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. Author manuscript; available in PMC: 2022 May 6.
Published in final edited form as: J Endocrinol. 2021 May 6;249(3):163–175. doi: 10.1530/JOE-21-0047

Lower threshold to NFκB activity sensitizes murine β-cells to streptozotocin

Clyde J Wright 1, Sarah McKenna 1, Robyn De Dios 1, Brit H Boehmer 1, Leanna Nguyen 1, Sankar Ghosh 2, Jeryl Sandoval 1, Paul J Rozance 1
PMCID: PMC8113150  NIHMSID: NIHMS1693589  PMID: 33764312

Abstract

The β-cell response to injury may be as critical for the development of diabetes as the specific insult. In the current study, we use streptozotocin (STZ) to injure the β-cell in order to study the response with a focus on NFκB. MIN6 cells were exposed to STZ (0.5–8mM, 0–24h) ±TNFα (100ng/mL) and ±IκBβ siRNA to lower the threshold to NFκB activation. Cell viability was determined by trypan blue exclusion. NFκB activation was determined by expression of the target genes Nos2 and Cxcl10, localization of the NFκB proteins p65 and p50, and expression and localization of the NFκB inhibitors, IκBβ and IκBα. There was no NFκB activation in MIN6 cell exposed to STZ (2 mM) alone. However, knocking down IκBβ expression using siRNA resulted in STZ-induced expression of NFκB target genes and increased cell death, while co-incubation with STZ and TNFα enhanced cell death compared to either exposure alone. Adult male IκBβ−/− and wild type (WT) mice were exposed to STZ and monitored for diabetes. The IκBβ−/− mice developed hyperglycemia and diabetes more frequently than controls following STZ exposure. Based on these results we conclude that STZ exposure alone does not induce NFκB activity. However, lowering the threshold to NFκB activation by co-incubation with TNFα or lowering IκBβ levels by siRNA sensitizes the NFκB response to STZ and results in a higher likelihood of developing diabetes in vivo. Therefore, increasing the threshold to NFκB activation through stabilizing NFκB inhibitory proteins may prevent β-cell injury and the development of diabetes.

Keywords: β-cell, NFκB, IκB, Streptozotocin, Diabetes, Nos2, Cxcl10

Introduction

Diabetes mellitus (DM), whether type 1 or type 2, results from an inability of the pancreatic β-cells to secrete sufficient amounts of insulin to appropriately regulate blood glucose concentrations appropriately. This inability is due in part, to β-cell injury. While this is more obvious in the pathogenesis of type 1 diabetes, it also plays an important role in the pathogenesis of type 2 diabetes. A wide range of stimuli can injure β-cells, but the β-cell response to these injurious stimuli may be just as critical for the development of β-cell failure and diabetes as the specific insult. Streptozotocin (STZ) is commonly used to injure the β-cells both in vitro and in vivo in order to study the injury response and their contributions to the pathogenesis of diabetes. STZ is thought to cause β-cell injury through one of three mechanisms: (1) accumulation of DNA damage due to methylation by STZ’s methylnitrosourea moiety,(Eleazu, et al. 2013) (2) nitric oxide (NO) production(Delaney, et al. 1995) resulting from liberation of NO from STZ (Turk, et al. 1993), and (3) production of reactive oxygen species such as hydrogen peroxide.(Friesen, et al. 2004; Gille, et al. 2002) While STZ induces injury, the cellular response to the injury determines whether the β-cell survives or not and therefore determines the physiologic implications of the exposure. Thus, the signaling events downstream of STZ exposure has been an area of intense study.

The inducible transcription factor NFκB regulates the cellular response to various stressors. The central role played by NFκB in the cellular response to these stressors has earned it the moniker of “master regulator the inflammatory response.”(Ghosh, et al. 1998; Hayden and Ghosh 2008; Rahman and Fazal 2011) With that in mind, NFκB activity has been studied in the context β-cell injury in response to various insults and in the pathogenesis of diabetes.(Patel and Santani 2009) In β-cells, NFκB activity upregulates the expression or pro-inflammatory cytokines and pro-apoptotic factors.(Cardozo, et al. 2001; Eizirik and Mandrup-Poulsen 2001) It is largely believed that NFκB activity contributes to β-cell injury through one of these two mechanisms.

In models of STZ-induced β-cell injury, blocking NFκB activity has a protective effect. STZ-induced DM is nearly completely abrogated in mice where NFκB activity is inhibited in the β-cell.(Eldor, et al. 2006) Similar results have been observed in mice with global disruption of NFκB activity.(Lamhamedi-Cherradi, et al. 2003; Mabley, et al. 2002) However, these previous studies did not specifically test whether STZ induced NFκB activity. In fact, data directly linking STZ exposure to NFκB activity in β-cells are lacking. While NFκB activity has been reported in islets isolated from STZ exposed mice,(Lgssiar, et al. 2004) these assays were performed three days after the completion of a prolonged, 5-day STZ exposure. An acute, direct stimulatory effect of STZ on NFκB activity in β-cells was not assessed.

Thus, whether STZ directly activates NFκB in β-cells is a critical gap in our knowledge. A better understanding of the NFκB-regulated innate immune signaling events downstream of STZ-induced injury could help target therapies meant to attenuate a variety of β-cell injuries. It could be argued that therapeutic approaches would be different if NFκB activation specific to β-cell contributes to injury, versus NFκB activation in cell populations separate from, or in addition to β-cells.

