Patients with T2DM (n=110) or PDM (n=113) were recruited between January 2016 and December 2019 in the Department of Endocrinology and the Center for Severe Acute Pancreatitis (SAP) at the Jinling Hospital, Medical school of Nanjing University. Blood samples from healthy populations, which are difficult to collect, were not included since the aim of this study was to investigate the role of chemerin-9 in DM. T2DM patients were 69 male and 41 female with a mean age of 48.6±2.1 years. PDM patients were 82 male and 31 female with a mean age of 45.3±1.8 years. The diagnosis of T2DM or PDM was verified according to the Expert Committee on Diagnosis and Classification of Diabetes Mellitus (12). The underlying cause of PDM was either acute or chronic pancreatitis. To evaluate the association between chemerin levels and IR status, the patients with T2DM or PDM were further divided into two groups, with (IR>1) and without (IR≤1) IR, and serum samples (10 ml) were collected for subsequent ELISA analysis. Written informed consent for the use of serum samples was obtained from all patients enrolled in this study. The study was approved by The Ethics Committee of the Jinling Hospital (Nanjing, China).

Serum levels of chemerin (ml063020 for mice, ml058526 for human) and serum glucose (ml057865 for mice, ml063205 for human) and insulin (DCM076-8) were analyzed using ELISA kit from Shanghai Enzyme-linked Biotechnology Co., Ltd Serum levels of IL-1 (SEA057Mu) and TNF-α (SEA133Mu) were analyzed ELISA kits from Cloud-Clone Corp.

C57BJ/6J mice (age, 8 weeks; weight, 18–22 g; female; n=24) obtained from the Experimental Animal Institute of Jinling Hospital were used to establish a model of PDM. Mice were housed under a 12-h light/dark cycle at 23±1°C with a relative humidity of 50±5%. Mice in the PDM model group (n=8) were injected peritoneally with arginine (16) (350 µg/g/day) for 42 days. Mice in PDM group or the control diet group (n=8) were fed with a normal diet for 42 days. Mice in the high-fat diet group (n=8) to model T2DM were fed with a high-fat diet, for 42 days; the composition of the control diet and high-fat diet is listed in Table I. The health and behavior of mice were checked every 3 days. Mice were anesthetized with an intraperitoneal injection of chloral hydrate (250 mg/kg) prior to collecting blood (200 µl/mouse) through the tail vein at day 0 and at 21 and 42 days post-modeling to measure fasting serum glucose (FPG) and postprandial serum glucose (PPG), the levels of chemerin, interleukin (IL)-1, and tumor necrosis factor (TNF)-α, as well as the homeostatic model assessment of insulin resistance (HOMA-IR). Following blood collection at 42 days, the mice were euthanized by 100% CO₂ inhalation administered at 30% volume/minute. Death was verified by cervical dislocation and the end-point weight of the mice was 25–27 g. Subsequently, pancreatic head tissue was collected for RNA extraction and hematoxylin and eosin (H&E) staining.

To evaluate the impact of the CMKLR1 agonist, chemerin-9, on PDM, peritoneal injections of phosphate buffer solution (PBS, 200 µl, Control group, n=8) or chemerin-9 (4 µg in 200 µl PBS, n=8) or were performed every day for 42 days, as previously described (17). Blood samples were collected at day 0 and 42. The in vivo animal experimental protocols were approved by the Ethics Committee of Jinling Hospital (Nanjing, China).

Total RNA from pancreatic head tissue samples (40 mg) was extracted using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. qPCR was subsequently performed using the SYBR Green PCR Master mix including reverse transcriptase, buffer, dNTPs (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Thermal cycling of RT-qPCR was set as: 95°C for 5 min, 95°C for 10 sec and 60°C for 20 sec, repeated for 40 cycles. Primers were obtained from Tiangen Biotech Company. The following primer pairs were used for qPCR: PDX1 forward, 5′-GCGAGATGCTGGCAGACCTCT-3′ and reverse, 5′-GGCAGACCTGGCGGTTCACAT-3′; GLUT2 forward, 5′-CAATTTCATCATCGCCCTCT-3′ and reverse, 5′-TGCAGCAATTTCGTCAAAAG-3′; and β-actin forward, 5′-TCACTGAGGATGAGGTGGAAC-3′ and reverse, 5′-TCAGTCGCTCCAGGTCTTCACG-3′. The 2^−ΔΔCq method was used to analyze the relative expression of mRNAs (18). β-actin was used as an internal reference control. The relative levels of mRNAs was normalized to the internal reference gene β-actin.

The pancreatic tissues were fixed in 10% formalin solution for 24 h at room temperature, and were processed by routine histological tissue preparation. All specimens were embedded in wax and sectioned at 4 µm. The sections were stained with hematoxylin (0.5%, 5 mins at room temperature) and eosin (0.5%, 1 min at room temperature) for pathological histological examination.

