Abstract
Objective
Progressive beta-cell dysfunction is a hallmark of type 2 diabetes (T2D). Increasing evidence indicates that over-stimulating proinsulin synthesis causes proinsulin misfolding and impairs insulin maturation and storage in db/db mice. However, defective insulin maturation in patients with T2D remains unknown.
Methods
We examined intra-islet and intra-cellular distributions of proinsulin and insulin and proinsulin to insulin ratio in the islets of patients with T2D. The expression of transcription factor NKX6.1 and dedifferentiation marker ALDH1A3, as well as glucagon, were detected by immunofluorescence.
Results
We identified a novel subgroup of beta cells expressing only proinsulin but not insulin. Importantly, significantly increased proinsulin positive and insulin negative (PI+/INS−) cells were evident in T2D, and this increase was strongly correlated with levels of hemoglobin A1C (HbA1c) in T2D and prediabetes. The percentages of beta cells expressing prohormone convertase 1/3 and carboxypeptidase E were not reduced. Indeed, while proinsulin displayed a higher degree of co-localization with the golgi markers GM130/TGN46 in control beta cells, it appeared to be more diffused within the cytoplasm and less co-localized with GM130/TGN46 in PI+/INS− cells. Furthermore, the key functional transcription factor NKX6.1 markedly decreased in the islets of T2D, especially in the cells with PI+/INS−. The decreased NKX6.1+/PI+/INS+ was strongly correlated with levels of HbA1c in T2D. Almost all PI+/INS− cells showed absence of NKX6.1. Moreover, the percentages of PI+/INS− cells expressing ALDH1A3 were elevated along with an increased acquisition of glucagon immunostaining.
Conclusion
Our data demonstrate defective insulin maturation in patients with T2D.
Introduction
Insulin plays a central role in the regulation of glucose homeostasis. The biosynthesis of insulin starts with its precursor-preproinsulin, which undergoes translocation across the membrane of the endoplasmic reticulum (ER) and is cleaved by signal peptidase to form proinsulin. Proinsulin rapidly folds and forms three highly conserved native disulfide bonds in the ER, then traffics through the golgi to the late secretory granules where it is processed by prohormone convertases (PC1/3 and PC2) and carboxypeptidase E (CPE) to form mature insulin (1). In pancreatic beta cells, proinsulin synthesis alone accounts for up to 30–50% of total cellular protein synthesis upon glucose stimulation (2, 3). The folding of this large amount of proinsulin put tremendous pressure and challenge on the ER folding machinery. In turn, proinsulin folding is sensitive to alteration of the ER environment in beta cells (4). In fact, accumulating evidence indicates that up to 10–15% of newly synthesized proinsulin fails to form native disulfide bonds in the ER of beta cells even under normal physiological conditions (5, 6, 7, 8). That is to say, a modest amount of misfolded proinsulin was produced as a natural by-product in the synthesis of proinsulin in normal pancreatic islets.
Type 2 diabetes (T2D) is the most common form of diabetes mellitus. Insulin resistance and relative shortage of insulin are thought to play a crucial role in the pathogenesis of T2D. To compensate for insulin resistance, beta cells are forced to synthesize and secret more insulin. Our recent studies showed that compensatory proinsulin synthesis induced by peripheral insulin resistance could significantly increase the total amount of misfolded proinsulin that is tightly linked to the development and progression of diabetes in leptin receptor-deficient db/db mice (9, 10, 11). The misfolded proinsulin can interact with co-expressed bystander proinsulin via proinsulin dimerization interface, forming disulfide-linked proinsulin complexes, through which impairing folding of the newly synthesized proinsulin and limiting proinsulin ER export, leading to decreased mature insulin and onset of diabetes (12, 13, 14, 15). Accumulation of misfolded proinsulin can also disturb ER protein homeostasis, induce ER stress and beta-cell apoptosis (16, 17, 18). Indeed, it has been reported that an increase in ER volume density and elevated ER stress markers are found during the development and progression of T2D in humans and in db/db diabetic mice (19, 20). ER homeostasis is critical for cellular function and cell survival. High/prolonged ER stress-induced functional beta cells mass decreasing plays a role in the development of T2D (21, 22). Loss of beta cell identity and subsequent transition to other islet cell types were indicated as a possible explanation for beta-cell mass reduction (23). Dedifferentiation of beta cells was determined by the reduction of several critical islet transcription factors and other genes important for glucose-stimulated insulin secretion (GSIS) (24). In addition, the loss of key transcription factors MafA, NKX6.1, and Pdx1 has been reported to be associated with beta cell dysfunction (25). These studies indicate that misfolded proinsulin aggregating and subsequent ER stress may lead to defective insulin maturation in the development and progression of diabetes in murine models.
Diabetes in humans is far more complex than that in mice, and the mouse model has always been questioned for poor recapitulation of human diseases. However, few studies have investigated whether these changes in mouse islets are also present in humans. Herein, using immunostaining with anti-proinsulin and insulin antibodies in pancreatic islets from human subjects, we identified a novel subgroup of functionally exhausting beta cells expressing only proinsulin but not insulin (PI+/INS−). Importantly, significantly increased PI+/INS− cells were evident in patients with T2D, and this increase was strongly correlated with levels of HbA1c. While proinsulin displayed a higher degree of co-localization with the Golgi markers GM130/TGN46 in control beta cells, it appeared to be more diffused within the cytoplasm in PI+/INS− cells. Furthermore, in these PI+/INS− cells, the key functional transcription factor NKX6.1 was markedly decreased, whereas beta cell dedifferentiation marker ALDH1A3 was increased. These data reveal defective insulin maturation and its correlation with HbA1c in patients with T2D.
