blot analysis using rabbit antisera against murine Acrp30 and [I-125]-labeled anti-rabbit IgGs. Each gel contained four standards of purified mouse Acrp30 at four different concentrations to generate absolute values and ensure linearity and reproducibility of the signal. Two-way ANOVA using sex and fat depots as independent variables identified significant (P < 0.05) effects of sex in each depot as well as a significant effect of depot in the case of gonadal WAT. A representative Western blot is shown below the bar graph. Subcut., subcutaneous. *Significant (P < 0.05) effect of sex; †significant (P < 0.05) effect of depot.

A: The effects of neonatal CX on Acrp30 levels in adult male Long-Evans rats. Blood was sampled from adult male rats (100 days old) that were either intact (Male; body weight = 488 ± 20, n = 10), castrated at 2–4 days after birth (CX-Neonate; body weight = 379 ± 9, n = 10), or castrated at 60–70 days after birth (CX-Adult; body weight = 436 ± 19, n = 10). Plasma samples from sham-operated animals (S-CX-Neonate and S-CX-Adult) and ovariectomized female (Female) rats were included for comparison. Plasma was analyzed for Acrp30 by Western blot analysis as previously described. Plasma leptin values were 5.5 ± 0.7 (Male), 9.2 ± 0.9 (CX-Neonate), 4.7 ± 0.5 (CX-Adult), and 7.6 ± 0.8 ng/ml (Female), respectively. One-way ANOVA using effect of treatment as the independent variable identified a significant (P < 0.05) effect of neonatal CX. The effect of sex was not significant between adult females and males that underwent neonatal CX. *Significant (P < 0.05) effect of neonatal CX. B: The effects of CX and glucocorticoid treatment on Acrp30 levels. Mice were 6 months old at the start of the experiment. CX and prednisolone (PRDSN) pellet implantation were performed at the same time. Slow-release pellets of 2.1 mg · kg⁻¹ · day⁻¹ prednisolone were implanted, and blood was sampled for Acrp30 28 days later. Blood was sampled from intact mice (CTL; n = 16), castrated (CX; n = 8), prednisolone-treated (PRDSN; n = 13) and the combination of CX and prednisolone-treatment (CX & PRDSN; n = 11). One-way ANOVA using effect of treatment as the independent variable identified significant (P < 0.05) effects of prednisolone treatment as well as the combination of prednisolone treatment and CX. *Significant (P < 0.05) effects of prednisolone treatment as well as the combination of prednisolone treatment and CX. C: Effects of CR and aging in male mice. Blood samples were obtained for Acrp30 measurement at 5 months (YOUNG; n = 5) and after 24–30 months (OLD; n = 5). F1 mice (C57BLxBALB) used here were maintained at The Jackson Laboratories. At 2 months after birth, calorie-restricted mice (▪) started receiving 60% of the baseline intake of the ad libitum-fed group (light bars). Feed intake and spillage were measured routinely for all mice, and adjustments in feed allotments were made to ensure that calorie-restricted mice received 60% of their individual baseline intake. Blood samples were obtained for Acrp30 measurement at 5 months (YOUNG) and after 24–30 months (OLD). Two-way ANOVA using effects of CR and age as the independent variables identified a significant (P < 0.05) effect of calorie-restriction. *Significant (P < 0.05) effect of calorie-restriction. D: Effects of caloric-restriction and aging on male Acrp30 mRNA levels. Gonadal fat pads from the mice used in panel C were used for RNA isolation and Northern blot analysis. mRNA levels for Acrp30 remain unchanged upon normalization with a β-actin probe. AL, ad libitum-fed.

