1,25(OH)2D3 promotes insulin secretion through the classical pyroptosis pathway in vitro and vivo

doi:10.21203/rs.3.rs-2633066/v1

Download PDF

Research Article

1,25(OH)2D3 promotes insulin secretion through the classical pyroptosis pathway in vitro and vivo

https://doi.org/10.21203/rs.3.rs-2633066/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Purpose: To study the effect of pyroptosis on pancreatic β cell function in diabetes. And explore whether 1,25(OH)₂D₃ interferes with the progression of type 2 diabetes through pyroptosis signaling pathway.

Methods: We explored the relationship between pyroptosis and diabetes and the role of 1,25(OH)₂D₃in this process. In vivo and vitro, we constructing a diabetes model combined with 1,25(OH)₂D₃intervention. Detected the insulin secretion level, the release level of inflammatory factors and the expression level of pyroptosis-related proteins.

Results: In vivo or vitro, we have found that 1,25(OH)₂D₃can reduce the level of inflammation, reduce the occurrence of pyroptosis by reducing the expression levels of pyroptosis-related proteins NF-κB, NLRP3, GSDMD, and caspase-1. It can protect pancreatic β cell from T2DM damage and increase insulin secretion. Inhibiting the expression of GSDMD can hinder the progress of cell pyroptosis, reduce the expansion of inflammatory response and protect pancreatic cells from damage.

Conclusion: In the occurrence of T2DM, pancreatic cells are induced by pyroptosis through the classical pathway, resulting in reduced insulin secretion. 1,25(OH)₂D₃ can be used as a potential adjuvant to inhibit pyroptosis of pancreatic cells through pyroptosis classical signaling pathway, reduce inflammatory response and relieve diabetes symptoms.

25(OH)2D3

pyroptosis

inflammation

diabetes

The metabolic disease diabetes mellitus (DM) affects many people worldwide. However, the pathogenesis of DM has not been fully elucidated. Previous studies have found that inflammation is closely related to the development of DM, but the inflammation-related molecular mechanisms underlying DM are still unclear. Therefore, it is important to explore the role of inflammation-related molecular mechanisms in DM^[1].

Pyroptosis is a type of programmed cell death mediated by the activation of the caspase family. It is accompanied by cell swelling and rupture, release of cell contents, activation of inflammasomes, and release of inflammatory factors^{[2, 3]}. GSDMD is a member of gasdermins family and an executor of pyroptosis. Activated GSDMD binds to the cell plasma membrane, oligimerizes to form pores, and destroys the integrity of the cell membrane. This leads to an increase in intracellular osmotic pressure and release of inflammatory substances in the cell, which promote inflammation^{[4, 5]}. Previous studies have identified two types of pyroptosis: canonical (dependent on caspase-1) and non-canonical (caspase-4, -5, and − 11) pyroptosis^[6]. Although insulin resistance is thought to be related to inflammation, further research is needed to determine whether damage caused by pancreatic cell pyroptosis leads to decreased insulin secretion ^[7].

Previous studies have found that vitamin D (VD) inhibits the secretion of inflammatory cytokines from pancreatic β cells in diabetic mice, but whether VD affects pancreatic β cell pyroptosis remains to be explored^[8]. This experiment aimed to explore the effects of pancreatic β cell pyroptosis on DM and of 1,25(OH)₂D₃ on the pyroptosis of pancreatic β cells in DM.

2.1 Cell culture

Mouse insulinoma β cells (MIN6 cells) were purchased from Shanghai Bioleaf Biotech Co., Ltd. (Shanghai, China). MIN6 cells were cultured in DMEM medium containing 10% FBS and placed in 5% CO₂ in a 37°C cell incubator. When the cells grew and converged to 80%, they were trypsinized and passaged. The experiment was divided into 8 groups. Normal cultured MIN6 cells (NC group), GSDMD siRNA transfected MIN6 cells (siRNA group). STZ stimulated normal cultured MIN6 cells and GSDMD siRNA transfected MIN6 cells (STZ group, siRNA-STZ group). 1,25(OH)₂D₃ interfered with normal cultured MIN6 cells and GSDMD siRNA transfected MIN6 cells (NC-VD group, siRNA-VD group). 1,25(OH)₂D₃ interfered with STZ-stimulated normal cultured MIN6 cells and GSDMD siRNA-transfected MIN6 cells (STZ-VD group, siRNA-STZ-VD group).

