Hyperglycemia during fetal development disturbs extracellular matrix (ECM) synthesis and deposition patterns, which disrupts organogenesis and adult organ function. Although the ECM cooperates in pancreas development, little is known about the effects of hyperglycemia on the pancreatic ECM during development. This study investigates the effect of severe maternal hyperglycemia on ECM composition and endocrine pancreas development in E19.0 mouse fetuses. Deposition patterns of pan-laminin, laminin (alpha 1 and gamma 1 chains) and integrin alpha 3 were evaluated by immunostaining. The proliferative index of islet cells and alpha and beta cell distribution were evaluated by PCNA, glucagon and insulin immunostaining, respectively. Pdx1 and Pax4 expressions were analyzed by RT-qPCR. While for pan-laminin and laminin (alpha1 and gamma1 chains) deposition was weaker in the endocrine pancreas of hyperglycemic mothers’ fetuses, integrin alpha 3 deposition in the basement membrane was increased. The proliferative index of endocrine cells was lower in the hyperglycemic group, while the beta-cell area was increased. In addition, there was a tendency towards lower Pdx1 and increased Pdx4 expression. These data suggest that maternal hyperglycemia alters fetal endocrine pancreas morphogenesis by modifying peri-islet basement membrane molecule patterns, promoting a decrease in endocrine cell proliferation associated with changes in the expression of important growth factors for the beta cells differentiated and the proliferative state.

OBJECTIVES:Reactive oxygen species (ROS) are short-lived and act in a site-specific manner, underscoring the importance of identifying the subcellular localization of their sources. ROS-generating NADPH oxidases (NOX) regulate pancreatic beta cell (dys)function. However, their subcellular localization and cytokine-mediated regulation in these cells remain largely unknown. We characterized the expression, subcellular localization and time-dependent cytokine-induced regulation of NOX isoforms in beta cells. METHODS:Isoforms were studied via RT-qPCR, immunoblotting and immunofluorescence in rat islets and beta cell lines. RESULTS:Beta cells express DUOX1 and DUOX2 proteins and Duoxa2 transcripts; lacking Duoxa1 expression. In INS-1E cells, NOX1 and DUOX1 localize in the endoplasmic reticulum (ER); DUOX2 in insulin vesicles; and NOX2 and NOX4 in vesicles, ER and plasma membrane. In INS-1E, cytokines increased expression of Nox1 and Duox1 at 4-8 h (returning to baseline at 16 h) and Nox2 and p47phox at 8 h (persisting until 24 h). Duox(a)2, p67phox and p40phox were downregulated and DUOX1 upregulated at 16-24 h. CONCLUSION:The absence of Duoxa1 in beta cells might lead to DUOX1 mismatching, impairing its trafficking and activity. NOXs in beta cells are diverse in subcellular localization and cytokine-induced regulation, suggesting their isoform-specific involvement in beta cell function, stress and apoptosis.

HNF4α is important for beta cells' ability to adequately secrete insulin in response to glucose concentration and endoplasmic reticulum (ER) homeostasis. In humans, HNF4α mutations are responsible for Diabetes mellitus subtype MODY1, which has an age-determining onset. Additionally, in other forms of DM, there is evidence that sex can influence beta cell dysfunction, with possible involvement of ER stress pathways. Thus, we assessed the influence of sex and age on beta-cell dysfunction induced by HNF4α absence. We used an animal model with specific beta cells KO of HNF4α, induced after birth (Ins. CRE HNF4αloxP/loxP). Glucose intolerance is observed after 10 d of KO induction, at 50 d of age, with KO males (MKO) displaying more severe glucose intolerance than KO females (FKO). The percentage of insulin-positive cells in KO mice islets is lower compared to Control at all ages evaluated, with MKO mice showing a more pronounced decline at later ages compared to FKO. Both KO groups exhibited reduced beta cell mass and increased α-cell mass, which was more pronounced in MKO. ER stress was induced in both KO groups; however, ER stress-mediated apoptosis was observed only in MKO. FKO mice show evidence of beta cell differentiated state loss. In summary, beta cell loss in HNF4α-KO is influenced by sex and age, involves induction of ER stress, and is more severe in males, where ER stress-induced beta cell death is observed. Partial protection observed in females seems to involve dedifferentiation of beta cells.

