Hyperglycemia is a typical symptom of diabetes. High glucose induces apoptosis of islet β cells. While autophagy functions in cytoprotection and autophagic cell death. The interaction between autophagy and apoptosis is important in the modulation of the function of islet β cells. Vitamin B3 can induce autophagy and inhibit islet β apoptosis.
The mechanism of vitamin B3-mediated protective effect on the function of islet β cells was explored by the method of western blot, immunofluorescence and flow cytometry.
In the present study, high glucose stress increased the apoptosis rate, while vitamin B3 reduced the apoptosis rate. The effect of vitamin B3 on autophagy flux under normal and high glucose stress was also investigated. Vitamin B3 increased the number of autophagosomes and increased the light chain (LC)3-II/LC3-I ratio. In contrast, vitamin B3 decreased sequestosome 1 (SQSTM1)/p62 protein expression and inhibited the phosphorylation of mammalian ribosomal protein S6 kinase β-1 (p70S6K/S6K1), which was a substrate of mammalian target of rapamycin (mTOR) under normal and high glucose stress. To further verify the protective effect of vitamin B3 on apoptosis, we treated islet β cell RIN-m5F with autophagy inhibitor 3-methyladenine (3-MA). Vitamin B3 decreased the apoptosis rate under high glucose stress, while the inhibition of apoptosis by vitamin B3 was blocked after adding 3-MA.
Our data suggested that vitamin B3 reduced the apoptosis rate of β cells, possibly through inducing autophagy under high glucose stress.
Vitamin B3, referred to as niacin (NA) or niacinamide (NAM) (
Diabetes is categorized as type I, type II, and gestational (
Islet β cells are important cells in the human body that regulate blood glucose levels (
The current focus on pharmacological and nutritional research in the treatment of diabetes has shifted to the use of bioactive substances from natural sources (
Vitamin B3 (8060) was purchased from Beijing Solarbio Biological Technology Co., Ltd. (Shanghai, China). Anti-light chain 3 (LC3) polyclonal antibody (PM036), anti-LC3 monoclonal antibody (M186-3), and anti-sequestosome 1 (SQSTM1)/p62 (PM045) antibody were obtained from Medical Biological Laboratory (MBL, Japan). Anti-p70S6K (2708) and anti-phosphorylated p70S6K (9206) were obtained from Cell Signaling Technology (Beverly, MA). Anti-3-phosphoglyceraldehyde dehydrogenase (GAPDH) antibody (ZB002) was purchased from YTHX Biotechnology Co., Ltd. (Beijing, China). Propidium iodide (PI)-annexin V/fluorescein isothiocyanate (FITC) for flow cytometry was purchased from BD Biotechnology Research Co., Ltd. Goat anti-mouse IgG (1070-05) and goat anti-rabbit (4050-05) antibodies were purchased from Southern Biotechnology Company (Birmingham, UK). Alexa Fluor 488 Goat anti-Rabbit (A11034) antibody was obtained from Invitrogen Life Technology (Shanghai, China).
RIN-m5F cells were cultured in Roswell Park Memorial Institute-1640 medium with 10% fetal bovine serum (FBS) (BI, Israel) at 37°C, 5% CO2.
The RIN-m5F cells were incubated in 96-well plates for 24 h (37°C, 5% CO2). Then, cells were added with 10 μL Cell Counting Kit-8 (CCK-8) solution to each well after the cells were treated in different ways. Finally, the absorbance at 450 nm of each well was measured by a microplate reader. Each experiment was repeated at least three times.
RIN-m5F cells were cultured in 24-well plates with round cover glasses. The cells were washed with phosphate-buffered saline (PBS) three times and then soaked with 4% paraformaldehyde for 10 min. The cells were sealed with PBS containing 10% FBS for 30 min after washing three times with PBS. The cells were washed with PBS three times after incubation with an anti-LC3 antibody at 37°C for 1 h. Then, the cells were incubated with Alexa Fluor 488 goat anti-rabbit antibody at 37°C for 1 h and finally sealed with a fluorescence quencher. Images were observed with a confocal microscope (Zeiss LSM 710, Germany). Each experiment was repeated at least three times.
