Original article | DOI: 10.26402/jpp.2021.4.03

H.S. KIM1, S.S. JOO1, Y.-M. YOO2

THE CORRELATION BETWEEN AMYLIN AND INSULIN BY TREATMENT WITH
2-DEOXY-D-GLUCOSE AND/OR MANNOSE IN RAT INSULINOMA INS-1E CELLS

1College of Life Science, Gangneung-Wonju National University, Gangneung, Gangwon-do, Republic of Korea; 2East Coast Life Sciences Institute, College of Life Science, Gangneung-Wonju National University, Gangneung, Gangwon-do, Republic of Korea
Amylin 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.
Key words:
amylin, insulin, 2-deoxy-d-glucose, mannose, INS-1E cells, type 2 diabetes, antioxidant enzymes, endoplasmic reticulum stress markers, reactive oxygenspecies

INTRODUCTION

Amylin or islet amyloid polypeptide (IAPP) is a 37-amino acid peptide synthesized and secreted with insulin by the pancreatic β cells (1). Amylin, a circulating glucoregulatory hormone, regulates glucose homeostasis (1, 2). A role for human amylin in the pathogenesis of type 2 diabetes (T2D) in causing insulin resistance or inhibiting insulin synthesis and secretion has been suggested by in vitro and in vivo studies (3-6). These studies are consistent with the effect of endogenous amylin on pancreatic β-cell to modulate and/or restrain insulin secretion, providing the first evidence that human amylin plays a role in modulating insulin secretion (7). The effect of endogenous amylin on insulin and glucose homeostasis was examined in human renal failure (8), which indicated that chronic elevations of amylin had no apparent impact of decreasing peripheral insulin action or secretion compared to healthy lean and obese, non-uremic subjects with normal or impaired glucose tolerance.

Some studies demonstrated that amylin influenced insulin secretion in isolated islet cells or pancreatic perfusion preparations (5, 6, 9-13). Moreover, other studies in rat soleus muscle and skeletal muscle showed the effect of amylin on insulin-stimulated glucose homeostasis (14, 15). However, the data supporting these studies have not been consistent (16-19), and in many studies, these effects were associated with physiological amylin concentrations (14, 15, 17, 20), which may cast doubt on the physiological relevance of the findings. The physiological role of amylin is also controversial in insulin secretion and insulin sensitivity. An in vivo amylin receptor antagonist study in rats reported that endogenous physiological amylin affected insulin secretion (15).

Hyperglycemia, dyslipidemia, inflammation, and autoimmunity influence the function of pancreatic beta cells, causing decreased insulin production and insulin secretion. Pancreatic beta-cell dysfunction reduces cell survival and insulin sensitivity, inducing diabetes mellitus (21-24). Decreases in insulin production/secretion related to diabetes and obesity have been associated with a reduction in β cell mass from apoptosis in animal models and human patients (25-27).

The glucose analog 2-deoxy-d-glucose (2-DG) is metabolized by hexokinase and acts as a glycolysis inhibitor (28). 2-DG triggers glucose deprivation without altering other nutrients or metabolic pathways (29) and then induces endoplasmic reticulum (ER) stress (30). Mannose is a monosaccharide as a nutritional supplement. Therefore, we attempted to understand the interaction between amylin and insulin synthesis/secretion using 2-DG and mannose. Here, we reported the correlation between amylin and insulin in rat insulinoma INS-1E cells by treatment with 2-DG and/or mannose. Also, we described p-ERK, antioxidant enzymes (Cu/Zn- SOD, Mn-SOD, and catalase), and ER stress markers (cleaved caspase-12, CHOP, p-SAPK/JNK, and BiP/GRP78).

MATERIALS AND METHADS

Cell culture

The INS-1E cells, a clonal pancreatic β-cell line received from Prof. Claes B. Wollheim (Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland), were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 11 mM glucose supplemented with 10 mM HEPES (pH 7.3), 10% heat-inactivated fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA), 50 µM β-mercaptoethanol, 1 mM sodium pyruvate, 50 µg/mL penicillin, and 100 µg/mL streptomycin at 37°C with 5% CO2 in a humidified incubator. The INS-1E cells on a 100 mm culture plate (Corning Inc., NY, USA) at a density of 8 × 106 were cultured in RPMI 1640 medium plus 2% heat-inactivated FBS with/without 2-DG (5, 10 mM) (Sigma-Aldrich, St. Louis, MO, USA) in the presence/absence of 1 mM mannose (Sigma-Aldrich, St. Louis, MO, USA) at 37°C with 5% CO2 for 24 or 48 h.

Cell viability assay

Cell survival was determined using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc. Rockville, MD, USA), according to the manufacturer’s protocol. The INS-1E cells were cultured in 96-well plates (Corning Inc., NY, USA) at a density of 5 × 103/well. The cells were treated with the 10 µl kit solution, incubated for 30 min and their absorbance was measured at 450 nm. The percentage of viable cells per sample was calculated by: viability (%) = [(total signal-background signal)/control signal] × 100.