We hypothesized that STZ would induce NFκB activation in β-cells resulting in the expression of NFκB target genes previously associated with injury (Cxcl10, Nos2 and Tnf). In contrast to our hypothesis, we could find no evidence that STZ exposure activates NFκB signaling in the insulin secreting MIN6 cell line. However, direct TNFα exposure induced immediate and robust NFκB activation demonstrating an intact signaling cascade in MIN6 cells as assessed by IκB inhibitory protein degradation and expression of Cxcl10, Nos2 and Tnf. Importantly, exposing MIN6 cells to STZ and TNFα increased Cxcl10, Nos2 and Tnf expression when compared to TNFα exposure alone, demonstrating that STZ exposure increased susceptibility to cytokine induced injury. Furthermore, by lowering the threshold to NFκB activation through silencing expression of the NFκB inhibitory protein IκBβ, STZ exposure alone induced expression of Cxcl10, Nos2 and Tnf and increased STZ-induced cell death. Finally, we demonstrated that adult male IκBβ−/− mice were more susceptible to STZ-induced diabetes, further supporting a mechanistic role of a lower threshold to NFκB activation increasing pancreatic β-cell death sensitivity to injury.

Methods

Cell Culture and Exposures

MIN6 immortalized murine insulinoma cells (gift of Dr. Richard Benninger, University of Colorado), passage 27–40, were cultured in DMEM (Corning) containing 4.5 g/L (25 mM) glucose and L-glutamine, supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 1% Antibiotic-Antimycotic, and .102 mM bME. MIN6 cells (70% confluent) were exposed to 0.5 mM-8mM Streptozotocin (Sigma-Aldrich) prepared in sterile citrate buffer (pH 4.5). To induce inflammatory stress, 70% confluent MIN6 were exposed to 10 ng/ml TNFα (Sigma-Aldrich).

Gene Expression Analysis

Total RNA was isolated using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using the verso kit (ThermoFisher Scientific) according to the manufacturer’s instructions. Relative RNA levels were evaluated by quantitative real-time PCR using the TaqMan gene expression system (Applied Biosystems). Gene expression was assessed with predesigned exon-spanning primers using the StepOnePlus Real Time PCR System (Applied Biosystems). Primer list is available in Table 1. Relative quantitation was performed via normalization to the endogenous control 18S, and then to the appropriate experimental control using the cycle threshold (ΔΔCt) method.

Table 1.

Primer List

Primer Catalog #
18s Mm03928990_g1
Cxcl10 Mm00445235_m1
Nos2 Mm00440502_m1
Tnf Mm00443258_m1
Nfkbib (IκBβ) Mm00456849_m1
Nfkbiα (IκBα) Mm00477798.m1
Chuk (IKKα) Mm00432529_m1
Ikbkb (IKKβ) Mm01222247_m1
Rela (p65) Mm00501346_m1
Nfkb1 (p50) Mm00476361_m1
Ins1 Mm01950294_s1
Ins2 Mm00731595_gh
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Protein isolation from cultured cells

To assess expression of NFκB pathway proteins, MIN6 whole-cell lysate was obtained using MPER (ThermoFisher Scientific) according to the manufacturer’s instructions. To assess NFκB activation following genotoxic or inflammatory stress exposure, cytosolic and nuclear extracts were obtained from MIN6 cells using the NE-PER kit (ThermoFisher Scientific) according to the manufacturer’s instructions.

Immunoblot analysis

Lysates and cytosolic and nuclear extracts were electrophoresed on a 4–12% polyacrylamide gel (Invitrogen) and proteins were transferred to an Immobilon membrane (Millipore) and stained with the Revert 700 Total Protein Stain (LI-COR) according to manufacturer’s instructions. Membranes were blotted with antibodies in Table 2. Total protein was assessed Densitometric analysis was performed using ImageStudio (LiCor).

Table 2.

Antibody List

Antibody Company Catalog #
IKKα Cell Signaling 2682
IKKβ (2C8) Cell Signaling 2370
p65 (D14E12) XP Cell Signaling 8242
p50 Abcam 32360
IκBβ Invitrogen PA1-32136
IκBα (L35A5) Cell Signaling 4814
GAPDH (D16H11) XP Cell Signaling 5174
HDAC1 (10E2) Cell Signaling 5356
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Transfection with IκBβ siRNA

To silence IκBβ expression, 70% confluent MIN6 cells were transfected with siRNA against IκBβ (Dharmacon) using Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher Scientific)

Cell viability by trypan blue exclusion

To assess cell viability, cells were trypsinized, pelleted, resuspended in a 1:1 mixture of PBS and 0.5% trypan blue (Corning), and manually counted using a hemocytometer. The numbers of live (unstained) cells were expressed as a ratio of total (stained and unstained) cells counted.

Islet isolations

Mouse islet isolation was conducted under a protocol approved by the University of Colorado Institutional Animal Care and Use Committee. Male C57BL/6 (WT) or IκBβ−/− mice were anesthetized and pancreata were inflated via the common bile duct with an enzyme solution [CIzyme RI (Vitacyte)]. Following digestion (37°C), islets were isolated by density centrifugation [Lympholyte 1.1 (Cedarlane)] and then removed. Islets were then handpicked under a microscope and aliquoted into microcentrifuge tubes HBSS +5% calf serum.