Subjects and methods
Patients and human pancreas
Human pancreatic paraffin sections and tissue were obtained from a cadaveric donor in Tianjin First Central Hospital following a standard operational procedure. The use of the human pancreatic paraffin section was approved by Tianjin First Central Hospital Medical Ethics Committee (NO.2016N082KY) and informed consent was obtained from each donor. All the subjects in the non-diabetic, prediabetic, or diabetic groups were age-, gender- and BMI-matched. The individual clinical information was summarized in Supplementary Table 1 (see section on supplementary materials given at the end of this article).
Antibodies and reagents
The following primary antibodies were used: a mouse MAB (B-C junction) that recognizes a human proinsulin B-Chain-C-peptide junction was raised by Abmart (Shanghai, China). Guinea pig anti-insulin (dilution 1:5000) and rabbit anti-protein disulfide isomerase (PDI, dilution 1:200) were a gift kindly provided by Dr Peter Arvan (University of Michigan, Ann Arbor, MI, USA). Rabbit polyclonal anti-GM130 (ab52649; dilution 1:200)) and anti-PC1/3 (ab220363; dilution 1:200) were from Abcam. Rabbit polyclonal anti-carboxypeptidase E (GTX33060; dilution 1:400) was purchase from GeneTex (Irvine, CA, USA). Rabbit polyclonal anti-NKX6.1 (NBP2-15339; dilution 1:200) was purchased from Novus Biologicals (Centennial, CO, USA). Rabbit polyclonal anti-ALDH1A3 (NBP2-15339; dilution 1:200) was from Sigma. A mouse MAB GAPDH (AC033; dilution 1:2000 was from Abclonal (Wuhan, China). Anti-TGN46 antibody (13573-1-AP; dilution1:50) was purchased from Proteintech (Wuhan, China), and anti-PC2 (14013T; dilution 1:200) was from Cell Signaling Technology.
Goat anti-mouse IgG Alexa Fluor 345, goat anti-mouse IgG horseradish peroxidase (HRP), and goat anti-guinea pig IgG HRP were purchased from Jackson ImmunoReserach. Goat anti-rabbit IgG Alexa Fluor 488 and goat anti-guinea pig IgG Alexa Fluor 555 were from Thermo Fisher Scientific. Detection antibodies were reconstituted in water with glycerol according to the manufacturer's instructions, stored at –20°C and used diluted at 1:500 in the immunofluorescence experiment and 1:5000 in Western blotting. Reagents for buffers, goat serum for blocking, mounting media with or without DAPI were from Southern Biotech (Birmingham, AL, USA). DTT and complete proteinase inhibitor were purchased from Millipore Sigma (Burlington, MA, USA). 4–12% Bis-Tris Gradient Gels were from Thermo Fisher Scientific.
Immunofluorescence
Human pancreas was fixed in 10% formalin, embedded in paraffin, and serially sectioned longitudinally (with 5 µm/each section). The slides were put in xylene for 20 min for deparaffinization. They were then transferred to 100, 95, 90, 80, and 70% ethanol sequentially for rehydration. After rehydration, antigen retrieval was performed by heating slides in preheated citrate buffer (pH 6.0) using a microwave oven to improve the immune detection of various proteins: three cycles for 5 min at medium power level followed by a 5 min break, cooled to room temperature for 1 h. The slides were blocked using 2% goat serum, followed by incubation of the primary antibodies cocktail diluted in PBS overnight at 4°C. After washing in PBS, slides were incubated with the detection antibodies for 1 h at room temperature. Slides were mounted using mounting media with or without DAPI. The sections were dried at room temperature overnight protecting from light.
Western blotting
Human pancreatic tissues were cryopreserved in –80°C refrigerator. The tissues were homogenized by cryogenic tissue grinder in ice-cold lysis buffer containing RIPA (25 mM Tris–HCl, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.2% sodium deoxycholate) and proteinase inhibitor, then centrifuged at 16,200 g at 4°C for 10 min. Collected supernatants were boiled for 5 min with sample loading buffer. Protein was resolved on 4–12% NuPage gradient gels (Thermo Fisher Scientific ) and transferred to nitrocellulose membranes. After blocked in 5% skim milk at room temperature for 1 h, membranes were incubated with primary antibodies at 4°C overnight followed by goat-anti-mouse-HRP polyclonal antibodies as secondary antibodies. Clinx was used for the chemiluminescent visualization of proteins. Results were quantified by grey value analysis (Image J).
Imaging
Immunofluorescence-stained slides were imaged at 40× and 100× magnification using a Carl Zeiss confocal microscope Axio Imager M2 and the Zeiss ZEN microscope software (Oberkochen, Germany). The number of cells with positive immunoreactivity for various antibodies was counted and calculated by two independent researchers in a blind manner, and the results were confirmed by the cell counter function of ImageJ software. For each subject, we analyzed at least 30 random islets. The Manders overlap coefficient (MOC) was calculated for establishing the extent of two proteins co-localization.
Statistical analysis
Data were presented as median (interquartile range) and as percentages. For statistical analysis of the groups, the Kruskal–Wallis analysis was used for continuous variables. Pearson correlation analysis was performed for investigating the correlation between two continuous variables. Pearson’s correlation coefficient (r) was used to estimate the strength of the association between the two continuous variables. All of the statistical analyses were conducted with IBM SPSS 22 software.