The discovery of insulin was a true landmark in biomedical research and provided a framework for the understanding of many pivotal mechanisms in cell biology throughout the twentieth century, as insulin for instance was the first major protein to have its amino acid sequence and 3D structure resolved. The elucidation of the processes that regulate and mediate insulin secretion has also contributed with crucial mechanistic insights on the pathways that lead to both type 1 and type 2 diabetes mellitus. More than 100 years after the discovery of this hormone, an overview of the present knowledge on insulin output from β-cells should be timely to the general researcher interested in the mechanisms that couple glucose stimulation to insulin secretion. Although the mechanisms underlying insulin secretion have been exhaustively studied and understood in animal models, the last decades have shown that important differences can be identified compared to human β-cells. Additionally, despite both reactive oxygen species as well as the immune system have been initially implicated in β-cell dysfunction and the progression to diabetes, increasing evidence indicates that both can also have physiological effects for proper insulin secretion. Given this background, this brief review focused on discussing various means by which glucose elicits insulin secretion by the β-cells, particularly on the modulatory role of redox balance and inflammation on β-cell function and/or demise, also drawing attention to the specific mechanisms connecting glucose stimulation to insulin secretion in humans.

HNF4alpha is important for beta cells' ability to adequately secrete insulin in response to glucose concentration and endoplasmic reticulum (ER) homeostasis. In humans, HNF4alpha mutation is responsible for Diabetes mellitus subtype MODY1, which has age determined onset. In addition, in other types of DM, there are evidences that gender can influence beta cell dysfunction, with possible involvement of ER stress pathways. Thus, we assessed the influence of gender and age on beta cell dysfunction induced by HNF4alpha absence. We used an animal model with specific beta cells KO for HNF4alpha, induced after birth (Ins. CRE HNF4alpha loxP/loxP). Glucose intolerance is observed after 10 days of KO induction, at 50 days of age, with KO males (MKO) showing greater glucose intolerance than KO females (FKO). Percentage of insulin-positive cells in KO mice islets is lower compared to Control at all ages evaluated, with MKO having a lower percentage at later ages compared to FKO. Both KO groups have reduced beta cell mass and increased alpha-cell mass, which is higher in MKO. ER stress is induced in both KO groups. However, ER stress-mediated apoptosis is observed only in MKO. FKO shows evidence of beta cell differentiated state loss. Thus, loss of beta cells in HNF4alpha KO is influenced by gender and age, involves induction of ER stress, and is more pronounced in males, where ER stress-induced beta cell death is observed. Partial protection observed in females seems to involve dedifferentiation of beta cells. ### Competing Interest Statement The authors have declared no competing interest.

Type I Diabetes mellitus (T1D) is characterized by a specific destruction of β-cells by the immune system. During this process pro-inflammatory cytokines are released in the pancreatic islets and contribute for β-cells demise. Cytokine-induced iNOS activation, via NF-κB, is implicated in induction of β-cells death, which includes ER stress activation. Physical exercise has been used as an adjunct for better glycemic control in patients with T1D, since it is able to increase glucose uptake independent of insulin. Recently, it was observed that the release of IL-6 by skeletal muscle, during physical exercise, could prevent β-cells death induced by pro-inflammatory cytokines. However, the molecular mechanisms involved in this beneficial effect on β-cells are not yet completely elucidated. Our aim was to evaluate the effect of IL-6 on β-cells exposed to pro-inflammatory cytokines. Pre-treatment with IL-6 sensitized INS-1E cells to cytokine-induced cell death, increasing cytokine-induced iNOS and Caspase-3 expression. Under these conditions, however, there was a decrease in cytokines-induced p-eIF2-α but not p-IRE1expression, proteins related to ER stress. To address if this prevention of adequate UPR response is involved in the increase in β-cells death markers induced by IL-6 pre-treatment, we used a chemical chaperone (TUDCA), which improves ER folding capacity. Use of TUDCA increased cytokines-induced Caspase-3 expression and Bax/Bcl-2 ratio in the presence of IL-6 pre-treatment. However, there is no modulation of p-eIF2-α expression by TUDCA in this condition, with increase of CHOP expression. Treatment with IL-6 alone is not beneficial for β-cells, leading to increased cell death markers and impaired UPR activation. In addition, TUDCA has not been able to restore ER homeostasis or improve β-cells viability under this condition, suggesting that other mechanisms may be involved.