Western blot analysis

Cells were harvested and resuspended in 20 mM Tris-HCl buffer (pH 7.4) containing 0.1 mM phenylmethylsulfonyl fluoride, 5 µg/mL aprotinin, 5 µg/mL pepstatin A, 1 µg/mL chymostatin, 5 mM Na3VO4 and 5 mM NaF. The whole-cell lysate was followed by centrifugation at 13,000 × g for 10 min at 4°C. Protein concentrations were determined by using the BCA assay (Sigma, St Louis, MO, USA). Proteins (40 µg) or media (20 µL) were separated via 10% Gradi-Gel II gradient PAGE (ELPIS-Biotech, Daejeon, Republic of Korea) and then transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with antibodies. The amylin antibody (catalog no. LS-C352341; 1:1000) was provided from LifeSpan BioSciences, Inc. (Seattle, WA, USA). Insulin (catalog no. sc-9168; 1:500), phospho-ERK (catalog no. sc-7380; 1:500), ERK (catalog no. sc-93; 1:500), Cu/Zn-SOD (catalog no. sc-271014; 1:500), Mn-SOD (catalog no. sc-137254; 1:500), C/EBP-homologous protein (CHOP; catalog no. sc-575; 1:500) and GAPDH (catalog no. sc-25778; 1:500) obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Catalase (catalog no. 12980; 1:1000), cleaved caspase-12 (catalog no. 2202; 1:1000), phospho-stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK; catalog no. 9251; 1:1000), and BiP/GRP78 (catalog no. 3177; 1:1000) were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). Subsequently, the membranes were incubated with anti-mouse IgG (catalog no. 7076; 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-rabbit IgG secondary antibodies conjugated to HRP (catalog no. 7074; 1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h in room temperature. Protein bands were detected with chemiluminescent substrate (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and then measured using ImageJ software (version 1.37; National Institute of Health) and were normalized to GAPDH.

Immunofluorescence staining

INS-1E cells grown on culture slides (BD Falcon Labware, REF 354108 corning, NY, USA) were permeabilized and fixed in methanol at –20°C for 3 min. Cells were washed with phosphate-buffered saline (PBS), blocked with 10% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 10 min, and incubated with primary antibody in blocking buffer for 1 h at room temperature (RT). Cells were hybridized with secondary antibodies for 1 h at RT. The coverslips were mounted on glass slides using Vectashield mounting medium (Vector Labs Inc., Burlingame, CA, USA). The following primary antibodies were used: amylin antibody (catalog no. LS-C352341; 1:500) and insulin (catalog no. sc-8033; 1:50). The following secondary antibodies were used: Alexa 594 (red)-conjugated anti-rabbit IgG (Vector Laboratories Inc.) and fluorescein isothiocyanate (green)-labeled anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Cells stained with 4,6-diamidino-2-phenylindole (DAPI, Santa Cruz Biotechnology, Dallas, TX, USA) for 10 min.

Statistical analysis

Significant differences were detected by using ANOVA followed by Tukey’s test for multiple comparisons. The analysis was performed using the Prism Graph Pad v4.0 (Graph Pad Software, San Diego, CA, USA). Values are expressed as means ± SEM of at least three separate experiments, in which case a representative result is depicted in the figures. P values < 0.05 were considered statistically significant.

RESULTS

The relationship between amylin and insulin

Initially, we investigated whether 24 h and 48 h treatment with 2-DG affected amylin/insulin synthesis by the cells and amylin/insulin secretion into the media. Cell viability was not changed by 24 h treatment with 2-DG and/or mannose (Fig. 1A). However, cell survival was significantly decreased by 48 h treatment with 5 and 10 mM 2-DG and was not changed by the other treatments (Fig. 2A).

Figure 1
Fig. 1. 2-DG influences amylin/insulin synthesis in cells and amylin/insulin secretion in media for 24 h. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with/without 2-DG (5, 10 mM) in the presence/absence of 1 mM mannose for 24 h at 37°C with 5% CO2. Cell viability assay was performed by Cell Counting Kit-8 (A). Amylin and insulin proteins were then analyzed by Western blot (B). The relative amounts of amylin and insulin protein (C) were quantified as described in Materials and methods. Data represent the mean ± SEM of three experiments. ***p < 0.001 vs. 2% FBS in cells; #p < 0.05, ###p < 0.001 vs. 2% FBS in media; $$$p < 0.001, insulin in cells vs. insulin in media. N.D., no detection of amylin in media.

In the 24 h treatment, the synthesis of amylin in the cells was significantly decreased with 2-DG and/or mannose compared to the control cells without 2-DG or mannose (Fig. 1B and 1C). However, no secretion of amylin into the media was detected by Western blot analysis (Fig. 1B and 1C). The synthesis of insulin by the cells was significantly decreased with 2-DG and/or mannose compared to the control cells without 2-DG or mannose, and secretion of insulin into the media was significantly increased with 2-DG and/or mannose (Fig. 1B and 1C). The synthesis of insulin by the cells was significantly increased compared to the secretion of insulin into the media (Fig. 1B and 1C).

In the 48 h treatment, the synthesis of amylin by the cells was significantly decreased by only 10 mM 2-DG treatment (Fig. 2B and 2C). The secretion of amylin into the media was significantly increased by 5 and 10 mM 2-DG treatment and significantly decreased by treatment with mannose only and 2-DG plus mannose compared to the control cells without 2-DG or mannose treatment (Fig. 2B and 2C). The synthesis of amylin by the cells and the secretion of amylin into the media showed significant decreases (Fig. 2B and 2C). The synthesis of insulin by the cells and the secretion of insulin into the media were significantly decreased with 2-DG and/or mannose compared to the control cells without 2-DG or mannose (Fig. 2B and 2C). The synthesis of amylin in the cells vs. the secretion of amylin into the media showed a significant increase (Fig. 2B and 2C).

