. 2022 Nov:65:101582.

doi: 10.1016/j.molmet.2022.101582. Epub 2022 Aug 24.

RIPK1 and RIPK3 regulate TNFα-induced β-cell death in concert with caspase activity

Christopher J Contreras¹, Noyonika Mukherjee², Renato C S Branco³, Li Lin³, Meghan F Hogan⁴, Erica P Cai³, Andrew A Oberst⁵, Steven E Kahn⁴, Andrew T Templin⁶

Affiliations

¹ Division of Endocrinology, Department of Medicine, Roudebush VA Medical Center and Indiana University School of Medicine, Indianapolis, IN, USA.
² Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA.
³ Lilly Diabetes Center of Excellence, Indiana Biosciences Research Institute, Indianapolis, IN, USA.
⁴ Division of Metabolism, Endocrinology and Nutrition, Department of Medicine, VA Puget Sound Health Care System and University of Washington, Seattle, WA, USA.
⁵ Department of Immunology, University of Washington, Seattle, WA, USA.
⁶ Division of Endocrinology, Department of Medicine, Roudebush VA Medical Center and Indiana University School of Medicine, Indianapolis, IN, USA; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA; Lilly Diabetes Center of Excellence, Indiana Biosciences Research Institute, Indianapolis, IN, USA; Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN, USA. Electronic address: templin@iu.edu.

PMID: 36030035
PMCID: PMC9464965
DOI: 10.1016/j.molmet.2022.101582

RIPK1 and RIPK3 regulate TNFα-induced β-cell death in concert with caspase activity

Christopher J Contreras et al. Mol Metab. 2022 Nov.

. 2022 Nov:65:101582.

doi: 10.1016/j.molmet.2022.101582. Epub 2022 Aug 24.

Authors

Christopher J Contreras¹, Noyonika Mukherjee², Renato C S Branco³, Li Lin³, Meghan F Hogan⁴, Erica P Cai³, Andrew A Oberst⁵, Steven E Kahn⁴, Andrew T Templin⁶

Affiliations

¹ Division of Endocrinology, Department of Medicine, Roudebush VA Medical Center and Indiana University School of Medicine, Indianapolis, IN, USA.
² Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA.
³ Lilly Diabetes Center of Excellence, Indiana Biosciences Research Institute, Indianapolis, IN, USA.
⁴ Division of Metabolism, Endocrinology and Nutrition, Department of Medicine, VA Puget Sound Health Care System and University of Washington, Seattle, WA, USA.
⁵ Department of Immunology, University of Washington, Seattle, WA, USA.
⁶ Division of Endocrinology, Department of Medicine, Roudebush VA Medical Center and Indiana University School of Medicine, Indianapolis, IN, USA; Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN, USA; Lilly Diabetes Center of Excellence, Indiana Biosciences Research Institute, Indianapolis, IN, USA; Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN, USA. Electronic address: templin@iu.edu.

PMID: 36030035
PMCID: PMC9464965
DOI: 10.1016/j.molmet.2022.101582

Abstract

Objective: Type 1 diabetes (T1D) is characterized by autoimmune-associated β-cell loss, insulin insufficiency, and hyperglycemia. Although TNFα signaling is associated with β-cell loss and hyperglycemia in non-obese diabetic mice and human T1D, the molecular mechanisms of β-cell TNF receptor signaling have not been fully characterized. Based on work in other cell types, we hypothesized that receptor interacting protein kinase 1 (RIPK1) and receptor interacting protein kinase 3 (RIPK3) regulate TNFα-induced β-cell death in concert with caspase activity.

Methods: We evaluated TNFα-induced cell death, caspase activity, and TNF receptor pathway molecule expression in immortalized NIT-1 and INS-1 β-cell lines and primary mouse islet cells in vitro. Our studies utilized genetic and small molecule approaches to alter RIPK1 and RIPK3 expression and caspase activity to interrogate mechanisms of TNFα-induced β-cell death. We used the β-cell toxin streptozotocin (STZ) to determine the susceptibility of Ripk3^+/+ and Ripk3^-/- mice to hyperglycemia in vivo.

Results: Expression of TNF receptor signaling molecules including RIPK1 and RIPK3 was identified in NIT-1 and INS-1 β cells and isolated mouse islets at the mRNA and protein levels. TNFα treatment increased NIT-1 and INS-1 cell death and caspase activity after 24-48 h, and BV6, a small molecule inhibitor of inhibitor of apoptosis proteins (IAPs) amplified this TNFα-induced cell death. RIPK1 deficient NIT-1 cells were protected from TNFα- and BV6-induced cell death and caspase activation. Interestingly, small molecule inhibition of caspases with zVAD-fmk (zVAD) did not prevent TNFα-induced cell death in either NIT-1 or INS-1 cells. This caspase-independent cell death was increased by BV6 treatment and decreased in RIPK1 deficient NIT-1 cells. RIPK3 deficient NIT-1 cells and RIPK3 kinase inhibitor treated INS-1 cells were protected from TNFα+zVAD-induced cell death, whereas RIPK3 overexpression increased INS-1 cell death and promoted RIPK3 and MLKL interaction under TNFα+zVAD treatment. In mouse islet cells, BV6 or zVAD treatment promoted TNFα-induced cell death, and TNFα+zVAD-induced cell death was blocked by RIPK3 inhibition and in Ripk3^-/- islet cells in vitro. Ripk3^-/- mice were also protected from STZ-induced hyperglycemia and glucose intolerance in vivo.