A: Effects of OVX and elevated estrogen exposure on plasma Acrp30. Blood was sampled from adult FVB mice at 55 days of age that were intact (CTL; n = 5), mice that underwent bilateral OVX 7 days after birth or were sham operated (SOVX; n = 5 each), mice that underwent bilateral OVX at 45 days old (ADULTS) or were sham operated (S-OVX; n = 5 each), and adult mice that underwent a combination of bilateral OVX and E2 implants (OVX & E2) or vehicle (n = 5 each). One-way ANOVA using effect of treatment as the independent variable identified significant (P < 0.05) effects of bilateral OVX during infancy and adulthood as well as the combination of bilateral OVX and E2 treatment. *Significant (P < 0.05) effects of bilateral OVX during infancy and adulthood; **significant (P < 0.05) effects of the combination of bilateral OVX and E2 treatment. B: Effects of CR and aging in female mice. Blood samples were obtained for Acrp30 measurement at 5 months (YOUNG; n = 5) and after 24–30 months (OLD; n = 5). F1 mice (C57BLxBALB) used here were maintained at The Jackson Laboratories. At 2 months after birth, calorie-restricted mice (▪) started receiving 60% of the baseline intake of the ad libitum-fed group (light bars). Feed intake and spillage were measured routinely for all mice, and adjustments in feed allotments were made to ensure that calorie-restricted mice received 60% of their individual baseline intake. Blood samples were obtained for Acrp30 measurement at 5 months (YOUNG) and after 24–30 months (OLD). Two-way ANOVA using effects of CR and age as the independent variables identified a significant (P < 0.05) effect of CR only in young females. *Significant (P < 0.05) effect of CR. C: Effects of CR and aging on female Acrp30 mRNA levels. Gonadal fat pads from the mice used in B were used for RNA isolation and Northern blot analysis. mRNA levels for Acrp30 remain unchanged upon normalization with a β-actin probe. AL, ad libitum-fed.

A: Acrp30 levels in 3T3-L1 adipocytes after overnight E2 exposure. Top panel: Intracellular Acrp30 levels in 3T3-L1 adipocytes after overnight E2 exposure in the presence ([cjs2108]) or absence (▪) of the ER antagonist (ICI 182,780). Differentiated 3T3-L1 adipocytes (day 8) were exposed to 10% FCS containing 0, 1, 10, and 100 ng/ml of E2 with or without 100 nmol/l ICI 182,780 for 12 h. Cells were then washed twice with cold PBS and lysed in TNET buffer. Equivalent amounts (50 μg) of total protein extract were analyzed by Western blot analysis as previously described. Numbers on the y-axis represent arbitrary units. Bottom panel: Extracellular Acrp30 levels. Cells were treated as described in the top panel; after the 12-h treatment, the medium was replaced with serum-free Dulbecco’s modified Eagle’s medium, and cells were allowed to secrete for an additional 3 h in the continued presence of the indicated E2 concentrations. The medium was subsequently analyzed by Western blot analysis for the levels of Acrp30. B: ER expression in differentiated adipocytes (day 8), confluent murine 3T3-L1 preadipocytes (day 0), and uterus. Cell lysates were incubated with protein A-Sepharose or rabbit anti-murine ER immune or preimmune sera. Extracts were analyzed by enhanced chemiluminescence on 10% SDS-PAGE for ER using a mouse monoclonal antibody. Total uterine protein extract was included as a positive control. MWM, molecular weight markers. C: Estrogen directly represses Acrp30 levels in 3T3-L1 adipocytes. Mature (day 8) 3T3-L1 adipocytes were incubated for 12 h in either the presence or absence of 100 ng/ml of E2 and 300 μmol/l cycloheximide (CHX), as indicated. Total RNA was subsequently extracted and analyzed by Northern blot analysis with a probe for mouse Acrp30. 18S and 28S RNA were visualized on a separate gel to ensure equal loading. CTL, control.

A: Circulating Acrp30 levels during gestation and lactation. Serial blood samples were obtained from young female FVB (n = 5) mice before mating; during the first, second, or third trimesters of pregnancy; and after 1 week of lactation. One-way ANOVA using the effect of the individual stage of reproduction as the independent variable identified significant (P < 0.05) effects in the second and third trimesters of pregnancy. *Significant (P < 0.05) effects. **Significant effect evident with 1 week of lactating compared with the last trimester of pregnancy. B: Circulating Acrp30 levels during gestation in response to CR. Samples were obtained from tail vein in the morning of days 12.5 and 18.5 of pregnancy in C57BL mice. On day 12.5, animals were separated into two groups, with one group receiving ∼50% (▪; n = 6) of the calories consumed by the ad libitum-fed group (light bars; n = 6). Two-way ANOVA using effects of CR and stage of pregnancy as the independent variables identified a significant (P < 0.05) effect of pregnancy but not CR. *Significant (P < 0.05) effect of pregnancy.