Figure 2
Fig. 2. 2-DG influences amylin/insulin synthesis in cells and amylin/insulin secretion in media for 48 h. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with/without 2-DG (5, 10 mM) in the presence/absence of 1 mM mannose for 48 h at 37°C with 5% CO2. Cell viability assay was performed by Cell Counting Kit-8 (A). Amylin and insulin proteins were then analyzed by Western blot (B). The relative amounts of amylin and insulin protein (C) were quantified as described in MATERIALS AND METHODS. Data represent the mean ± SEM of three experiments. ***p < 0.001 vs. 2% FBS in cells; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. 2% FBS in media; @@@p < 0.001, amylin in cells vs. amylin in media; $$$p < 0.001, insulin in cells vs. insulin in media; %%%p < 0.001, amylin in cells vs. insulin in cells; &&&p < 0.001, amylin in media vs. insulin in media. Arrowheads are three amylin protein bands identified in 15 – 30 kDa size.

Most importantly, the synthesis of amylin in the cells vs. the synthesis of insulin in the cells and the secretion of amylin into the media vs. the secretion of insulin into the media showed a relative correlation, respectively (Fig. 2C). Immunofluorescence staining data showed that insulin and amylin were co-localized in cells. Relative fluorescence intensity was not significant with 10 mM 2-DG for 24 h, but intracellular amylin was significantly increased compared to intracellular insulin with 10 mM 2-DG for 48 h (Fig. 3). This result was consistent with the cellular results by treating the 10 mM 2-DG for 24 h and 48 h in Figs. 1B, 1C, 2B and 2C, respectively.

Figure 3 Fig. 3. Immunofluorescence staining of insulin and amylin production in INS-1E cells with 10 mM 2-DG for 24 h and 48 h. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with 10 mM 2-DG at 37°C with 5% CO2. Immunofluorescence staining was described in MATERIALS AND METHODS. Scale bars, 25 µm. ***p < 0.001, insulin in cells vs. amylin in cells.

The effect of p-ERK and antioxidant enzymes

p-ERK and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase were investigated with 2-DG and/or mannose for 24 h and 48 h. In the 24 h treatment, the levels of p-ERK did not change, and Cu/Zn-SOD was significantly increased with 2-DG only and 2-DG-plus mannose compared to the control cells without 2-DG or mannose (Fig. 4). Mn-SOD was significantly decreased, and catalase was significantly increased with 2-DG and/or mannose (Fig. 4).

Figure 4
Fig. 4. The effect of p-ERK and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase for 24 h. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with/without 2-DG (5, 10 mM) in the presence/absence of 1 mM mannose for 24 h at 37°C with 5% CO2. p-ERK and antioxidant proteins were then analyzed by Western blot (A). The relative amounts of p-ERK and antioxidant proteins (B) were quantified as described in MATERIALS AND METHODS. Data represent the mean ± SEM of three experiments. **p < 0.01, ***p < 0.001 vs. 2% FBS; %%%p < 0.001, 2-DG vs. 2-DG-plus mannose.

In the 48 h treatment, p-ERK was significantly increased by treatment with 5 mM 2-DG and 10 mM 2-DG-plus mannose and significantly decreased with 10 mM 2-DG compared to the control cells without 2-DG or mannose (Fig. 5). Mn-SOD and Cu/Zn-SOD were significantly reduced with 5 mM 2-DG and mannose and increased with 10 mM 2-DG compared to the control cells without 2-DG or mannose (Fig. 5). Catalase was significantly decreased with 2-DG and/or mannose compared to the control cells without 2-DG or mannose (Fig. 5).

Figure 5
Fig. 5. The effect of p-ERK and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase for 48 h. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with/without 2-DG (5, 10 mM) in the presence/absence of 1 mM mannose for 48 h at 37°C with 5% CO2. p-ERK and antioxidant proteins were then analyzed by Western blot (A). The relative amounts of p-ERK and antioxidant proteins (B) were quantified as described in MATERIALS AND METHODS. Data represent the mean ± SEM of three experiments. **p < 0.01, ***p < 0.001 vs. 2% FBS.

The effect of ER stress markers: cleaved caspase-12, CHOP, p-SAPK/JNK, and BiP/GRP78

ER stress markers including cleaved caspase-12, CHOP, p-SAPK/JNK, and BiP/GRP78 were analyzed following treatment with 2-DG and/or mannose. In 24 h treatment, cleaved caspase-12 was significantly increased with 2-DG and 2-DG-plus mannose compared to the control cells without 2-DG or mannose (Fig. 6). Treatment with 2-DG vs. 2-DG-plus mannose showed significant decreases. p-SAPK/JNK was significantly increased with 2-DG and 10 mM 2-DG-plus mannose and significantly decreased with 2-DG plus mannose compared to 2-DG alone (Fig. 6). CHOP was significantly increased with 2-DG and significantly reduced with 2-DG plus mannose compared to 2-DG alone (Fig. 6). BiP/GRP78 was significantly reduced with 2-DG and/or mannose except for 10 mM 2-DG treatment and significantly decreased with 2-DG plus mannose compared to 2-DG alone (Fig. 6).

Figure 6
Fig. 6. The effect of ER stress markers including cleaved caspase-12, CHOP, p-SAPK/JNK, and BiP/GRP78 for 24 h. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with/without 2-DG (5, 10 mM) in the presence/absence of 1 mM mannose for 24 h at 37°C with 5% CO2. ER stress markers were then analyzed by Western blot (A). The relative amounts of ER stress markers (B) were quantified as described in MATERIALS AND METHODS. Data represent the mean ± SEM of three experiments. **p < 0.01, ***p < 0.001 vs. 2% FBS; %%p < 0.01, %%%p < 0.001, 2-DG vs. 2-DG-plus mannose.

