Stearoyl CoA desaturase is a gatekeeper that protects human beta cells against lipotoxicity and maintains their identity

Aims/hypothesis During the onset of type 2 diabetes, excessive dietary intake of saturated NEFA and fructose lead to impaired insulin production and secretion by insulin-producing pancreatic beta cells. The majority of data on the deleterious effects of lipids on functional beta cell mass were obtained either in vivo in rodent models or in vitro using rodent islets and beta cell lines. Translating data from rodent to human beta cells remains challenging. Here, we used the human beta cell line EndoC-βH1 and analysed its sensitivity to a lipotoxic and glucolipotoxic (high palmitate with or without high glucose) insult, as a way to model human beta cells in a type 2 diabetes environment. Methods EndoC-βH1 cells were exposed to palmitate after knockdown of genes related to saturated NEFA metabolism. We analysed whether and how palmitate induces apoptosis, stress and inflammation and modulates beta cell identity. Results EndoC-βH1 cells were insensitive to the deleterious effects of saturated NEFA (palmitate and stearate) unless stearoyl CoA desaturase (SCD) was silenced. SCD was abundantly expressed in EndoC-βH1 cells, as well as in human islets and human induced pluripotent stem cell-derived beta cells. SCD silencing induced markers of inflammation and endoplasmic reticulum stress and also IAPP mRNA. Treatment with the SCD products oleate or palmitoleate reversed inflammation and endoplasmic reticulum stress. Upon SCD knockdown, palmitate induced expression of dedifferentiation markers such as SOX9, MYC and HES1. Interestingly, SCD knockdown by itself disrupted beta cell identity with a decrease in mature beta cell markers INS, MAFA and SLC30A8 and decreased insulin content and glucose-stimulated insulin secretion. Conclusions/interpretation The present study delineates an important role for SCD in the protection against lipotoxicity and in the maintenance of human beta cell identity. Data availability Microarray data and all experimental details that support the findings of this study have been deposited in in the GEO database with the GSE130208 accession code. Electronic supplementary material The online version of this article (10.1007/s00125-019-05046-x) contains peer-reviewed but unedited supplementary material, which is available to authorised users.


Introduction
Type 2 diabetes develops as a consequence of a combination of insulin resistance of peripheral tissues and progressive decrease of functional pancreatic beta cell mass. This deficit is manifested by inadequate and insufficient insulin secretion in response to increased circulating glucose levels [1,2]. Insulin resistance often precedes the development of type 2 diabetes, but it is now well established that pancreatic beta cell failure is a sine qua non condition for hyperglycaemia and type 2 diabetes to develop [1,2].
NEFA represent an important source of energy for pancreatic beta cells in the normal state but can induce beta cell dysfunction and death when present in excessive levels during a prolonged period [1][2][3]. Chronic availability of fatty acids causes cell death and dysfunction in rodent beta cell lines [4,5], isolated rodent islets and primary beta cells [6,7], and animal models of diabetes [3,8]. Several studies pointed out that the degree of NEFA saturation is important since saturated NEFA (e.g. palmitate or stearate) cause marked apoptosis, whereas unsaturated NEFA (e.g. palmitoleate or oleate) are much less cytotoxic and protect against saturated NEFAmediated toxicity [7,[9][10][11]. The chronic adverse effects of saturated NEFA on beta cell function and viability are potentiated by the presence of hyperglycaemia, a phenomenon that is particularly seen in rodent beta cells and that has been termed 'glucolipotoxicity' [12,13]. Numerous studies have suggested different mechanisms by which NEFA mediate beta cell dysfunction and death such as endoplasmic reticulum (ER) stress [14], increased intracellular triacylglycerol [15], reactive oxygen species (ROS) [16,17], inflammation [14] and de novo synthesis of ceramide [15].
So far, the vast majority of data on the role of NEFA in beta cells has been derived from rodent models, either primary islets or rat and mouse beta cell lines [4,[18][19][20], with a more limited number of investigations performed using primary human islets [10,14,15,[21][22][23][24][25][26]. This is mainly due to the limited access to human islet preparations, which not only contain variable numbers of beta cells from one preparation to the other, but are also contaminated with non-endocrine cells such as exocrine tissue [27].