Results
Proportion of proinsulin positive and insulin negative (PI+/INS−) cells were elevated in the patients with T2D
Large amounts of proinsulin molecules accumulation in the endoplasmic reticulum of beta cells have been proved as an early event of type 2 diabetes in the mouse model (9). We assessed whether increased proinsulin accumulation and defective insulin maturation also occur in beta cells of patients with T2D or prediabetes. The clinical features of all studied subjects were summarized in Table 1. Result in Fig. 1A illustrated the presence of cells that only expressed proinsulin but not insulin (PI+/INS−) in all three groups. The percentage of the PI+/INS− cells among all proinsulin positive (PI+) cells was determined in islets from all donors individually. It was found that the percentage of these cells elevated in islets from T2D (Fig. 1A), which reached 15.04%, a significant increase over the non-DM group (15.04% vs 2.51%, P < 0.01), and near 2.1-fold increase over the prediabetes group (15.04% vs 7.17%, P < 0.05). Notably, the proportion of PI+/INS− cells in islets from patients with prediabetes slightly increased compared to that in control subjects, even though there was no significant difference (Fig. 1B), suggesting that this change may develop gradually during the development of T2D. We then examined the association between the HbA1c levels and proportion of PI+/INS− cells and found the positive correlation between these two groups of variables (Fig. 1C, r = 0.67, P < 0.0001). To further confirm the results, we also evaluated the ratio of proinsulin to insulin using the total lysates from the whole pancreas of the cadaveric donors. There was an obvious elevation of the ratio of proinsulin to insulin in the pancreas from patients with prediabetes and diabetes (Fig. 1D and E), suggesting an impaired insulin maturation in these two groups.
Elevated proportion of proinsulin+ insulin− (PI+/INS−) cells in islets from patients with diabetes or prediabetes. (A) Pancreas from healthy subjects, patients with prediabetes (pre-DM), and diabetes (DM) stained with proinsulin (green), insulin (red), and DAPI (blue) are shown. Yellow box showed a specific area of the islet which was enlarged and represented. Yellow arrows were used to point out the PI+/INS− cells and white arrows to demonstrate beta cells with proinsulin concentrated pattern. (B) Proportion of PI+/INS− cells in non-diabetes (n = 11), prediabetes (n = 6), and diabetes (n = 14) group. Data present medians (IQR). Kruskal–Wallis statistical analysis was performed. *P < 0.05, **P < 0.01. (C) Correlation of PI+/INS− cells proportion with the level of HbA1c. The r of Pearson correlation and P value are shown. (D) Western blotting was performed to detect proinsulin and insulin relative expression in pancreatic tissue from three groups. (E) The result from (D) was quantified. The ratio of proinsulin to insulin was calculated and data were presented from three independent experiments in non-DM, pre-DM, and DM. Data are expressed as means ± s.d. One-way ANOVA analysis was performed. **P < 0.01, ***P < 0.001. PI, proinsulin; DM, diabetes. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
Elevated proportion of proinsulin+ insulin− (PI+/INS−) cells in islets from patients with diabetes or prediabetes. (A) Pancreas from healthy subjects, patients with prediabetes (pre-DM), and diabetes (DM) stained with proinsulin (green), insulin (red), and DAPI (blue) are shown. Yellow box showed a specific area of the islet which was enlarged and represented. Yellow arrows were used to point out the PI+/INS− cells and white arrows to demonstrate beta cells with proinsulin concentrated pattern. (B) Proportion of PI+/INS− cells in non-diabetes (n = 11), prediabetes (n = 6), and diabetes (n = 14) group. Data present medians (IQR). Kruskal–Wallis statistical analysis was performed. *P < 0.05, **P < 0.01. (C) Correlation of PI+/INS− cells proportion with the level of HbA1c. The r of Pearson correlation and P value are shown. (D) Western blotting was performed to detect proinsulin and insulin relative expression in pancreatic tissue from three groups. (E) The result from (D) was quantified. The ratio of proinsulin to insulin was calculated and data were presented from three independent experiments in non-DM, pre-DM, and DM. Data are expressed as means ± s.d. One-way ANOVA analysis was performed. **P < 0.01, ***P < 0.001. PI, proinsulin; DM, diabetes. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
Elevated proportion of proinsulin+ insulin− (PI+/INS−) cells in islets from patients with diabetes or prediabetes. (A) Pancreas from healthy subjects, patients with prediabetes (pre-DM), and diabetes (DM) stained with proinsulin (green), insulin (red), and DAPI (blue) are shown. Yellow box showed a specific area of the islet which was enlarged and represented. Yellow arrows were used to point out the PI+/INS− cells and white arrows to demonstrate beta cells with proinsulin concentrated pattern. (B) Proportion of PI+/INS− cells in non-diabetes (n = 11), prediabetes (n = 6), and diabetes (n = 14) group. Data present medians (IQR). Kruskal–Wallis statistical analysis was performed. *P < 0.05, **P < 0.01. (C) Correlation of PI+/INS− cells proportion with the level of HbA1c. The r of Pearson correlation and P value are shown. (D) Western blotting was performed to detect proinsulin and insulin relative expression in pancreatic tissue from three groups. (E) The result from (D) was quantified. The ratio of proinsulin to insulin was calculated and data were presented from three independent experiments in non-DM, pre-DM, and DM. Data are expressed as means ± s.d. One-way ANOVA analysis was performed. **P < 0.01, ***P < 0.001. PI, proinsulin; DM, diabetes. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
General characteristic of the study group. Values are presented as mean ± s.d., unpaired Student's t-test and Chi-square test were performed for comparison.