2.2 Animals

GSDMD^-/-C57BL/6J was customized by GemPharmatech Co., Ltd. All mice were housed in the barrier facility of the Experimental Animal Center of Soochow University. The conditions were: temperature 22 ± 2°C, humidity 55 ± 5°C, light-dark cycle 12/12 h (license No. (Su) SYXK2017-0043)). The experiment was divided into 6 groups. Control group: WT group, GSDMD^-/- group, model group: DM group, GSDMD^-/--DM group, 1,25(OH)₂D₃ intervention group: DM-VD group, GSDMD^-/--DM-VD group.

DM-VD group and GSDMD^-/--DM-VD group were intraperitoneally injected with 1,25(OH)₂D₃ at 5 µg/kg every other day. WT group, GSDMD^-/- group, DM group, and GSDMD^-/--DM group were intraperitoneally injected with the same volume of peanut oil. The changes of physiological indicators (diet, water, body weight) and fasting blood glucose (FBG) of each group were recorded during the 1,25(OH)₂D₃ intervention. The serum samples of each group were collected and the expression levels of glycated hemoglobin (GHbA1c), insulin (INS), 25(OH)D, and inflammatory factors IL-18 and IL-1β were detected by ELISA. Mice were sacrificed by CO₂ asphyxiation, and the pancreas were analyzed of pathological changes and the expression of pyroptosis-related proteins.

2.3 Modeling of T2DM mice

Male mice aged 4–6 weeks were selected and fed with high-fat diet for 4 weeks. Fasting for 12 h, STZ was injected by intraperitoneal (The injection volume is 100 mg/kg). On the 3rd, 5th, and 7th days, the fasting blood glucose values of each group were detected. When the fasting blood glucose values were ≥ 16.7 mmol/L for two consecutive times, the model was considered successful.

2.4 CCK-8 detects cell viability

After cells had adhered to the wall and converged to 70–80%, the trypsinized cells were diluted to a cell suspension with a density of 5 × 10³, seeded in a 96-well plate with a volume of 100 µl/well, and cultured in 5% CO₂ in a 37°C cell incubator. Different concentrations of 1,25(OH)₂D₃ (5 × 10^− 9, 5 × 10^− 8, 5 × 10^− 7, and 5 × 10^− 6 mmol/L) were added to the cells for 24 h. After the intervention, cell wells were washed with PBS and fresh medium was added. Then, 10 µl of CCK-8 reagent (Dojindo, Kumamoto, Japan ) was added to each well and the cells were incubated for 2 h. A microplate reader was used to measure the absorbance of each reaction well at 450 nm.

2.5 Lactate dehydrogenase (LDH) release detection

We used the Lactate Dehydrogenase Cytotoxicity Detection Kit protocol for LDH release detection (Beyotime, China). An appropriate quantity of cells was seeded in a 96-well cell culture plate (100 µl/well) to attain a cell density of < 80–90%. Then, 10 µl of LDH release reagent was added to the sample maximum enzyme activity control well 1 h before the scheduled detection. A pipette was used to mix the solution, and the solution was incubated for 1 h. Then, the mixture was centrifuged at 400 g for 5 min in a multi-well plate centrifuge, and the supernatant was transferred to a new 96-well plate; 60 µl of LDH detection working reagent was added to each well, and the plate was incubated at room temperature for 30 min in the dark. A microplate reader was used to detect the absorbance of each reaction well at 490 nm.