In the 48 h treatment, cleaved caspase-12 was significantly increased with 10 mM 2-DG and was significantly decreased with 5 mM 2-DG and 10 mM 2-DG-plus mannose compared to the control cells without 2-DG or mannose (Fig. 7). p-SAPK/JNK was significantly increased with 2-DG and 10 mM 2-DG-plus mannose except for 5 mM 2-DG treatment (Fig. 6). CHOP was significantly increased with 2-DG and significantly decreased with 2-DG plus mannose compared to 2-DG alone (Fig. 7). BiP/GRP78 was significantly increased with 2-DG and significantly decreased with mannose and 2-DG-plus mannose. Also, the levels were significantly reduced with 2-DG compared to 2-DG plus mannose (Fig. 7).

Figure 7
Fig. 7. The effect of ER stress markers including cleaved caspase-12, CHOP, p-SAPK/JNK, and BiP/GRP78 for 48 h. INS-1E cells were incubated in RPMI 1640 medium supplemented with 2% FBS with/without 2-DG (5, 10 mM) in the presence/absence of 1 mM mannose for 48 h at 37°C with 5% CO2. ER stress markers were then analyzed by Western blot (A). The relative amounts of ER stress markers (B) were quantified as described in MATERIALS AND METHODS. Data represent the mean ± SEM of three experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs. 2% FBS; %%%p < 0.001, 2-DG vs. 2-DG-plus mannose.

DISCUSSION

The size of the amylin protein detected by Western blot analysis needs addressing. Kim et al. (31) identified human amylin oligomers in pancreatic β cells and detected 6 – 20 kDa-sized amylin oligomers by performing Western blot analysis. They also reported that the exposure of INS-1 cells to the autophagy inhibitor 3-methyladenine markedly increased the accumulation of human amylin protein, but there was no similar effect on murine amylin. Our previous study identified the expression/oligomerization of 15 – 30 kDa-sized murine amylin in pancreatic INS-1E cells treated with bafilomycin A1 (a potent inhibitor of cellular autophagy) and MG132 (proteasome inhibitor) (24). Amylin oligomerization/expression was decreased in ER stress conditions caused by thapsigargin, tunicamycin-BafA1, or MG132 treatment. In particular, the survival of cells treated with Baf A1 and MG132 was reduced, indicating that amylin oligomerization/expression may act as a survival factor, thereby improving the viability of pancreatic b-cells (24). Burillo et al. (32) showed that amylin generated a band of 16 kDa in human amylin over-expressing INS-1E cells, which was thought to be an amylin tetramer. These results were obtained in vitro, but it is difficult to confirm the formation of amylin in vivo. If a similar mechanism exists, it could facilitate the access of aggregated human amylin protein to extra-pancreatic regions including the brain, and could be a plausible explanation for the higher incidence of cognitive decline and Alzheimer’s disease among T2D (33, 34). Figs. 1 and 2 of this study show 10 – 30 kDa-sized amylin oligomers bands in cells detected following 24 h treatment and 15 – 30 kDa-sized bands following 48 h treatment, indicating that different the amylin protein sizes seen in Western blot analysis may depend upon the cell viability and the protein synthesis by treatment with chemicals. Significantly, the amylin protein band in media was not detected in 24 h treatment condition, and three amylin 10 – 30 kDa-sized protein bands were identified by treatment with 5 and 10 mM 2-DG treatment for 48 h (three arrowheads in Fig. 2B). The detection of three amylin protein bands was associated with decreased cell viability and increased ER stress by treatment with 5 and 10 mM 2-DG for 48 h. Also, it suggests that amylin protein can oligomerize differently in cells and media.

This study demonstrated the change in amylin and insulin relationship by treatment with 2-DG and/or mannose for 48 h in rat INS-1E cells. Four possible hypotheses could help to understand the correlation between amylin and insulin. First, the amylin mRNA levels in islet cells at different glucose concentrations, especially high glucose concentrations (16.7 mM), might be affected by increased rates of transcription and/or decreased rates of amylin mRNA degradation (35). Gasa et al. (36) showed that the rat islet content of both amylin and insulin mRNA was higher (3- and 1.6-fold, respectively) in high-glucose concentrations (16.7 mM) compared to basal glucose concentrations (5.5 mM) for 24 h. This result showed that amylin and insulin mRNA were both relatively stable (apparent half-life > 24 h) in pancreatic islet cells (37). Also, amylin and insulin genes have similar beta cell-specific promoters, suggesting that the two genes might share the same transcriptional regulators (38). However, the tissue specificity (39, 40) and differential response of the two genes in rats treated with dexamethasone or streptozotocin (41, 42) support that their expression may differ. These studies showed that glucose concentrations significantly affected the increased expression of amylin in normal pancreatic beta-cells. An inhibitor of beta-cell glucokinase mannoheptulose and glucose analog 2-DG completely blocked the effect of glucose on amylin mRNA, suggesting that glycolysis is necessary for amylin mRNA accumulation.

