Vascular endothelial growth factor B regulates insulin secretion in β cells of type 2 diabetes mellitus mice via PLCγ and the IP3R‑evoked Ca2+/CaMK2 signaling pathway
- Authors:
- Published online on: September 5, 2023 https://doi.org/10.3892/mmr.2023.13084
- Article Number: 197
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Copyright: © Li et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Diabetes is a metabolic disease characterized by chronic hyperglycemia caused by multiple etiologies, which is caused by defects in insulin secretion and/or utilization (1). Insulin secretion and its regulation play an important role in glucose metabolism and homeostasis (2). The abnormal and incontrollable insulin secretion in β cells is closely related to the occurrence of diabetes, but its molecular mechanism has not been fully understood and remains need to be further clarified. In recent years, the role of VEGFB in regulating lipid and glucose metabolism has attracted extensive attention. The present study found that VEGFB is related to total cholesterol (TC), triglyceride (TG), and glycosylated hemoglobin (GHb) in T2DM patients (3). In type 2 diabetes mellitus (T2DM) mice, systemic inhibition of VEGFB improves glucose tolerance and insulin sensitivity (4). Specific VEGFB overexpression in rats can ameliorate diabetes by improving insulin action (5). Targeted overexpression of the VEGFB signal can improve some key factors that promote the development of T2DM, including glucose tolerance, abnormal lipid metabolism and β cell function. Therefore, VEGFB may become an important regulatory approach in the development of T2DM.
Insulin secretion is a complex process, and calcium channels on β cell membrane play an important regulatory role during this process (6). The change in intracellular Ca2+ concentration is closely related to insulin secretion (7). Previous studies have shown that abnormal insulin secretion in patients with diabetes may be related to the dysfunction of the intracellular calcium signaling pathway (8). At present and to the best of the authors' knowledge, the pathophysiological mechanism of abnormal insulin secretion remains unclear. Although it has been revealed that VEGFB can regulate insulin secretion by affecting fatty acid content, its specific regulatory mechanism also needs to be studied in the future. VEGFB transduces signals through the protein kinase C pathway (9). Therefore, can VEGFB stimulate the release of Ca2+ through phosphatidylinositol 3-kinase, phospholipase C-1, GTPase activating protein, and other signal proteins after it combines with VEGFR1. The answer to these scientific questions will help to further analyze the pathogenesis of diabetes and provide a theoretical basis for the precise treatment for it.
The potential molecular mechanism of abnormal insulin secretion in β cells of T2DM mice were examined. VEGFB knockout (KO) or overexpression can inhibit or activate phospholipase C gamma (PLCγ) and inositol 1,4,5-triphosphate receptor IP3R signaling pathway in a VEGFR1-dependent way. Then, the change of PLCγ/IP3R caused by VEGFB/VEGFR1 will alter the expression of key factors on the calcium/calmodulin signaling pathways such as PPP3CA. KO or overexpression of VEGFB can cause altered insulin secretion by changing the calcium concentration in β cells and affect the glucose tolerance and insulin sensitivity of T2DM mice. The present study demonstrated that VEGFB can regulate insulin secretion via PLCγ and the IP3R-evoked Ca2+/CaMK2 signaling pathway.
Materials and methods
Experimental animals
The experiments on mice were approved (IACUC approval no: 2022-210) by the animal ethics committee of Binzhou Medical University (Yantai, China). All mice were raised at 24°C, 12/12-h light/dark, 50% humidity). C57BL/6 male mice (n=6) (age, 4 weeks-old; weight, 16–18 g), were selected into 5 experimental groups: wild-type (WT), streptozocin (STZ)-WT, STZ-KO, adeno-associated virus (AAV)-control, and AAV–VEGFB186 group. VEGFB+/+ mice fed standard diet (SD, Rodent Diet with 10% kcal% fat, Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd.) were named the WT group (n=6). A total of four groups of mice induced by STZ and high-fat diet (HFD, Rodent Diet with 60% kcal% fat, Research Diets, Inc.) were T2DM models. WT group was not induced with STZ. VEGFB+/+ T2DM mice were named STZ-WT (n=6), and VEGFB−/− T2DM mice were named STZ-KO (n=6). VEGFB+/+ T2DM mice injected with AAV targeting VEGFB186 were named the AAV–VEGFB186 group (n=6), and mice in the AAV-control group were injected with non-targeting VEGFB186 and were regarded as the negative control (n=6). WT, STZ-WT and STZ-KO groups of mice were euthanized and measured in the 24th week. Some mice developed complications of T2DM after 32 weeks due to the longer course of T2DM, therefore the latter two groups were in the 32nd week (10–13). In the animal experiment, all mice were administered 3% isoflurane, and sacrificed by cervical dislocation after blood collection from the eyeball. Pancreatic tissues of mice were removed and fixed with 4% paraformaldehyde or 2.5% glutaraldehyde.