Isolation of mRNA, cDNA synthesis and analysis of isolated islets

Freshly isolated islets were spun down for 5 minutes at 4,000 × g at 4°C. Media was carefully removed, and the islets were resuspended in RLT buffer (Qiagen). Islet mRNA was collected from suspension using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Islet mRNA was assessed for purity and concentration using the NanoDrop (ThermoFisher Scientific), and cDNA synthesized using the Verso cDNA synthesis Kit (ThermoFisher Scientific). Relative mRNA levels were evaluated by quantitative real-time PCR using exon spanning primers, TaqMan gene expression and StepOnePlus Real-Time PCR System (Applied Biosystems). Relative quantitation was performed via normalization to the endogenous control 18S using the cycle threshold (ΔΔCt) method.

Isolation of protein and Western blot analysis of isolated islets

Freshly isolated islets were spun down for 5 minutes at 4,000 × g at 4°C. Media was carefully removed, and the islets were resuspended in T-PER (ThermoFisher Scientific). Lysates were electrophoresed on a 4–12% polyacrylamide gel (Invitrogen) and proteins were transferred to an Immobilon membrane (Millipore) and stained with the Revert 700 Total Protein Stain (LI-COR) according to manufacturer’s instructions. Membranes were blotted with antibodies in Table 2. Blots were imaged using the LiCor Odyssey imaging system and densitometric analysis was performed using ImageStudio (LiCor).

In vivo streptozotocin induced β-cell injury

Diabetes was established using a model of multiple low dose streptozotocin exposure. Male C57BL/6 (WT) or IκBβ−/− mice (6–9 weeks old) were both divided into three groups. Two groups were exposed to streptozotocin (40 or 50 mg/kg dissolved in sterile citrate buffer pH 4.5, Sigma-Aldrich) and a third group was exposed to vehicle (sterile citrate buffer) by intraperitoneal (IP) injection for 5 consecutive days. Fasting blood glucose was measured prior to injection and for 8 weeks following injections on days 2, 5, 7, 14, 21, 28, 42, and 56 with the Nova Statstrip glucometer (Nova Biomedical) using blood obtained from the tail vein following fasting for 6 hours. Diabetes was defined as a fasting blood glucose concentration greater than 200 mg/dL measured on two consecutive blood draws.

Statistical Analysis

For comparison between treatment groups, the null hypothesis that no difference existed between treatment means was tested by Student’s t-test for parametric data and by the Mann-Whitney U for nonparametric or by ANOVA for multiple groups with multiple comparisons corrected using Dunnett’s test. Normality was tested using the Shapiro-Wilk normality test. All outliers have been included in data analysis. Differences in survival without diabetes was determined by the Mantel-Cox test (Prism, GraphPad Software, Inc). Statistical significance was defined as p<0.05.

Results

MIN6 cell and isolated islets express proteins responsible for NFκB signaling

Previous reports have demonstrated that MIN6 cells express IκBα and p65.(Bernal-Mizrachi, et al. 2002; Chang, et al. 2003; Fan, et al. 2015; Jia, et al. 2013; Kim, et al. 2005; Sarkar, et al. 2009; You, et al. 2018) However, data demonstrating that other key proteins responsible for NFκB signaling are expressed in the insulinoma MIN6 cell line are lacking. Importantly, whole cell lysates from MIN6 cells confirmed the presence of the NFκB activating kinases IKKα and IKKβ, the NFκB inhibitory proteins IκBα and IκBβ and the NFκB subunits p50 and p65 (Fig. 1A). Similarly, we detected expression of these proteins in islets isolated from adult male mice (Fig. 1B). These results confirm expression of these key signaling proteins in MIN6 cells and isolated islets.

Figure 1. MIN6 cell and isolated islets express proteins responsible for NFκB signaling.

Figure 1.

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Representative Western blots showing (A) MIN6 and (B) isolated islet expression of the NFκB activating kinases: IKKα and IKKβ; NFκB subunits: p50 and p65 and cRel; inhibitory proteins: IκBα and IκBβ; GAPDH as loading control.

STZ does not induce NFκB signaling in MIN6 cells as assessed by IκB cytosolic degradation and NFκB subunit nuclear translocation

Having demonstrated that MIN6 express key NFκB signaling proteins, we next sought to determine whether STZ exposure directly induced NFκB signaling and activation. We used a dose of STZ (2 mM) known to induce a response in MIN6 cells without causing overwhelming cell death.(Bollard, et al. 2018) MIN6 cells demonstrated no evidence of STZ-induced degradation of the NFκB inhibitory proteins IκBα or IκBβ at short (.25–4 hours; Fig. 2A and C) or long (8–24 hours; Fig. 2B and C) exposure durations. Consistent with this, there was no evidence of nuclear translocation of the NFκB subunits p65 or p50 at short (.25–4 hours; Fig. 2D and F) or long (8–24 hours; Fig. E and F) exposure durations. These results demonstrate an absence of NFκB signaling in MIN6 cell exposed to STZ (2 mM) for .25–24 hours. Results were similar for an exposure to a lower dose of STZ (1 mM, data not shown).