| Non-DM (n = 11) | Pre-DM (n = 6) | DM (n = 14) | P value | |
|---|---|---|---|---|
| Age (years) | 47.18 ± 7.57 | 47.83 ± 13.27 | 51.71 ± 8.72 | 0.4373 |
| Gender (M/F) | 9/2 | 4/2 | 12/2 | 0.6092 |
| BMI (kg/m2) | 25.48 ± 2.80 | 27.92 ± 7.76 | 26.19 ± 3.25 | 0.5407 |
| HbA1c | 5.22 ± 0.25 | 5.90 ± 0.25 | 7.29 ± 1.07 | <0.0001 |
| Section location (neck/tail) | 3/8 | 2/4 | 4/10 | 0.9647 |
DM, diabetes mellitus; M, male; F, female; ; HbA1c, hemoglobin A1c.
The proinsulin in beta cells from T2D appeared to be more diffused and less co-localized with golgi markers
The conversion from proinsulin to insulin requires cleavages at both junctions of the connecting segment linking the B and A chains of proinsulin to release insulin and C-peptide. This process relies on prohormone convertases PC1/3 and PC2 to recognize and cleave in the conjunction sites and carboxypeptidase E (CPE) to remove remaining residues. While the long-standing theory of the proinsulin process was supported by the fact that primary mouse beta cells process proinsulin sequentially by PC1/3 and then PC2, there is no definitive evidence to support that proinsulin is processed by both PC1/3 and PC2 in human beta cells. To examine whether defective insulin maturation we observed in Fig. 1 was associated with impairments of proinsulin processing enzymes, we firstly conducted immunofluorescent staining with PC1/3 and CPE antibodies for pancreas sections from the control and diabetes subjects. Both immunostaining images and counted results support that the percentages of proinsulin positive cells expressing PC1/3 or CPE were not reduced in patients with T2D and prediabetes (Fig. 2A, B, C and D). However, it remained to be further determined whether quantities of PC1/3 and CPE proteins were reduced in these two groups. We then examined the expression of PC2 and found that PC2 was not abundantly expressed in human beta cells and mostly expressed in proinsulin negative cells in the islets from the controls. However, more anti-PC2 immunoreactivity was found in proinsulin positive cells in patients with T2D (Supplementary Fig. 1), consisting with a recent report from Ramzy et al. revealing that PC2 did not appear to be the primary driver of proinsulin processing in healthy human islets, but it could potentially contribute to a compensatory response in islets from patients with T2D (26).
Immunofluorescent staining analyzed PC1/3 and CPE expression in islets from control and diabetes subjects. (A) Pancreatic sections of control and diabetes patients were immunostained with proinsulin (white), insulin (red), and PC1/3 (green). (B) Quantitative analysis of PC1/3 in (A) was presented as PI+PC1/3+ cells/PI+ cells percentage (n = 3). (C) Immunostaining with proinsulin (white), CPE (green), and insulin (red) of islets was performed in control and diabetes patients. (D) Quantitative analysis of CPE presented as PI+CPE+ cells/PI+ cells in control (n = 3) and diabetes (n = 3) patients. Data are expressed as medians (IQR). Mann–Whitney analysis was performed and no significant difference was found among the two groups. PI, proinsulin; PC1/3, prohormone convertases; CPE, carboxypeptidase E. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
Immunofluorescent staining analyzed PC1/3 and CPE expression in islets from control and diabetes subjects. (A) Pancreatic sections of control and diabetes patients were immunostained with proinsulin (white), insulin (red), and PC1/3 (green). (B) Quantitative analysis of PC1/3 in (A) was presented as PI+PC1/3+ cells/PI+ cells percentage (n = 3). (C) Immunostaining with proinsulin (white), CPE (green), and insulin (red) of islets was performed in control and diabetes patients. (D) Quantitative analysis of CPE presented as PI+CPE+ cells/PI+ cells in control (n = 3) and diabetes (n = 3) patients. Data are expressed as medians (IQR). Mann–Whitney analysis was performed and no significant difference was found among the two groups. PI, proinsulin; PC1/3, prohormone convertases; CPE, carboxypeptidase E. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
Immunofluorescent staining analyzed PC1/3 and CPE expression in islets from control and diabetes subjects. (A) Pancreatic sections of control and diabetes patients were immunostained with proinsulin (white), insulin (red), and PC1/3 (green). (B) Quantitative analysis of PC1/3 in (A) was presented as PI+PC1/3+ cells/PI+ cells percentage (n = 3). (C) Immunostaining with proinsulin (white), CPE (green), and insulin (red) of islets was performed in control and diabetes patients. (D) Quantitative analysis of CPE presented as PI+CPE+ cells/PI+ cells in control (n = 3) and diabetes (n = 3) patients. Data are expressed as medians (IQR). Mann–Whitney analysis was performed and no significant difference was found among the two groups. PI, proinsulin; PC1/3, prohormone convertases; CPE, carboxypeptidase E. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
Proinsulin trafficking from the ER to the golgi is an upstream and critical step to initiate proinsulin to insulin processing. Notably, proinsulin distribution appeared to be more diffused in beta cells from diabetes subjects while it tended to display a concentrated pattern in control (Figs 1A and 3A, white arrow). To define the subcellular localization of the concentrated proinsulin in beta cells, the pancreatic sections were double-stained with proinsulin and the ER or the Golgi markers. Examination of histological features revealed the co-localization of concentrated proinsulin with Golgi markers, including cis-Golgi marker GM130 and trans-Golgi marker TGN46 (Fig. 3A and B). Although proinsulin appeared to be more diffused within the cytoplasm in PI+/INS-− cells, given a technical challenge of immunostaining of the ER marker PDI that was essentially ubiquitous throughout the pancreas, it remained to be further determined whether the diffused proinsulin was indeed co-localized in the ER (Supplementary Fig. 2). Furthermore, the median MOC of proinsulin protein and TGN46 was 44% in control, whereas this index reduced to 27% in T2D islets (Fig. 3C), reflecting a reduced degree of proinsulin-Golgi co-localization in the diabetes group.