In this study, we sought to investigate lipotoxicity in a recently engineered functional human beta cell line, EndoC-βH1 [28]. This line represents a precious tool to study human beta cells in pathophysiological conditions [29]. As an example, EndoC-βH1 cells react to cytokine exposure in a similar manner to primary human beta cells [30]. Moreover, this cell line is suitable for drug screening [31].
Human islet culture Pancreases were obtained with informed written consent and processed with the approval of the local ethics committee of the University of Pisa. Human islets were isolated at the University of Pisa, Italy, using collagenase digestion and density gradient purification from heartbeating organ donors [34]. The organ donors (three men, five women, age 67 ± 8 years [mean ± SD], BMI 27.3 ± 4.0 kg/m 2 , cause of death cerebral haemorrhage in six, stroke in one and post-anoxic encephalopathy in one) did not have a medical history of diabetes. Human islets were cultured in Ham's F-10 medium as described [14]. Beta cell purity, evaluated by insulin immunocytochemistry in dispersed islet cells, was 47 ± 10%. Information on human islets is available in the Human Islets checklist in the ESM.
Human induced pluripotent stem cell culture and differentiation into beta cells The previously described human induced pluripotent stem cell (iPSC) line HEL115.6 [35] was differentiated into beta cells using a seven-stage protocol that makes use of monolayer culture on Matrigel-coated plates up to pancreatic progenitor stage 4 and then moves the cells to suspension culture until the last stage of beta cell differentiation [35]. Stage 7 aggregates contained 41 ± 14% beta cells (assessed by insulin immunocytochemistry).
Assessment of cell death Live/dead cells were counted following Trypan Blue staining. Caspase 3/7 activity assays were performed using the Promega Apo-ONE Homogenous caspase-3/7 Assay kit as described [36] (Promega, Charbonières-les-Bains, France). As another method for apoptosis detection, cells were stained with the Hoechst 33342 (5 μg/ml, Sigma-Aldrich) and propidium iodide (PI, 5 μg/ml, Sigma-Aldrich) and counted by fluorescence microscopy [37]. The xCELLigence system (ACEA Biosciences, San Diego, CA, USA), which is based on the continuous real-time monitoring of cell adhesion, was used for real-time and label-free monitoring of cell viability and growth [38]. Briefly, EndoC-βH1 cells were seeded into 96-well E-plates coated with extracellular matrix and fibronectin (50,000 cells/ well), transfected with siRNA, treated with NEFA or BSA 72 h later and monitored for up to 72 h.

Insulin content and glucose-stimulated insulin secretion
Insulin content and glucose-stimulated insulin secretion (GSIS) were measured as described [39].
RNA isolation, reverse transcription, qRT-PCR and transcriptomic analyses qRT-PCR was performed as described [32]. ACTB or PPIA transcript levels were used as housekeeping genes for normalisation. Primer sequences are listed in ESM Table 2. Global transcriptomic analyses were performed using the Affymetrix 2.0ST gene chip as described [32] (Affymetrix-Thermofisher, Courtaboeuf, France).
Human IAPP promoter analysis The 797 bp upstream sequence of the IAPP gene, which encodes islet amyloid polypeptide (IAPP), was extracted from NCBI Map viewer/Ace view, and scanned for the presence of SOX9 binding motifs using MatInspector (Genomatix software, https://www. genomatix.de/, access date: 3 January 2019; [40]). Results are presented in ESM Table 3.
Immunoblotting Western blots were performed as described [32] using the following antibodies diluted in TBS 3% BSA 0. Statistical analyses Graphs were constructed by using PRISM6 software (GraphPad, San Diego, CA, USA). Quantitative data are presented as the mean ± SD from three independent experiments. Results were analysed by oneway ANOVA with post hoc Tukey testing for multiple conditions or by t test if only two conditions were being tested (two-tailed). Randomisation and blinding were not carried out. A p value less than 0.05 was considered significant.

