Adipocyte iron regulates adiponectin and insulin sensitivity

Abstract

Iron overload is associated with increased diabetes risk. We therefore investigated the effect of iron on adiponectin, an insulin-sensitizing adipokine that is decreased in diabetic patients. In humans, normal-range serum ferritin levels were inversely associated with adiponectin, independent of inflammation. Ferritin was increased and adiponectin was decreased in type 2 diabetic and in obese diabetic subjects compared with those in equally obese individuals without metabolic syndrome. Mice fed a high-iron diet and cultured adipocytes treated with iron exhibited decreased adiponectin mRNA and protein. We found that iron negatively regulated adiponectin transcription via FOXO1-mediated repression. Further, loss of the adipocyte iron export channel, ferroportin, in mice resulted in adipocyte iron loading, decreased adiponectin, and insulin resistance. Conversely, organismal iron overload and increased adipocyte ferroportin expression because of hemochromatosis are associated with decreased adipocyte iron, increased adiponectin, improved glucose tolerance, and increased insulin sensitivity. Phlebotomy of humans with impaired glucose tolerance and ferritin values in the highest quartile of normal increased adiponectin and improved glucose tolerance. These findings demonstrate a causal role for iron as a risk factor for metabolic syndrome and a role for adipocytes in modulating metabolism through adiponectin in response to iron stores.

Figures

Figure 1. Serum ferritin levels are inversely associated with serum adiponectin ferritin levels and predict presence or absence of MetS.

(A) Serum adiponectin and ferritin levels were measured and correlated in a cohort of subjects with normal ferritin. P = 0.0003. (B) Ferritin levels in the same subjects subdivided by diabetes status. *P < 0.0004. (C) Adiponectin levels in the same subjects subdivided by diabetes status. *P < 0.0012. (D) Ferritin levels in the obese subjects without MetS (white bars) or with MetS, without or with diabetes (gray and black bars, respectively). *P < 0.03 for the diabetic subgroup compared with either other group. (E) Adiponectin levels in the subset of the subjects described D in whom adiponectin levels were determined. *P < 0.03 for either MetS subgroup compared with the non-MetS group.

Figure 2. Serum adiponectin and adipocyte mRNA levels decrease with dietary iron overload and with iron treatment in 3T3-L1 cells.

(A) Tfrc mRNA quantified by RT-PCR and normalized to cyclophilin in collagenased adipocytes from epididymal fat pads of mice fed normal chow (NC) or a high-iron diet (HI) for 8 weeks. *P < 0.05. (B) Serum adiponectin levels were measured in 7-month-old 129/SvEvTac background mice following 2 months of being fed low-iron (7 mg/kg carbonyl iron), normal chow (330 mg/kg), or high-iron (20 g/kg) diets. *P = 0.004, low iron vs. normal chow; ‡P = 0.0002, high iron vs. normal chow. (C) Adiponectin mRNA levels in isolated epididymal adipocytes from mice fed high-iron diet or normal chow. P = 0.07. (D) Body weights were determined in C57BL6/J mice after 8 weeks on normal chow or high-iron diets, and body composition was determined by magnetic resonance imaging (n = 6/group or 8/group, *P < 0.05, ‡P < 0.01). (E) Body weights were determined in mice with knockout of the adiponectin gene compared with those of controls after 8 weeks on normal chow or high-iron diets (n = 5–12/group, ‡P < 0.001). (F) Euglycemic hyperinsulinemic clamps were performed on WT mice on normal or high-iron diets. Glucose infusion rates (GIRs) trended lower when normalized to total body weight (P = 0.22) but differed significantly when normalized to lean body mass (‡P < 0.0005).

Figure 3. Transcriptional regulation of adiponectin by iron.

