Insulin demand regulates β cell number via the unfolded protein response
01 Oct 2015-Journal of Clinical Investigation (American Society for Clinical Investigation)-Vol. 125, Iss: 10, pp 3831-3846
TL;DR: A stem cell-independent model of tissue homeostasis is defined, in which differentiated secretory cells use the UPR sensor to adapt organ size to meet demand, suggesting that therapeutic UPR modulation has potential to expand β cell mass in people at risk for diabetes.
Abstract: Although stem cell populations mediate regeneration of rapid turnover tissues, such as skin, blood, and gut, a stem cell reservoir has not been identified for some slower turnover tissues, such as the pancreatic islet. Despite lacking identifiable stem cells, murine pancreatic β cell number expands in response to an increase in insulin demand. Lineage tracing shows that new β cells are generated from proliferation of mature, differentiated β cells; however, the mechanism by which these mature cells sense systemic insulin demand and initiate a proliferative response remains unknown. Here, we identified the β cell unfolded protein response (UPR), which senses insulin production, as a regulator of β cell proliferation. Using genetic and physiologic models, we determined that among the population of β cells, those with an active UPR are more likely to proliferate. Moreover, subthreshold endoplasmic reticulum stress (ER stress) drove insulin demand-induced β cell proliferation, through activation of ATF6. We also confirmed that the UPR regulates proliferation of human β cells, suggesting that therapeutic UPR modulation has potential to expand β cell mass in people at risk for diabetes. Together, this work defines a stem cell-independent model of tissue homeostasis, in which differentiated secretory cells use the UPR sensor to adapt organ size to meet demand.
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TL;DR: A droplet-based, single-cell RNA-seq method is implemented to determine the transcriptomes of over 12,000 individual pancreatic cells from four human donors and two mouse strains and provides a resource for the discovery of novel cell type-specific transcription factors, signaling receptors, and medically relevant genes.
Abstract: Although the function of the mammalian pancreas hinges on complex interactions of distinct cell types, gene expression profiles have primarily been described with bulk mixtures. Here we implemented a droplet-based, single-cell RNA-seq method to determine the transcriptomes of over 12,000 individual pancreatic cells from four human donors and two mouse strains. Cells could be divided into 15 clusters that matched previously characterized cell types: all endocrine cell types, including rare epsilon-cells; exocrine cell types; vascular cells; Schwann cells; quiescent and activated stellate cells; and four types of immune cells. We detected subpopulations of ductal cells with distinct expression profiles and validated their existence with immuno-histochemistry stains. Moreover, among human beta- cells, we detected heterogeneity in the regulation of genes relating to functional maturation and levels of ER stress. Finally, we deconvolved bulk gene expression samples using the single-cell data to detect disease-associated differential expression. Our dataset provides a resource for the discovery of novel cell type-specific transcription factors, signaling receptors, and medically relevant genes.
1,046 citations
TL;DR: It is concluded that the normally high rate of insulin production suppresses β cell proliferation in a cell-autonomous manner after reduced insulin production independently of hyperglycemia.
165 citations
Cites background from "Insulin demand regulates β cell num..."
...Hyperglycemia and ‘‘insulin demand’’ have been suggested to directly mediate b cell proliferation in other models (Porat et al., 2011; Sharma et al., 2015)....
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...Recently, Alonso and colleagues reported that mild ER stress is associated with increased b cell proliferation in the context of sustained hyperglycemia, and that this effect could be reversed by ATF6 and IRE1 inhibitors, but not an inhibitor of the PERK pathway (Sharma et al., 2015)....
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TL;DR: Current understanding from rodent and human studies of the progression of β cell responses during the development of MetS, as well as recent findings addressing the complexity ofβ cell identity and heterogeneity within the islet during disease progression are focused on.
Abstract: In a society where physical activity is limited and food supply is abundant, metabolic diseases are becoming a serious epidemic. Metabolic syndrome (MetS) represents a cluster of metabolically related symptoms such as obesity, hypertension, dyslipidemia, and carbohydrate intolerance, and significantly increases type 2 diabetes mellitus risk. Insulin resistance and hyperinsulinemia are consistent characteristics of MetS, but which of these features is the initiating insult is still widely debated. Regardless, both of these conditions trigger adverse responses from the pancreatic β cell, which is responsible for producing, storing, and releasing insulin to maintain glucose homeostasis. The observation that the degree of β cell dysfunction correlates with the severity of MetS highlights the need to better understand β cell dysfunction in the development of MetS. This Review focuses on the current understanding from rodent and human studies of the progression of β cell responses during the development of MetS, as well as recent findings addressing the complexity of β cell identity and heterogeneity within the islet during disease progression. The differential responses observed in β cells together with the heterogeneity in disease phenotypes within the patient population emphasize the need to better understand the mechanisms behind β cell adaptation, identity, and dysfunction in MetS.