Cell culture and treatment
Mouse islet β cell line Min6 was purchased from the Procell Life Science&Technology Company and was cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) medium containing 10% fetal bovine serum, and 1% Penicillin/streptomycin (P/S). The temperature inside the incubator was 37°C and the gas proportion was 5% CO2. Min6 cells were adhered in the six-well plate to the confluence of 50% and grouped into negative control (NC) and silencing (SI) groups. Cells in the NC group were treated with non-targeting VEGFB sequence (5′UUCUCCGAACGUGUCACGUTT3′, 3′ ACGUGACACGUUCGGAGAATT 5′) while SI groups were treated with VEGFB KO sequence [VEGFB small interfering RNA (siRNA): 5′GAACACAGCCAATGTGAAT 3′] in the SI group. JetPRIME and Jet buffer (Polyplus-transfection® Inc., United States) were used to transfect the sequence within 48 h at room temperature and then the cells in two groups were detected the efficiency of transfection by reverse transcription-quantitative (RT-q) PCR and western blot analysis.
VEGFB KO mouse
VEGFB KO mouse model was constructed by CRISPR/Cas 9 technology. The work was undertaken by the Saiye (Guangzhou) Biotechnology Co., Ltd. The gRNA sequences were as follows: gRNA-1, 5′-AAGGGCTCCGTCCTTGAGTCAGG-3′; and gRNA-2, 5′-CAGGGGATGACTTATGGGCCAGG-3′. The wild-type mice were not transfected with any control construct. A total of two pairs of primers were used for the PCR cycle and the sequences were as follows: primer 1 forward, 5′-TCTCAAGGTTGGCGGAAGTGG-3′ and reverse, 5′-CAAACTCACCATGTCACCAAGGAG-3′; and primer 2 forward, 5′-TCTCAAGGTTGGCGGAAGTGG-3′ and reverse, 5′-TTGGGATCACGCAAGATAAGGG-3′. Mice genotypes were identified by 12% agarose gel electrophoresis and visualized by ethidium bromide, VEGFB+/+ and VEGFB−/− mice were screened for the T2DM model. In the present study, the protein and mRNA levels of VEGFB expression in VEGFB+/+ and VEGFB−/− mice were detected in the pancreas (Fig. 1A-D).
T2DM mouse model
The mice were fed with HFD from the 8th week. STZ was intraperitoneally injected twice at a dose of 30 mg/kg within the 15–16th weeks, with an interval of 1 day between the two injections (14,15). The blood glucose of mice was measured at 0, 3, 10 and 30 days after injection of STZ. Four weeks after STZ injection, the mice with fasting blood glucose (FBG) ≥11.1 mmol/l were defined as the T2DM model (Fig. 1E).
Overexpression of VEGFB in T2DM mouse
The AAV vector was purchased from the OBiO Technology (Shanghai) Corp., Ltd. AAV-CAG-VEGFB186-P2A-EGFP-3×FLAG-WPRE was regarded as an overexpression vector and AAV-CAG-EGFP-3×FLAG-WPRE was a control vector. A total of eight weeks after injection with STZ, the mice whose FBG was ≥16.7 mmol/l were prepared for intraperitoneal AAV infection into the pancreas. The virus titer was controlled >1.0×1012 on each side (Fig. 1F-I).
Measurement of weight, FBG and postprandial blood glucose (PBG)
From the 8th week, the three indicators were measured at a fixed time every week. The mice were not fed within 12 h and FBG was examined. After the mice were administered a resumption of diet for 2 h, the PBG was measured. The blood was drawn from the tail vein by using a Roche blood glucose meter (Roche Diabetes Care, Inc.) for FBG and PBG measurement.