Conclusions: RIPK1 and RIPK3 regulate TNFα-induced β-cell death in concert with caspase activity in immortalized and primary islet β cells. TNF receptor signaling molecules such as RIPK1 and RIPK3 may represent novel therapeutic targets to promote β-cell survival and glucose homeostasis in T1D.

Keywords: Caspase; RIPK1; RIPK3; TNFα; Type 1 diabetes; β-cell death.

Published by Elsevier GmbH.

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Conflict of interest statement

All authors declare that no competing interests exist.

Figures

**Figure 1**
**NIT-1 and INS-1 β cells express components of TNFα pathway signaling**. A) Schematic of TNFα death signaling pathways as described in non-islet cell types. B)*Tnfrsf1a*, *Ripk1*, *Casp8*, *Ripk3* and *Mlkl* RNA expression were quantified in NIT-1 (circles), INS-1 (squares), and NIH-3T3 (triangles) cells, normalized to *18S* rRNA levels, and expressed as ΔCT of the gene of interest (n = 3). C) NIT-1 and INS-1 β cells express TNFR1, RIPK1, CASP8, RIPK3 and MLKL protein, as visualized by duplicate immunoblot analysis of 20 μg of protein from total cell lysates.

**Figure 2**
**NIT-1 and INS-1 β cells are susceptible to RIPK1- and cIAP-mediated TNFα-induced cell death**. A Sartorius IncuCyte S3 live cell imaging and analysis instrument was used to monitor cell death via quantification of Sytox green positive cells. Cell culture treatment conditions are indicated by color (black: vehicle, blue: 40 ng/mL TNFα, red: 5 μM BV6, purple: TNFα+BV6) and β-cell lines are indicated by shapes (NIT-1 CTL: circles, NIT-1 RIPK1Δ: triangles, INS-1: squares). A) Cell death was monitored for 24 h in NIT-1 CTL and NIT-1 RIPK1Δ cells following TNFα treatment, and B) cell death was quantified 24 h post treatment (n = 6). C) Percent NIT-1 CTL cell death was quantified 24 h after treatment with vehicle or TNFα using the Sartorius IncuCyte S3 advanced label-free classification analysis software module, as described (n = 6). D) NIT-1 CTL and NIT-1 RIPK1Δ cell caspase 3/7 activity was quantified 24 h after treatment and expressed relative to vehicle treated NIT-1 CTL cells (n = 6). E) NIT-1 CTL and NIT-1 RIPK1Δ cells were treated with BV6, TNFα or a combination thereof, then F) cell death was quantified 4 h post treatment (n = 5–6). For comparisons between cell lines, cell death was reported as Sytox green positive cell objects normalized to phase positive cell area for each time point. G) Percent NIT-1 CTL cell death was quantified 4 h after treatment with BV6, TNFα or a combination thereof using the Sartorius IncuCyte S3 advanced label-free classification analysis software module (n = 4–5). H) NIT-1 CTL and NIT-1 RIPK1Δ cell caspase 3/7 activity was quantified 4 h after treatment and expressed relative to vehicle treated NIT-1 CTL cells (n = 4–6). I) Genomic DNA was isolated from NIT-1 CTL cells following treatment with vehicle or TNFα+BV6 for 4 h, then visualized on an agarose gel. J) For INS-1 cells, cell death was monitored for 24 h following treatment with BV6, TNFα or a combination thereof, then K) cell death was quantified 24 h post treatment (n = 5). For comparisons within a cell line, cell death was reported as Sytox green positive cell objects relative to time t = 0. L) Percent INS-1 cell death was quantified 24 h after treatment using the Sartorius IncuCyte S3 advanced label-free classification analysis software module, as described (n = 4). M) Representative IncuCyte images illustrate Sytox green positive INS-1 cells 24 h post treatment. Data are presented as mean ± SEM and were analyzed by one-way ANOVA followed by Sidák post-test and multiple comparisons correction. ∗*p <* 0.05; ∗∗*p <* 0.01; ∗∗∗*p <* 0.001 as indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