The second possible link between glucose and amylin/insulin is calcium homeostasis in pancreatic β-cells. Glucose controls calcium concentrations across many cell compartments, including the ER, mitochondria, nucleus, and cytosol. Calcium plays a significant role in the physiological insulin release from pancreatic b-cells (43, 44). Therefore, glucose stimulates insulin secretion and subcellular calcium concentration changes in pancreatic β-cells. The role of calcium in regulating insulin gene expression in b-cells has been controversial. While an increase in extracellular glucose concentration elevated the insulin mRNA levels in the beta-cell line HIT cells (45), glucose-induced stimulation inhibited insulin gene transcription in primary islet cells after calcium withdrawal (46). Fatty acids induced the expression of amylin mRNA and protein through calcium dependence but did not affect amylin mRNA stability in pancreatic β-cells (47). Fatty acids regulate amylin and insulin expression differently but induce both amylin and insulin secretion by β-cells. Glucose metabolism involving intracellular calcium signals has been reported to stimulate amylin expression and release in β-cells. Amylin and insulin genes were not co-regulated in cultured human pancreatic islets (13, 35, 36, 48, 49).

The third hypothesis is that amylin and insulin were potentiated by cAMP in pancreatic beta-cells. Forskolin or dibutyryl cAMP increases amylin mRNA levels, suggesting that cAMP may mediate some glucose effects on amylin gene expression (35). However, there were similar or no effects of cAMP on insulin gene expression in RIN-5F (50), beta TC3 cells (51), and HIT-T5 cells (45).

The last hypothesis is that amylin and insulin are regulated coordinately in response to the condition of hypoglycemia, fasting, and streptozotocin (52-55). Amylin is synthesized and secreted at a level of approximately 0.5 – 1.0% compared to the molar ratio of insulin in vivo and in vitro. The biosynthetic experiments confirm that amylin synthesis in rat islets occurs at a rate approximately 100-fold lower than insulin and suggest that amylin mRNA is less efficiently translated than insulin mRNA. These findings indicate that a relatively small amount of amylin production and secretion may not influence the toxicity of rat pancreatic beta cells, compared to the human beta cells toxicity by overexpressing human amylin.

The glucose analog 2-DG acts as a glycolytic inhibitor, leading to ER stress and an unfolded protein response (29, 30, 56, 57). 2-DG-induced ER stress has been shown to activate autophagy and then influence insulin synthesis and secretion. Kim et al. (58) and Kim and Yoo (59) demonstrated that 2-DG decreased insulin production through ER stress and/or autophagy in INS-1E cells. Our previous report showed that ER stress in the presence of thapsigargin decreased intracellular insulin biosynthesis and the extracellular secretion of insulin in INS-1E cells (60). Also, in the present study, 2-DG increased CHOP, BiP/GRP78, and cleaved caspase-12, affecting insulin and amylin levels in INS-1E cells. Therefore, 2-DG treatment can regulate insulin synthesis at the cellular level through physiological and biochemical changes in ER homeostasis.

GRP78/BiP is localized in the ER and its expression is increased by environmental stresses in many types of cells. Other studies have shown that conditions including glucose deprivation, 2-DG treatment, hypoxia, or an increase in intracellular calcium concentration induced GRP78/BiP expression (61-64). GRP78/BiP protein can be regulated by several cellular stresses that perturb ER function and homeostasis, including tunicamycin, thapsigargin, and calcium ionophore A23187 (65). 2-DG is an incredibly potent inducer of GRP78/BiP protein expression (66). Kim et al. (60) reported that melatonin significantly regulated insulin production by expressing GRP78/BiP protein, which induced ER stress in rat insulinoma INS-1E cells treated with 2-DG or 2-DG plus melatonin. Therefore, GRP78/BiP accompanied by ER stress is crucial in insulin biosynthesis and secretion in rat pancreatic b-cells (67).

2-DG-mediated glucose deprivation can stimulate reactive oxygen species (ROS) production and block the upregulation of cellular antioxidant potential (68). In the present study, cell viability in 2-DG and/or mannose treatment for 24 h was not changed (Fig. 1A), but cell survival in the treatment of 5 and 10 mM 2-DG for 48 h was significantly decreased and was not influenced by the other treatments (Fig. 2A). The levels of p-ERK did not change, and Cu/Zn-SOD in 2-DG only and 2-DG-plus mannose treatment for 24 h was significantly increased. Mn-SOD in 2-DG and/or mannose treatment for 24 h was significantly decreased, and catalase was significantly increased (Fig. 4). p-ERK in 5 mM 2-DG and 10 mM 2-DG-plus mannose treatment for 48 h was significantly increased and significantly reduced in 10 mM 2-DG treatment. Mn-SOD and Cu/Zn-SOD in 5 mM 2-DG and mannose treatment for 48 h were significantly reduced and increased in 10 mM 2-DG treatment (Fig. 5). Therefore, these results suggest that 2-DG influenced antioxidant enzymes against ROS through a mechanism involving mitochondrial oxidative phosphorylation (OXPHOS) (data not shown).

Glucose-stimulated insulin secret (GSIS) is associated with mitochondrial function rather than ER stress (69, 70). Insulin secretion in media was increased compared to insulin production in cells or amylin secretion in media under 2-DG and/or mannose treatment for 48 h. This result suggests that the decrease in insulin production in cells is involved with the reduction in cell viability through mitochondrial OXPHOS (data not shown).

In conclusion, we reported 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, this result can provide primary data for the hypothesis that the interrelationship between amylin and insulin may be involved with the human amylin toxicity in pancreatic beta cells.

Acknowledgments: This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A1A01060627).

Conflict of interest: None declared.