2.6 Cell transfection

Lipofectamine 2000 and GSDMD siRNA (GenePharma, Shanghai, China) were diluted to the appropriate concentration using the serum-free medium Opti-MEM. The solution was allowed to stand at room temperature for 5 min and mixed uniformly. Then, the solution was allowed to stand for 20 min to form a transfection complex. The medium was removed and replaced with pre-warmed fresh cell culture medium (1500 µl/well). Then, 500 µl of the transfection complex was added to each well, shaken gently, and placed back into the cell incubator. The cell transfection efficiency, mRNA expression level, and protein expression level were tested at 6, 24–72, and 48–92 h, respectively.

2.7 Glucose tolerance test in mice

Before the experiment, the mice in each group were fasted for 12 hours. After intragastric administration of 2.0 g/kg of glucose, the blood glucose values (BG) of mice in each group at 0 h, 0.5 h, 1 h and 2 h were detected by tail clipping. Calculate the area under the curve (AUC) to reflect the change of glucose tolerance. AUC = 1/4 (BG 0 h) + 1/2 (BG 0.5 h) + 3/4 (BG 1h) + 1/2 (BG 2h).

2.8 Insulin resistance index(HOMA-IR)

After 1,25(OH)₂D₃ intervention, the mice were fasting for 12 hours before the experiment. The FBG values of each group were obtained by tail-cutting. Orbital vein was used to obtain blood of each group and centrifugation to collect serum. ELISA detects fasting insulin (FINS) levels and calculates HOMA-IR index. HOMA-IR=(FBG×FINS) /22.5.

2.9 Enzyme linked immunosorbent assay (ELISA)

The levels of IL-18, IL-1β, and INS in the supernatant of MIN6 cells and mouse serum were detected using the manufacturer’s instructions (Shanghai Jining Industrial Co., Ltd).

2.10 Western blot analysis

After the intervention, the cells were lysed, and the proteins were collected by centrifugation. Animal tissue were lysed to extraction protein. The protein concentration was quantified using a BCA assay (Beyotime, Jiangsu, China). We loaded 15 µl of the sample per well and applied constant-voltage electrophoresis (80 V). After using a Rainbow Marker, the voltage was switched to 120 V. The PVDF membrane (Millipore, Billerica, MA, USA) was activated in a methanol solution, and the protein samples were transferred onto the PVDF membrane using an electric transfer on ice. The membrane was blocked using 5% skim milk at room temperature for 2 h and incubated with the primary antibodies at 4°C overnight. After the incubation, the membrane was washed three times with TBST solution and incubated with the secondary antibodies (anti-rabbit IgG; Abcam, Cambridge, UK) at room temperature for 2 h. The protein bands were detected using ECL reagents (Beyotime), and Bio-Rad's ChemiDoc XRS + imaging system was used to take photographs. Image J was used for gray value analysis. The primary antibodies used in the experiment were anti-NLRP3 (1:1000, Abcam), anti-NF-κB (1:1000, Abcam), anti-GSDMD (1:1000, Abcam), anti-Caspase-1 (1:1000, Abcam), and anti-GAPDH (1:1000, Abcam).

2.11 Hematoxylin-Eosin stain

Routine deparaffinization of pancreatic tissue paraffin block sections. Hematoxylin stained for 10 min, eosin stained for 20 s, washed with water. Dehydrated with xylene and transparent, then permanently mounted with neutral gum.

2.12 Statistical analyses

GraphPad Prism 7.0, Image J、SPSS21.0 was used for analysis. Data are expressed as mean ± standard deviation. Data were analyzed using an unpaired t-test. One-way ANOVA for comparison of multiple groups of data. Differences were considered statistically significant at p < 0.05.

