LEAP2 ameliorates high fat diet-impaired glucose tolerance and enhances glucose-stimulated insulin secretion from β cells through PPARγ signal pathway

To the Editor: Type 2 diabetes (T2D) is an endocrine system disease, characterized by persistent hyperglycemia, and is one of the most common and fastest growing diseases in the world.[1] Although significant progresses have been made in understanding the pathology of T2D with abnormal insulin-secretion profiles, the molecular and cellular mechanisms of β cells remain largely uncertain. Moreover, the liver-expressed antimicrobial peptide 2 (LEAP2) may antagonize the inhibition effect of ghrelin on insulin secretion by inhibiting the ghrelin receptor (growth hormone secretagogue receptor, GHSR). The regulation of glucose homeostasis by the ghrelin–LEAP2–GHSR axis remains largely unknown. It is hypothesized that the ghrelin–LEAP2–GHSR axis may regulate the insulin–glucose homeostasis in diabetic condition. In this study, a high-fat diet (HFD) combined with multiple injections of low-dose streptozotocin (STZ) was used to construct a mouse model of T2D. The effects of exogenous LEAP2 in T2D and normal mice were tested. Eight-week-old C57BL/6J mice were provided by Department of Laboratory Animal, Central South University (Hunan, China). All animal experiments were approved by the Ethics Committee for Animal Experiments of Central South University. The in vitro effects of LEAP2 were studied in cultured MIN6 cells (passage from 10 to 20, kindly provided by Prof. Jingjing Zhang, The Second Xiangya Hospital of Central South University, Changsha, Hunan, China), and the potential molecular mechanisms involved in altered insulin secretion were investigated. Firstly, mice were intraperitoneally injected with LEAP2 (30 μg/kg daily) for 14 days after establishment of the T2D mouse model [Figure 1A]. LEAP2 treatment had no significant effects on body weight and food intake in the control and T2D mice [Figure 1B and Supplementary Figure 1A, https://links.lww.com/CM9/C78]. The fasting blood glucose and basal insulin levels in T2D mice showed typical changes in overt diabetes with an increase in fasting blood glucose and a decrease in insulin levels. There were no significant changes in the fasting blood glucose and insulin levels between the T2D + LEAP2 mice and the T2D mice, suggesting that the treatment adopted in this experiment (30 μg/kg, treatment for 14 days) had no significant effects on the fasting blood glucose and the fasting insulin secretion from the pancreatic islets in such T2D mice [Supplementary Figure 1B,C, https://links.lww.com/CM9/C78]. LEAP2 treatment, although did not change fasting glucose and insulin levels, significantly improved glucose tolerance, without affecting the insulin sensitivity or final insulin levels in T2D mice [Figure 1C, D and Supplementary Figure 1D, E, https://links.lww.com/CM9/C78]. After HFD and STZ treatment, the pancreatic islets of T2D mice are damaged and shrinked compared to control mice [Figure 1E]. An histological examination of the pancreas revealed that the LEAP2 treatment partially reversed the loss of islet mass and structural damage in T2D mice, no significant changes in pancreatic islet structure were observed in LEAP2 treatment alone, indicating that a 2-week LEAP2 treatment improved the morphology of pancreatic islets in T2D mice [Figure 1E]. The LEAP2 treatment increased the mass of the islets relative to the total pancreas in T2D + LEAP2 mice [Supplementary Figure 1F,G, https://links.lww.com/CM9/C78]. These results demonstrate that LEAP2 improved glucose tolerance with probably better glucose-stimulated insulin secretion and reversed the remaining islets damaged by HFD and repeated STZ.Figure 1: LEAP2 improved the glucose tolerance and pancreatic islet morphology in vivo and promoted GSIS in vitro. (A) Experimental protocol for constructing the mouse T2D model and LEAP2 treatment. (B) Determination