160 citations
Cites background from "Insulin demand regulates β cell num..."
...(39) suggested that induction of UPR functioned as a sensor of insulin demand and induced β cell proliferation through the activation of ATF6 in mouse and human islets....
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TL;DR: Exploring the roles and the potential therapeutic effects of antioxidants in the process of β cell regeneration would provide novel perspectives to preserve and/or expand pancreatic β cell mass for the treatment of T2D.
Abstract: Pancreatic β cell neogenesis and proliferation during the neonatal period are critical for the generation of sufficient pancreatic β cell mass/reserve and have a profound impact on long-term protection against type 2 diabetes (T2D). Oxidative stress plays an important role in β cell neogenesis, proliferation, and survival under both physiological and pathophysiological conditions. Pancreatic β cells are extremely susceptible to oxidative stress due to a high endogenous production of reactive oxygen species (ROS) and a low expression of antioxidative enzymes. In this review, we summarize studies describing the critical roles and the mechanisms of how oxidative stress impacts β cell neogenesis and proliferation. In addition, the effects of antioxidant supplements on reduction of oxidative stress and increase of β cell proliferation are discussed. Exploring the roles and the potential therapeutic effects of antioxidants in the process of β cell regeneration would provide novel perspectives to preserve and/or expand pancreatic β cell mass for the treatment of T2D.
138 citations
Cites background from "Insulin demand regulates β cell num..."
...Significantly, increased number of β cells is observed in response to elevated demand of insulin in several rodentmodels of obesity anddiabetes, and the main mechanism underlying this is the proliferation of fully differentiated β cells [2, 38, 39]....
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TL;DR: It is suggested that human β-cells transition between states with high rates of biosynthesis to fulfill the body’s insulin requirements to maintain normal blood glucose levels and UPR-mediated recovery from ER stress due to high insulin production.
Abstract: Proinsulin is a misfolding-prone protein, making its biosynthesis in the endoplasmic reticulum (ER) a stressful event. Pancreatic β-cells overcome ER stress by activating the unfolded protein response (UPR) and reducing insulin production. This suggests that β-cells transition between periods of high insulin biosynthesis and UPR-mediated recovery from cellular stress. We now report the pseudotime ordering of single β-cells from humans without diabetes detected by large-scale RNA sequencing. We identified major states with 1) low UPR and low insulin gene expression, 2) low UPR and high insulin gene expression, or 3) high UPR and low insulin gene expression. The latter state was enriched for proliferating cells. Stressed human β-cells do not dedifferentiate and show little propensity for apoptosis. These data suggest that human β-cells transition between states with high rates of biosynthesis to fulfill the body's insulin requirements to maintain normal blood glucose levels and UPR-mediated recovery from ER stress due to high insulin production.
132 citations
Cites background or result from "Insulin demand regulates β cell num..."
...INSUPR State Promotes Human b-Cell Proliferation Mouse b-cells proliferate under conditions of UPR activation and low insulin production (43,44)....
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...This is similar to the situation in mouse b-cells (43,44)....
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References
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TL;DR: It is demonstrated that chemical chaperones enhance the adaptive capacity of the ER and act as potent antidiabetic modalities with potential application in the treatment of type 2 diabetes.
Abstract: Endoplasmic reticulum (ER) stress is a key link between obesity, insulin resistance, and type 2 diabetes. Here, we provide evidence that this mechanistic link can be exploited for therapeutic purposes with orally active chemical chaperones. 4-Phenyl butyric acid and taurine-conjugated ursodeoxycholic acid alleviated ER stress in cells and whole animals. Treatment of obese and diabetic mice with these compounds resulted in normalization of hyperglycemia, restoration of systemic insulin sensitivity, resolution of fatty liver disease, and enhancement of insulin action in liver, muscle, and adipose tissues. Our results demonstrate that chemical chaperones enhance the adaptive capacity of the ER and act as potent antidiabetic modalities with potential application in the treatment of type 2 diabetes.
2,267 citations
"Insulin demand regulates β cell num..." refers background in this paper
...Taken with the caveats that small-molecule inhibitors may have off-target effects on other pathways or tissues and that TUDCA may reduce metabolic demand in high-demand states, such as HFDfed or db/db mice (36), these results suggest that UPR activation may be required for insulin demand or ER stress–driven β cell proliferation in vivo....
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TL;DR: DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels and was detected after 15 min of induction and identified using DIGE preparatively.
Abstract: We describe a modification of two-dimensional (2-D) polyacrylamide gel electrophoresis that requires only a single gel to reproducibly detect differences between two protein samples. This was accomplished by fluorescently tagging the two samples with two different dyes, running them on the same 2-D gel, post-run fluorescence imaging of the gel into two images, and superimposing the images. The amine reactive dyes were designed to insure that proteins common to both samples have the same relative mobility regardless of the dye used to tag them. Thus, this technique, called difference gel electrophoresis (DIGE), circumvents the need to compare several 2-D gels. DIGE is reproducible, sensitive, and can detect an exogenous difference between two Drosophila embryo extracts at nanogram levels. Moreover, an inducible protein from E. coli was detected after 15 min of induction and identified using DIGE preparatively.