3.1 Effect of 1,25(OH)₂D₃ on insulin secretion from Min6 cells.

After the cells were treated with 1,25(OH)₂D₃ for 24 h, CCK-8 was used to detect the absorbance at 450 nm. Compared to the control group, cells treated with 1,25(OH)₂D₃ had significantly increased proliferation of MIN6 cells. A concentration of 5 × 10^− 7 mmol/L of 1,25(OH)₂D₃ was superior to other doses in terms of the effects on the proliferation of MIN6 cells (p < 0.001; Fig. 1A). Therefore, the concentration of 5 × 10^− 7 mmol/L was selected for subsequent experiments. ELISA was used to detect the secretion of insulin level. Compared to the NC group, cells in the STZ group had a significantly decreased insulin level (p < 0.001; Fig. 1B). In addition, 1,25(OH)₂D₃ significantly increased the insulin secretion from MIN6 cells (p < 0.001; Fig. 1B).

3.2 1,25(OH)₂D₃ inhibits pyroptosis of MIN6 cells

Compared to the control group, STZ stimulation increased the LDH secretion level from MIN6 cells, while 1,25(OH)₂D₃ significantly reduced LDH secretion (p < 0.01; Fig. 2A). Western blot analysis showed that compared to the control group, the expression of pyroptosis-related proteins NF-κB, NLRP3, GSDMD, and caspase-1 was significantly increased in the STZ group (p < 0.01; Fig. 2B, C). Compared to the STZ group, the expression level of pyroptosis-related proteins in the STZ-VD group was significantly decreased (p < 0.001; Fig. 2B, C). ELISA was used to detect the release of inflammatory factors, which showed that STZ significantly increased the release of inflammatory factors IL-18 and IL-1β from MIN6 cells. In contrast, the expression of IL-1β and IL-18 in the STZ-VD group was significantly reduced compared to the STZ group (p < 0.001; Fig. 2D, E).

Western blot analysis showed that inhibiting the expression of GSDMD protein downregulated the expression of NLRP3 and NF-κB. Compared to the siRNA group, the protein expression level was higher in the siRNA-STZ group (p < 0.01; Fig. 2B, C). In addition, 1,25(OH)₂D₃ effectively reduced the increased expression level of pyroptosis-related proteins caused by STZ, which is consistent with the aforementioned results. However, there was no significant difference in the expression level of caspase-1 protein in response to inhibition of GSDMD protein (p > 0.05; Fig. 2B, C). ELISA was used to detect the release of inflammatory factors, which showed that compared to the siRNA group, the expression levels of inflammatory factors were significantly decreased in the siRNA-STZ group (p < 0.001; Fig. 2D, E). Compared to the siRNA group, the expression level of the inflammatory factor IL-1β in the siRNA-STZ-VD group was significantly decreased (p < 0.001; Fig. 2D). The expression level of the inflammatory factor IL-18 in siRNA-STZ-VD group was slightly decreased but without statistical significance (p > 0.05; Fig. 2E).

3.3 1,25(OH)₂D₃ promotes insulin secretion and improves glucose metabolism in T2DM mice

Detect insulin secretion in each group, compared with control group, the insulin secretion in model group was significantly increased (p < 0.0001; Fig. 3A). Compared with DM group, the insulin secretion in DM-VD group was significantly decreased (p < 0.05; Fig. 3A). Compared with GSDMD^-/--DM group, the insulin secretion in GSDMD^-/--DM-VD group also decreased, but was not significant (p > 0.05; Fig. 3A). Compared with DM group, the insulin secretion in GSDMD^-/--DM group was decreased, but there was no significant difference (p > 0.05; Fig. 3A).

Detect of glucose metabolism indexes showed that compared with control group, the model group had significant increases in fasting blood glucose, insulin resistance index, glycosylated hemoglobin, and glucose tolerance AUC indexes (p < 0.0001; Fig. 3B, C, D). After 1,25(OH)₂D₃ intervention, glucose metabolism indexes decreased to varying degrees. Compared with DM group, the glucose metabolism indexes of the GSDMD^-/--DM group was also decreased (p < 0.05; Fig. 3D).