The proinsulin in beta cells from diabetes patients tends to more diffused and less co-localized with the Golgi markers. (A and B) Representative images of pancreatic islets from control and diabetes patients stained with proinsulin (green), GM130 (in panel A)/TGN46 (in panel B) (Golgi apparatus maker, red), and DAPI (blue) are shown. (C) Dot plot showing distribution of the MOC (a measure of proinsulin–TGN46 co-localization) in individual islets from control and patients with T2D. ****P < 0.0001. (D) Proportion of beta cells containing proinsulin co-localized with Golgi in Non-DM (n = 11), pre-DM (n = 6), and DM (n = 14) group. Data present medians (IQR). Kruskal–Wallis statistical analysis was performed. **P < 0.01. (E) The correlation of proinsulin-Golgi co-localizing cells ratio with the level of HbA1c in patients (Pearson analysis). (F) The proportion of beta cells with proinsulin-Golgi co-localization in the PI+/INS− or PI+/INS+ cells were analyzed. *P < 0.05, **P < 0.01. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
The proinsulin in beta cells from diabetes patients tends to more diffused and less co-localized with the Golgi markers. (A and B) Representative images of pancreatic islets from control and diabetes patients stained with proinsulin (green), GM130 (in panel A)/TGN46 (in panel B) (Golgi apparatus maker, red), and DAPI (blue) are shown. (C) Dot plot showing distribution of the MOC (a measure of proinsulin–TGN46 co-localization) in individual islets from control and patients with T2D. ****P < 0.0001. (D) Proportion of beta cells containing proinsulin co-localized with Golgi in Non-DM (n = 11), pre-DM (n = 6), and DM (n = 14) group. Data present medians (IQR). Kruskal–Wallis statistical analysis was performed. **P < 0.01. (E) The correlation of proinsulin-Golgi co-localizing cells ratio with the level of HbA1c in patients (Pearson analysis). (F) The proportion of beta cells with proinsulin-Golgi co-localization in the PI+/INS− or PI+/INS+ cells were analyzed. *P < 0.05, **P < 0.01. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
The proinsulin in beta cells from diabetes patients tends to more diffused and less co-localized with the Golgi markers. (A and B) Representative images of pancreatic islets from control and diabetes patients stained with proinsulin (green), GM130 (in panel A)/TGN46 (in panel B) (Golgi apparatus maker, red), and DAPI (blue) are shown. (C) Dot plot showing distribution of the MOC (a measure of proinsulin–TGN46 co-localization) in individual islets from control and patients with T2D. ****P < 0.0001. (D) Proportion of beta cells containing proinsulin co-localized with Golgi in Non-DM (n = 11), pre-DM (n = 6), and DM (n = 14) group. Data present medians (IQR). Kruskal–Wallis statistical analysis was performed. **P < 0.01. (E) The correlation of proinsulin-Golgi co-localizing cells ratio with the level of HbA1c in patients (Pearson analysis). (F) The proportion of beta cells with proinsulin-Golgi co-localization in the PI+/INS− or PI+/INS+ cells were analyzed. *P < 0.05, **P < 0.01. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
We then assessed the percentage of the beta cells containing proinsulin co-localized with the Golgi markers in islets of controls and patients with T2D or prediabetes. We found an approximately 37% reduction in the diabetes group compared to that of the control (Fig. 3D, 24.15% vs 38.04%, P < 0.01) and 24% decrease compared to that of prediabetes group (Fig. 3D, 24.15% vs 31.63%, P < 0.01). In addition, the percentage of beta cells with a Golgi co-localized proinsulin pattern was negatively correlated with HbA1c level of subjects (Fig. 3E, r = –0.67, P < 0.0001). We defined the beta cells expressing both proinsulin and insulin as PI+/INS+ cells. The proinsulin pattern in the PI+/INS− or PI+/INS+ beta cells from all three groups was then analyzed. It was found that the PI+/INS− cells hardly appeared to display a Golgi co-localized proinsulin pattern (Fig. 3F, left panel). The proinsulin of PI+/INS+ cells from diabetes patients tended to be less co-localized with Golgi markers compared to that in control and prediabetes subjects (Fig. 3F, right panel). These data indicated that defective ER to golgi trafficking may contribute to impaired insulin maturation in the beta cells of T2D.