of rate of body weight change after LEAP2 treatment in T2D and control mice (n = 7). (C,D) Measurement of blood glucose during IGTT after LEAP2 treatment in T2D and control mice (n = 5–7). (E) Representative hematoxylin and eosin staining images of pancreases after LEAP2 treatment in T2D and control mice (n = 5). From left to right: Control group, standard chow diet mice were intraperitoneally injected with saline; LEAP2 group, standard chow diet mice were intraperitoneally injected with LEAP2 (30 μg/kg); T2D group, T2D mice were intraperitoneally injected with saline; T2D+LEAP2 group, T2D mice were intraperitoneally injection with LEAP2 (30 μg/kg). ​All treatments were carried out at the same time in the experiment, after 14 days of continuous treatment with LEAP2 or saline. Scale bars = 50 μm. (F) LEAP2 significantly promoted MIN6 cells insulin secretion in the conditions of 3 mmol/L (Control and LEAP2 group) or 18 mmol/L (HG and HG + LEAP2 group) glucose (n = 3). (G) Measurement of intracellular Gck levels treated by LEAP2 in MIN6 cells (n = 6). (H) Measurement of intracellular ATP level treated by LEAP2 in MIN6 cells (n = 6). (I) Measurement of intracellular Ca2+ level treated by LEAP2 in MIN6 cells (n = 6). (J,K) PPARγ level in MIN6 cells treated with LEAP2 by Western blotting (n = 3). (L) Measurement of insulin content in MIN6 cells treated with LEAP2 or GW9662 (n = 3). (M) Measurement of Gck content in MIN6 cells treated with LEAP2 or GW9662 (n = 3). (N) GHSR siRNA significantly downregulated GHSR expression in mRNA levels (n = 3). (O) Measurement of insulin content in MIN6 cells treated with LEAP2 after knockdown of GHSR (n = 3). * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. All data are presented as the mean ± SD. AUC: Area under the curve; HFD: High-fat diet; HG: High glucose; GHSR: Growth hormone secretagogue receptor; IGTT: Intraperitoneal glucose tolerance test; LEAP2: Liver-expressed antimicrobial peptide 2; ns: Not significant; prot: Protein; PPAR: Poly (ADP-ribose) polymerase; siRNA: small interfering RNA; SD: Standard deviation; STZ: Streptozotocin; T2D: Type 2 diabetes.The above-mentioned observations were performed in vivo, and the direct effects of LEAP2 on the pancreatic β cells were then investigated in vitro. Using the MIN6 cell line (derived from tumors arising in transgenic mice expressing the SV40 T antigen under control of the insulin promoter) as an in vitro experimental model without ghrelin, the cytotoxic effects of LEAP2 were not observed at a concentration of 1 μmol/L for 2 h using the Cell Counting Kit-8 (CCK-8) [Supplementary Figure 2A, https://links.lww.com/CM9/C78]. To investigate the effects of LEAP2 on GSIS in the MIN6 cells, glucose concentrations of 3 mmol/L and 18 mmol/L were selected in this experiment. The results showed that insulin secretion in the high glucose (HG, 18 mmol/L) group increased significantly compared with that in the control (low glucose, 3 mmol/L) group. Compared with the HG group, the insulin secretion of the HG + LEAP2 group was significantly increased [Figure 1F]. Furthermore, we investigated the molecular mechanisms of LEAP2 in promoting the GSIS; The quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to measure the expression of insulin synthesis-related genes (Ins2, Pdx-1, Mafa) and insulin secretion-related genes (Glut2, Gck, Ucp2, Kir6.2, Sur1) in MIN6 cells. Compared with the control group, no significant differences were found on the mRNA expression of insulin synthesis-related genes by LEAP2 treatment [Supplementary Figure 2B–D, https://links.lww.com/CM9/C78]. However, LEAP2 significantly increased the mRNA expression of Sur1 and Gck, but not Glut2, Kir6.2, and Ucp2 in MIN6 cells [Supplementary Figure 2E–I, https://links.lww.com/CM9/C78]. Gck is the first rate-limiting enzyme in the process of glucose metabolism. Its hydrolysis rate directly affects the subsequent oxidative decomposition of glucose and the insulin secretion.