REFERENCES

  1. Hay DL, Chen S, Lutz TA, Parkes DG, Roth JD. Amylin: pharmacology, physiology, and clinical potential. Pharmacol Rev 2015; 67: 564-600.
  2. Lutz TA. Control of energy homeostasis by amylin. Cell Mol Life Sci 2012; 69: 1947-1965.
  3. Cooper GJ. Amylin compared with calcitonin gene-related peptide: structure, biology, and relevance to metabolic disease. Endocr Rev 1994; 15: 163-201.
  4. Ludvik B, Lell B, Hartter E, Schnack C, Prager R. Decrease of stimulated amylin release precedes impairment of insulin secretion in type II diabetes. Diabetes 1991; 40: 1615-1619.
  5. Silvestre RA, Peiro E, Degano P, Miralles P, Marco J. Inhibitory effect of rat amylin on the insulin responses to glucose and arginine in the perfused rat pancreas. Regul Pept 1990; 31: 23-31.
  6. Wang ZL, Bennet WM, Ghatei MA, Byfield PG, Smith DM, Bloom SR. Influence of islet amyloid polypeptide and the 8-37 fragment of islet amyloid polypeptide on insulin release from perifused rat islets. Diabetes 1993; 42: 330-335.
  7. Mather KJ, Paradisi G, Leaming R, et al. Role of amylin in insulin secretion and action in humans: antagonist studies across the spectrum of insulin sensitivity. Diabetes Metab Res Rev 2002; 18: 118-126.
  8. Ludvik B, Clodi M, Kautzky-Willer A, et al. Increased levels of circulating islet amyloid polypeptide in patients with chronic renal failure have no effect on insulin secretion. J Clin Invest 1994; 94: 2045-2050.
  9. Kogire M, Ishizuka J, Thompson JC, Greeley GH Jr. Inhibitory action of islet amyloid polypeptide and calcitonin gene-related peptide on release of insulin from the isolated perfused rat pancreas. Pancreas 1991; 6: 459-463.
  10. Peiro E, Degano P, Silvestre RA, Marco J. Inhibition of insulin release by amylin is not mediated by changes in somatostatin output. Life Sci 1991; 49: 761-765.
  11. Furnsinn C, Leuvenink H, Roden M, Nowatny P, Waldhausl W. Inhibition of glucose-induced secretion by amylin in rats in vivo. Diabetologia 1993; 35: A29.
  12. Degano P, Silvestre RA, Salas M, Peiro E, Marco J. Amylin inhibits glucose-induced insulin secretion in a dose-dependent manner. Study in the perfused rat pancreas. Regul Pept 19932; 43: 91-96.
  13. Fehmann HC, Weber V, Goke R, Goke B, Arnold R. Cosecretion of amylin and insulin from isolated rat pancreas. FEBS Lett 1990; 262: 279-281.
  14. Frontoni S, Choi SB, Banduch D, Rossetti L. in vivo insulin resistance induced by amylin primarily through inhibition of insulin-stimulated glycogen synthesis in skeletal muscle. Diabetes 1991; 40: 568-573.
  15. Young AA, Gedulin B, Wolfe-Lopez D, Greene HE, Rink TJ, Cooper GJ. Amylin and insulin in rat soleus muscle: dose responses for cosecreted noncompetitive antagonists. Am J Physiol 1992; 263: E274-E281.
  16. Fehmann HC, Weber V, Goke R, Goke B, Eissele R, Arnold R. Islet amyloid polypeptide (IAPP; amylin) influences the endocrine but not the exocrine rat pancreas. Biochem Biophys Res Commun 1990; 167: 1102-1108.
  17. Nagamatsu S, Carroll RJ, Grodsky GM, Steiner DF. Lack of islet amyloid polypeptide regulation of insulin biosynthesis or secretion in normal rat islets. Diabetes 1990; 39: 871-874.
  18. Nagamatsu S, Nishi M, Steiner DF. Effects of islet amyloid polypeptide (IAPP) on insulin biosynthesis or secretion in rat islets and mouse beta TC3 cells. Biosynthesis of IAPP in mouse beta TC3 cells. Diabetes Res Clin Pract 1992; 15: 49-55.
  19. Broderick CL, Brooke GS, DiMarchi RD, Gold G. Human and rat amylin have no effects on insulin secretion in isolated rat pancreatic islets. Biochem Biophys Res Commun 1991; 177: 932-938.
  20. Ohsawa H, Kanatsuka A, Yamaguchi T, Makino H, Yoshida S. Islet amyloid polypeptide inhibits glucose-stimulated insulin secretion from isolated rat pancreatic islets. Biochem Biophys Res Commun 1989; 160: 961-967.
  21. Cernea S, Dobreanu M. Diabetes and beta cell function: from mechanisms to evaluation and clinical implications. Biochem Med 2013; 23: 266-280.
  22. Donath MY, Dalmas E, Sauter NS, Boni-Schnetzler M. Inflammation in obesity and diabetes: islet dysfunction and therapeutic opportunity. Cell Metab 2013; 17: 860-872.
  23. Kaczmarek P, Skrzypski M, Pruszynska-Oszmalek E, et al. Chronic orexin-A (hypocretin-1) treatment of type 2 diabetic rats improves glucose control and beta-cell functions. J Physiol Pharmacol 2017; 68: 669-681.
  24. Jung EM, Yoo YM, Jeung EB. Melatonin influences the expression and oligomerization of amylin in rat INS-1E cells. J Physiol Pharmacol 2019; 70: 695-703.