Nearly absence of the transcription factor NKX6.1 expression in PI+/INS− beta cells
In human pancreatic islets, the homeobox protein NKX6.1 is a transcription factor and is essential for maintaining the functional and molecular traits of mature beta cells (27). Since the PI+/INS− cells had a defect in insulin maturation, we, therefore, examined the expression and intra-cellular localization of NKX6.1 in the islet beta cells from patients with prediabetes and T2D. It is noticed that NKX6.1 was mostly located in the nucleus in non-DM beta cells, while NKX6.1 tended to be expressed in the cytoplasm instead of the nucleus, or even totally absent in the beta cells from patients with diabetes (Fig. 4A and B), which is consistent with the previous reports in human and mice (28, 29). The yellow arrows indicated the PI+/INS− cells with NKX6.1 nuclear absence. In addition, the NKX6.1 was also decreased in the PI+/INS+ beta cells of patients with prediabetes or T2D (Fig. 4C, right panel). The reduced NKX6.1+/PI+/INS+ was strongly correlated with HbA1c level in T2D patients (Fig. 4D, r = –0.72, P = 0.0079). Interestingly, NKX6.1 could be barely detected in PI+/INS− beta cells among all three groups (Fig. 4C, left panel). Taken together, the shift of NKX6.1 from the nucleus to the cytoplasm and the reduced expression in beta cells is a molecular feature in the progression to type 2 diabetes. Particularly, the PI+/INS− subset beta cells, which were defined and focused in this study, showed nearly the absence of NKX6.1.
The different expression of beta-cell transcription factor NKX6.1 in islets from control and patients with prediabetes or diabetes. (A) Immunostaining with proinsulin (white), insulin (green), and NKX6.1 (red) was performed in pancreatic sections from non-diabetes, prediabetes, and diabetes groups. Yellow arrows were used to demonstrate the PI+/INS− cells with NKX6.1 nuclear absence. (B) Quantitative analysis of nuclear localization of NKX6.1 in control group (n = 3), pre-diabetes (n = 4), and diabetes patients (n = 5). (*P < 0.05, Kruskal–Wallis test). (C) Ratio of NKX6.1+ cells/PI+INS− cells and NKX6.1+ cells/PI+INS+ cells in non-diabetes (n = 3), prediabetes (n = 4), and diabetes (n = 5) group. (*P < 0.05, Kruskal–Wallis test). (D) Correlation between the ratio of NKX6.1+ cells/PI+INS+ with HbA1c level in patients. (Pearson correlation analysis). PI, proinsulin; INS, insulin. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
The different expression of beta-cell transcription factor NKX6.1 in islets from control and patients with prediabetes or diabetes. (A) Immunostaining with proinsulin (white), insulin (green), and NKX6.1 (red) was performed in pancreatic sections from non-diabetes, prediabetes, and diabetes groups. Yellow arrows were used to demonstrate the PI+/INS− cells with NKX6.1 nuclear absence. (B) Quantitative analysis of nuclear localization of NKX6.1 in control group (n = 3), pre-diabetes (n = 4), and diabetes patients (n = 5). (*P < 0.05, Kruskal–Wallis test). (C) Ratio of NKX6.1+ cells/PI+INS− cells and NKX6.1+ cells/PI+INS+ cells in non-diabetes (n = 3), prediabetes (n = 4), and diabetes (n = 5) group. (*P < 0.05, Kruskal–Wallis test). (D) Correlation between the ratio of NKX6.1+ cells/PI+INS+ with HbA1c level in patients. (Pearson correlation analysis). PI, proinsulin; INS, insulin. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
The different expression of beta-cell transcription factor NKX6.1 in islets from control and patients with prediabetes or diabetes. (A) Immunostaining with proinsulin (white), insulin (green), and NKX6.1 (red) was performed in pancreatic sections from non-diabetes, prediabetes, and diabetes groups. Yellow arrows were used to demonstrate the PI+/INS− cells with NKX6.1 nuclear absence. (B) Quantitative analysis of nuclear localization of NKX6.1 in control group (n = 3), pre-diabetes (n = 4), and diabetes patients (n = 5). (*P < 0.05, Kruskal–Wallis test). (C) Ratio of NKX6.1+ cells/PI+INS− cells and NKX6.1+ cells/PI+INS+ cells in non-diabetes (n = 3), prediabetes (n = 4), and diabetes (n = 5) group. (*P < 0.05, Kruskal–Wallis test). (D) Correlation between the ratio of NKX6.1+ cells/PI+INS+ with HbA1c level in patients. (Pearson correlation analysis). PI, proinsulin; INS, insulin. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
Up-regulated ALDH1A3 expression and glucagon expression in PI+/INS− cells
Insulin-producing beta cells become dedifferentiated during diabetes progression. The factors, such as metabolic inflexibility or cellular stress can trigger progression from beta-cell dysfunction to beta-cell dedifferentiation (30, 31). The isoform of the enzyme aldehyde dehydrogenase 1 family member A3 (ALDH1A3) is regarded as a marker of cellular dedifferentiation (29). Since PI+/INS− cells lost the most important function of beta cells to produce mature insulin, we, therefore, evaluated the expression of dedifferentiation marker ALDH1A3. Interestingly, an increased expression level of ALDH1A3 was found in PI+/INS− cells (Fig. 5A, yellow arrow). We then calculated the proportion of ALDH1A3+ cells in PI+/INS− or PI+/INS+ beta cells among the control, prediabetes, and diabetes groups, respectively. The percentage of ALDH1A3+ cells in PI+/INS− cells was dramatically elevated compared to that in PI+/INS+ beta cells from each group. However, the proportion of ALDH1A3+ cells in PI+/INS− cells was not significantly different among the three groups (Fig. 5B). Since the PI+/INS− cells displayed an increased expression of dedifferentiation marker, we then asked whether these cells could become glucagon-producing cells. It is worth noting that some of the PI+/INS− cells were indeed associated with the acquisition of glucagon staining (Fig. 6A). The percentage of proinsulin and glucagon double-positive (PI+GCG+) cells increased in T2D islets (Fig. 6B).
