Serum progranulin concentrations may be associated with macrophage infiltration into omental adipose tissue

Abstract

Objective: Progranulin is an important molecule in inflammatory response. Chronic inflammation is frequently associated with central obesity and associated disturbances; however, the role of circulating progranulin in human obesity, type 2 diabetes, and dyslipidemia is unknown.

Research design and methods: For the measurement of progranulin serum concentrations, we developed an enzyme-linked immunosorbent assay (ELISA). Using this ELISA, we assessed circulating progranulin in a cross-sectional study of 209 subjects with a wide range of obesity, body fat distribution, insulin sensitivity, and glucose tolerance and in 60 individuals with normal (NGT) or impaired (IGT) glucose tolerance or type 2 diabetes before and after a 4-week physical training program. Progranulin mRNA and protein expression was measured in paired samples of omental and subcutaneous adipose tissue (adipocytes and cells of the stromal vascular fraction) from 55 lean or obese individuals. Measurement of Erk activation and chemotactic activity induced by progranulin in vitro was performed using THP-1-based cell migration assays.

Results: Progranulin serum concentrations were significantly higher in individuals with type 2 diabetes compared with NGT and in obese subjects with predominant visceral fat accumulation. Circulating progranulin significantly correlates with BMI, macrophage infiltration in omental adipose tissue, C-reactive protein (CRP) serum concentrations, A1C values, and total cholesterol. Multivariable linear regression analyses revealed CRP levels as the strongest independent predictor of circulating progranulin. The extent of in vitro progranulin-mediated chemotaxis is similar to that of monocyte chemoattractant protein-1 but independent of Galpha. Moreover, in type 2 diabetes, but not in IGT and NGT individuals, physical training for 4 weeks resulted in significantly decreased circulating progranulin levels.

Conclusions: Elevated progranulin serum concentrations are associated with visceral obesity, elevated plasma glucose, and dyslipidemia. We identified progranulin as a novel marker of chronic inflammation in obesity and type 2 diabetes that closely reflects omental adipose tissue macrophage infiltration. Physical training significantly reduces elevated circulating progranulin in patients with type 2 diabetes.

FIG. 1.

Progranulin serum concentrations in NGT individuals and patients with type 2 diabetes (T2D). Circulating progranulin in men (n = 12) and women (n = 18) with NGT (A) and in individuals with either type 2 diabetes (n = 124) or NGT (n = 30) (B). Data are means ± SE.

FIG. 2.

Relationship between progranulin serum concentrations and anthropometric and metabolic parameters. Correlation between circulating progranulin and A1C (r² = 0.25, P < 0.01) (A), circulating CRP (r² = 0.4, P < 0.001) (B), BMI (r² = 0.2, P < 0.05) (C), and total cholesterol serum concentration (r² = 0.2, P < 0.05) (D). Data are log transformed to achieve normal distribution. hsCRP, high-sensitivity C-reactive protein.

FIG. 3.

Progranulin serum concentrations in visceral obesity. A: Circulating progranulin in lean, visceral obese, and subcutaneous (SC) obese individuals with NGT. Abdominal visceral and subcutaneous fat areas were calculated using computed tomography (CT) scans at the level of L4–L5 in the cohort of paired visceral and subcutaneous adipose tissue donors. Visceral obesity was defined as >0.4 ratio of visceral to subcutaneous fat area as determined by CT scan. Data are means ± SE. *P < 0.05 between the groups. B: Correlation between circulating progranulin and macrophage infiltration into omental adipose tissue (r² = 0.5, P < 0.001). Data are log transformed to achieve normal distribution. Progranulin mRNA (C) and protein (D) expression in visceral and subcutaneous (SC) adipose tissue is shown. A representative Western blot is shown. E: Paraffin section of visceral adipose tissue of a normal glucose control individual. Immunostaining with a polyclonal anti-progranulin antibody. Arrows indicate progranulin-positive cytosol of adipocytes and cells of the stroma vascular fraction. F: Progranulin mRNA expression in isolated adipocytes and SVF. AU, arbitrary units. (Please see http://dx.doi.org/10.2337/db08-1147 for a high-quality digital representation of this figure.)

FIG. 4.

Chemotactic activity exerted by recombinant human progranulin. A: Erk activation by recombinant human progranulin in MCF10A cells. To examine the signal of phospho-p44/42 MAPK and p44/42 MAPK, after serum starvation for 24 h, MCF10A cells were stimulated with 500 ng/ml progranulin. Cell lysates were prepared at different time points as indicated and subjected to Western blot. B: Similar results have been obtained after treatment of THP-1 with 100 ng/ml progranulin. C: Transwell migration assay using THP-1 cells was conducted as described in research design and methods. THP-1 cells were placed into upper chambers, setting out to cell migration to lower chambers harboring no progranulin, different concentrations of progranulin (10, 100, and 250 ng/ml), or boiled progranulin. After 16 h, migrated cells were counted. D: Chemotactic effect of human Progranulin-FLAG in THP-1 cells. Cell migration of THP-1 monocytic leukemia cells was evaluated in disposable 24-well transwell polystyrene membrane with 8-μm pore size. hProgranulin-FLAG (100 ng/ml) was diluted in RPMI 1640 supplemented with 0.5% FBS and was then added to the low chamber. 0, 10, 50, 100, and 250 ng/ml hProgranulin were added into upper chamber together with THP-1 cells (2 × 10⁵ cells per well). After 16-h incubation at 37°C in 5% CO₂ humidified atmosphere, migration cells in the lower chamber were counted. Migrated cells in two separate fields per well from duplicate wells were enumerated on a hemacytometer by means of light microscopy. UC, upper chamber; LC, lower chamber. E and F: Transwell migration assay comparing human progranulin (hPrgn) and human MCP-1 (hMCP-1) using PTX-treated or PD98059-treated THP-1 cells.

FIG. 5.

Effect of 4 weeks of intensive exercise program on progranulin serum concentrations in NGT individuals and patients with IGT or type 2 diabetes (T2D). A: Circulating progranulin in groups of NGT (n = 20), IGT (n = 20), and type 2 diabetes (n = 20). B: Circulating progranulin before, immediately after, and 24 and 48 h after an acute exercise circle training to exhaustion for 60 min in 20 healthy volunteers. Data are means ± SE. *P < 0.05 between baseline and after 4 weeks of intensive physical training. **P < 0.01 between baseline and 48 hours after acute exercise.