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Chemerin regulates β-cell function in mice

Although various function of chemerin have been suggested, its physiological role remains to be elucidated. Here we show that chemerin-deficient mice are glucose intolerant irrespective of exhibiting reduced macrophage accumulation in adipose tissue. The glucose intolerance was mainly due to increased hepatic glucose production and impaired insulin secretion. Chemerin and its receptor ChemR23 were expressed in β-cell. Studies using isolated islets and perfused pancreas revealed impaired glucose-dependent insulin secretion (GSIS) in chemerin-deficient mice. Conversely, chemerin transgenic mice revealed enhanced GSIS and improved glucose tolerance. Expression of MafA, a pivotal transcriptional factor for β-cell function, was downregulated in chemerin-deficient islets and a chemerin-ablated β-cell line and rescue of MafA expression restored GSIS, indicating that chemerin regulates β-cell function via maintaining MafA expression. These results indicate that chemerin regulates β-cell function and plays an important role in glucose homeostasis in a tissue-dependent manner.


Glucose measurements and intraperitoneal glucose tolerance test
For the intraperitoneal glucose tolerance test, 50% glucose (1.5g/kg body weight) was injected intraperitoneally after a 15-h fasting. Blood was collected from the tail vein at 0, 30, 60, 90 and 120 min after the injection. For insulin measurements, blood was collected at 0, 15 and 30 min after the injection.

Hormone assays
The serum was separated by centrifugation and stored at -80 and assayed using mouse insulin ELISA kits (Shibayagi Inc. Japan), mouse leptin ELISA kits (R&D systems, USA), and mouse serum adiponectin kit (Otsuka assay, Japan). Serum total cholesterol, triglyceride, and NEFA were assayed using Cholesterol E test WAKO, Triglyceride E test WAKO, and NEFA C test WAKO kit from WAKO.

Euglycemic Hyperinsulinemic Clamp Studies
Euglycemic Hyperinsulinemic Clamp Studies were performed as previously described 3 .
Briefly, five to seven days before the clamp, mice were anesthetized with sodium pentobarbital (80-100 mg/kg, ip) and a catheter was inserted into the right internal jugular vein for infusion. The analysis was performed under nonstressful conditions with conscious mice that had been deprived of food for 4 h. The

Islet morphometry analysis, immunohistochemistry and immunofluorescence
The pancreas was isolated, fixed in 4% paraformaldehyde and embedded in paraffin.

Consecutive 4
m sections were cut and mounted on slides. The percentage islet area was quantified using Image J software as described previously 5,6 . 8-weeks-old male C57BL/6J mice were used for the analysis of chemerin and ChemR23 expression.
Immunohistochemistry and immunofluorescence were performed as previously described 7,8 using polyclonal pig anti-swine insulin antibody at a 1: 40 dilution (DakoCytomation, Denmark) as the primary antibody. A macrophage marker, Mac3 staining was performed as previously described 3 .

Quantitative real time PCR and immunoblotting
Quantitative real-time PCR (QRPCR) was performed, as previously described 9,10 , on a LightCycler system using the FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Switzerland). Each value was normalized to 36B4 or -actin expression. The sequence of primers is listed below. Immunoblotting analysis was performed as previously described 8,11 .

Islet isolation and batch incubation
Mouse pancreatic islet isolation and batch incubation were performed as previously described 12

MIN6 cells and knockdown of chemerin and ChemR23
Mouse cell line, MIN6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 25 mM glucose supplemented with 15% (v/v) heat-inactivated fetal calf serum under humidified conditions of 5% CO 2 , 95% air at 37 °C 14 . After 6h of starvation, the indicated agents were added, incubated for 3 h and chemerin expression was analyzed by QRPCR. For the knockdown of chemerin or ChemR23 in MIN6 cells, shRNA expression vector, pcPURm6i 15 containing target sequence for mouse chemerin or ChemR23 was transfected by reverse transfection method into MIN6 cells using Lipofectamine 2000 reagent (Invitrogen) as previously described 16,17 , according to the manufacturer's instruction. The target sequence for knock down of chemerin and ChemR23 are as follows; Chemerin, CKD-2:GGAGAAGGCGGAAATGCCCT, CKD-3:AGACCAACTGCCCCAAGAA, CKD -5:AAGCCGGAGTGCACAATCA and CHEMR23, CRKD-1:GCCACAACATGTACACCAG, CRKD-6:GTGCCAAGCTCTG TCTTCA, CRKD-8:GCATGAACCCCATTCTGTA. The vector plasmid was mixed with Lipofectamine 2000 in Opti-MEM I medium and incubated at room temperature for 20 minutes. MIN6 cells were stripped by trypsin-EDTA and incubated in antibiotic-free DMEM with 15% FBS before transfection. The transfection mixture was added to the cells in suspension status. The transfection efficiency was verified using GFP expression vector. Then, the cell were seeded to 96 well plates and incubated for approximately 8 h at 37°C and 5% CO 2 before being replaced with normal culture medium. Measurement of insulin secretion and content was performed as previously described 18 , 48 h after the transfection. The values of insulin were normalized by protein content.

Statistics and data analysis
Results are represented as mean SEM from at least 3 different experiments performed independently, unless stated otherwise. The data were analyzed using an unpaired 2-tailed Student's t test, analysis of variance (ANOVA), or Mann-Whitney u-test as appropriate. A P value of less than 0.05 was considered significant.