Glucose and insulin levels were measured in the mice on day 21 and 42 following fasting for 16 h. For postprandial glucose, fasting mice were injected with 1–1.5 mg/g of glucose and blood samples were collected after 120 min. The concentrations of serum glucose and insulin were determined using ELISA assays. Following obtaining the fasting glucose and insulin levels, the HOMA-IR was calculated according to the following formula: HOMA-IR=[fasting glucose (nmol/l) × fasting insulin (µU/l)]/22.5.

Statistical analyses were performed using the SPSS version 22.0 (IBM Corp.) and GraphPad Prism version 5.0 (GraphPad Software, Inc.) software. Quantitative data are presented as the mean ± SEM with at least three independent repeats. Statistical significance between groups was determined using the Student's t-test or one-way ANOVA with post hoc Tukey HSD (Honestly Significant Difference) Test. P<0.05 was considered to indicate a statistically significant difference.

The role of chemerin in IR in patients with T2DM or PDM was explored by determining whether an association between the level of chemerin and IR status is present in these subjects. Compared with patients with T2DM with an IR≤1, patients with an IR>1 exhibited a significantly decreased level of chemerin (P<0.05; Fig. 1A). Patients with PDM with an IR>1 had a significantly lower level of chemerin compared with those with IR ≤1 (P<0.05; Fig. 1B). The level of chemerin in patients with PDM was significantly lower compared with levels in patients with T2DM (P<0.05; Fig. 1C). Together, these data suggested a close association of chemerin levels and IR in patients with DM (T1/2), and suggested that chemerin may serve a crucial role in the pathogenesis of PDM, which is characterized by impaired glucose tolerance and IR.

A mouse model of PDM was generated to further evaluate the role between chemerin and IR. Subsequent H&E staining revealed that compared with mice fed the high-fat diet or the control group, mice in the arginine group exhibited a focal enlargement of the interlobular septum in the pancreatic head and a minor increase in the number of white blood cells in, or around, the pancreatic lobules (data not shown). Compared with control group, the high-fat diet group showed pancreatic alveolar atrophy and interstitial fibrosis. Necrosis of glandular cells and bleeding were also absent in the pancreatic head region (Fig. 2). PDM mice had significantly higher fasting blood glucose levels and HOMA-IR at 42 days post-modeling compared to the high-fat diet and control group (P<0.05; Fig. 3A and B). Notably, following days, PDM model mice demonstrated a significant increase in the levels of FPG and PPG (P<0.05; Fig. 3C) compared with the high-fat diet or control group. Together, these data indicated that arginine injection for 42 days successfully induced PDM in mice, resulting in higher FPG levels, impaired glucose tolerance, and enhanced IR.

The levels of circulating chemerin, IL-1, and TNF-α in PDM mice, high-fat diet mice and the control group were measured by ELISA. PDM mice exhibited significantly reduced levels of chemerin at 42 days compared with mice fed a high-fat or control diet (P<0.05; Fig. 4A), in addition to significantly elevated levels of IL-1 and TNF-α compared with the high-fat diet and control group at 42 days (both P<0.05; Fig. 4B and C). Compared with control group, mice in high-fat diet group also showed decreased level of chemerin, as well as increased level of IL-1 and TNF-a (P<0.05, Fig. 4A-C). Subsequent RT-qPCR analysis of GLUT2 and PDX1 mRNA expression levels in the pancreatic head tissues collected from each group revealed that PDM mice exhibited significantly lower levels of both genes compared with mice in the high-fat diet and control group (P<0.05; Fig. 4D and E). These results indicated chemerin may exert protective role in the pathogenic process of PDM.

To further clarify the role of chemerin in PDM, the model mice were treated with chemerin-9, a classical agonist of CMKLR1. Before treatment, mice in control and chemerin-9 group showed similar level in fasting glucose, postprandial glucose and HOMA-IR (Fig. 5A-C). The treatment significantly decreased the FPG levels, PPG levels and HOMA-IR compared with the mice in the control group (all P<0.05; Fig. 5A-C). In addition, chemerin-9 treatment significantly increased the levels of chemerin compared with the control treatment group (P<0.05; Fig. 6A). No measurable change was reported in the serum levels of IL-1 or TNF-α between the chemerin-9 treatment and the control before or after treatment (Fig. 6B and C). In addition, the mRNA expression levels of GLUT2 and PDX1 were significantly increased following chemerin-9 treatment PDM mice compared with the control treatment (both P<0.05; Fig. 6D and E). These results indicate targeting chemerin may represent a novel therapeutic strategy for PDM.