• Nutrition Research and Practice
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• v.12 no.3
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• pp.183-190
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• 2018

BACKGROUND/OBJECTIVE: This study was designed to investigate how a Portulaca oleracea L. extract (POE) stimulates insulin secretion in INS-1 pancreatic ${\beta}-cells$. MATERIALS/METHOD: INS-1 pancreatic ${\beta}-cells$ were incubated in the presence of various glucose concentrations: 1.1 or 5.6, 16.7 mM glucose. The cells were treated with insulin secretagogues or insulin secretion inhibitor for insulin secretion assay using an insulin ELISA kit. In order to quantify intracellular influx of $Ca^{2+}$ caused by POE treatment, the effect of POE on intracellular $Ca^{2+}$ in INS-1 pancreatic ${\beta}-cells$ was examined using Fluo-2 AM dye. RESULTS: POE at 10 to $200{\mu}g/mL$ significantly increased insulin secretion dose-dependently as compared to the control. Experiments at three glucose concentrations (1.1, 5.6, and 16.7 mM) confirmed that POE significantly stimulated insulin secretion on its own as well as in a glucose-dependent manner. POE also exerted synergistic effects on insulin secretion with secretagogues, such as L-alanine, 3-isobutyl-1-methylxanthine, and especially tolbutamide, and at a depolarizing concentration of KCl. The insulin secretion caused by POE was significantly attenuated by treatment with diazoxide, an opener of the $K{^+}_{ATP}$ channel (blocking insulin secretion) and by verapamil (a $Ca^{2+}$ channel blocker). The insulinotropic effect of POE was not observed under $Ca^{2+}$-free conditions in INS-1 pancreatic ${\beta}-cells$. When the cells were preincubated with a $Ca^{2+}$ fluorescent dye, Fluo-2 (acetoxymethyl ester), the cells treated with POE showed changes in fluorescence in red, green, and blue tones, indicating a significant increase in intracellular $Ca^{2+}$, which closely correlated with increases in the levels of insulin secretion. CONCLUSIONS: These findings indicate that POE stimulates insulin secretion via a $K{^+}_{ATP}$ channel-dependent pathway in INS-1 pancreatic ${\beta}-cells$.

• Nutrition Research and Practice
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• v.8 no.5
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• pp.494-500
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• 2014

BACKGROUND/OBJECTIVES: This study investigated whether Padina arborescens extract (PAE) protects INS-1 pancreatic ${\beta}$ cells against glucotoxicity-induced apoptosis. MATERIALS/METHODS: Assays, including cell viability, lipid peroxidation, generation of intracellular ROS, NO production, antioxidant enzyme activity and insulin secretion, were conducted. The expressions of Bax, Bcl-2, and caspase-3 proteins in INS-1 cells were evaluated by western blot analysis, and apoptosis/necrosis induced by high glucose was determined by analysis of FITC-Annexin V/PI staining. RESULTS: Treatment with high concentrations of glucose induced INS-1 cell death, but PAE at concentrations of 25, 50 or $100{\mu}g/ml$ significantly increased cell viability. The treatment with PAE dose dependently reduced the lipid peroxidation and increased the activities of antioxidant enzymes reduced by 30 mM glucose, while intracellular ROS levels increased under conditions of 30 mM glucose. PAE treatment improved the secretory responsiveness following stimulation with glucose. The results also demonstrated that glucotoxicity-induced apoptosis is associated with modulation of the Bax/Bcl-2 ratio. When INS-1 cells were stained with Annexin V/PI, we found that PAE reduced apoptosis by glucotoxicity. CONCLUSIONS: In conclusion, the present study indicates that PAE protects against high glucose-induced apoptosis in pancreatic ${\beta}$ cells by reducing oxidative stress.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.7
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• pp.1060-1065
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• 2003