Results
EndoC-βH1 cells are resistant to palmitate toxicity We first analysed the effect of palmitate on EndoC-βH1 cell viability. We did not observe lipotoxicity associated with morphological changes or obvious cell death (characterised by floating cells or debris) in EndoC-βH1 cells treated with 0.4 mmol/l palmitate (C16:0). The concept of glucolipotoxicity, i.e. the deleterious effects of combined elevated glucose and NEFA concentrations, prompted us to study EndoC-βH1 cell viability following both high glucose and NEFA exposure. The efficiency of HG (30 mmol/l) treatment was validated by TXNIP mRNA upregulation ( [39] and data not shown). Remarkably, we did not observe cell toxicity after palmitate incubation at low glucose (5.6 mmol/l) or HG (Fig. 1a). To strengthen our investigation, we measured caspase 3/7 cleavage as another marker of cells undergoing apoptosis. Accordingly, we did not observe changes in caspase 3/7 cleavage activity upon palmitate exposure (Fig. 1b). We then quantified PARP cleavage, another apoptosis-related measurement. Thapsigargin induced cell apoptosis as determined by increased PARP cleavage, but this was not the case with palmitate ( Fig. 1c, d). Finally, to survey the effects of palmitate over a prolonged period of time (up to 72 h) in real time, we used the xCELLigence system. Palmitate treatment did not decrease cell proliferation/survival, but, in fact, it increased it in a time-dependent manner (Fig. 1e).
These data indicate that long chain saturated NEFA such as palmitate, with or without HG, do not induce glucolipotoxicity in EndoC-βH1 cells.

SCD is involved in EndoC-βH1 protection against lipotoxicity
Real-time monitoring using xCELLigence suggested that palmitate may in fact increase cell proliferation/survival (Fig. 1). Palmitate can either enter the mitochondrial NEFA β-oxidation pathway, or be elongated and then desaturated to be incorporated into neutral lipids, two pathways known to be protective to cells (Fig. 2a, [13,14]). We tested whether altering the enzymes involved in palmitate metabolism modifies the effects of NEFA on EndoC-βH1 cells. We performed knockdown using siRNA against: CPT1A, the rate-limiting-step enzyme of NEFA βoxidation; ELOVL6, which elongates palmitate into stearate; and SCD and SCD5, which desaturate palmitate or stearate into palmitoleate (C16:1) or oleate (C18:1), respectively. Each siRNA was specific and efficient (>50% downregulation in the mRNA target) (ESM Fig. 1a). siRNA-transfected EndoC-βH1 cells were next treated with palmitate ± HG. Upon CPT1A and ELOVL6 knockdown, palmitate did not induce caspase 3/7 cleavage (Fig.  2b). But upon SCD knockdown (Fig. 2c,d, ESM Fig. 1a), palmitate treatment increased caspase 3/7 cleavage in EndoC-βH1 cells (Fig. 2b). To rule out off-target effects, we used three other siRNAs targeting different regions of the SCD mRNA (ESM Table 1, ESM Fig. 1b), and these consistently sensitised EndoC-βH1 cells to palmitate-induced apoptosis measured by Hoechst 33342 and PI staining (ESM Fig. 1c). Of note, upon SCD5 knockdown, another SCD isoform expressed by human beta cells, palmitate ± HG did not induce toxicity (Fig. 2b). Moreover, palmitate ± HG treatment of βH1-SCD KD cells decreased cell survival as measured by cell morphology, cell counts and xCELLigence (Fig. 2e, g). Similar results were obtained with stearate (C18:0), another long chain saturated NEFA (ESM Fig. 2a, b). Of note, real-time qPCR quantification indicated that, in EndoC-βH1 cells, SCD mRNA expression was high (C t~1 9) when compared with other enzymes implicated in saturated NEFA metabolism (CPT1A: C t~2 6; ELOVL6: C t~2 4; SCD5: C t~2 5). Its expression was also high in human islets and in iPSC-derived beta cells, with an increase in the last stage of human beta cell maturation in this in vitro model of pancreatic endocrine cell development (ESM Fig. 3).
Thus, SCD, an enzyme that catalyses a rate-limiting step in the synthesis of unsaturated NEFA, is involved in EndoC-βH1 cell protection against (gluco)lipotoxicity induced by palmitate and stearate.