A high caloric intake, rich in saturated fats, greatly contributes to the development of obesity, which is the leading risk factor for type 2 diabetes (T2D). A persistent caloric surplus increases plasma levels of fatty acids (FAs), especially saturated ones, which were shown to negatively impact pancreatic β-cell function and survival in a process called lipotoxicity. Lipotoxicity in β-cells activates different stress pathways, culminating in β-cells dysfunction and death. Among all stresses, endoplasmic reticulum (ER) stress and oxidative stress have been shown to be strongly correlated. One main source of oxidative stress in pancreatic β-cells appears to be the reactive oxygen species producer NADPH oxidase (NOX) enzyme, which has a role in the glucose-stimulated insulin secretion and in the β-cell demise during both T1 and T2D. In this review, we focus on the acute and chronic effects of FAs and the lipotoxicity-induced β-cell failure during T2D development, with special emphasis on the oxidative stress induced by NOX, the ER stress, and the crosstalk between NOX and ER stress.

Modern lifestyles, including lack of physical activity and poor nutritional habits, are driving the rapidly increasing prevalence of obesity and type 2 diabetes. Increased levels of free fatty acids (FFAs), particularly saturated FFAs, in obese individuals have been linked to pancreatic β-cell failure. This process, termed lipotoxicity, involves activation of several stress responses, including ER stress and oxidative stress. However, the molecular underpinnings and causal relationships between the disparate stress responses remain unclear. Here we employed transgenic mice, expressing a genetically-encoded cytosolic H2O2 sensor, roGFP2-Orp1, to monitor dynamic changes in H2O2 levels in pancreatic islets in response to chronic palmitate exposure. We identified a transient increase in H2O2 levels from 4 to 8 h after palmitate addition, which was mirrored by a concomitant decrease in cellular NAD(P)H levels. Intriguingly, islets isolated from NOX2 knock-out mice displayed no H2O2 transient upon chronic palmitate treatment. Furthermore, NOX2 knockout rescued palmitate-dependent impairment of insulin secretion, calcium homeostasis and viability. Chemical inhibition of NOX activity protected islets from palmitate-induced impairment in insulin secretion, however had no detectable impact upon the induction of ER stress. In summary, our results reveal that transient NOX2-dependent H2O2 production is a likely cause of early palmitate-dependent lipotoxic effects.

In type 1 diabetes (T1D) development, proinflammatory cytokines (PIC) released by immune cells lead to increased reactive oxygen species (ROS) production in β-cells. Nonetheless, the temporality of the events triggered and the role of different ROS sources remain unclear. Isolated islets from C57BL/6J wild-type (WT), NOX1 KO and NOX2 KO mice were exposed to a PIC combination. We show that cytokines increase O2•− production after 2 h in WT and NOX1 KO but not in NOX2 KO islets. Using transgenic mice constitutively expressing a genetically encoded compartment specific H2O2 sensor, we show, for the first time, a transient increase of cytosolic/nuclear H2O2 in islet cells between 4 and 5 h during cytokine exposure. The H2O2 increase coincides with the intracellular NAD(P)H decrease and is absent in NOX2 KO islets. NOX2 KO confers better glucose tolerance and protects against cytokine-induced islet secretory dysfunction and death. However, NOX2 absence does not counteract the cytokine effects in ER Ca2+ depletion, Store-Operated Calcium Entry (SOCE) increase and ER stress. Instead, the activation of ER stress precedes H2O2 production. As early NOX2-driven ROS production impacts β-cells’ function and survival during insulitis, NOX2 might be a potential target for designing therapies against early β-cell dysfunction in the context of T1D onset.

