Introduction
Vitamin B3, referred to as niacin (NA) or niacinamide (NAM) (Chaykin, 1967), has a broad role in human health, such as attenuating skin aging and hyperpigmentation, as a potential drug to treat heart failure, promoting differentiation of the retinal pigment epithelium, protect neuronal cells, atherosclerosis, hypercholesterolemia, and diabetes (Abdellatif et al., 2021; Boo, 2021; Denu, 2005; Hazim et al., 2022; Mastropasqua et al., 2022). Diabetes is a syndrome characterized by hyperglycemia. A recent study showed that vitamin B3 promotes the release of C peptide and insulin, protects pancreatic β cell function, and reduces the risk of diabetes (Yilmaz et al., 2017); however, the associated molecular mechanism remains to be explored.
Diabetes is categorized as type I, type II, and gestational (Kitabchi et al., 2009). Type II diabetes mellitus (T2DM) is characterized by insulin resistance, which leads to a disorder of blood glucose metabolism. Due to insulin resistance, islet β cells have to secrete more insulin, eventually resulting in islet β cell failure (Gasmi et al., 2021; Marasco and Linnemann, 2018). Type I diabetes mellitus (T1DM) occurs from the damage of the β-cells mediated by the autoimmune process. Numerous experiments report that autophagy regulates glucose homeostasis and insulin resistance, averts β cell dysfunction, improves insulin sensitivity, and protects β cells from apoptosis during diabetes (Barlow and Thomas, 2015; Ebato et al., 2008; Li et al., 2018; Lim et al., 2018; Muralidharan and Linnemann, 2021; Zhang et al., 2015; Zhou et al., 2022). Autophagy is a conserved intracellular self-protection mechanism that functions directly in cell death and survival (Booth et al., 2014; Ploumi et al., 2022). Autophagy can be activated under various physiological conditions and is involved in the regulation of energy metabolism, stress response, pathogen clearance, and tumor occurrence and development (Shen et al., 2015). For example, it helps host cells degrade accumulated proteins, damaged mitochondria, and other aging or disordered organelles (Klionsky et al., 2021; Tu et al., 2013). Then small molecules such as amino acids can be produced, which is conducive to cell growth and ensures cell survival (Barlow and Thomas, 2015; Ebato et al., 2008; Ploumi et al., 2022; Zhang et al., 2015). Autophagy is essential, and up-regulation of autophagy activity may help to prevent the progression of diabetes. In diabetic human islets, progressive deterioration in β-cell function, reduction of glucose-stimulated insulin secretion, decreased β-cell mass, and increased β-cell apoptosis have been found (Del Prato et al., 2007; Butler et al., 2003; Sakuraba et al., 2002; Noa et al., 2007; Sidarala et al., 2020; UKPDS-Group, 1998).
Islet β cells are important cells in the human body that regulate blood glucose levels (Guo et al., 2019). Cell homeostasis is inseparable from autophagy. When autophagy is defective, the loss of the function of islet β cells can eventually lead to diabetes (Lytrivi et al., 2020; Yang et al., 2019). During hyperglycemia in patients with diabetes, normal autophagy of islet β cells can eliminate the large aggregation of ubiquitinated proteins (Jung, 2016). Insulin is produced mainly intracellularly in islet β cells. Under normal conditions, islet β cells are damaged or undergo apoptosis, further reducing insulin secretion (Chung et al., 2021). Studies have shown many reasons for islet β cell apoptosis, such as damage to the autoimmune system and imbalance of the autophagy regulation mechanism (Masini et al., 2009).
The current focus on pharmacological and nutritional research in the treatment of diabetes has shifted to the use of bioactive substances from natural sources (Wu et al., 2021). Diet intervention and exercise are the best programs for diabetes management. Vitamin B3, a natural product found in food, is an indispensable nutrient for humans and animals (Denu, 2005; Hrubsa et al., 2022). In this study, we constructed a hyperglycemic cell model and stressed islet β cells with vitamin B3 to explore the mechanism of vitamin B3 in the role of promoting the release of insulin. Our study also suggests that protecting Islet β cells by regulating autophagy through dietary vitamin B3 is an effective way of preventing the onset of diabetes.
Materials and Methods
Reagents and antibodies
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).