Isolation of islet cells
A total of three mice in each group were used to isolate islets, and 100–150 islet cell clusters could be collected from each mouse. The pancreas was removed, and the peripheral adipose tissue was isolated and placed in Hank's buffer after the mice's death. Collagenase P (0.5 mg/ml; Roche) was injected through the pancreatic duct and digested for 10 min after complete expansion of the pancreas. Hank's buffer was pre-cooled at 4°C to stop digestion, and cell mass was selected under the stereoscopic microscope (Olympus Corporation).
Western blot analysis
Lysates consisting of 1% cocktail RIPA (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) and PMSF (cat. no. 36978; Gibco; Thermo Fisher Scientific, Inc.) were added to the cells from islet cell clusters on ice for 30 min. The loading buffer (cat. no. D1020-5; Beijing Solarbio Science & Technology Co., Ltd.) was added to the supernatant and heated after centrifugation. Protein samples (20 µg protein /lane) were transferred onto PVDF membranes after separating in 10% SDS-PAGE gel. After blocking with 5% skimmed milk at room temperature for 1 h, membranes were incubated with primary antibodies at 4°C (Table I). After 12 h, membranes were incubated with secondary antibodies (1:5,000, cat. no. S0001, Affinity, HRP) at room temperature for 2 h. Optical density was detected after samples were treated with enhanced chemiluminescence reaction (Tanon 5200; Tanon Science & Technology Co., Ltd.). The blots were performed densitometric analysis by ImageJ software (version 1.52a, National Institutes of Health)
RT-qPCR
Total RNA was collected from islet cell clusters with TriQuick Reagent (cat. no. R100; Beijing Solarbio Science & Technology Co., Ltd.). RNA-easy Isolation Reagent (Vazyme Biotech Co., Ltd.) reverse transcription and real-time detection were accomplished with TB Green Premix Ex Taq II (Takara Bio, Inc.) fluorescence quantitative kit on PCR QuantStudio 3 (Thermo Fisher Scientific, Inc.). The thermo cycling conditions of RT-qPCR were as follows: Initial denaturation at 95°C for 30 sec; then 40 cycles were conducted at 95°C for 5 sec and 60°C for 34 sec; the dissolution process was performed at 95°C, 60°C and 95°C for 15 sec, 1 min and 15 sec, respectively in the end. The primer sequences were as follows: VEGFB forward, 5′-GCTGGGCACTAGTTGTTTG-3′ and reverse, 5′-AGCCACCAGAAGAAAGTGG-3′; and β-actin forward, 5′-CATCCGTAAAGACCTCTATGCCAAC-3′ and reverse, 5′-ATGGAGCCACCGATCCACA-3′. The 2−ΔΔCq method was used to quantify the expression of mRNA by using β-actin as an internal reference gene (16).
ELISA and colorimetry
Serums from five mice were collected for measurement of blood glucose (cat. no. ml016964), GHb (cat. no. ml063816) and insulin (cat. no. ml001983) content with a microplate reader (BioTek Corp.) by ELISA. A standard curve was established according to the measured value of the standard and the sample content was calculated. TG (cat. no. A110-1-1), and TC (cat. no. A111-1-1; both from Nanjing Jiancheng Bioengineering Institute) were detected by the colorimetry method according to the manufacturer's instructions.
Hematoxylin and eosin (H&E) Staining
Pancreatic tissues of 3 mice in each group were fixed with 4% paraformaldehyde at 4°C for 12 h. After dehydration by an automatic dehydrator, tissue was embedded with paraffin, and sliced into 5-µm sections. The section was dewaxed to water with xylene and stained with hematoxylin for 5 min and eosin for 1 min at room temperature. After sealing with neutral glue, the images of the samples were acquired by the optical microscope (OLYMPUS-DP27; Olympus Corporation).
Transmission electron microscopy
The pancreas tissues of 3 mice in each group were fixed with 2.5% glutaraldehyde solution and 1% osmic acid at 4°C for 12 h. Tissues were subjected to mixed treatment with entrapment agent and acetone (v/v=1/2) after dehydration with gradient alcohol, pure entrapment agent-permeated and embedded. Subsequently, they were sliced with Reichert ultra-thin microtome (70 nm). Lead citrate solution and uranyl acetate 50% ethanol saturated solution were used for staining at room temperature for 10 min, respectively. A transmission electron microscope (JEM-1400; JEOL, Ltd.) was used to observe and capture images of the sections.