NEW FINDINGS:What is the central question of this study? Type 1 diabetes mellitus (T1D) leads to hyperglycaemia owing to pancreatic β-cell destruction by the immune system. Physical exercise has been shown to have potentially beneficial protective roles against cytokine-induced pancreatic β-cell death, but its benefits are yet to be proved and should be understood better, especially in the islet environment. What is the main finding and its importance? Physical exercise protects against β-cell loss in a well-described animal model for T1D, induced by multiple low doses of streptozotocin. This seems to be related to reduced cytokine-induced β-cell death and increased islet cell proliferation. Contributions of islet neogenesis and/or transdifferentiation of pancreatic non-β-cells into β-cells cannot be excluded. ABSTRACT:Physical exercise has beneficial effects on pancreatic β-cell function and survival in a pro-inflammatory environment. Although these effects have been linked to decreased islet inflammation and modulation of pro-apoptotic pathways, little is known about the islet microenvironment. Our aim was to evaluate the effects of physical exercise in islet histomorphology in a mouse model of type 1 diabetes mellitus induced by multiple low doses of streptozotocin. As expected, induction of type 1 diabetes mellitus led to β-cell loss and, consequently, decreased islet area. Interestingly, although the decrease in islet area was not prevented by physical exercise, this was not the case for the decrease in β-cell mass. This was probably related to induction of β-cell regeneration, because we observed increased proliferation and regeneration markers, such as Ki67 and Pcna, in islets of trained mice. These were found in the central and peripheral regions of the islets. An increase in the percentage of α- and δ-cells in these conditions, combined with an increase in proliferation and Pax4 labelling in peripheral regions, suggest that β-cell regeneration might also occur by transdifferentiation. This agrees with the presence of cells double stained for insulin and glucagon only in islets of diabetic trained mice. In addition, this group had more extra-islet insulin-positive cells and islets associated with ducts than diabetic mice. Physical exercise also decreased nuclear factor-κB activation in islet cells of diabetic trained compared with diabetic untrained mice, indicating a decrease in pro-inflammatory cytokine-induced β-cell death. Taken together, these findings indicate that preservation of β-cell mass induced by physical exercise involves an increase in β-cell replication and decrease in β-cell death, together with islet neogenesis and islet cell transdifferentiation.

Modern lifestyles, including lack of physical activity and poor nutritional habits, are driving the rapidly increasing prevalence of obesity and type 2 diabetes. Increased levels of free fatty acids (FFAs), particularly saturated FFAs, in obese individuals have been linked to pancreatic β-cell failure. This process, termed lipotoxicity, involves activation of several stress responses, including ER stress and oxidative stress. However, the molecular underpinnings and causal relationships between the disparate stress responses remain unclear. Here we employed transgenic mice, expressing a genetically-encoded cytosolic H2O2 sensor, roGFP2-Orp1, to monitor dynamic changes in H2O2 levels in pancreatic islets in response to chronic palmitate exposure. We identified a transient increase in H2O2 levels from 4 to 8 h after palmitate addition, which was mirrored by a concomitant decrease in cellular NAD(P)H levels. Intriguingly, islets isolated from NOX2 knock-out mice displayed no H2O2 transient upon chronic palmitate treatment. Furthermore, NOX2 knockout rescued palmitate-dependent impairment of insulin secretion, calcium homeostasis and viability. Chemical inhibition of NOX activity protected islets from palmitate-induced impairment in insulin secretion, however had no detectable impact upon the induction of ER stress. In summary, our results reveal that transient NOX2-dependent H2O2 production is a likely cause of early palmitate-dependent lipotoxic effects.

ARHGAP21 is a RhoGAP protein implicated in the modulation of insulin secretion and energy metabolism. ARHGAP21 transient-inhibition increase glucose-stimulated insulin secretion (GSIS) in neonatal islets; however, ARHGAP21 heterozygote mice have a reduced insulin secretion. These discrepancies are not totally understood, and it might be related to functional maturation of beta cells and peripheral sensitivity. Here, we investigated the real ARHGAP21 role in the insulin secretion process using an adult mouse model of acute ARHGAP21 inhibition, induced by antisense. After ARHGAP21 knockdown induction by antisense injection in 60-day old male mice, we investigated glucose and insulin tolerance test, glucose-induced insulin secretion, glucose-induced intracellular calcium dynamics, and gene expression. Our results showed that ARHGAP21 acts negatively in the GSIS of adult islet. This effect seems to be due to the modulation of important points of insulin secretion process, such as the energy metabolism (PGC1α), Ca2+ signalization (SYTVII), granule-extrusion (SNAP25), and cell-cell interaction (CX36). Therefore, based on these finds, ARHGAP21 may be an important target in Diabetes Mellitus (DM) treatment.