3.4 1,25(OH)₂D₃ inhibits the pyroptosis of pancreatic cells in T2DM mice

Compared with WT group, the expressions of pyroptosis-related proteins NF-κB, GSDMD, and caspase-1 in DM group were significantly increased (p < 0.001, p < 0.0001; Fig. 4A, B), and the expression of NLRP3 protein was increased but has no significant. Compared with DM group, the expressions of pyroptosis-related proteins NLRP3, NF-κB, GSDMD and caspase-1 in DM-VD group were significantly decreased (p < 0.01, p < 0.001, p < 0.0001; Fig. 4A, B). In GSDMD^-/- mice, the expressions of pyroptosis-related proteins NLRP3, NF-κB, and caspase-1 decreased overall (p < 0.05, p < 0.0001; Fig. 4A, B). Compared with the DM group, the expression of the pyroptosis-related proteins in the GSDMD^-/--DM group were significantly decreased (p < 0.05, p < 0.0001; Fig. 4A, B).

ELISA was used to detected the secretion of inflammatory factors IL-18 and 1L-1β in the serum of each group. Compared with the WT group, the secretion of inflammatory factors IL-18 and 1L-1β of the mice in the DM group increased significantly (p < 0.001, p < 0.0001; Fig. 4C, D). Compared with DM group, the secretion of inflammatory factors IL-18 and 1L-1β in the DM-VD group was significantly decreased (p < 0.01; Fig. 4C, D). Compared with GSDMD^-/- group, the release of inflammatory factors IL-18 and 1L-1β in GSDMD^-/--DM mice were increased. After 1,25(OH)₂D₃ intervention, the release of inflammatory factors in the GSDMD^-/--DM-VD group was significantly decreased, but there was no statistical difference in the inflammatory factor IL-18 (p > 0.05; Fig. 4D). Compared with DM group, the release of inflammatory factors in the GSDMD^-/--DM group were decreased, but the decrease of IL-1β had no statistical difference (p > 0.05; Fig. 4C, D).

3.5 1,25(OH)₂D₃ improves pancreatic tissue damage in T2DM mice

Results of HE staining found that the shape of the islets of WT group was normal and the cells in the islets were evenly dispersed and the outline was clear (Fig. 5A). The islets of DM group had infiltration of inflammatory cells, the morphology of islets changed and the distribution of cells in the islets was disordered (Fig. 5B). The shape of islets of DM-VD group was normal, with less inflammatory cell infiltration and cells are more even distribution (Fig. 5C). The cells in the islets of GSDMD^-/- group were evenly dispersed and the islet morphology was normal (Fig. 5D). Islets in GSDMD^-/--DM group were normal in shape, with a small amount of inflammatory cell infiltration, and the distribution of cells was disordered (Fig. 5E). GSDMD^-/--DM-VD group had less inflammatory cell infiltration and cells distribution more uniform in islets (Fig. 5F).

DM is a metabolic disease characterized by a high blood glucose level. The morbidity of DM in adults has continued to increase. Many studies have reported a close relationship between inflammation and DM. The inflammasome NLRP3 can be activated by metabolic signaling molecules such as glucose and saturated fatty acids. Once NLRP3 is activated, cells secrete a large number of pro-inflammatory cytokines, which affect glucose tolerance and insulin resistance, leading to DM progression. In addition, a high level of IL-1β may reduce insulin sensitivity in obese individuals^{[9, 10]}. In our study, we used STZ to stimulate MIN6 cells to establish a DM model. We found that STZ stimulation could significantly reduce the level of insulin secretion in MIN6 cells (p < 0.001) and increase the levels of inflammatory factors IL-18 and IL-1β in the cell supernatant (p < 0.001), suggesting that inflammation is involved in the development of DM. Long-term chronic inflammation can damage pancreatic β cells and affect insulin secretion.