Long chain saturated NEFA modulate the expression of stressrelated genes in βH1-SCD KD cells We next analysed in βH1-SCD KD cells the effects of palmitate (± HG) treatments on the expression of genes previously found to be upregulated by palmitate in human islets, such as genes related to ER stress (ATF3, DDIT3, spliced variant of XBP1) and inflammation (IL8, TNF) and also IAPP [14]. When EndoC-βH1 cells were transfected with a control siRNA, we did not observe upregulation of the aforementioned genes upon palmitate treatment (± HG), confirming the lack of lipotoxicity. However, palmitate treatment of βH1-SCD KD cells induced ATF3, DDIT3,  spliced XBP1, IL8, TNF and IAPP mRNAs (Fig. 3a-d, f-h). Similar inductions were observed with stearate (C18:0) treatment (ESM Fig. 4). ER stress marker DDIT3 was also induced at the protein level (Fig. 3e). Of note, ER stress-and inflammation-related gene expression was already induced upon SCD knockdown by itself, suggesting that the inhibition of endogenous NEFA desaturation is sufficient to elicit mild ER and inflammatory stress; exogenous palmitate or stearate treatment further enhanced these inductions (Fig. 3b-d, f-g, ESM Fig. 4) [23].
These data indicate that following SCD knockdown, EndoC-βH1 cells respond to palmitate and stearate in a way similar to that observed in human islets.
Palmitate-induced IAPP upregulation in βH1-SCD KD cells requires SOX9 IAPP is upregulated in several dysfunctional beta cell models. Genomatix analysis suggested eight potent SOX9 binding sites in the human IAPP promoter (Fig. 4a and ESM Table 3). SOX9 is a transcription factor expressed in pancreatic progenitors and in duct cells in the adult pancreas . Data represent the means ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 relative to control as indicated on the graph but also in beta cells upon dedifferentiation [32,[42][43][44].
Here, we observed that SOX9 expression was significantly upregulated in palmitate-treated βH1-SCD KD cells at the mRNA and protein levels (Fig. 4b, c). To study SOX9 involvement in IAPP induction, we prevented SOX9 induction using siRNA in βH1-SCD KD cells (Fig. 4c-e) and then treated these cells with palmitate + HG. Under this setting, IAPP induction by palmitate + HG was abolished (Fig. 4f).
Our data thus demonstrate that upregulation of IAPP by palmitate + HG requires the induction of the beta cell dedifferentiation marker SOX9. The key in (a) is also applicable to (b-d) and (f-h). Data represent the means ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 relative to control as indicated on the graph Dedifferentiation is observed upon SCD knockdown We next investigated other described beta cell dedifferentiation markers [32,42]. We observed HES1 and MYC upregulation in palmitate-treated βH1-SCD KD cells (Fig.  5a, b). At the same time, the expression of the beta cell-specific markers INS, MAFA and SLC30A8 sharply decreased (Fig. 5c-e). Surprisingly, their expression was already downregulated in βH1-SCD KD cells alone (without palmitate treatment) (Fig. 5c-f), suggesting that SCD depletion is sufficient to induce EndoC-βH1 cell dedifferentiation. RNA microarray analysis indicated the downregulation of additional beta cell markers such as G6PC2, SLC2A2 and FOXO1 in βH1-SCD KD cells (Fig.  5g), further supporting beta cell dedifferentiation [  x-axis conditions below (f) also apply to (d, e) and the key above (d) also applies to (e, f). Data represent the means ± SD of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 relative to control as indicated on the graph Diabetologia (2020) 63:39 -409 42,45]. We did not observe any upregulation of nonbeta cell endocrine cell markers such as GCG or SST or exocrine markers such as HNF1B and PTF1A (ESM Fig. 5). Finally, insulin content decreased following SCD downregulation (Fig. 5h). Moreover, GSIS was reduced by 38% in βH1-SCD KD cells (Fig. 5i).
Induction of inflammation and ER stress in βH1-SCD KD cells is reduced by oleate and palmitoleate treatment SCD is the rate-limiting enzyme that catalyses the production of palmitoleate/oleate from palmitate/stearate. MS analysis indicated that SCD knockdown in EndoC-βH1 cells decreased basal oleate concentrations with a significant decrease in the oleate/stearate ratio (Table 1). Of note, we did not observe a decrease in basal palmitoleate concentrations after SCD knockdown compared with siCTRL ( Table 1), suggesting that SCD is primarily transforming stearate into oleate in EndoC-βH1 cells. Moreover, elongation of C16 into C18 NEFA by ELOVL6 might be an important step for long chain fatty acid metabolism in EndoC-βH1 cells. Remarkably, ELOVL6 is slightly upregulated upon SCD knockdown (ESM Fig. 1). However, co-transfection of SCD and ELOVL6 siRNAs did not reverse dedifferentiation, inflammation and ER stress, suggesting that the degree of NEFA saturation is more important than length in conferring toxicity (data not shown).