Figure 2. STZ does not induce NFκB signaling in MIN6 cells as assessed by IκB cytosolic degradation and NFκB subunit nuclear translocation.

Figure 2.

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(Α-B) Representative Western blots showing IκBα and IκBβ in cytosolic extracts from MIN6 cells exposed to STZ (2mM) at (A) short time points (.25–4 hours) and (B) longer time points (8–24 hours). (C) Densitometry ratio to control of IκB⍺ and IκBβ provided. Values shown as means ± SEM; n=6/time point. (D-E) Representative Western blots showing p65 and p50 in nuclear extracts from MIN6 cells exposed to STZ (2mM) at (D) short time points (.25–4 hours) and (E) longer time points (8–24 hours). (F) Densitometry ratio to control of p65 and p50 provided. Values shown as means ± SEM; n=6/time point.

TNFα induces NFκB signaling in MIN6 cells as assessed by IκB cytosolic degradation and NFκB subunit nuclear translocation

We next interrogated NFκB activity in MIN6 cells exposed to TNFα by assessing the temporal relationship between exposure and the degradation of the NFκB inhibitory proteins IκBα and IκBβ. Using a TNFα exposure known to induce NFκB as measured by IκBα degradation within one hour of exposure,(Chang et al. 2003) we sought to more precisely determine the degradation profile of the main cytosolic NFκB inhibitors IκBα and IκBβ (Fig. 3). Of note, TNFα exposure induced an oscillatory pattern of IκBα degradation and reaccumulation (Fig. 3A and B). This oscillatory pattern of IκBα degradation and reaccumulation is consistent with previous reports demonstrating that IκBα is an NFκB target gene and that the pattern of degradation and reaccumulation is evidence of TNFα-mediated NFκB activity (Hoffmann, et al. 2002). In addition, and similar to other cell lines,(Rao, et al. 2010) TNFα induced a later and sustained pattern of IκBβ degradation (Fig. 3A and C). These results demonstrate that MIN6 cells are susceptible to TNFα-induced NFκB signaling, and that signaling proceeds through both IκBα and IκBβ degradation. Having noted TNFα-induced IκB degradation, we next evaluated for NFκB subunit nuclear translocation. Within 60 minutes of exposure, nuclear levels of p65 and p50 had increased significantly (Fig. 3C and D). These results demonstrate that in MIN6 cells, TNFα exposure induces both IκB degradation and NFκB nuclear translocation.

Figure 3. TNFα induces NFκB signaling in MIN6 cells as assessed by IκB cytosolic degradation.

Figure 3.

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(Α) Representative Western blots showing IκBα and IκBβ in cytosolic extracts from MIN6 cells exposed to TNFα (100 ng/ml, .25–4 hours). (B) Densitometry ratio to control of IκB⍺ and IκBβ. Values shown as means ± SEM; n=4/time point. *=p<0.05 vs. unexposed control (C) Representative Western blots showing IκBα and IκBβ in nuclear extracts from MIN6 cells exposed to TNFα (100 ng/ml, 1 and 5 hours) (B) Densitometry ratio to control of p65 and p50. Values shown as means ± SEM; n=4/time point. *=p<0.05 vs. unexposed control.

TNFα, and TNFα plus STZ, but not STZ alone, induces expression of Cxcl10, Nos2 and Tnf in MIN6 cells

We next sought to determine whether STZ or TNF exposure induced the expression of NFκB target genes known to be associated with STZ-induced β-cell death in vivo. With this in mind, we assessed the expression of Cxcl10 (IP10)(Burke, et al. 2016; Jiang, et al. 2017; Martin, et al. 2007; Schulthess, et al. 2009) Nos2 (iNOS) (Baker, et al. 2001; Heimberg, et al. 2001; Jiang et al. 2017) and Tnf (TNFα) (Ishizuka, et al. 1999; Kagi, et al. 1999; Sarkar et al. 2009; Stephens, et al. 1999). Consistent with a lack of NFκB activation, STZ did not induce expression of either Cxcl10 (Fig. 4A), Nos2 (Fig. 4B) or Tnf (Fig. 4C). In contrast, and consistent with an induction of NFκB activation, TNFα exposure induced a significant increase of Cxcl10 (Fig. 4A), Nos2 (Fig. 4B) and Tnf (Fig. 4C). Having failed to demonstrate an effect of STZ alone on the induction of Cxcl10, Nos2 or Tnf expression, we asked whether STZ and TNFα would sensitize cells resulting in expression of these known mediators of β-cell injury. In contrast to STZ exposure alone, and consistent with a sensitizing effect of STZ and TNFα on induced gene expression, STZ plus TNFα exposure induced a significant increase of Cxcl10 (Fig. 4A), Nos2 (Fig. 4B) and Tnf (Fig. 4C). Importantly, the NFκB inhibitor BAY 11–7085 completely abrogated the effect of STZ on TNFα-mediated gene expression (Fig. 4A, B and C). These results demonstrate that STZ alone does not induce the expression of Cxcl10, Nos2, or Tnf, however, STZ can act with TNFα to significantly increase the expression of these factors associated with STZ-induced β-cell death in vivo. Furthermore, inhibition of NFκB activity attenuates this effect.

Figure 4. TNFα, and TNFα plus STZ, but not STZ alone, induces expression of Cxcl10 and Nos2 in MIN6 cells.