2,220 citations
"Insulin demand regulates β cell num..." refers background in this paper
...pH 4-10 isoelectric focusing strips, and separated by SDS-PAGE (66)....
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...Samples were boiled in SDS-PAGE loading buffer for 5–10 minutes....
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...After separation on SDS-PAGE, proteins were transferred to PVDF membrane, and the membrane was then blocked for 1–2 hours with 5% skim milk powder containing PBS and 0.1% Tween-20....
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...Pancreatic islets were isolated and hand-picked on ice in ICM containing 1% FBS. Islet lysates from 4 mice in each condition were pooled, labeled with Cy3 or Cy5 (University of Pittsburgh Proteomics Core), combined, focused to 70,000 volt-hours (IPGphor power source, Bio-Rad) on Immobiline pH 4-10 isoelectric focusing strips, and separated by SDS-PAGE (66)....
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TL;DR: This work introduces a method for genetic lineage tracing to determine the contribution of stem cells to a tissue of interest and suggests that terminally differentiated β-cells retain a significant proliferative capacity in vivo and casts doubt on the idea that adult stem cells have a significant role in β-cell replenishment.
Abstract: How tissues generate and maintain the correct number of cells is a fundamental problem in biology. In principle, tissue turnover can occur by the differentiation of stem cells, as is well documented for blood, skin and intestine, or by the duplication of existing differentiated cells. Recent work on adult stem cells has highlighted their potential contribution to organ maintenance and repair. However, the extent to which stem cells actually participate in these processes in vivo is not clear. Here we introduce a method for genetic lineage tracing to determine the contribution of stem cells to a tissue of interest. We focus on pancreatic beta-cells, whose postnatal origins remain controversial. Our analysis shows that pre-existing beta-cells, rather than pluripotent stem cells, are the major source of new beta-cells during adult life and after pancreatectomy in mice. These results suggest that terminally differentiated beta-cells retain a significant proliferative capacity in vivo and cast doubt on the idea that adult stem cells have a significant role in beta-cell replenishment.
2,103 citations
"Insulin demand regulates β cell num..." refers background in this paper
...Lineage-tracing studies show that the primary means of generating new β cells in adult mice is proliferation of fully differentiated, mature β cells (10, 11)....
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TL;DR: It is concluded that ER overload in beta cells causes ER stress and leads to apoptosis via Chop induction and a new therapeutic approach for preventing the onset of diabetes by inhibiting Chop induction or by increasing chaperone capacity in the ER is suggested.
Abstract: Overload of pancreatic β cells in conditions such as hyperglycemia, obesity, and long-term treatment with sulfonylureas leads to β cell exhaustion and type 2 diabetes. Because β cell mass declines under these conditions, apparently as a result of apoptosis, we speculated that overload kills β cells as a result of endoplasmic reticulum (ER) stress. The Akita mouse, which carries a conformation-altering missense mutation (Cys96Tyr) in Insulin 2, likewise exhibits hyperglycemia and a reduced β cell mass. In the development of diabetes in Akita mice, mRNAs for the ER chaperone Bip and the ER stress–associated apoptosis factor Chop were induced in the pancreas. Overexpression of the mutant insulin in mouse MIN6 β cells induced Chop expression and led to apoptosis. Targeted disruption of the Chop gene delayed the onset of diabetes in heterozygous Akita mice by 8–10 weeks. We conclude that ER overload in β cells causes ER stress and leads to apoptosis via Chop induction. Our findings suggest a new therapeutic approach for preventing the onset of diabetes by inhibiting Chop induction or by increasing chaperone capacity in the ER.
904 citations
"Insulin demand regulates β cell num..." refers background in this paper
...To determine whether ER stress applied directly to the β cell increases β cell proliferation in vivo, we studied heterozygous Ins2C96Y/+ (herein referred to as Akita) mice, in which a mutant proinsulin (1 of 4 insulin alleles) causes β cell ER stress due to improper disulfide formation (32, 33)....
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TL;DR: Evidence that endoplasmic reticulum (ER) stress occurs in type 2 diabetes and is required for aspects of the underlying beta cell failure is provided.
Abstract: Aims/hypothesis
Increased lipid supply causes beta cell death, which may contribute to reduced beta cell mass in type 2 diabetes. We investigated whether endoplasmic reticulum (ER) stress is necessary for lipid-induced apoptosis in beta cells and also whether ER stress is present in islets of an animal model of diabetes and of humans with type 2 diabetes.
769 citations
"Insulin demand regulates β cell num..." refers background in this paper
...Furthermore, islets from people with diabetes show evidence of decompensated ER stress (31, 64)....
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