We next asked whether oleate or palmitoleate supplementation could reverse some phenotypic traits observed in βH1-SCD KD cells. Treatment of βH1-SCD KD cells with oleate and palmitoleate reduced the effects of palmitate/HG on caspase 3/7 cleavage activity that was paralleled by an absence of induction of IL8 and ATF3 (Fig. 6a-c). Finally, in the absence of palmitate/HG, while oleate and palmitatoleate did not reverse the INS, MAFA or SLC30A8 downregulation observed upon SCD knockdown (Fig. 6d-f), the induction of inflammation (IL8, TNF) and ER stress (spliced XBP1, ATF3) markers was reduced (Fig. 6g-j).

Discussion
Chronically elevated saturated NEFA levels can impair the function of pancreatic beta cells. The mechanisms involved in beta cell lipotoxicity induced by saturated NEFA are the subject of active investigations because of its association with the development of type 2 diabetes [2,3]. However, our knowledge of how saturated NEFA act on human beta cells and induce diabetes is limited. Defining these mechanisms could help to develop new strategies to prevent beta cell dysfunction and death in type 2 diabetes. Rodent models have been useful to better understand the mechanisms of NEFAinduced beta cell dysfunction. However, differences exist between human and rodent beta cells in response to NEFA [21,46,47]. For example, palmitate differentially affects protein acetylation in rodent and human beta cells [47]. Remarkably, human islets appear to be more resistant to apoptosis than rodent RIN1046-38, INS-1 or Min6 cell lines [21,46,48,49]. It is thus of major importance to develop human beta cell models of lipotoxicity. As access to primary human islet preparations is limited and variability exists from one human islet preparation to the other [27], we recently developed functional human beta cell lines [28,50] and tested here their use in modelling human beta cell lipotoxicity.
Rat and mouse beta cells are highly sensitive to palmitate treatment that induces dysfunction and apoptosis [3]. On the other hand, previous data indicated that treatment of EndoC-βH1 cells with palmitate does not induce lipotoxicity under standard culture conditions [51,52]. Our current data further confirm this. By investigating saturated NEFA metabolism and its related enzymes through knockdown using siRNA, we identified SCD as the main brake on palmitate toxicity. SCD is highly expressed in primary human beta cells ( [50,51] and the present study). Interestingly, elevated SCD levels have been shown to protect against saturated NEFA in a number of cell types, including the mouse beta cell line MIN6 cells and human islets [21,48,49]. The working hypothesis is that SCD rapidly desaturates palmitate/stearate into palmitoleate/oleate, and thus decreases their toxicity. Five different SCDs (SCD1-5) have been described in the mouse while there are only two in humans (SCD and SCD5) [53]. It is noteworthy that SCD5 is predominantly expressed in the human brain and pancreatic islets (beta and delta cells), human beta cell lines and pancreatic ductal cells ( [53,54] and the present study). Even though SCD5 has been shown to desaturate NEFA [55], our data indicate that, while SCD knockdown induces lipotoxicity in EndoC-βH1 cells upon palmitate treatment, this is not the case upon SCD5 knockdown. This suggests that, in human beta cells, SCD plays the dominant role in the desaturation of long chain saturated NEFA. Another possibility is that products of SCD and SCD5 are used for differential lipogenic reactions. Indeed, SCD is known to play a central role in the synthesis of neutral lipids such as triacylglycerol, which are protective for beta cells [11]. In contrast, in neuronal cells overexpressing SCD5, triacylglycerol and phosphatidylethanolamine formation was reduced whereas de novo synthesis of phosphatidylcholine and cholesteryl esters was increased [55]. Additional analyses are needed to unravel SCD5 function in human beta cells. Interestingly, SCD5 is involved in neuronal cell proliferation and differentiation [55] and in survival of MCF-7 cells, in which cancer-associated fibroblasts induced the expression of SCD5 [56].