Immunofluorescence
After paraffin removal with xylene, gradient hydration with ethanol and antigen repair with sodium citrate and 3% H2O2 solution-eliminated endogenous peroxidase activity, 5% goat serum (cat. no. SL038, Solarbio) was used for blocking at 37°C for 30 min. Then the antibody mixture of insulin (1:200; cat. no. 66198-1-ig; ProteinTech Group, Inc.) and glucagon (1:100; cat. no. ab92517; Abcam) was added dropwise and incubated for 12 h in a 4°C wet box. The next day, the mixture of fluorescent goat anti-rabbit IgG/TRITC (1:100; cat. no. ZF-0317; OriGene Technologies, Inc.) and goat anti-mouse IgG/FITC (1:100; cat. no. ZF-0314; OriGene Technologies, Inc.) was added and incubated for 1 h. Afterwards, it was stained with 10 µg/ml DAPI, rinsed, blocked, and stored at 4°C without light after washing with PBS. Images were captured by a confocal laser scanning microscope (LSM880; Zeiss AG).
Oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPITT)
During the OGTT, mice were not fed within 12 h and then ravaged with 40% glucose at the dose of 2 mg/kg. During the IPITT, the mice were injected intraperitoneally with 0.5 UI/kg insulin after fasting for 6 h. Blood glucose at 0, 15, 30, 60, 90 and 120 min was detected.
Islet secretion function index
FBG, fasting insulin (FINS), insulin increment (ΔI30), and glucose increment (ΔG30) at 30 min in OGTT, 1 and 2 h of PBG of five mice in each group were detected. Insulin secretion index of the steady-state model (HOMA-β)=FINS ×20/(FBG-3.5); Islet β cells secretion index (ΔI30/ΔG30)=the ratio of insulin increment to glucose increment in OGTT at 30 min; modified β cells function index=(FINS × FBG)/(PBG 1 h + PBG 2h-2FBG).
Glucose stimulation
Min6 cell line and islet cells from 3 mice in each group were used for the detection. Fresh islets and Min6 cells were cultured overnight in the sugar-free medium at 37°C. After washing, they were cultured with 2.8 mmol/l low-sugar medium at 37°C for 2 h, and incubation medium was collected to detect insulin (Shanghai Enzyme-linked Biotechnology Co., Ltd.; cat. no. ml001983) and intracellular Ca2+ content (Shanghai Enzyme-linked Biotechnology Co., Ltd.; cat. no. ml058009). And then 16.7 mmol/l high-sugar medium was replaced for the incubation.
Calcium content analyses
Intracellular calcium content was detected according to the manufacturer's instructions (cat. no. ml058009). Diluted standard and samples were added to the 96-well plate with 50 µl, and then the antibodies were added with 50 µl. The membrane plate was covered, gently shaken and mixed, and incubated at 37°C for 1 h. The enzyme HRP was added after washing with buffer three times and incubated at 37°C for 30 min. A total of 50 µl of substrates A and B was added to each well, gently shaken and mixed, and incubated at 37°C for 10 min without light. The OD value was measured at a wavelength of 450 nm after adding 50 µl of termination solution.
Proteomic analysis
Islet cells of five VEGFB+/+ and VEGFB−/− mice were isolated. Meanwhile, islet cells of five VEGFB+/+STZ and VEGFB−/−STZ mice were isolated for proteomic analysis. PBS containing protease inhibitors and phosphatase inhibitors were used to treat cells. And then homogenated in a denatured buffer containing urea, HEPES. The Bradford assay (Bio-Rad Laboratories, Inc.) was used to examine the protein content. DL-Dithiothreitol solution and Iodoacetic amide solution were added. Trypsin/Lys-C (FUJIFILM Wako Pure Chemical Corporation) was added so that the final concentration of the sample digestion buffer was 5% (w/w) trypsin/protein. Trifluoroacetic acid and acetonitrile (ACN) were used for column washing. The peptide elution fractions were labeled with 6-plex TMT reagent and then the labeled peptide was acidified with formic acid (pH 2.5), and the sample was filtered and desalted through C18 Stage-tips, and completely dried in a vacuum centrifuge. Peptides were dissolved and separated by RPLC-MS using the EASY-nLC 1000 system (Thermo Fisher Scientific, Inc.). The peptide was washed at 250 l/min with ACN concentrated from 4–100%. All results of data were analyzed by using a QExactive plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Inc.). The mass spectrometer was operated in the positive ion module to obtain the investigation mass spectrum with 7000 resolution and the successive high collision dissociation fragmentation spectrum. The bioinformatics tools used to analyze the heatmap were HIPLOT (hiplot.com.cn/home/index.html) and Gene Set Analysis Toolkit (https://www.webgestalt.org).