(A) Media adiponectin levels in 3T3-L1 cells 12 hours following 12-hour pretreatment. P < 0.0001. (B) RT-PCR quantification of adiponectin mRNA levels in 3T3-L1 adipocytes treated with no iron or 100 μM FeSO4 for 24 hours, normalized to cyclophilin A. *P = 0.02. (C) Adiponectin promoter-driven luciferase activity in the presence or absence of 100 μM FeSO4. ‡P = 0.0025. (D) Western blot for acetylated FOXO1 (Ac-FOXO1), phosphorylated FOXO1 (P-FOXO1), total FOXO1 (Tot-FOXO1), and β-actin in 3T3-L1 adipocytes treated with no iron or 100 μM FeSO4 for 8 hours. (E) Quantitation of Western blots (total n = 6 independent determinations) normalized to β-actin. *P < 0.05. (F) Quantitation of Western blots for phosphorylated AKT in 3T3-L1 adipocytes treated with no iron or 100 μM FeSO4 for 8 hours and insulin (10 nM) for 1 hour. (G) ChIP showing FOXO1 occupancy of adiponectin promoter FOXO1 sites and PPRE and PPARγ occupancy of PPRE in 3T3-L1 adipocytes (n = 3 experiments each assayed in duplicate, *P < 0.05). (H) Immunoprecipitation of 3T3-L1 adipocyte extracts, treated overnight in the presence or absence of 100 μM FeSO4, by antibodies to FOXO1, followed by immunoblotting for FOXO1 (t-FOXO1) and C/EBPα (0.58 ± 0.15 density units for control, 0.61 ± 0. 26 density units for iron-treated extracts, P = 0.93).

Figure 4. Functional expression of ferroportin in adipocytes.

(A) Fpn1 mRNA levels quantified by RT-PCR in 3T3-L1 adipocytes exposed to different concentrations of iron (FeSO4) in the culture medium. *P < 0.05 compared with 0.1 mM, ‡P < 0.01, P < 0.0001 for overall trend. (B) FPN1 protein levels in 3T3-L1 adipocytes, as detected by immunoblotting in adipocytes treated with no iron, 100 μM FeSO4, or 1 μg/ml hepcidin for 8 hours. (C) Fpn1 mRNA in adipose tissue and spleen from WT (Fpn1fl/fl) and Fpn1–/– (AP2 Cre ferroportin knockout) mice. ‡P < 0.001. (D) Tfrc mRNA quantified by RT-PCR and normalized to cyclophilin in collagenased adipocytes from epididymal fat pads of WT and Fpn1–/– mice (n = 10–14/group, ‡P < 0.001). (E) Adiponectin mRNA in the adipocytes used in E. ‡P < 0.01. (F) Serum adiponectin in WT and Fpn1–/– mice (n = 9–12/group, *P < 0.01). (G) High molecular weight (HMW) adiponectin determined as a percentage of total in the same group depicted in F. (H) Glucose tolerance testing of WT and Fpn1–/– mice (n = 5–6/group, *P < 0.05 for individual glucose values). (I) Body composition by magnetic resonance imaging in WT and Fpn1–/– mice on normal chow or high-iron diets (n = 11–20/group, *P < 0.05).

Figure 5. Adiponectin in mouse and human hemochromatosis.

(A) Tfrc mRNA levels in isolated adipocytes from WT and Hfe–/– mice on normal chow, normalized to cyclophilin A (n = 5/group, *P = 0.05). (B) Serum adiponectin levels in male (*P = 0.04) and female (P = 0.06) subjects with HH compared with non-HH sibling controls. (C) Serum adiponectin levels plotted as a function of BMI for subjects with HH (black circles) and non-HH sibling controls (white circles). Sexes were combined for linear regression analysis of HH (P = 0.87) and control subjects (P = 0.03). (D) Fasting glucose levels in WT, Hfe–/–, and Hfe–/–:APN–/– double-knockout mice (n = 5–7/group, *P = 0.006 by ANOVA).

Figure 6. Phlebotomy improves glucose tolerance.

Human men with impaired glucose tolerance and serum ferritin levels in the highest quartile of normal underwent phlebotomy to decrease their ferritin values to the lowest quartile of normal. (A) Results of oral glucose tolerance testing before (open symbols) and approximately 6 months after initiation of phlebotomy (closed symbols). The integrated area under the glucose curve for the 120-minute test is shown. Squares represent individual values, and circles represent the means. (*P < 0.03 by paired t test). (B) Insulin secretory capacity (AIRg) and (C) Si were determined from FSIVGTT performed before and after phlebotomy. (D) The disposition index was calculated from the data in B and C. *P < 0.05 by paired t test.