Cell culture
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.
Cell viability assay
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.
Immunofluorescence staining
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.
Western blotting
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.
Apoptosis analysis
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.
Discussion
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 (Kitazawa et al., 2021; Taylor et al., 2021). Research on the dietary intervention of diabetes has been increasing rapidly, with multiple natural and nutritional products thought to benefit the treatment of diabetes. For example, mangiferin significantly reduces glucose levels and prevents tissue damage in animal models of diabetes (Wu et al., 2021). Intervention with low-calorie Mediterranean-style and low-carbohydrate dietary regimens can effectively reduce insulin resistance, alleviate fasting hyperinsulinemia, and recover the function of β cells in patients with T2DM (Bolla et al., 2019; Karatzi and Manios, 2021). Vitamin B3, a natural nutrient in foods, can also recover the function of β cells by promoting the release of C-peptide and insulin, reducing the risk of type I diabetes mellitus (T1DM) (Yilmaz et al., 2017). Vitamin B3 is also involved in regulating several redox and non-redox reactions of cell energy metabolism (Dudev and Lim, 2010), which can prevent oxidative stress and improve cell survival rate, and may function in ameliorating T2DM (Yilmaz et al., 2017). Vitamin B3 has been reported to induce autophagy via an mTOR-dependent pathway, regulating the expression of autophagy-related ATG5 protein, blocking oxidative stress, and inhibiting the activity of silent mating type information regulation 2 homolog 1 (SIRT1) (Li et al., 2019; Maiese, 2021; Oblong et al., 2020; Shen et al., 2017b). Here, we found that vitamin B3 had a limited protective effect on β islet cells. However, the molecular mechanism was unknown.
Hyperglycemia is a typical symptom of diabetes. High glucose can cause complications of diabetes, such as diabetic nephropathy and cardiovascular disease (Hou et al., 2022; Kang et al., 2020a). It also induces ferroptosis by facilitating glutathione peroxidase 4 ubiquitination, promotes necroptosis via inhibition activity of aldehyde dehydrogenases 2, increases NETosis, apoptosis, and pyroptosis (Han et al., 2022; Kang et al., 2020b; Menegazzo et al., 2015; Zhang et al., 2021). Thus, high glucose-induced apoptosis of β cells and the mechanism needed to be explored.
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 (Ding and Choi, 2015). The targets of mTOR are classical nutritional pathways that regulate autophagy by forming two complexes, mTOR complex 1 (mTORC1) and mTORC2. MTORC1 inhibits the activity of Unc-51-like autophagy activating kinase (ULK1) through direct phosphorylation of ULK1, thus inhibiting the expression of autophagy-related proteins LC3-II and Beclin-1 (Dehdashtian et al., 2018). When glucose is insufficient, AMPK promotes autophagy by directly activating ULKl through the phosphorylation of Ser 317 and Ser 777 (Gödel et al., 2011). In contrast, increased mTOR or protein kinase B (AKT) activity under high glucose results in ULKl phosphorylation and inhibits the activity of ULK1, thereby disrupting the interaction between ULK1 and AMPK and then blocking autophagy (Kubli and Gustafsson, 2014; Yao et al., 2016). Natural products and diet interventions have been reported to effectively alleviate diabetes through the mTOR signaling pathway. For example, isorhamnetin and metformin ameliorate insulin resistance associated with T2DM through the mTOR signaling pathway (Taylor et al., 2021). A ketogenic diet (KD) lowers diabetic hyperketonemia by regulating glucose metabolism and suppressing insulin-like growth factor-1 and phosphoinositide 3-kinase/AKT/mTOR pathways (Kumar et al., 2021). Nicotinamide, as an inhibitor of sirtuin pathways, has been shown to protect hepatocytes against palmitate-induced lipotoxicity via SIRT1-dependent autophagy induction. Nicotinamide can also inhibit the phosphorylated activity of mTOR (Maiese, 2020; Shen et al., 2017a). In the present study, we found that vitamin B3-induced autophagy might occur via an mTOR-dependent pathway, and the protection of islet cells is limited. However, partial recovery of β-cell function is also of great significance in alleviating diabetes. Therefore, its mechanism and in vivo study need to be further explored.