Statistical analysis
SPSS 22.0 statistical software (IBM Corp.) was used to analyze all data. The results were shown as the mean ± SD. One-way ANOVA followed by Dunnett's post hoc test was used, while comparisons between two groups were assessed using paired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.
Results
VEGFB regulates glucolipid metabolism and insulin sensitivity in T2DM mice
From the 14th week, the weight of mice fed HFD was higher than those of mice fed SD, and the weight of STZ-KO mice was higher in comparison with STZ-WT mice in the 18th week (Fig. 1J). There was no significant difference between SD and HFD feeding except in the 22nd and 24th week (Fig. 1K). In the 24th week, the TC and TG of STZ-KO mice were significantly higher than those of STZ-WT mice (Fig. 1L and M). When the T2DM mice were administered AAV injection, the weight and food intake of AAV–VEGFB186 mice were decreased from the 30th week (Fig. 1N and O). And the TC and TG contents were decreased when compared with STZ mice (Fig. 1P and Q).
From the 18th week, the FBG and PBG of T2DM mice were increased. In STZ-KO mice, FBG was increased from the 22nd week and PBG was increased from the 18th week compared with STZ-WT mice (Fig. 2A and B). Under the HFD condition, the serum glucose and GHb of STZ-KO mice were higher than those of STZ-WT mice in the 24th week (Fig. 2C and D). OGTT and IPITT revealed that the ability to regulate blood glucose in T2DM mice and the efficiency of glucose uptake and utilization promoted by insulin decreased. Blood glucose of STZ-KO mice was higher than that of STZ-WT mice with the stimulation of glucose and insulin. At the same time, the area under the curve also increased (Fig. 2E-H).
Compared with the Con group, the body weight and blood glucose of mice in the AAV group decreased significantly from the 24th week, especially the decrease in PBG, indicating that VEGFB has a therapeutic effect on blood glucose in T2DM mice (Fig. 2I and J). In addition, serum glucose and GHb were significantly lower in the 32nd week (Fig. 2K and L). The OGTT and ITT results demonstrated that the glucose tolerance and insulin sensitivity of AAV–VEGFB186 mice increased compared with the WT group but decreased compared with control group (Fig. 2M-P).
VEGFB affects insulin secretion of islet β cells in T2DM mice
Compared with WT, the serum insulin and insulin secretion function of T2DM mice was lower, while the serum insulin and insulin secretion function of the STZ-KO group was significantly lower than those of the STZ-WT group (Fig. 3A-D). After AAV injection, the serum insulin and insulin secretion function of AAV–VEGFB186 mice were higher than AAV-control mice (Fig. 3E-H).
The morphological changes of pancreatic islets were observed by H&E staining. It is not easy to distinguish multiple cell types in the islets under H&E staining. The endocrine cells in the islets mainly include A cells (~20% of the total islet cells), B cells (~75% of the total islet cells), D cells (~5% of the total islet cells), and others such as PP cells and D1 cells (17,18). The islets of mice in the WT group were round or oval with clear boundaries and close arrangement between islet cells. The size of islets in T2DM mice was not homogeneous, and some islets showed atrophy and volume reduction. β cells, with a large number, were in the center of the islet, while α cells, with a small number, were in the periphery of the pancreas islet. The nucleus of β cells in WT was circular and intact, and secretory vesicles could also be observed. The volume of β cells and the number of mitochondria decreased in T2DM mice (Fig. 4A). After the injection of AAV–VEGFB186, the size of the islet was improved (Fig. 4B).
The number of islet cells and the density of β cells in STZ-KO mice were lower than those of STZ-WT mice, and meanwhile, the density of mature and immature secretory vesicles of STZ-KO mice decreased significantly (Fig. 4C-F). Compared with AAV-control, the number of islet cells, the density of β cells, and secretory vesicles in AAV–VEGFB186 mice were higher (Fig. 4G-J).