The expression of dedifferentiation marker ALDH1A3 in islets from control, prediabetes, and diabetes patients. (A) Representative sections of immunofluorescence staining for proinsulin (white), ALDH1A3 (green), and insulin (red) of islets from control and patients with prediabetes or diabetes. Yellow arrows indicate the PI+/INS− cells with ALDH1A3 stained positive. (B) The proportion of ALDH1A3+ cells in PI+/INS− and PI+/INS+ beta cells. Data present as medians (IQR). n = 5 in control group and diabetes group; n = 3 in prediabetes group. (*P < 0.05, Kruskal–Wallis test). PI, proinsulin; INS, insulin. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
The expression of dedifferentiation marker ALDH1A3 in islets from control, prediabetes, and diabetes patients. (A) Representative sections of immunofluorescence staining for proinsulin (white), ALDH1A3 (green), and insulin (red) of islets from control and patients with prediabetes or diabetes. Yellow arrows indicate the PI+/INS− cells with ALDH1A3 stained positive. (B) The proportion of ALDH1A3+ cells in PI+/INS− and PI+/INS+ beta cells. Data present as medians (IQR). n = 5 in control group and diabetes group; n = 3 in prediabetes group. (*P < 0.05, Kruskal–Wallis test). PI, proinsulin; INS, insulin. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
The expression of dedifferentiation marker ALDH1A3 in islets from control, prediabetes, and diabetes patients. (A) Representative sections of immunofluorescence staining for proinsulin (white), ALDH1A3 (green), and insulin (red) of islets from control and patients with prediabetes or diabetes. Yellow arrows indicate the PI+/INS− cells with ALDH1A3 stained positive. (B) The proportion of ALDH1A3+ cells in PI+/INS− and PI+/INS+ beta cells. Data present as medians (IQR). n = 5 in control group and diabetes group; n = 3 in prediabetes group. (*P < 0.05, Kruskal–Wallis test). PI, proinsulin; INS, insulin. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
The percentage of proinsulin and glucagon double-positive (PI+GCG+) cells increased in islets of patients with T2D. (A) Representative image of islets from control (non-DM) and T2D (DM) subjects. Insulin is shown in blue, proinsulin in green, and glucagon in red. The islet area marked in the yellow box is enlarged in the right panel. Yellow arrows indicate glucagon (GCG) positive staining in PI+/INS− beta cell. (B) The percentage of PI+GCG+ cells in total PI+ cells was calculated and showed as medians (IQR). n = 4 in both groups. PI, proinsulin; GCG, glucagon. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
The percentage of proinsulin and glucagon double-positive (PI+GCG+) cells increased in islets of patients with T2D. (A) Representative image of islets from control (non-DM) and T2D (DM) subjects. Insulin is shown in blue, proinsulin in green, and glucagon in red. The islet area marked in the yellow box is enlarged in the right panel. Yellow arrows indicate glucagon (GCG) positive staining in PI+/INS− beta cell. (B) The percentage of PI+GCG+ cells in total PI+ cells was calculated and showed as medians (IQR). n = 4 in both groups. PI, proinsulin; GCG, glucagon. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
The percentage of proinsulin and glucagon double-positive (PI+GCG+) cells increased in islets of patients with T2D. (A) Representative image of islets from control (non-DM) and T2D (DM) subjects. Insulin is shown in blue, proinsulin in green, and glucagon in red. The islet area marked in the yellow box is enlarged in the right panel. Yellow arrows indicate glucagon (GCG) positive staining in PI+/INS− beta cell. (B) The percentage of PI+GCG+ cells in total PI+ cells was calculated and showed as medians (IQR). n = 4 in both groups. PI, proinsulin; GCG, glucagon. A full color version of this figure is available at https://doi.org/10.1530/EJE-21-0144.
Citation: European Journal of Endocrinology 185, 4; 10.1530/EJE-21-0144
Discussion
Recent studies conducted in mouse models revealed that compensatory proinsulin synthesis in response to insulin resistance can induce a marked increase of misfolded proinsulin that accumulates in the ER, impairing proinsulin to insulin maturation, and causing insulin deficiency during the onset and development of T2D (9). In this study, with immunofluorescence staining, we have identified a novel subset of PI+/INS− beta cells in which proinsulin was more diffused within the cytoplasm and lost concentrated Golgi staining (Figs 1, 3 and Supplementary Fig. 2). Compared to PI+/INS+ beta cells, PI+/INS− cells displayed an absence of NKX6.1 staining but enhanced ALDH1A3 expression, and some of them were associated with the acquisition of glucagon staining (Figs 3, 4, 5 and 6). These results suggest that although the PI+/INS− cells could still actively synthesize proinsulin, they lost the most important function of beta cells to produce mature insulin, and PI+/INS− cells may be an early signature of beta-cell dysfunction in patients with T2D.