Diabetes Mellitus (DM) is a metabolic disorder with multiple etiology and characterized by chronic hyperglycemia. It results from total (Type 1) or partial (Type 2) depletion of β cells, associated or not with insulin resistance (IR). Any Processes that promote the functional β-cell mass regeneration may be a target for DM treatment. HNF4α is an important transcription factor for pancreatic β cells because, beyond the control of the insulin gene, it regulates the expression of genes related to glucose metabolism, it is crucial in increasing beta cell mass in response to stress, such as in pregnancy and it is important to β-cell fate. However, little is known of its involvement in pancreatic β-cell apoptosis and regeneration. Based on the fact that HNF4α (1) participates in cell death processes, (2) controls processes of compensatory increase of β-cell mass, and (3) acts directly in regenerative processes, we aimed to unravel possible mechanisms, dependent of HNF4α, which can be used to treat Diabetes Mellitus. First we analyzed, in vitro, the consequence of HNF4α knockdown in human β-cell line EndoC-βH1 in the gene expression of several markers to islet-cell fate and in the cytokine-induced apoptosis. We observed that the siRNA for HNF4α reduced the expression of genes important to β-cell fate (PDX-1, NKX2.2, MAF-A, UCN-3 and KIR) and increased the expression of genes not-related with β-cell (NGN3, SOX9 and GLUCAGON). In additional, HNF4α knockdown prevented the cytokine-induced Caspase-3 cleavage in human EndoC-βH1 and rat INS-1E cell line. Then, we addressed our focus to the HNF4α ability to promote β-cell expansion in response to a metabolic demand. Here we investigated the HNF4α role in the IR-induced β-cell proliferation. We used WildType (C) and Knockout (K) animals for HNF4-alpha (HNF4αloxP/loxP;Ins.Cre). After CRE-recombinase activation the induction of Knockout was confirmed by ipGTT. To induce an IR, animals C and K were treated (D) or not-treated (S) with 100 mg/kg of dexamethasone for 5 consecutive days. Then, we analyzed GSIS, pancreatic morphometry (IHC) and expression of genes involved in pancreatic islet cell proliferation/transdifferentiation. We observed that indeed Dexa induced an IR and a β-cell mass expansion in CD mice, which were not observed in KD mice. Moreover, Dexa induced, in CD mice, an increase in genes involved in islet-cell expansion (Glucagon, Somatostatin, PDX1, PAX4, HHEX, NGN3) and this increased were abolished in KD mice. So, we observed that HNF4α seems to be important in the β-cell fate since its knockdown induces a non-β-cell gene expression in a β-cell line. Moreover, HNF4α acts in the β-cell death process and is involved in the Dexa-induced β-cell mass expansion. Taking together, HNF4α may be a target to prevent β-cell apoptosis and to induce β-cell regeneration. Financial Support: FAPESC/CNPq/CAPES/FAPESP.

Type 1 diabetes is caused by an autoimmune assault that induces progressive beta-cell dysfunction and dead. Pro-inflammatory cytokines, such as interleukin 1 beta (IL1B), tumor necrosis factor (TNF) and interferon gamma (IFNG) contribute for beta-cell death, which involves the activation of the nuclear factor kappa B (NFκB) and c- Jun N-terminal kinase (JNK). Prolactin (PRL), a physiological mediator for beta-cell proliferation, was shown to protect beta cells against cytokines pro-apoptotic effects. We presently investigated the mechanisms involved in the protective effects of prolactin against cytokine-induced beta-cell death. The findings obtained indicate that STAT3 activation is involved in the anti-apoptotic role of PRL in rat beta cells. PRL prevents the activation of JNK via AKT and promotes a shift from expression of pro- to anti-apoptotic proteins downstream of the JNK cascade. Furthermore, PRL partially prevents the activation of NFκB and the transcription of its target genes IkBa, Fas, Mcp1, A20 and Cxcl10 and also decreases NO production. On the other hand, the pro-survival effects of PRL do not involve modulation of cytokine-induced endoplasmic reticulum stress. These results suggest that the beneficial effects of PRL in beta cells involve augmentation of anti-apoptotic mechanisms and, at the same time, reduction of pro-apoptotic effectors, rendering beta cells better prepared to deal with inflammatory insults. The better understanding of the pro-survival mechanisms modulated by PRL in beta cells can provide tools to prevent cell demise during an autoimmune attack or following islet transplantation.