During pyroptosis, NLRP3 inflammasome is activated by pathogen-related and damage-related molecular patterns. Once activated, NLRP3 promotes polymerization of the adaptor protein ASC, recruits caspase-1, and promotes the dimerization and activation of caspase-1. Activated caspase-1 cleaves the precursors of IL-18 and IL-1β and activates GSDMD. Activated GSDMD binds to the plasma membrane to form pores, thereby triggering a series of inflammatory cascades^{[11, 12]}. As a nuclear transcription factor with extensive biological activity, NF-κB regulates the expression of multiple genes, such as those encoding for cytokines, transcription factors, and oxidative stress-related enzymes. It is also involved in a variety processes of pathological processes, such as inflammation, oxidative stress, and apoptosis. Some studies have found that the NF-κB signaling pathway is closely related to inflammation and affects insulin resistance^{[13, 14]}. In our study, we found that STZ stimulation can upregulate the expression levels of NF-κB, NLRP3, GSDMD, and caspase-1 (p < 0.01), as well as promote the release of LDH, IL-18, and IL-1β (p < 0.01). And we found the same trend in vivo experiments. This suggested that pyroptosis affects the development of DM. Pyroptosis increases the release of inflammatory factors and cell contents, thereby promoting the inflammatory response, affecting the function of pancreatic β cells, and aggravating DM.

VD can protect cells from oxidative damage mediated by hyperglycemia in DM. Some epidemiological studies have shown that circulating 1,25(OH)₂D₃ levels are negatively correlated with the occurrence of DM^{[15, 16]}. Other studies have reported that VD has anti-inflammatory effects mediated by an altered balance between pro- and anti-inflammatory cytokines, thereby limiting metabolic inflammation^[17]. However, the correlation between VD and DM needs further research. In vivo and vitro, we explored the relationship between 1,25(OH)₂D₃ and diabetic pyroptosis and found that 1,25(OH)₂D₃ treatment can increase insulin secretion from damaged cells (p < 0.05) as well as downregulate the increased expression of pyroptosis-related proteins (NF-κB, NLRP3, GSDMD, and caspase-1) (p < 0.001) caused by STZ stimulation. It can also decrease the release of LDH and inflammatory factors (IL-18 and IL-1β) from the cell supernatant (p < 0.01), suggesting that 1,25(OH)₂D₃ inhibits pyroptosis, reduces the release of inflammatory factors, and protects the pancreatic β cells. HE staining results found that the degree of pancreatic tissue damage decreased after 1,25(OH)₂D₃ intervention.

Furthermore, we explored the effects of 1,25(OH)₂D₃ on pyroptosis by transfecting MIN6 cells with GSDMD siRNA and use GSDMD^-/- mice to make T2DM model. We found that the inhibition of GSDMD expression downregulated the expression levels of NF-κB and NLRP3 proteins and significantly reduced the increased release of inflammatory factors caused by STZ stimulation. GSDMD is an executor of pyroptosis. Inhibiting the expression of GSDMD can prevent the formation of pores in the cell plasma membranes, prevent the leakage of cell contents and inflammatory factors, and reduce cell damage. Interestingly, inhibiting the expression of GSDMD led to an increased expression level of caspase-1, with no significant difference between the groups. Therefore, STZ acts on GSDMD to induce pyroptosis in vitro. Inhibiting the expression of GSDMD leads to reduced expression level of inflammasomes and pyroptosis. However, the increased level of caspase-1 suggests that the inhibition of pyroptosis activates the apoptosis signaling pathway. This needs to be studied further.