  25. Prentki M, Nolan CJ. Islet beta cell failure in type 2 diabetes. J Clin Invest 2006; 116; 1802-1812.
  26. Marrif HI, Al-Sunousi SI. Pancreatic β cell mass death. Front Pharmacol 2016; 7: 83. doi: 10.3389/fphar.2016.00083
  27. Billert M, Jasaszwili M, Strowski M, Nowak KW, Skrzypski M. Adropin suppresses insulin expression and secretion in INS-1E cells and rat pancreatic islets. J Physiol Pharmacol 2020; 71: 99-104.
  28. Sols A, Crane RK. The inhibition of brain hexokinase by adenosinediphosphate and sulfhydryl reagents. J Biol Chem 1954; 206: 925-936.
  29. Aghaee F, Pirayesh Islamian J, Baradaran B. Enhanced radiosensitivity and chemosensitivity of breast cancer cells by 2-deoxy-d-glucose in combination therapy. J Breast Cancer 2012; 15: 141-147.
  30. Xi H, Kurtoglu M, Liu H, et al. 2-Deoxy-D-glucose activates autophagy via endoplasmic reticulum stress rather than ATP depletion. Cancer Chemother Pharmacol 2011; 67: 899-910.
  31. Kim J, Cheon H, Jeong YT, et al. Amyloidogenic peptide oligomer accumulation in autophagy-deficient b cells induces diabetes. J Clin Invest 2014; 124: 3311-3324.
  32. Burillo J, Fernandez-Rhodes M, Piquero M, et al. Human amylin aggregates release within exosomes as a protective mechanism in pancreatic β cells: pancreatic β-hippocampal cell communication. Biochim Biophys Acta Mol Cell Res 2021; 1868: 118971. doi: 10.1016/j.bbamcr.2021.118971
  33. Zhang Y, Song W. Islet amyloid polypeptide: Another key molecule in Alzheimer’s pathogenesis? Prog Neurobiol 2017; 153: 100-120.
  34. Biessels GJ, Despa F. Cognitive decline and dementia in diabetes mellitus: mechanisms and clinical implications. Nat Rev Endocrinol 2018; 14: 591-604.
  35. Gasa R, Gomis R, Casamitjana R, Rivera F, Novials A. Glucose regulation of islet amyloid polypeptide gene expression in rat pancreatic islets. Am J Physiol Endocrinol Metab 1997; 272: E543-E549.
  36. Gasa R, Gomis R, Casamitjana R, Novials A. Signals related to glucose metabolism regulate islet amyloid polypeptide (IAPP) gene expression in human pancreatic islets. Regul Pept 1997; 68: 99-104.
  37. Subash-Babu P, Ignacimuthu S, Alshatwi AA. Nymphayol increases glucose-stimulated insulin secretion by RIN-5F cells and GLUT4-mediated insulin sensitization in type 2 diabetic rat liver. Chem Biol Interact 2015; 226: 72-81.
  38. German MS, Moss LG, Wang J, Rutter WJ. The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes. Mol Cell Biol 1992; 12: 1777-1788.
  39. Madsen OD, Nielsen JH, Michelsen B, et al. Islet amyloid polypeptide and insulin expression are controlled differently in primary and transformed islet cells. Mol Endocrinol 1991; 5: 143-148.
  40. Nicholl CG, Bhatavdekar JM, Mak J, Girgis SI, Legon S. Extra-pancreatic expression of the rat islet amyloid polypeptide (amylin) gene. J Mol Endocrinol 1992; 9: 157-163.
  41. Bretherton-Watt D, Ghatei MA, Bloom SR, et al. Altered islet amyloid polypeptide (amylin) gene expression in rat models of diabetes. Diabetologia 1989; 32: 881-883.
  42. Mulder H, Ahren B, Sundler F. Islet amyloid polypeptide (amylin) and insulin are differentially expressed in chronic diabetes induced by streptozotocin in rats. Diabetologia 1996; 39: 649-657.
  43. Draznin B. Intracellular calcium, insulin secretion, and action. Am J Med 1988; 85: 44-58.
  44. Klec C, Ziomek G, Pichler M, Malli R, Graier WF. Calcium Signaling in b-cell physiology and pathology: a revisit. Int J Mol Sci 2019; 20: 6110. doi: 10.3390/ijms20246110
  45. Goodison S, Kenna S, Ashcroft SJ. Control of insulin gene expression by glucose. Biochem J 1992; 285: 563-568.
  46. German MS, Moss LG, Rutter WJ. Regulation of insulin gene expression by glucose and calcium in transfected primary islet cultures. J Biol Chem 1990; 265: 22063-22066.
  47. Qi D, Cai K, Wang O, et al. Fatty acids induce amylin expression and secretion by pancreatic beta-cells. Am J Physiol Endocrinol Metab 2010; 298: E99-E107.
  48. Gasa R, Gomis R, Casamitjana R, Novials A. High glucose concentration favors the selective secretion of islet amyloid polypeptide through a constitutive secretory pathway in human pancreatic islets. Pancreas 2001; 22: 307-310.
  49. Kahn SE, D’Alessio DA, Schwartz MW, et al. Evidence of co-secretion of islet amyloid polypeptide and insulin by beta-cells. Diabetes 1990; 39: 634-638.
  50. Welsh M, Nielsen DA, MacKrell AJ, Steiner DF. Control of insulin gene expression in pancreatic beta-cells and in an insulin-producing cell line, RIN-5F cells. I. Effects of glucose and cyclic AMP on the transcription of insulin mRNA. J Biol Chem 1985; 260: 13585-13589.