RIN-m5F cells were cultured in 6-well plates. Then, the medium was removed, and the cells were washed with PBS three times. The cells were treated with 2% sodium dodecyl sulfate (SDS; 200 μL per well). Then, the extract was heated at 100°C for 10 min and mixed with 6× protein loading buffer (Transgen; J21020). The proteins in this extract were separated with sodium dodecyl sulfate-polyacrylamide gel (4% stacking gel, 15% separating gel, 50 V for 30 min, 90 V for 120 min) electrophoresis after the extract was heated for 10 min at 100°C and then transferred to a nitrocellulose membrane (250 mA, 30–60 min). The membranes were sealed with 5% skimmed milk powder for 1 h and incubated overnight with primary antibody at 4°C. The membranes were incubated with a secondary antibody for 1 h after washing with PBST (PBS plus 0.2% Tween-20) three times. Then the membranes were visualized, and images were acquired with a luminescent image analyzer (Model: Image Quant LAS4000 Mini, Serial No. 3614294; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) after incubation for a few minutes with WesternBrightTM ECL chemiluminescent HRP substrate (SuperSignal West Dura, 32106, Thermo Pierce). Each experiment was repeated at least three times.
Annexin V-FITC-PI Apoptosis Detection Kit was used for the analysis of apoptotic cells by flow cytometry. Rin-m5F cells were cultured in 12-well plates for 24–36 h, then washed with PBS and digested with trypsin. The cells were obtained by centrifugation and suspended in 100 μL binding buffer (10e6 cells/mL). Each tube was stained with 5 μL annexin V-FITC and 5 μL PI for 15 min, and then 400 μL binding buffer was added. The intensity of these cells was analyzed with flow cytometry (Beckman MoFlo XDP, USA). Each experiment was repeated at least three times.
We observed that 20 mM glucose increased the apoptosis rate from 8.46% to 18.71%, while 20 μM vitamin B3 reduced the apoptosis rate of RIN-m5F cells from 18.71% to 11.03% (
Our data showed that 20 μM vitamin B3 had no effect on the cell viability of RIN-m5F cells, while 40 and 80 μM vitamin B3 decreased the cell viability of RIN-m5F cells from 89.68% to 78.91% and 53.06%, respectively (
Glucose at 10 and 20 mM had no effect on cell viability; however, after treatment with 30 mM glucose, cell viability decreased (
To test the effect of vitamin B3 on autophagy under high glucose stress, the cells were treated with 20 μM vitamin B3 under high glucose stress for 36 h. The number of autophagosomes increased with the increased dose of vitamin B3 under high glucose stress (
Our data showed that vitamin B3 reduced the total apoptosis rate of β cells from 18.05% to 10.25% under high glucose stress (
Diabetes is one of the most prevalent diseases in the world and is a serious public health threat. Drug therapy and dietary interventions are effective ways to treat diabetes. Metformin, sulfonylureas, insulin, and other drugs can significantly relieve T2DM with a hypoglycemic effect, but they cannot prevent islet cell failure (
Hyperglycemia is a typical symptom of diabetes. High glucose can cause complications of diabetes, such as diabetic nephropathy and cardiovascular disease (
Autophagy is regulated by different signaling pathways, including the mTOR/AMP-activated protein kinase (AMPK)-dependent and -independent signaling pathways. Hyperglycemia can activate the mTOR signaling pathway by inhibiting autophagy (
type II diabetes mellitus
type I diabetes mellitus
AMP-activated protein kinase
mTOR complex 1
mTOR complex 2
insulin-like growth factor-1
phosphoinositide 3-kinase
protein kinase B
Ketogenic diet
This paper was supported by the
Yanyang Wu and Yu Zhang conceived and designed the experiments; Yu Zhang performed the experiments; Yu Zhang and Xi’an Zhou analyzed the data; Yanyang Wu and Dongbo Liu contributed reagents/materials/analysis tools; Yu Zhang wrote the paper.
All data generated or analyzed during this study are included in this published article (and its supplementary information files).
Not applicable.
The authors declare that they have no conflicts of interest to report regarding the present study.