In summary, STZ stimulation damages pancreatic β cells and induces pyroptosis. It promotes the formation of pores in the plasma membrane, upregulates the protein expression levels of NLRP3, NF-κB, caspase-1, and GSDMD in the pyroptosis signaling pathway, and increases the release of inflammatory factors. Treatment with 1,25(OH)₂D₃ reduces cell damage caused by STZ stimulation. It can downregulate the protein expression levels of NLRP3, NF-κB, caspase-1, and GSDMD in the pyroptosis signaling pathway, reduce the release level of inflammatory factors, and inhibit pyroptosis. Our experiments have demonstrated that pyroptosis is involved in the development of DM, and 1,25(OH)₂D₃ plays an important role in DM by acting on the canonical pyroptosis pathway. In addition, 1,25(OH)₂D₃ inhibits the expression of pyroptosis-related proteins and inflammatory factors to protect the pancreatic β cells and increase insulin secretion.

Funding

This work was supported by National Natural Science Foundation of China (grant numbers 31970505).

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Ethics approval

This study was approved by the Ethics Committee of Suzhou University.

Acknowledgments

We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

Jo E K, Kim J K, Shin D M, et al. Molecular mechanisms regulating NLRP3 inflammasome activation. Cell Mol Immunol. 2016;13(2):148-159. https://doi.org/10.1038/cmi.2015.95
Wang Y, Zhu X, Yuan S, et al. TLR4/NF-κB Signaling Induces GSDMD-Related Pyroptosis in Tubular Cells in Diabetic Kidney Disease. Front Endocrinol (Lausanne). 2019;10603. https://doi.org/10.3389/fendo.2019.00603
Shi J, Zhao Y, Wang K, et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature. 2015;526(7575):660-665. https://doi.org/10.1038/nature15514
Zeng C, Wang R, Tan H. Role of Pyroptosis in Cardiovascular Diseases and its Therapeutic Implications. Int J Biol Sci. 2019;15(7): 1345-1357. https://doi.org/10.7150/ijbs.33568
Zhao W, Yang H, Lyu L, et al. GSDMD, an executor of pyroptosis, is involved in IL-1β secretion in Aspergillus fumigatus keratitis. Exp Eye Res. 2021;202108375. https://doi.org/10.1016/j.exer.2020.108375
Aglietti R A, Dueber E C. Recent Insights into the Molecular Mechanisms Underlying Pyroptosis and Gasdermin Family Functions. Trends Immunol. 2017;38(4):261-271. https://doi.org/10.1016/j.it.2017.01.003
Zhu M J, Kang Y, Xue Y, et al. Red raspberries suppress NLRP3 inflammasome and attenuate metabolic abnormalities in diet-induced obese mice. J Nutr Biochem. 2018;5396-103. https://doi.org/10.1016/j.jnutbio.2017.10.012
Lips P, Eekhoff M, van Schoor N, et al. Vitamin D and type 2 diabetes. J Steroid Biochem Mol Biol. 2017;173280-285. https://doi.org/10.1016/j.jsbmb.2016.11.021
Ding S, Xu S, Ma Y, et al. Modulatory Mechanisms of the NLRP3 Inflammasomes in Diabetes. Biomolecules. 2019;9(12):850. https://doi.org/10.3390/biom9120850
Reynolds C M, McGillicuddy F C, Harford K A, et al. Dietary saturated fatty acids prime the NLRP3 inflammasome via TLR4 in dendritic cells-implications for diet-induced insulin resistance. Mol Nutr Food Res. 2012;56(8):1212-1222. https://doi.org/10.1002/mnfr.201200058
Monteleone M, Stanley A C, Chen K W, et al. Interleukin-1β Maturation Triggers Its Relocation to the Plasma Membrane for Gasdermin-D-Dependent and -Independent Secretion. Cell Rep. 2018;24(6):1425-1433. https://doi.org/10.1016/j.celrep.2018.07.027
Yu Z W, Zhang J, Li X, et al. A new research hot spot: The role of NLRP3 inflammasome activation, a key step in pyroptosis, in diabetes and diabetic complications. Life Sci. 2020;240117138. https://doi.org/10.1016/j.lfs.2019.117138
Hu S, Dai J, Chen X. Vitamin D reduces autophagy by regulating NF-κB resistance to Aspergillus fumigatus infection. Gene. 2020;753144819. https://doi.org/10.1016/j.gene.2020.144819
Luo C, Yang H, Tang C, et al. Kaempferol alleviates insulin resistance via hepatic IKK/NF-κB signal in type 2 diabetic rats. Int Immunopharmacol. 2015;28(1):744-750. https://doi.org/
He D, Wang Y, Liu R, et al. 1,25(OH)(2)D(3) Activates Autophagy to Protect against Oxidative Damage of INS-1 Pancreatic Beta Cells. Biol Pharm Bull. 2019;42(4):561-567. https://doi.org/10.1248/bpb.b18-00395
Guo X, Lin H, Liu J, et al. 1,25-Dihydroxyvitamin D attenuates diabetic cardiac autophagy and damage by vitamin D receptor-mediated suppression of FoxO1 translocation. J Nutr Biochem. 2020;80108380. https://doi.org/10.1016/j.jnutbio.2020.108380
Garbossa S G, Folli F. Vitamin D, sub-inflammation and insulin resistance. A window on a potential role for the interaction between bone and glucose metabolism. Rev Endocr Metab Disord. 2017;18(2):243-258. https://doi.org/ 10.1007/s11154-017-9423-2