The prevalence of diabetes has reached 8.8% in worldwide population and is predicted to increase up to 10.4% by 2040. Thus, there is an urgent need for the development of means to treat or prevent this major disease. Due to its role in inflammatory responses, several studies demonstrated the importance of the transcription factor nuclear factor-κB (NF-κB) in both type 1 diabetes (T1D) and type 2 diabetes (T2D). The two major NF-κB pathways are the canonical and the non-canonical. The later pathway is activated by the NF-κB-inducing kinase (NIK) that triggers p100 processing into p52, which forms with RelB its main dimer. Cytokines mediating the activation of this pathway are present in the serum of T1D and T2D patients. Conversely, limited information is available regarding the role of the alternative pathway on diabetes development and β-cell fate. In the present review, we will briefly describe the involvement of NF-κB on diabetes pathology and discuss new studies indicating an important role for the non-canonical NF-κB activation in β-cell function and survival. The non-canonical NF-κB pathway is emerging as a novel potential target for the development of therapeutic strategies to treat or prevent diabetes.

Activation of the transcription factor nuclear factor kappa B (NFkB) contributes to β-cell death in type 1 diabetes (T1D). Genome-wide association studies have identified the gene TNF-induced protein 3 (TNFAIP3), encoding for the zinc finger protein A20, as a susceptibility locus for T1D. A20 restricts NF-κB signaling and has strong antiapoptotic activities in β-cells. Although the role of A20 on NF-κB inhibition is well characterized, its other antiapoptotic functions are largely unknown. By studying INS-1E cells and rat dispersed islet cells knocked down or overexpressing A20 and islets isolated from the β-cell-specific A20 knockout mice, we presently demonstrate that A20 has broader effects in β-cells that are not restricted to inhibition of NF-κB. These involves, suppression of the proapoptotic mitogen-activated protein kinase c-Jun N-terminal kinase (JNK), activation of survival signaling via v-akt murine thymoma viral oncogene homolog (Akt) and consequently inhibition of the intrinsic apoptotic pathway. Finally, in a cohort of T1D children, we observed that the risk allele of the rs2327832 single nucleotide polymorphism of TNFAIP3 predicted lower C-peptide and higher hemoglobin A1c (HbA1c) levels 12 months after disease onset, indicating reduced residual β-cell function and impaired glycemic control. In conclusion, our results indicate a critical role for A20 in the regulation of β-cell survival and unveil novel mechanisms by which A20 controls β-cell fate. Moreover, we identify the single nucleotide polymorphism rs2327832 of TNFAIP3 as a possible prognostic marker for diabetes outcome in children with T1D.

Insulin-secreting pancreatic β-cells are extremely dependent on their endoplasmic reticulum (ER) to cope with the oscillatory requirement of secreted insulin to maintain normoglycemia. Insulin translation and folding rely greatly on the unfolded protein response (UPR), an array of three main signaling pathways designed to maintain ER homeostasis and limit ER stress. However, prolonged or excessive UPR activation triggers alternative molecular pathways that can lead to β-cell dysfunction and apoptosis. An increasing number of studies suggest a role of these pro-apoptotic UPR pathways in the downfall of β-cells observed in diabetic patients. Particularly, the past few years highlighted a cross talk between the UPR and inflammation in the context of both type 1 (T1D) and type 2 diabetes (T2D). In this article, we describe the recent advances in research regarding the interplay between ER stress, the UPR, and inflammation in the context of β-cell apoptosis leading to diabetes.