Increased ratios of proinsulin to insulin (PI/I) or proinsulin to C-peptide (PI/C) in the blood have been considered as signs of beta-cell dysfunction in patients with T2D and T1D (32, 33). However, due to the limitation of human islets, the changes of PI/I ratio in the islets of patients with T1D and T2D remain to be further determined. To date, most studies focusing on possible proinsulin trafficking/processing defects in islets of patients with diabetes were based on in situ immunostaining. Several groups have reported alterations of proinsulin-insulin co-localization and identification of the subset of beta cells with proinsulin-enriched and insulin-poor staining in the patients with T1D, suggesting a defect in proinsulin conversion or dysfunction in vesicular trafficking (34, 35, 36). In the current study, we found that an increase in subpopulation of PI+/INS− cells along with an elevated PI/I ratio in the patients with T2D and prediabetes (Fig. 1), suggesting a defect in insulin maturation in diabetes. There was a study reporting that processing enzymes of proinsulin might be decreased in some of the islets in T2D (37). In this report, we found that the percentages of beta cells expressing PC1/3 or CPE were not changed in the patients with T2D (Fig. 2). As for PC2, we found that it was mostly expressed in proinsulin-negative cells in the islets of non-diabetic controls. However, there was a trend of an increase of PC2 expression in proinsulin positive cells in the islets of patients with T2D (Supplementary Fig. 1). This was consistent with an observation from a recent study showing that low immunoreactive PC2 in non-diabetic islet beta cells and increased expression of PC2 in beta cells from donors with T2D (26). Although our data cannot rule out decreases in absolute expression of proinsulin processing enzymes within each cell, together, these data suggest that insulin maturation defects we observed in this study are unlikely caused by alterations in percentages of cells expressing processing enzymes.
Insulin deficiency and insulin resistance are two main pathogenesis components of T2D. However, insulin resistance alone does not lead to diabetes. In insulin-resistant individuals, failure of beta-cell compensation is linked to the onset and progression of the disease. The ER protein homeostasis serves as a central role in maintaining beta-cell function. An imbalance between the increasing metabolic demand for insulin production and the capacity of the ER to properly fold proinsulin can cause ER stress by the aberrant retention of misfolded proinsulin in the ER (2). In addition, misfolded proinsulin can also interact with bystander proinsulin in the ER, blocking the ER export of bystander proinsulin (13). Increased proinsulin biosynthesis and misfolding along with defective insulin maturation have been concurrently presented in insulin-resistant db/db mouse model (38). Consistently, in this study, we found that an increase of PI+/INS− cells in the patients with T2D and prediabetes (Fig. 1). In addition, proinsulin in PI+/INS− cells displayed more diffused pattern that did not co-localize with Golgi markers (Fig. 3 and Supplementary Fig. 2), suggesting that proinsulin may have a defect in intracellular trafficking from the ER to Golgi in PI+/INS− cells. In vivo and vitro studies showed that attenuating beta cell proinsulin synthesis can improve proinsulin maturation, insulin production, and insulin secretion (39, 40). These data suggest that the ER disturbance with accumulated misfolded proinsulin impairs proinsulin to insulin maturation and may accelerate the progression from compensated insulin resistance to an overt T2D.
During the natural history of beta-cell adaptation and failure in T2D, loss of normal beta-cell function arguably precedes the loss of beta-cell mass. The beta-cell dedifferentiation was usually defined as loss of several specific gene expressions along with upregulation of genes not typically expressed in mature beta cells, including those expressed in islet progenitor cells. NKX6.1 is one of the genes thought to be essential for the development and maintenance of functional beta cells. Moreover, the genome-wide association study (GWAS) has indicated that a variation in NKX6.1 is associated with a higher risk of T2D in East Asians (41, 42). Studies from animal models also showed that loss of NKX6.1 results in a failure to synthesize and secrete insulin and subsequently developing rapid-onset diabetes (43). In our study, the expression of NKX6.1 significantly reduced in the islets of T2D, especially in the cells with PI+/INS−. Furthermore, we found a negative correlation between the proportion of NKX6.1+ cells in PI+INS+ beta cells with HbA1c level (Fig. 4), which was consistent with the previous report (43). ALDH1A3 is enriched in progenitor cells in the human fetal pancreas and is considered as a marker of dedifferentiation of beta cells (28, 44). The elevation of PI+/INS− with upregulated ALDH1A3 contributes to the increased abundance of ALDH1A3 observed in pancreatic islets from T2D. In addition to a decrease of NKX6.1 and an increase of ALDH1A3, more beta cells in the islets of T2D displayed double immunostaining of proinsulin and glucagon (Fig. 6). These data support the notion that even though PI+/INS− cells still actively synthesized proinsulin, this subpopulation of beta cells may start to lose some features of beta cells and undergo a transition to endocrine progenitor cells.
In summary, our studies along with previous reports highlighted features that the accumulation of misfolded proinsulin molecules in the ER impaired insulin maturation, thereby leading to an increase of functionally exhausted beta cells. Our results support the notion that a progressive dysfunction of beta cells may start from a gradually increased proinsulin accumulation in the ER that disturbs ER protein homeostasis and impairs intracellular proinsulin trafficking, leading to defect in insulin maturation and diabetes.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/EJE-21-0144.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this study.
Funding
This work was supported by the National Key R&D Program of China 2019YFA0802502 and research grants from the National Natural Science Foundation of China 81620108004, 81830025, 81700699. Dr Shusen Wang’s lab was supported by the National Key R&D Program of China 2020YFA0803704 and National Natural Science Foundation of China 81870535 and 82070805.
Author contribution statement
M L: conceptualization, supervision and writing/editing manuscript; S W, Y H, J Z and T L: investigation, validation, writing and data curation; J W, N L, J Y and R L: development of methodology. All co-authors involved in data analysis, editing and reviewing the manuscript.
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