To determine whether dietary fatty acids affect pancreatic $\beta$-cell function, the INS-1 $\beta$-cells and the pancreatic islets isolated from Zucker obese (fa/fa) rats were cultured with stearic acid and conjugated linoleic acid (CLA). As a result, DNA fragmentation laddering was substantially decreased in the INS-1 $\beta$-cells and the isolated pancreatic islets cultured with 2 mM CLA compared to those cultured with stearic acid. To investigate the mechanism by which CLA alleviates cell apoptosis under DNA fragmentation assay, we examined mRNA expressions of apoptosis-related proteins including Bax and Bcl-2 associated with cell death agonist and antagonist, respectively, in both INS-1 cells and islets cultured with 2 mM fatty acids. Bax mRNA expression was not altered by either stearic acid or CLA, whereas Bcl-2 mRNA expression was enhanced by CLA when compared to the stearic acid cultures. However, there were no changes in cell apoptosis and apoptotic-regulating gene products in either INS-1 cells or isolated islets treated with or without 2 mM CLA. It is concluded that CLA maintains $\beta$-cell viability via increased Bcl-2 expression compared to the stearic acid cultures, which may help to alleviate, at least somewhat, the onset of NIDDM in the physiological status. More detailed study is still needed to elucidate the effect of CLA on the prevention of fatty acid-induced $\beta$-cell apoptosis.

• Journal of Life Science
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• v.34 no.1
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• pp.9-17
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• 2024

Diabetes mellitus (DM) is one of the main global health problems. Chronic exposure to hyperglycemia can lead to cellular dysfunction that may become irreversible over time, a process that is termed glucose toxicity. Our perspective about glucose toxicity as it pertains to the pancreatic β-cell is that the characteristic decreases in insulin secretion are caused by regulated apoptotic gene expression. In this study, we examined whether ferulic acid protects INS-1 pancreatic cells against high glucose-induced apoptosis. High glucose concentration (30 mM) induced glucotoxicity and death of INS-1 pancreatic β cells. However, treatment with 1, 5, 10, or 20 μM ferulic acid increased the cell viability in a concentration-dependent manner. Treatment with ferulic acid dose-dependently decreased the intracellular levels of reactive oxygen species, thiobarbituric acid reactive substances, and nitric oxide in INS-1 pancreatic β cells pretreated with high glucose. These effects influence the apoptotic pathway, increasing the expression of the anti-apoptotic protein Bcl-2 and reducing the levels of pro-apoptotic proteins, including Bax, cytochrome C, and caspase 9. Annexin V/propidium iodide staining indicated that ferulic acid significantly reduced high glucose-induced apoptosis. These results demonstrate that ferulic acid is a potential therapeutic agent to protect INS-1 pancreatic β cells against high glucose-induced apoptosis.

• Biomolecules & Therapeutics
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• v.28 no.2
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• pp.163-171
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• 2020

Silibinin exhibits antidiabetic potential by preserving the mass and function of pancreatic β-cells through up-regulation of estrogen receptor-α (ERα) expression. However, the underlying protective mechanism of silibinin in pancreatic β-cells is still unclear. In the current study, we sought to determine whether ERα acts as the target of silibinin for the modulation of antioxidative response in pancreatic β-cells under high glucose and high fat conditions. Our in vivo study revealed that a 4-week oral administration of silibinin (100 mg/kg/day) decreased fasting blood glucose with a concurrent increase in levels of serum insulin in high-fat diet/streptozotocin-induced type 2 diabetic rats. Moreover, expression of ERα, NF-E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) in pancreatic β-cells in pancreatic islets was increased by silibinin treatment. Accordingly, silibinin (10 μM) elevated viability, insulin biosynthesis, and insulin secretion of high glucose/palmitate-treated INS-1 cells accompanied by increased expression of ERα, Nrf2, and HO-1 as well as decreased reactive oxygen species production in vitro. Treatment using an ERα antagonist (MPP) in INS-1 cells or silencing ERα expression in INS-1 and NIT-1 cells with siRNA abolished the protective effects of silibinin. Our study suggests that silibinin activates the Nrf2-antioxidative pathways in pancreatic β-cells through regulation of ERα expression.