VEGFB regulates insulin secretion through Ca2+/CaMK2 and its association with PPP3CA
A total of 2,034 proteins were identified in the islets of VEGFB+/+ and VEGFB−/− mice, of which 100 proteins were different between the two groups. A total of 34 upregulated and 12 downregulated proteins were analyzed among these differential proteins in the islets of mice (Fig. 5A). A total of 1,722 proteins were identified in the islets of STZ-WT and STZ-KO mice, of which 43 proteins were different between the two groups among these differential proteins (Fig. 5B). The heatmap showed that PPP3CA was associated with VEGFB in differential proteins (Fig. 5C). The differential proteins were analyzed by interpretative phenomenological analysis (IPA) method after VEGFB KO, which was mainly involved in cellular glucose homeostasis (Fig. 5D and E).
The protein expression of PPP3CA and CaMK2 was decreased in STZ-KO mice, while in AAV–VEGFB186 mice, the expression of PPP3CA and CaMK2 was increased (Fig. 5F-M). The intracellular Ca2+ and insulin were detected, and their contents in STZ-KO mice were lower than those of STZ-WT mice (Fig. 5N-Q), while in AAV–VEGFB186 mice, Ca2+ and insulin levels increased after glucose stimulation (Fig. 5R-U).
VEGFB/VEGFR1 affects the content of Ca2+ via the PLCγ/IP3R signaling pathway
In order to detect the effects of VEGFB on the PLCγ/IP3R signaling pathway, the expression of VEGFB, VEGFR1, PLCγ and IP3R was examined by western blot analysis. The results revealed that VEGFR1 protein expression was decreased as the VEGFB was knocked out, which suppressed the expression of the downstream proteins PLCγ and IP3R in STZ-KO mice (Fig. 6A-H). Moreover, the expression of VEGFR1, PLCγ and IP3R proteins were elevated in AAV–VEGFB186 mice with overexpressed VEGFB gene (Fig. 6I-P).
VEGFB/VEGFR1 affects the content of Ca2+ and insulin secretion via VEGFA in physiological state
In VEGFB+/+ and VEGFB−/− mice, the serum glucose content was decreased and serum insulin was increased after the VEGFB was knocked out (Fig. S1A and B). The calcium content and insulin secretion of islet cells in VEGFB−/− mice significantly increased after stimulation of 2.8 and 16.7 mM glucose (Fig. S1C-F). The expression of the VEGFR1 protein declined following the loss of the VEGFB gene while the expression of VEGFA and VEGFR2 protein increased. siRNA transfection in MIN6 was performed to detect the insulin secretion and calcium content as the expression of VEGFB was suppressed at the protein and mRNA levels (Fig. S1J and K). The ATP, calcium and insulin secretion were increased in the SI group with the stimulation of 2.8 and 16.7 mM glucose (Fig. S1L-Q). The expression of VEGFR1 was reduced and the expression levels of VEGFA and VEGFR2 were elevated in the SI group (Fig. S1R-T).
Discussion
VEGFB) is a glycoprotein with high metabolic activity, which has been a late discovery factor in VEGF families (19). However, its role in promoting angiogenesis is not ascertained (19,20). It was previously reported that VEGFB can regulate free fatty acid uptake in endothelial cells by adjusting fatty acid transporters (21). The expression of VEGFB and fatty acid transporters increased after binding with VEGFR1, which resulted in hyperglycemia (22). Paradoxically, a previous study reported that VEGFB suppressed inflammation related to obesity and ameliorated lipid homeostasis since it was transduced into obese mice (23). An increasing number of researchers were interested in deciphering the peculiar regulatory effect of VEGFB on lipid metabolism due to this controversial phenomenon. Numerous studies have identified that VEGFB can inhibit lipid deposition and improve lipid metabolism (24). VEGFB-deficient mice had white fat swelling and increased lipid accumulation. Fat-specific VEGFB inhibition could promote lipid deposition (25). However, the combination of VEGFB and IL22 proteins could reduce lipid deposition by suppressing fatty acid transporters (26). The findings of the present study were similar to the aforementioned studies. T2DM mice gained weight and elevated serum TC and TG after VEGFB KO. VEGFB186 overexpression improved lipid metabolism in T2DM mice.