Our study further shows that palmitate treatment of βH1-SCD KD cells induced the expression of genes related to inflammation (IL8, TNF) and ER stress (ATF3, DDIT3, spliced XBP1). Increased phospholipid saturation upon inhibition of SCD could contribute to enhance ER stress in the presence of palmitate, as observed in HeLa cells [57]. These saturated lipids reduce ER membrane fluidity, which may secondarily lead to ER Ca 2+ depletion, reduced protein folding and ER stress [37]. Palmitate also induced the expression of IAPP mRNA levels in βH1-SCD KD cells, as previously observed in human islets treated with palmitate [14].
Remarkably, we found that the expression of SOX9, a beta cell dedifferentiation marker [32,42,44], was induced by palmitate in βH1-SCD KD cells, as were HES1 and MYC. SOX9 activation was necessary for the induction of IAPP by palmitate. Of note, amyloid deposits were recently described surrounding dedifferentiated beta cells in individuals with type 2 diabetes [58]. We propose that beta cell dedifferentiation and induction of SOX9 expression represents an early step that enhances IAPP expression. Human IAPP is coexpressed and co-secreted with insulin. In type 2 diabetes patients, IAPP forms cytotoxic 'amyloid' plaques within islets [59,60]. This phenomenon is difficult to study in mice as rodent IAPP does not form amyloid fibres [59,60]. Palmitate-treated βH1-SCD KD cells may thus represent a new model to understand the regulation of IAPP expression and its potential to form deleterious amyloid fibres [60].
We observed that SCD knockdown by itself was sufficient to give rise to major phenotypes. It decreased the expression of central beta cell markers such as INS, MAFA and SLC30A8. These observations underline a new role for SCD in maintaining mature beta cell identity. It is noteworthy that SCD is also upregulated during beta cell maturation suggesting an important role in adult beta cell function and identity ( [61,62] and the present study). SCD knockdown reduced GSIS in EndoC-βH1 cells. Interestingly, it has been shown that extraction of NEFA with NEFA-free BSA from the plasma membrane of MIN6 cells reduced insulin secretion [63]. There, oleate was one of the most extracted NEFA, suggesting that its endogenous synthesis through SCD plays a central role in the regulation of insulin secretion in beta cells. SCD knockdown also induced markers of inflammation and ER stress in EndoC-βH1 cells. The beneficial effects of oleate compared with palmitic acid on insulin resistance and type 2 diabetes is well established [64]. In the present study, SCD knockdown decreased the ratio oleate/palmitate by 30%, suggesting that this reduction could contribute to the deleterious effect of palmitate in βH1-SCD KD cells. In keeping with this, the induction of inflammatory (IL8, TNF) and ER stress (spliced XBP1, ATF3) markers was rescued upon addition of oleate and palmitoleate, the products of SCD enzyme reactions. On the other hand, treatment with oleate and palmitoleate did not rescue the expression of beta cell differentiation markers. Future experiments will test whether other conditions of treatment with oleate or palmitoleate (different concentrations, longer exposure time) will reverse the dedifferentiation phenotype observed upon SCD knockdown. Taken together, we propose that SCD is a gatekeeper in human beta cells that protects against dedifferentiation, dysfunction, inflammation and ER stress. βH1-SCD KD cells thus represent an innovative model to discover pathways and molecules that maintain high levels of SCD and protect against such deleterious effects.
Many observations suggest that SCD is important for beta cell adaptation and compensation during type 2 diabetes development in rodents. Scd1 and Scd2 mRNA expression is induced in islets from prediabetic hyperinsulinaemic Zucker Diabetic Fatty rats and their expression decreases when diabetes develops [49]. Consistent with this observation, dietinduced obesity reduces Scd1 mRNA expression in rodent islets [65]. Moreover, while global knockout of Scd1 in mice improves insulin sensitivity, when introduced on the ob/ob background with leptin-deficiency, Scd1 deletion leads to a worsening of diabetes [66]. Importantly, SCD gene expression was lower in beta cell enriched tissue (obtained by laser capture microdissection) from individuals with type 2 diabetes compared with healthy donors [67]. We propose that, over time, in the course of type 2 diabetes progression, SCD expression by beta cells is first induced during compensation in response to insulin resistance, and as the duration of diabetes increases, SCD expression decreases leading to a decline in   cholesterol [68,69], will help us define new strategies to overcome beta cell dedifferentiation, dysfunction and death in type 2 diabetes. Our results described above will enable progress on this important topic using βH1-SCD KD as a human beta cell model.