• Journal of Life Science
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• v.31 no.11
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• pp.969-979
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• 2021

Type 2 diabetes is a serious chronic metabolic disease, and the goal of diabetes treatment is to keep blood glucose at a normal level and prevent complications from diabetes. Hyperglycemia is a key pathologic feature of type 2 diabetes that mainly results from insulin resistance and pancreatic β-cell dysfunction. Chronic exposure of β-cells to elevated glucose concentrations induces glucotoxicity. In this study, we examined whether an 80% ethanol extract of Oxya chinensis sinuosa Mishchenko (OEE) protected INS-1 pancreatic β-cells against glucotoxicity-induced apoptosis and oxidative stress. Pretreatment with a high concentration of glucose (high glucose = 30 mM) induced glucotoxicity and apoptosis of INS-1 pancreatic β cells. Treatment with OEE significantly increased cell viability. Treatment with 0.01-0.20 mg/ml OEE dose dependently decreased intracellular reactive oxygen species, lipid peroxidation, and nitric oxide levels and increased insulin secretion in high glucose-pretreated INS-1 β cells. OEE also significantly increased the activities of antioxidant enzymes in response to high-glucose-induced oxidative stress. Moreover, OEE treatment significantly reduced the expressions of pro-apoptotic proteins, including Bax, cytochrome C, caspase-3, and caspase-9, and increased anti-apoptotic Bcl-2 expression. Apoptotic cells were identified using Annexin-V/propidium iodide staining, which revealed that treatment with OEE significantly reduced high-glucose-induced apoptosis. These findings implicate OEE as a valuable functional food in protecting pancreatic β-cells against glucotoxicity-induced apoptosis and oxidative stress.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.1
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• pp.53-59
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• 2011

The secretion of insulin from pancreatic ${\beta}$-cells is triggered by the influx of $Ca^{2+}$ through voltage-dependent $Ca^{2+}$ channels. The resulting elevation of intracellular calcium ($[Ca^{2+}]_i$) triggers additional $Ca^{2+}$ release from internal stores. Less well understood are the mechanisms involved in $Ca^{2+}$ mobilization from internal stores after activation of $Ca^{2+}$ influx. The mobilization process is known as calcium-induced calcium release (CICR). In this study, our goal was to investigate the existence of and the role of caffeine-sensitive ryanodine receptors (RyRs) in a rat pancreatic ${\beta}$-cell line, INS-1 cells. To measure cytosolic and stored $Ca^{2+}$, respectively, cultured INS-1 cells were loaded with fura-2/AM or furaptra/AM. $[Ca^{2+}]_i$ was repetitively increased by caffeine stimulation in normal $Ca^{2+}$ buffer. However, peak $[Ca^{2+}]_i$ was only observed after the first caffeine stimulation in $Ca^{2+}$ free buffer and this increase was markedly blocked by ruthenium red, a RyR blocker. KCl-induced elevations in $[Ca^{2+}]_i$ were reduced by pretreatment with ruthenium red, as well as by depletion of internal $Ca^{2+}$ stores using cyclopiazonic acid (CPA) or caffeine. Caffeine-induced $Ca^{2+}$ mobilization ceased after the internal stores were depleted by carbamylcholine (CCh) or CPA. In permeabilized INS-1 cells,$Ca^{2+}$ release from internal stores was activated by caffeine, $Ca^{2+}$, or ryanodine. Furthermore, ruthenium red completely blocked the CICR response in perrneabilized cells. RyRs were widely distributed throughout the intracellular compartment of INS-1 cells. These results suggest that caffeine-sensitive RyRs exist and modulate the CICR response from internal stores in INS-1 pancreatic ${\beta}$-cells.

• Journal of Ginseng Research
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• v.47 no.4
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• pp.572-582
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• 2023

Background: Free fatty acid-induced lipotoxicity is considered to play an important role in pancreatic β-cell dysfunction. The effect of ginsenosides on palmitic acid-induced pancreatic beta-cells cell death and failure of glucose-stimulated secretion of insulin (GSIS) was evaluated in this study. Methods: Enzyme-linked immunosorbent assay kit for a rat insulin was used to quantify glucose-stimulated insulin secretion. Protein expression was examined by western blotting analysis. Nuclear condensation was measured by staining with Hoechst 33342 stain. Apoptotic cell death was assessed by staining with Annexin V. Oil Red O staining was used to measure lipid accumulation. Results: We screened ginsenosides to prevent palmitic acid-induced cell death and impairment of GSIS in INS-1 pancreatic β-cells and identified protopanaxadiol (PPD) as a potential therapeutic agent. The protection effect of PPD was likely due to a reduction in apoptosis and lipid accumulation. PPD attenuated the palmitic acid-induced increase in the levels of B-cell lymphoma-2-associated X/B-cell lymphoma 2, poly (ADP-ribose) polymerase and cleaved caspase-3. Moreover, PPD prevented palmitic acid-induced impairment of insulin secretion, which was accompanied by an increase in the activation of phosphatidylinositol 3-kinase, peroxisome proliferator-activated receptor γ, insulin receptor substrate-2, serine-threonine kinase, and pancreatic and duodenal homeobox-1. Conclusion: Our results suggest that the protective effect of PPD on lipotoxicity and lipid accumulation induced by palmitic acid in pancreatic β-cells.