[2,3] LEAP2 significantly increased the protein level of Gck in MIN6 cells [Figure 1G]. The functional analysis showed that 1 μmol/L of LEAP2 treatment led to a significant increase in adenosine 5′-triphosphate (ATP) content in MIN6 cells [Figure 1H]. The level of [Ca2+]i was detected by the Fluo-3-pentaacetoxymethyl ester (Fluo-3 AM) probe (catalog No. S1056, Beyotime, Shanghai, China) in MIN6 cells and was significantly increased by LEAP2 treatment [Figure 1I]. It was reported that activation of poly (ADP-ribose) polymerase (PPAR)γ increased the level of Gck in hepatocytes and islet β-cells.[4,5] Similarly, the LEAP2 (1 μmol/L) treatment of cells for 2 h increased the mRNA and protein levels of PPARγ in MIN6 cells [Figure 1J, K and Supplementary Figure 2J, https://links.lww.com/CM9/C78]. However, LEAP2 treatment had no effects on the protein expression of Akt and phosphorylated Akt in MIN6 cells [Supplementary Figure 2K,L, https://links.lww.com/CM9/C78] To investigate the activation of PPARγ in the secretory effects of LEAP2 on insulin secretion, MIN6 cells were incubated with the PPARγ-specific antagonist, GW9662 (catalog No. HY-16578, MedChemExpress, Shanghai, China), for 12 hours in the presence and absence of LEAP2. Compared with the control group, GW9662 significantly reduced the insulin and Gck content in MIN6 cells, and suppressed the increase in insulin secretion from MIN6 cells by LEAP2, and the increase in the protein level of Gck by LEAP2 [Figure 1L, M]. LEAP2 is a new endogenous antagonist of ghrelin receptor (GHSR) and the second ligand of GHSR.[6] In order to further confirm that the effect of LEAP2 on insulin secretion from the MIN6 cells was mediated by GHSR without ghrelin, small interfering RNA (siRNA) was used to suppress GHSR expression [Figure 1N and Supplementary Figure 3A,B, https://links.lww.com/CM9/C78]. After knockdown of GHSR in the MIN6 cells, the effect of LEAP2 on insulin secretion was significantly blocked [Figure 1O] with no significant differences in expression of the insulin-synthesis genes and insulin-secretion-related genes [Supplementary Figure 3C–I, https://links.lww.com/CM9/C78]. Interestingly, the basal insulin secretion of the MIN6 cells was also reduced by the knockdown of GHSR [Figure 1O]. In addition, the basal levels of PPARγ mRNA and protein expression without LEAP2 or ghrelin were also reduced by GHSR knockdown [Supplementary Figure 3J–L, https://links.lww.com/CM9/C78]. The increase in PPARγ mRNA and protein expression by LEAP2 treatment was significantly reduced after knockdown of GHSR [Supplementary Figure 3M–O, https://links.lww.com/CM9/C78]. GHSR is a G-protein coupled receptor with exceptionally high constitutive activity (up to 50% of maximal activation) in triggering intracellular signals in the absence of ghrelin or its analogs,[7] suggesting an important function of constitutive activation of GHSR in MIN6 β-cells. In conclusion, LEAP2 was demonstrated to improve glucose tolerance and pancreatic islet morphology in diabetic mice in vivo, and promote glucose-stimulated insulin secretion from MIN6 β-cells in vitro. LEAP2 increased ATP content and [Ca2+]i concentrations in MIN6 cells to enhance GSIS through signaling pathways involving the GHSR-PPARγ–Gck axis [Supplementary Figure 4, https://links.lww.com/CM9/C78]. This report has demonstrated that LEAP2 is a promising hormone treating compromised glucose tolerance in HFD-induced T2D mice. Moreover, LEAP2 promotes insulin secretion, through the GHSR–PPARγ–Gck axis. LEAP2 may serve as a potential drug target to reverse damaged insulin secretion, especially for compromised GSIS in T2D. This anti-diabetic effect of LEAP2 warrants further investigation in other diabetic models and other metabolic disorders such as obesity. Funding This research was supported by grants from the National Natural Science Foundation of China (Nos.81870059, 82070068). Conflicts of interest None.