**Figure 3**
**NIT-1 and INS-1 β cells are susceptible to TNFα-induced cell death when caspases are inhibited**. A) NIT-1 CTL cell death was monitored over 48 h as described, and B) quantified at 48 h post treatment (n = 3). Cell culture treatment conditions are indicated by color (black: vehicle, yellow: 50 μM zVAD, blue: 40 ng/mL TNFα, green: TNFα+zVAD) and β-cell lines are indicated by shapes (NIT-1 CTL: circles, INS-1: squares). C) Percent NIT-1 CTL cell death was quantified 48 h after treatment with zVAD, TNFα or a combination thereof using the Sartorius IncuCyte S3 advanced label-free classification analysis software module (n = 6). D) NIT-1 CTL cell caspase 3/7 activity was quantified 48 h post treatment and expressed relative to vehicle treated cells (n = 3). E) INS-1 cell death was monitored over 24 h, and F) quantified at 24 h post treatment (n = 7). G) Percent INS-1 cell death was quantified 24 h after treatment using the Sartorius IncuCyte S3 advanced label-free classification analysis software module, as described (n = 3). H) INS-1 cell caspase 3/7 activity was quantified 24 h post treatment and expressed relative to vehicle treated cells (n = 9). I) Representative IncuCyte images illustrate Sytox green positive INS-1 cells 24 h post treatment. J) Heatmap displaying genes with significantly different expression among groups. High expression is shown in dark red and low expression in light yellow (n = 3). K) Venn diagrams displaying (left) the number of genes up-regulated commonly by TNFα and TNFα+zVAD (301), up-regulated specifically by TNFα (435), or up-regulated specifically by TNFα+zVAD (225), and (right) the number of genes down-regulated commonly by TNFα and TNFα+zVAD (2932), down-regulated specifically by TNFα (1558), or down-regulated specifically by TNFα+zVAD (2188). **A-H)** Data are presented as mean ± SEM and were analyzed by one-way ANOVA followed by Sidák post-test and multiple comparisons correction. ∗*p <* 0.05; ∗∗*p <* 0.01; ∗∗∗*p <* 0.001 as indicated. **J,K)** Differentially expressed genes were identified at p < 0.05, p-values were corrected for false discovery rate using the Benjamini-Hochberg method, and corrected p < 0.05 was used to identify significantly different gene expression. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

**Figure 4**
**RIPK1 and cIAPs regulate TNFα-induced NIT-1 and INS-1 β-cell death when caspases are inhibited**. A) NIT-1 CTL and NIT-1 RIPK1Δ cell death was monitored over 48 h, and B) quantified at 48 h post treatment (n = 5–6). Cell culture treatment conditions are indicated by color (black: vehicle, green: 40 ng/ml TNFα + 50 μM zVAD, white: TNFα+zVAD + 5 μM BV6) and β-cell lines are indicated by shapes (NIT-1 CTL: circles, NIT-1 RIPK1Δ: triangles, INS-1: squares). C) Percent NIT-1 CTL cell death was quantified 48 h after treatment with vehicle, TNFα +zVAD or TNFα+BV6+zVAD using the Sartorius IncuCyte S3 advanced label-free classification analysis software module, as described (n = 6). D) NIT-1 CTL and RIPK1Δ cell caspase 3/7 activity was quantified 24 h post treatment and expressed relative to vehicle treated NIT-1 CTL cells (n = 4–5). E) Genomic DNA was isolated from NIT-1 CTL cells following treatment with vehicle or TNFα+BV6+zVAD for 8 h, then visualized on an agarose gel. F) INS-1 cell death was monitored over 24 h, and G) quantified at 24 h post treatment (n = 5–6). H) Percent INS-1 cell death was quantified 24 h after treatment using the Sartorius IncuCyte S3 advanced label-free classification analysis software module (n = 4). I) Representative IncuCyte images illustrate Sytox green positive INS-1 cells 24 h post treatment. Data are presented as mean ± SEM and were analyzed by one-way ANOVA followed by Sidák post-test and multiple comparisons correction. ∗*p <* 0.05; ∗∗*p <* 0.01; ∗∗∗*p <* 0.001 as indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