Figure 4.

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(A-C) Fold-increase in (A) Cxcl10, (B) Nos2, and (C) Tnf mRNA expression in MIN6 cells following BAY 11–7085 (1H, 20μM), STZ (2mM, 5 hours), TNFα (100 ng/ml, 5 hours), STZ (2mM) plus TNFα (100 ng/ml, 5 hours), or BAY 11–7085 pre-treatment (1H, 20μM) followed by STZ (2mM) TNFα (100 ng/ml, 5 hours). n=4–12/time point. *=p<0.05 vs. unexposed control, †=p<0.05 vs. TNFα exposed.

STZ induces expression of Cxcl1, Nos2 and Tnf in MIN6 cells more susceptible to NFκB signaling due to silencing of IκBβ expression

We next asked whether we could increase the MIN6 cell susceptibility to STZ by lowering the threshold to NFκB signaling by decreasing cellular expression of the IκB family of inhibitory proteins. Both MIN6 cells and isolated islets express the inhibitory proteins IκBα and IκBβ (Fig. 1). We found that TNFα exposure induced an early, transient and oscillatory pattern of IκBα degradation (Fig. 3A and B), while degradation of IκBβ occurred at later time points (Fig. 3A and C). The oscillatory nature of IκBα expression following NFκB activation is due to the fact that Nfkbia (gene name for IκBα) is an NFκB target gene (Sun, et al. 1993). Therefore, increases in Nfkbia expression are consistent with NFκB activity and the resulting increase in IκBα expression contributes to negative feedback inhibition of this innate immune signaling. To achieve a consistent and stable decrease in NFκB inhibitory protein expression, and to avoid the complication of minimizing the effectiveness of using siRNA to target expression resulting from dynamic changes in Nfkbia expression, we chose to target IκBβ expression. Thus, we transfected MIN6 cells with siRNA directed against the mRNA of the NFκB inhibitory protein IκBβ (gene name Nfkbib) to decrease its mRNA expression by over 50% (Fig. 5A), and significantly decreased protein expression by 40% (Fig. 5B and 5C). In cells transfected with siRNA directed against IκBβ, STZ exposure significantly induced the expression of both Cxcl10 (Fig. 5D), Nos2 (Fig. 5E) and Tnf (Fig. 5F). Importantly, and in contrast to Cxcl10 and Nos2, silencing IκBβ expression alone was enough to increase Tnf expression (Fig. 5F). These results demonstrate that Tnf expression can be induced by the increase in NFkB activity that occurs with silencing expression of NFκB inhibitory proteins. Of note, we observed very little added effect with STZ exposure at the exposure and time point tested. These results demonstrate that lowering the threshold to NFκB signaling by decreasing cellular expression of the IκBβ results in STZ-induced expression of NFκB target genes associated with β-cell death in vivo.

Figure 5. STZ induces expression of Cxcl10 and Nos2 in MIN6 cells more susceptible to NFκB signaling due to silencing of IκBβ expression.

Figure 5.

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(A) Relative mRNA expression of IκBβ (gene name Nfkbib) in MIN6 cells following transfection with IκBβ siRNA or exposure to vehicle (V) alone. Values are means + SEM. n=5–8. *, p<0.05 vs. untransfected control. (B) Representative Western blot showing IκBβ and GAPDH in whole cell lysates from MIN6 cells following transfection with IκBβ siRNA, GAPDH siRNA, or scramble siRNA (S), or exposed to vehicle (V) alone. Total protein staining shown as loading control. (C) Densitometry ratio to control of IκBβ provided. Values shown as means ± SEM; n=4–9/time point. (D-F) Fold change in STZ-induced (2mM, 5 hour) expression of (D) Cxcl10 (E) Nos2 and (F) Tnf in MIN6 cells following transfection with IκBβ siRNA. Values are expressed as a ratio of mRNA fold-change vs. control. Values are means + SEM. n=4–8/time point. *, p<0.05 vs. control.

TNFα plus STZ and Silencing of IκBβ expression increases sensitivity to STZ-induced cell death.

Having demonstrated that STZ and TNFα sensitized cells to induced expression of Cxcl10, Nos2 and Tnf in MIN6 cells, and was able to induce expression of Cxcl10, Nos2 and Tnf in MIN6 cells transfected with siRNA directed against the NFκB inhibitory protein IκBβ, we sought to assess the effect of these conditions on cell viability. Consistent with previous reports, both TNFα and STZ used at the doses in this study induced significant, but moderate levels of cell death (Fig. 6A). Furthermore, and consistent with increased expression of Cxcl10, Nos2, and Tnf, TNFα plus STZ induced a significant increase in cell death compared to STZ or TNFα alone (Fig. 6A) Additionally, across a wide range of STZ exposures (.5, 2, 4, 8 mM) transection with siRNA directed against the NFκB inhibitory protein IκBβ significantly increased MIN6 susceptibility to STZ-induced cell death (Fig. 6B). These results demonstrate that lowering the threshold to NFκB activation increases MIN6 susceptibility to STZ-induced cell death.

Figure 6. Silencing of IκBβ expression increases sensitivity to STZ-induced cell death.

Figure 6.