The increase in TG causes ectopic lipid deposition, damages β cell function and affects insulin secretion (27,28). A correlation was found between VEGFB and TC, TG and blood glucose in patients with T2DM. The plasma VEGFB levels in newly diagnosed patients with T2DM was closely related to glucose metabolism and insulin level (3). The VEGFB expression in the renal tissue of patients with diabetic nephropathy was positively correlated with the content of gamma-hydroxybutyric acid (26). The present study illustrated that blood glucose increased after VEGFB KO, as the growth of blood lipids in T2DM mice increased. VEGFB overexpression can improve glucolipid metabolism in T2DM mice. The regulatory effect of VEGFB on glucose metabolism may be related to the reduction of TG in T2DM mice.
The markers of T2DM pathogenesis include islet dysfunction and a reduced number of β cells (28). Glucotoxicity damages the β cell and impairs insulin secretion function (29). Decreased β cell function will influence insulin secretion although the number of β cells has a certain impact on T2DM (30). Hyperglycemia will worsen β cell damage, affecting insulin secretion (31). The present study revealed that loss of VEGFB exacerbated β cell population reduction in T2DM mice. However, VEGFB overexpression attenuates β cell injury in T2DM mice. These results of the present study indicated that VEGFB can alleviate β cell damage to insulin secretion. The insulin of T2DM mice decreased after VEGFB KO, and the evaluation index of insulin secretion decreased, indicating that VEGFB has an effect on basic and early insulin secretion function.
Insulin is released from insulin secretory vesicles in pancreatic β cells. Insulin secretory vesicles are divided into immature and mature vesicles (32). Proinsulin is encapsulated into immature insulin secretory vesicles with low electron density and has to undergo through a series of tight regulatory procedures to develop into mature insulin secretory vesicles (33). The mature secretory vesicles are composed of insulin, zinc and calcium crystals, containing dense core vesicles (34). Mature insulin secretory vesicles are stored in the vesicle pool or transported near the cell membrane (35). Insulin secretory vesicles fuse with the cell membrane to release insulin when the blood glucose level increases. The present study revealed that VEGFB can regulate insulin synthesis by affecting the number of immature vesicles in β cells. Concurrently, VEGFB can regulate insulin secretion by affecting the number of mature vesicles.
VEGFR, a specific VEGF receptor, elicits a variety of biological functions through a combination of corresponding VEGF. At present, the VEGFR family contains five members, and VEGFR1, VEGFR2 and VEGFR3 belong to receptor tyrosine kinases (36). The function of VEGFB on lipid homeostasis is strongly dependent on VEGFR1 (37). VEGFB could enhance fatty acid uptake by endothelial cells through VEGFR1 (21). VEGFR1 KO in mice with obesity and insulin resistance could decrease insulin secretion (23). The results of the present study revealed consistent VEGFR1 expression with VEGFB expression after VEGFB KO or overexpression in T2DM mice. Consequently, it was revealed that VEGFB combines with VEGFR1 to regulate insulin secretion in T2DM mice.
The intracellular VEGFR-mediated signal transduction is a complex process. The mechanism of the VEGFR1-mediated signaling pathway is not clear and remains a current research hotspot. The VEGFR1-mediated signaling pathway can activate numerous biological reactions. A previous study revealed that the VEGFR1-mediated signal transduction pathway could activate intracellular MAPK signal transduction by binding with PIGF (38). VEGFR1 suppression could inhibit the expression of its upstream PI3K/AKT signaling pathway (39). VEGFR1 is involved in the PKGI signaling pathway (40). Additionally, the combination of VEGFB and VEGFR1 could activate the intracellular PLCγ signal transduction (41). PLCγ activation produces IP3 (42). PLCγ and IP3 combination could effectively stimulate calcium efflux. PLCγ activation releases Ca2+, promotes β cell function, and improves insulin secretion to prevent the occurrence of hyperglycemia (43). It was observed in the present study that VEGFB/VEGFR1 could affect Ca2+ content in β cells by activating the PLCγ/IP3 signaling pathway to regulate insulin secretion.
Additionally, CaMK is the main mediator of calcium (44). The CaMK expressed in β cells is CaMK2 which is a multifunctional Ca2+/CaMK and is activated by glucose and other insulin secretagogues (45). It has the function of phosphorylating a variety of proteins and is crucial for insulin secretion. Moreover, CaMK2 needs to supplement the reserve vesicle pool in β cells after stimulation is completed (46). The present study is consistent with those revelations, showing that VEGFB can promote Ca2+ by activating the CaMK2 to regulate insulin secretion.