• Nutrition Research and Practice
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• v.15 no.3
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• pp.294-308
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• 2021

BACKGROUD/OBJECTIVES: Allomyrina dichotoma larva (ADL), one of the many edible insects recognized as future food resources, has a range of pharmacological activities. In a previous study, an ADL extract (ADLE) reduced the hepatic insulin resistance of high-fat diet (HFD)-induced diabetic mice. On the other hand, the associated molecular mechanisms underlying pancreatic beta-cell dysfunction remain unclear. This study examined the effects of ADLE on palmitate-induced lipotoxicity in a beta cell line of a rat origin, INS-1 cells. MATERIALS/METHODS: ADLE was administered to high-fat diet treated mice. The expression of apoptosis-related molecules was measured by Western blotting, and reactive oxidative stress generation and nitric oxide production were measured by DCH-DA fluorescence and a Griess assay, respectively. RESULTS: The administration of ADLE to HFD-induced diabetic mice reduced the hyperplasia, 4-hydroxynonenal levels, and the number of apoptotic cells while improving the insulin levels compared to the HFD group. Treatment of INS-1 cells with palmitate reduced insulin secretion, which was attenuated by the ADLE treatment. Furthermore, the ADLE treatment prevented palmitate-induced cell death in INS-1 cells and isolated islets by reducing the apoptotic signaling molecules, including cleaved caspase-3 and PARP, and the Bax/Bcl2 ratio. ADLE also reduced the levels of reactive oxygen species generation, lipid accumulation, and nitrite production in palmitate-treated INS-1 cells while increasing the ATP levels. This effect corresponded to the decreased expression of inducible nitric oxide synthase (iNOS) mRNA and protein. CONCLUSIONS: ADLE helps prevent lipotoxic beta-cell death in INS-1 cells and HFD-diabetic mice, suggesting that ADLE can be used to prevent or treat beta-cell damage in glucose intolerance during the development of diabetes.

• The Korean Journal of Physiology and Pharmacology
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• v.72 no.4
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• pp.517-528
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• 2021

Aamylin or islet amyloid polypeptide (IAPP) is a peptide synthesized and secreted with insulin by the pancreatic β-cells. A role for amylin in the pathogenesis of type 2 diabetes (T2D) by causing insulin resistance or inhibiting insulin synthesis and secretion has been suggested by in vitro and in vivo studies. These studies are consistent with the effect of endogenous amylin on pancreatic β-cells to modulate and/or restrain insulin secretion. Here, we reported the correlation between amylin and insulin in rat insulinoma inS-1e cells by treating 2-deoxy-ᴰ-glucose (2-DG) and/or mannose. Cell viability was not affected by 24 h treatment with 2-DG and/or mannose, but it was significantly decreased by 48 h treatment with 5 and 10 mm 2-DG. in the 24 h treatment, the synthesis of insulin in the cells and the secretion of insulin into the media showed a significant inverse association. in the 48-h treatment, amylin synthesis vs. the secretion and insulin synthesis vs. the secretion showed a significant inverse relation. The synthesis of amylin vs. insulin and the secretion of amylin vs. insulin showed a significant inverse relationship. The p-ERK, antioxidant enzymes (Cu/Zn-superoxide dismutase (SOD), Mn-SOD, and catalase), and endoplasmic reticulum (ER) stress markers (cleaved caspase-12, CHOP, p-SAPK/JNK, and BiP/GRP78) were significantly increased or decreased by the 24 h and 48 h treatments. These data suggest the relative correlation to the synthesis of amylin by cells vs. the secretion into the media, the synthesis of amylin vs. insulin, and the secretion of amylin vs. insulin under 2-DG and/or mannose in rat insulinoma INS-1E cells. Therefore, these results can provide primary data for the hypothesis that the amylin-insulin relationships may be involved with the human amylin toxicity in pancreatic beta cells.