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(A) Viability of MIN6 cells exposed to STZ (2mM, 24 hours), TNFα (100 ng/ml, 24 hours) or STZ (2mM) plus TNFα (100 ng/ml, 24 hours) as assessed by trypan blue. Values expressed as mean + SEM of at least four independent experiments for each group. *p<0.05 vs. control, †=p<0.05 vs. STZ exposed, #=p<0.05 vs. TNFα exposed. (B) Viability of MIN6 cells exposed to STZ (.5–8 mM, 24 hours) and MIN6 cells following transfection with IκBβ siRNA and then exposed to STZ as assessed by trypan blue. Values expressed as mean + SEM of at least four independent experiments for each group. *p<0.05 vs. untransfected, treatment-matched control. Survival expressed as a percentage above each bar.

Adult male IκBβ−/− mice are more sensitive to STZ-induced diabetes

Having demonstrated that increasing susceptibility to NFκB activation increased MIN6 susceptibility to STZ-induced cell death, we next asked whether this would have implications in vivo. For this study we used adult male IκBβ−/− mice [kind gift of Sankar Ghosh, previously published (Rao et al. 2010)]. First, we assessed mRNA and protein expression of key members of NFκB signaling in islets isolated from WT and IκBβ−/− mice. We found no difference in the mRNA expression of the NFκB activating kinases (Chuk, Ikbkb), the NFκB inhibitory protein Nfkbia, or NFκB subunits (Fig. 4D: Nfkb1, Rela) between WT and IκBβ−/− mice (Fig. 7A). As expected, we did not detect Nfkbib (IκBβ) expression in islets isolated from IκBβ−/− mice (Fig. 7A). Additionally, we found no difference in the corresponding protein expression of the NFκB activating kinases IKKα and IKKβ, the NFκB inhibitory protein IκBα the NFκB subunits p50 and p65 (Fig. 7B and C). As expected, we did not detect IκBβ expression in islets isolated from IκBβ−/− mice (Fig. 7B and C). Next, we used the multiple, low-dose model of STZ exposure to assess differences in susceptibility to developing diabetes (defined as a fasting blood glucose concentration greater than 200 mg/dL measured on two consecutive blood draws) between adult male WT and IκBβ−/− mice. In WT mice, a dose of 40 mg/kg did not induce diabetes (Fig. 7D). In contrast, and despite our small sample size, we found a significantly increased incidence of diabetes in IκBβ−/− mice exposed to this dose of STZ (Fig. 7E). The incidence of diabetes was significantly increased in both WT and IκBβ−/− mice exposed to a higher dose of 50 mg/kg (Fig. 7G and H). While not significantly different, at a dose of 50 mg/kg the IκBβ−/− mice met criteria at a median of 21 days, while WT were at a median of 28 days.

Figure 7. Adult male IκBβ−/− mice are more sensitive to STZ-induced diabetes.

Figure 7.

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(A) Baseline mRNA expression in pancreatic islet isolated from WT and IκBβ−/− mice of the NFκB activating kinases: Chuk, Ikbkb, NF-κB subunits: Nfkb1, Rela, NFκB inhibitory proteins: Nfkbia, Nfkbib Values expressed as mean + SEM of at least three independent experiments for each group. *p<0.05 vs. WT control. (B) Representative Western blots showing WT and IκBβ−/− isolated islet expression of the NFκB activating kinases: IKKα and IKKβ; NFκB subunits: p50 and p65 and cRel; inhibitory proteins: IκBα and IκBβ; GAPDH as loading control. (C) Densitometric analysis of Western blots of protein isolated from isolated islets of WT and IκBβ−/− (KO) mice for NFκB activating kinases: IKKα and IKKβ; NFκB subunits: p50 and p65 and cRel; inhibitory proteins: IκBα and IκBβ; All values normalized to GAPDH as loading control. N=4 per group. *p<0.05 vs. WT control. (D-E) Percent survival without diabetes (defined as defined as a fasting blood glucose concentration greater than 200 mg/dL measured on two consecutive blood draws) in (D) WT compared to WT treated with streptozotocin (40 mg/kg IP x 5 days) and (E) IκBβ−/− compared to IκBβ−/− treated with streptozotocin (40 mg/kg IP x 5 days) (N=5 per group. *p<0.05 vs. control. (F) Fasting blood glucose levels of unexposed WT, unexposed IκBβ−/−, WT treated with streptozotocin (40 mg/kg IP x 5 days), and IκBβ−/− treated with streptozotocin (40 mg/kg IP x 5 days) mice from Day 0 (prior to first exposure) through Day 58. Lines connect mean levels, with individual data fasting blood glucose data provided. The dotted line marks a fasting blood glucose concentration of 200 mg/dL. (G-H) Percent survival without diabetes (defined as defined as a fasting blood glucose concentration greater than 200 mg/dL measured on two consecutive blood draws) in (G) WT compared to WT treated with streptozotocin (50 mg/kg IP x 5 days) and (H) IκBβ−/− compared to IκBβ−/− treated with streptozotocin (50 mg/kg IP x 5 days). N=5 per group. *p<0.05 vs. control. (F) Fasting blood glucose levels of unexposed WT, unexposed IκBβ−/−, WT treated with streptozotocin (50 mg/kg IP x 5 days), and IκBβ−/− treated with streptozotocin (5#nop0 mg/kg IP x 5 days) mice from Day 0 (prior to first exposure) through Day 58. Lines connect mean levels, with individual data fasting blood glucose data provided. The dotted line marks a fasting blood glucose concentration of 200 mg/dL.