  51. Schuppin GT, Rhodes CJ. Specific co-ordinated regulation of PC3 and PC2 gene expression with that of preproinsulin in insulin-producing beta TC3 cells. Biochem J 1996; 313: 259-268.
  52. Steiner DF, Ohagi S, Nagamatsu S, Bell GI, Nishi M. Is islet amyloid polypeptide a significant factor in pathogenesis or pathophysiology of diabetes? Diabetes 1991; 40: 305-309.
  53. Inoue K, Hisatomi A, Umeda F, Nawata H. Relative hypersecretion of amylin to insulin from rat pancreas after neonatal STZ treatment. Diabetes 1992; 41: 723-727.
  54. Alam T, Chen L, Ogawa A, Leffert JD, Unger RH, Luskey KL. Coordinate regulation of amylin and insulin expression in response to hypoglycemia and fasting. Diabetes 1992; 41: 508-514.
  55. Pieber TR, Stein DT, Ogawa A, et al. Amylin-insulin relationships in insulin resistance with and without diabetic hyperglycemia. Am J Physiol 1993; 265: E446-E453.
  56. Jiang W, Zhu Z, Thompson HJ. Modulation of the activities of AMP-activated protein kinase, protein kinase B, and mammalian target of rapamycin by limiting energy availability with 2-deoxyglucose. Mol Carcinog 2008; 47: 616-628.
  57. Kurtoglu M, Gao N, Shang J, et al. Under normoxia, 2-deoxy-D-glucose elicits cell death in select tumor types not by inhibition of glycolysis but by interfering with N-linked glycosylation. Mol Cancer Ther 2007; 6: 3049-3058.
  58. Kim HS, Han TY, Yoo YM. Melatonin-mediated intracellular insulin during 2-deoxy-d-glucose treatment is reduced through autophagy and edc3 protein in insulinoma INS-1E cells. Oxid Med Cell Longev 2016; 2016: 2594703. doi: 10.1155/2016/2594703
  59. Kim HS, Yoo YM. Data of intracellular insulin protein reduced by autophagy in INS-1E cells. Data Brief 2016; 8: 1151-1156.
  60. Yoo YM. Melatonin-mediated insulin synthesis during endoplasmic reticulum stress involves HuD expression in rat insulinoma INS-1E cells. J Pineal Res 2013; 55: 207-220.
  61. Kuznetsov G, Bush KT, Zhang PL, Nigam SK. Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia. Proc Natl Acad Sci USA 1996; 93: 8584-8589.
  62. Lee AS. Mammalian stress response: induction of the glucose-regulated protein family. Curr Opin Cell Biol 1992; 4: 267-273.
  63. Li WW, Hsiung Y, Zhou Y, Roy B, Lee AS. Induction of the mammalian GRP78/BiP gene by Ca2+ depletion and formation of aberrant proteins: activation of the conserved stress-inducible grp core promoter element by the human nuclear factor YY1. Mol Cell Biol 1997; 17: 54-60.
  64. Yu Z, Luo H, Fu W, Mattson MP. The endoplasmic reticulum stress-responsive protein GRP78 protects neurons against excitotoxicity and apoptosis: suppression of oxidative stress and stabilization of calcium homeostasis. Exp Neurol 1999; 155: 302-314.
  65. Sasaya H, Utsumi T, Shimoke K, Nakayama H, Matsumura Y, Fukunaga K, et al. Nicotine suppresses tunicamycin-induced, but not thapsigargin-induced, expression of GRP78 during ER stress-mediated apoptosis in PC12 cells. J Biochem 2008; 144: 251-257.
  66. Kishi S, Shimoke K, Nakatani Y, et al. Nerve growth factor attenuates 2-deoxy-d-glucose-triggered endoplasmic reticulum stress-mediated apoptosis via enhanced expression of GRP78. Neurosci Res 2010; 66: 14-21.
  67. Zhang L, Lai E, Teodoro T, Volchuk A. GRP78, but not protein-disulfide isomerase, partially reverses hyperglycemia-induced inhibition of insulin synthesis and secretion in pancreatic (beta)-cells. J Biol Chem 2009; 284: 5289-5298.
  68. Pajak B, Siwiak E, Soltyka M, et al. 2-deoxy-d-glucose and its analogs: from diagnostic to therapeutic agents. Int J Mol Sci 2019; 21: 234. doi: 10.3390/ijms20246110
  69. Jhun BS, Lee H, Jin ZG, Yoon Y. Glucose stimulation induces dynamic change of mitochondrial morphology to promote insulin secretion in the insulinoma cell line INS-1E. PLoS One 2013; 8: e60810. doi: 10.1371/journal.pone .0060810
  70. Plecita-Hlavata L, Jaburek M, Holendova B, et al. Glucose-stimulated insulin secretion fundamentally requires H2O2 signaling by NADPH oxidase 4. Diabetes 2020; 69: 1341-1354.
R e c e i v e d : July 1, 2021
A c c e p t e d : August 30, 2021
Author’s address: Prof. Seong Soo Joo, Department of Marine Life Science, College of Life Science, Gangneung-Wonju National University, Gangneung, Gangwon-do 25457, Republic of Korea. e-mail: ssj66@gwnu.ac.kr
Prof. Yeong-Min Yoo, East Coast Life Sciences Institute, College of Life Science, Gangneung-Wonju National University, Gangneung, Gangwon-do 25457, Republic of Korea. e-mail: yyeongm@hanmail.net