PPP3CA was further analyzed and it was found that is associated with VEGFB in differential proteins through proteomics and bioinformatics. In 2008, Wang et al reported that PPP3CA modulates the VEGF-stimulated cell proliferation and signaling cascades in cells (47). PPP3CA is a serine/threonine phosphatase regulated by Ca2+/CaM (48,49). Gelernter et al found that PPP3CA encodes a calcium-dependent, calmodulin-stimulated protein phosphatase involved in calcium signaling (50). The secretion of Ca2+ depends on insulin resistance and type 2 diabetes. Insulin secretion pathways were reported to be activated by upregulating PPP3CA (51,52). PPP3CA can be involved in complications of diabetes (53). Therefore, it was confirmed that PPP3CA and CAMK2 variations are consistent in VEGFB regulation of insulin secretion in T2DM mice, which indicated that VEGFB may stimulate insulin secretion by activating Ca2+/CaM to accelerate substrate protein phosphorylation (Fig. 7).
Moreover, the present study revealed decreased blood glucose and increased insulin secretion in VEGFB−/− mice fed with SD in the 24th week, which was different from the variation in T2DM mice with VEGFB KO. This may be associated with the leverage function of VEGFB in maintaining homeostasis. VEGFB does not play an obvious biological function in a physiological state, while it plays as a safety guard in a pathological state (54–56). Therefore, it was hypothesized that the mechanisms of VEGFB that regulate insulin secretion are different under physiological and pathological conditions. VEGFB may participate in the regulation of insulin secretion through the VEGFA/VEGFR1 signaling pathway under physiological conditions, unlike the regulatory mechanism of VEGFB on insulin secretion in T2DM mice. The signal system of the VEGF family is complex, and the affinity and selectivity of members to different receptors are different. VEGFA can combine with VEGFR1 and VEGFR2, while VEGFB can only combine with VEGFR1 (57,58). The present study supports that VEGFR2 plays a dominant role in all receptors. Some researchers consider that VEGFR1 is a decoy receptor. In general, it not only transmits mitogenic signals but also blocks VEGF, thereby preventing VEGF from binding to VEGFR2. VEGFR1 can negatively regulate the VEGFR2 signaling pathway and promote VEGFR2 under certain pathological conditions (59). At present, the specific mechanism is not completely clear. VEGFR1 expression was downregulated in the present study, while VEGFA and VEGFR2 expression levels were upregulated after the loss of VEGFB in the islets β cell of mice fed with SD. Same results were acquired through the validation of the Min6 cell line. It was indicated that the increase of insulin secretion after VEGFB KO may be related to the VEGFA/VEGFR2 signaling pathway upregulation caused by the decreased VEGFR1 expression under physiological conditions (Fig. S1U). The specific mechanism remains unclear although it was validated in Min6 cells in the present study. In the future, the association between VEGFB and VEGFA/VEGFR2 shall be further validated by the authors using receptor blockers at the in vivo and in vitro levels.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
The present study was supported by the National Natural Science Foundation of China (grant no. 31771284), the Basic Research Project of Yantai Science and Technology Innovation and Development Plan (grant no. 2022JCYJ026) and the Natural Science Foundation of Shandong province (grant no. ZR202111250163).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The mass spectrometry proteomics data have been deposited to the Proteome Xchange Consortium via the PRIDE partner repository with the dataset identifier PXD043843 (https://www.ebi.ac.uk/pride/).
Authors' contributions
YQL, RRL and XL conceptualized and designed experiments, analyzed and interpreted data and drafted the article. FX, MZY, LHZ, QHW, WGJ and YNL designed and conducted experiments, acquired, analyzed and interpreted the data, and revised the article critically for intellectual content. All authors read and approved the final version of the manuscript. WGJ and YNL confirming the authenticity of all the raw data.
Ethics approval and consent to participate
The present study was reviewed and approved by the Institutional Review Board of Binzhou Medical University (Yantai, China). All procedures involving animals were reviewed and approved (IACUC approval no. 2022-210) by the Institutional Animal Care and Use Committee of the Medical Ethics Committee of Binzhou Medical University (Yantai, China).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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