Discussion

We found that key components of the NFκB signaling cascade are expressed in MIN6 cells and in isolated islets. We also determined that STZ exposure alone did not activate NFκB signaling in MIN6 cells. In contrast, TNFα exposure induced immediate and robust NFκB activation, as assessed by IκB inhibitory protein degradation and expression of Cxcl10, Nos2 and Tnf, all NFκB target genes previously demonstrated to be associated with β-cell injury. Importantly, exposing MIN6 cells to STZ and TNFα increased Cxcl10, Nos2 and Tnf expression. Furthermore, by lowering the threshold to NFκB activation through silencing expression of the NFκB inhibitory protein IκBβ, STZ exposure alone induced expression of Cxcl10, Nos2 and Tnf. This was associated with increased MIN6 cell susceptibility to STZ-induced cell death. Finally, we demonstrated that adult male IκBβ−/− mice were more susceptible to STZ-induced diabetes, further supporting a mechanistic role of a lower threshold to NFκB activation increasing pancreatic β-cell death sensitivity to injury.

These results are important because we show that STZ exposure alone does not induce NFκB signaling in MIN6 cells. Although we show that MIN6 have the full complement of NFκB activating kinases (IKKs), inhibitory proteins (IκBs) and activating subunits (p65 and p50), we could not detect STZ-induced NFκB activation as assessed by inhibitory protein degradation, activating subunit nuclear translocation or target gene expression. Previous studies have speculated that STZ induces NFκB activation through the production of reactive oxygen species(Los, et al. 1995; Schreck, et al. 1991), accumulation of DNA damage(McCool and Miyamoto 2012), or some other cellular stress/damage signaling possibly from resident islet macrophages that express IL1β and TNF⍺ transcripts(Calderon, et al. 2015). However, prior to this report, the data testing for a direct link between STZ exposure to NFκB activity in β-cells were lacking. Previous studies have shown that STZ exposures at higher (10 mM) doses and longer (48 hours) durations can induce NFκB activation in HEPG2 cells.(Raza and John 2012) Additionally, NFκB activity has been reported in islets isolated from STZ exposed mice.(Lgssiar et al. 2004) However, assays to assess NFκB activity were performed days (3) after the completion of a prolonged (5 days) STZ exposure, so a direct stimulatory effect was not assessed. Additionally, assessments were made in isolated islets after in vivo exposure STZ. Thus, the exposure was not limited to β-cells. NFκB activation in other cell types or in β-cells in response to cytokines released by macrophages like IL1β and TNF⍺ could not be excluded. Here, we show that in MIN6 cells, STZ did not directly induce NFκB activity as measured by IκB inhibitory protein degradation, NFκB subunit nuclear translocation, or by inducing NFκB target gene expression. These results support the hypothesis that STZ does not directly induce NFκB activity in β-cells.

This is an important observation, because previous studies have demonstrated that inhibiting NFκB activity in the setting of STZ exposure prevents β-cell apoptosis and provides nearly complete protection against the development of DM in vivo.(Eldor et al. 2006; Lamhamedi-Cherradi et al. 2003; Mabley et al. 2002) These findings have been reported in mice with global disruption of NFκB activity,(Lamhamedi-Cherradi et al. 2003; Mabley et al. 2002) and in mice with NFκB inhibition specifically targeted to the β-cell.(Eldor et al. 2006) Importantly, these previous studies did not specifically test whether STZ induced NFκB activity, but rather assessed the effect of inhibiting NFκB activity on the development of diabetes in the setting of STZ exposure. Our work provides mechanistic insights to these findings, demonstrating that the relationship between STZ and NFκB activity in β-cell may not be direct and likely includes additional stimulating factors.

In contrast to the lack of data directly linking STZ exposure to NFκB activity, multiple studies have shown that cytokine exposure induced NFκB activity in β-cell lines (Baker et al. 2001; Jiang et al. 2017; Kim et al. 2005) and isolated islets.(Giannoukakis, et al. 2000; Heimberg et al. 2001; Jiang et al. 2017) Furthermore, these studies demonstrate that inhibiting cytokine-induced NFκB activity attenuates injury in β-cell lines(Baker et al. 2001; Jiang et al. 2017) and isolated islets.(Cardozo et al. 2001; Giannoukakis et al. 2000; Heimberg et al. 2001; Jiang et al. 2017) Our studies complement the existing literature by interrogating the effect of adding cytokine exposure (TNFα) to STZ. We found that co-exposure significantly increased expression of NFκB target genes and cell death compared to STZ or TNFα exposure alone. These findings provide insight to the mechanisms underlying the role of NFκB in mediating the response to STZ in vivo, where a combination of the STZ induced insult and the endogenous cytokine response ultimately contribute to β-cell injury.