Download PDF

Version 1

posted

You are reading this latest preprint version

1,25(OH)2D3 promotes insulin secretion through the classical pyroptosis pathway in vitro and vivo

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Cell culture

2.2 Animals

2.3 Modeling of T2DM mice

2.4 CCK-8 detects cell viability

2.5 Lactate dehydrogenase (LDH) release detection

2.6 Cell transfection

2.7 Glucose tolerance test in mice

2.8 Insulin resistance index(HOMA-IR)

2.9 Enzyme linked immunosorbent assay (ELISA)

2.10 Western blot analysis

3 Results

3.1 Effect of 1,25(OH)₂D₃ on insulin secretion from Min6 cells.

3.2 1,25(OH)₂D₃ inhibits pyroptosis of MIN6 cells

3.3 1,25(OH)₂D₃ promotes insulin secretion and improves glucose metabolism in T2DM mice

3.4 1,25(OH)₂D₃ inhibits the pyroptosis of pancreatic cells in T2DM mice

3.5 1,25(OH)₂D₃ improves pancreatic tissue damage in T2DM mice

4 Discussion

Declarations

References

Status:

Version 1

1,25(OH)2D3 promotes insulin secretion through the classical pyroptosis pathway in vitro and vivo

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Cell culture

2.2 Animals

2.3 Modeling of T2DM mice

2.4 CCK-8 detects cell viability

2.5 Lactate dehydrogenase (LDH) release detection

2.6 Cell transfection

2.7 Glucose tolerance test in mice

2.8 Insulin resistance index(HOMA-IR)

2.9 Enzyme linked immunosorbent assay (ELISA)

2.10 Western blot analysis

3 Results

3.1 Effect of 1,25(OH)2D3 on insulin secretion from Min6 cells.

3.2 1,25(OH)2D3 inhibits pyroptosis of MIN6 cells

3.3 1,25(OH)2D3 promotes insulin secretion and improves glucose metabolism in T2DM mice

3.4 1,25(OH)2D3 inhibits the pyroptosis of pancreatic cells in T2DM mice

3.5 1,25(OH)2D3 improves pancreatic tissue damage in T2DM mice

4 Discussion

Declarations

References

Status:

Version 1

3.1 Effect of 1,25(OH)₂D₃ on insulin secretion from Min6 cells.

3.2 1,25(OH)₂D₃ inhibits pyroptosis of MIN6 cells

3.3 1,25(OH)₂D₃ promotes insulin secretion and improves glucose metabolism in T2DM mice

3.4 1,25(OH)₂D₃ inhibits the pyroptosis of pancreatic cells in T2DM mice

3.5 1,25(OH)₂D₃ improves pancreatic tissue damage in T2DM mice