**Figure 5**
**RIPK3 promotes TNFα-induced INS-1 cell death when caspases are inhibited**. A) NIT-1 CTL and NIT-1 RIPK3Δ cell death was quantified 48 h post treatment (n = 3). Cell culture treatment conditions are indicated by color (black: vehicle, blue: 40 ng/ml TNFα, green: 40 ng/ml TNFα + 50 μM zVAD) and β-cell lines are indicated by shapes (NIT-1 CTL: circles, NIT-1 RIPK3Δ: diamonds, INS-1: squares, INS-1 Empty: triangles, INS-1 mRIPK3: hexagons). B) NIT-1 CTL and RIPK3Δ cell caspase 3/7 activity was quantified 24 h post treatment and expressed relative to vehicle treated NIT-1 CTL cells (n = 4). C) INS-1 cell death was quantified 24 h post treatment relative to t = 0 (n = 4–9). Cell culture treatment conditions are indicated by color (black: vehicle, white: 5 μM GSK’872, blue: 40 ng/ml TNFα, light blue: TNFα+GSK’872, green: TNFα + 50 μM zVAD, light green: TNFα+zVAD + GSK’872). D) INS-1 cell caspase 3/7 activity was quantified 24 h post treatment and expressed relative to vehicle treated cells (n = 3). E)*Ripk3* RNA expression (n = 4) and F) RIPK3 protein expression were quantified in control (pcDNA3-Empty: triangles) and Ripk3 overexpressing (pcDNA3-mRipk3: hexagons) INS-1 cells. G) INS-1 pcDNA3-Empty and pcDNA3-mRipk3 cell death was quantified 24 h post TNFα or TNFα+zVAD treatment (n = 4). H) Immunoblot analysis of proteins immunoprecipitated with anti-RIPK3 following 24 h TNFα+zVAD treatment (n = 3). I) Immunoblot analysis of proteins immunoprecipitated with anti-MLKL following 24 h TNFα+zVAD treatment (n = 3). Data are presented as mean ± SEM and were analyzed by one-way ANOVA followed by Sidák post-test and multiple comparisons correction. ∗*p <* 0.05; ∗∗*p <* 0.01; ∗∗∗*p <* 0.001; ns, p > 0.05 as indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

**Figure 6**
**Mouse islets cells are susceptible to TNFα-induced cell death when cIAPs or caspases are inhibited**. A) Mouse islet cells express components of the TNFα signaling pathway at the RNA and B) protein levels. C) Mouse islet cell death was quantified 24 h post treatment relative to t = 0 (n = 8). Cell culture treatment conditions are indicated by color (black: vehicle, blue: 40 ng/ml TNFα, purple: TNFα + 5 μM BV6, green: TNFα + 50 μM zVAD, white: TNFα+BV6+zVAD. D) Mouse islet cell caspase 3/7 activity was quantified 24 h post treatment and expressed relative to vehicle treated cells (n = 4). E) Representative IncuCyte images illustrate Sytox green positive mouse islet cells 24 h post treatment. F) Mouse islet cell death was quantified 24 h after treatment with vehicle, TNFα or TNFα+zVAD in the absence (left) or in the presence (right) of the RIPK3 inhibitor GSK’872 (5 μM, n = 3–5). G) Mouse islet cell caspase 3/7 activity was quantified 24 h after treatment with vehicle, TNFα or TNFα+zVAD in the absence (left) or in the presence (right) of the RIPK3 inhibitor GSK’872 (n = 4). H) Mouse genotyping and islet immunoblot analysis confirmed loss of RIPK3 expression in Ripk3^−/− compared to Ripk3^+/+ mice. I) Basal cell death rate was quantified in Ripk3^+/+ and Ripk3^−/− mouse islet cells (n = 5–6). J) Ripk3^−/− mouse islet cell death was monitored for 24 h and quantified 24 h post treatment relative to t = 0 (n = 4). K) Mouse islet cell caspase 3/7 activity was quantified 24 h post treatment and expressed relative to vehicle treated cells (n = 4). Data are presented as mean ± SEM and were analyzed by Student's t-test or one-way ANOVA followed by Sidák post-test and multiple comparisons correction, as described. ∗*p <* 0.05; ∗∗*p <* 0.01; ns, p > 0.05 as indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

**Figure 7**
Ripk3^−/− mice are protected from STZ-induced hyperglycemia *in vivo*. A) Schematic of multiple low dose-streptozotocin (STZ) treatment and blood glucose monitoring. B) Prior to STZ treatment, glucose tolerance was evaluated by intraperitoneal glucose tolerance test (IPGTT) in Ripk3^+/+ and Ripk3^−/− mice (n = 6/group). C) 10 days after starting STZ treatment, glucose tolerance was evaluated by IPGTT in Ripk3^+/+ and Ripk3^−/− mice (n = 6/group). **D,E)** Non-fasting blood glucose was quantified in Ripk3^+/+ and Ripk3^−/− mice 0, 3, 7, 9 and 14 days after STZ treatment (n = 6/group), and data are displayed over time **(D)** and at individual time points **(E)**. Data are presented as mean ± SEM and were analyzed by Student's t-test. ∗*p <* 0.05; ∗∗*p <* 0.01; ∗∗∗*p <* 0.001; ns, p > 0.05; as indicated.

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RIPK1 and RIPK3 regulate TNFα-induced β-cell death in concert with caspase activity

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RIPK1 and RIPK3 regulate TNFα-induced β-cell death in concert with caspase activity

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