The rigor of the previous research showing that inhibition of NFκB activity in the setting of STZ or cytokine exposure can prevent β-cell is strong. In contrast, there are few reports testing whether increased NFκB activity or lowering the threshold to NFκB activation increases injury. It has been shown that increasing β-cell expression of the activating kinase IKKβ accelerates the development of diabetes in mice.(Salem, et al. 2014) Here, we contribute to this literature by showing decreasing expression of the NFκB inhibitory protein IκBβ increases STZ-induced cytokine expression and cell death in vitro, and accelerates the development of diabetes in vivo. It is important to note that any individual cell’s “threshold to activation” is dependent upon multiple factors, including the duration and the extent of the stimulating exposure, the expression of the receptor, adaptor proteins, activating kinases and inhibitory proteins. Here, we experimentally targeted expression of the NFκB inhibitory protein IκBβ as degradation of the cytosolic IκBs is the penultimate step of NFκB nuclear translocation and gene transcription. Importantly, in vivo work investigating the role of the threshold to activation must consider all aspects of the activation cascade beyond the cytosolic IκB expression. As an example, Li et all have recently demonstrated that islet specific overexpression of the NFκB activating kinase NIK leads to the spontaneous development of diabetes.(Li, et al. 2020) Similar to decreasing the expression of inhibitory proteins, overexpression of an activating kinase spontaneously lowers the threshold to activation. Similar to our results, Li et al found that β-cell injury was associated with increased expression of the NFκB target genes Cxcl10 and Tnf. Together with the previous reports demonstrating that inhibiting NFκB activity prevents β-cell injury (loss-of-function), and similar to the report by Li et al, our report shows that lowering the threshold to NFκB activity increases β-cell injury (gain-of-function), further supporting the role of this key innate immune signaling pathway in the pathogenesis of diabetes.

Our study has a number of limitations. The current study focused on NFκB signaling, and we did not interrogate other signaling pathways or transcription factors including AP-1 previously implicated in β-cell injury(Cnop, et al. 2005; Wellen and Hotamisligil 2005). We employed IκBβ−/− mice to interrogate the role of a lower threshold to NFκB activation in vivo. However, the effect on IκBβ is not specific to β-cells and the protective effect may be due to decreased cytokine expression from resident islet macrophages or other cell types.(Rao et al. 2010; Scheibel, et al. 2010) Additionally, as previous studies have shown that β-cell NFκB activation is stimulated by calcium flux,(Norlin, et al. 2005) we have targeted IκBβ as this NFκB inhibitory protein is known to be sensitive to intracellular calcium flux.(Biswas, et al. 2003; Biswas, et al. 2008; De Dios, et al. 2020) Of note, some reports demonstrate that inhibition of cytokine induced NFκB activity specifically through targeting IκBα increases β-cells apoptosis.(Chang et al. 2003; Kim et al. 2005; Kim, et al. 2007; Norlin et al. 2005) We have not interrogated whether our results differ from these because of the exposure (STZ vs. cytokine mix) or because we targeted IκBβ vs. IκBα. Studies to determine whether there is a difference with targeting IκBβ vs. IκBα in the β-cells are ongoing. This is an important distinction as NFκB signaling proceeds through phosphorylation and proteolysis of the IκB family of inhibitory proteins. Of the three cytoplasmic inhibitory proteins have been identified (IκBα, IκBβ and IκBε),(Hayden and Ghosh 2004) previous to this report only IκBα has been studied in the context β-cell NFκB activation. Importantly, by studying the inflammatory stress-induced NFκB transcriptome in other in vitro and in vivo systems lacking the expression of individual IκBs, much has been learned about how these inhibitory proteins differentially regulate NFκB activity and target gene expression.(Beg, et al. 1995; Hoffmann et al. 2002; Kearns, et al. 2006; Rao et al. 2010; Scheibel et al. 2010; Tergaonkar, et al. 2005) While we have demonstrated that IκBβ−/− mice are more susceptible to STZ-induced diabetes, more work needs to be done to determine whether increased expression of Cxcl10, Nos2, or Tnf contribute to this finding. These studies are in progress. Finally, while the reported findings are relevant to the pre-clinical study of DM using STZ, the relevance to various disease states remains to be determined. Whether the same observations would be made using a lower glucose media is unknown. Further work needs to be done to understand the implications of these differences on the β-cell response to injury.

We conclude that although β-cells possess all the key members in the NFκB signaling cascade, STZ exposure alone does not induce NFκB activity. However, with co-exposure to TNFα, expression of injurious NFκB target genes and cell death is increased. Furthermore, STZ-induced β-cell injury is increased both in vitro and in vivo by lowering the threshold to NFκB activation through decreased inhibitory protein expression. These results support further study of targeting the NFκB signaling cascade to prevent the development of β-cells injury and diabetes. Specifically, our results support the hypothesis that increasing the threshold to NFκB activation though stabilizing NFκB inhibitory protein stability will help prevent β-cells injury and the development of diabetes.

Acknowledgements

We would like to acknowledge the Pancreatic Islet Core at the Barbara Davis Center for Diabetes, University of Colorado for their assistance.

Funding:

This work was supported by a Pilot & Feasibility Study award (CJW) as part of the NIH/NIDDK funded UC Denver Diabetes Research Center (1P30DK116073-01A1, PI Lori Sussel). This work was supported by NIH grants R01HL132941 to CJW, R01DK088139 and R01HD093701 to PJR, T32HD007186 (BHB trainee, PJR PI), and R01AI139217 to SG.

Footnotes

Conflicts of interest: The authors declare no potential financial or ethical conflicts of interest.

Data Availability Statement:

The data used to support the findings of this study are included within the article.

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Data Availability Statement

The data used to support the findings of this study are included within the article.

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