Heterogeneous Expression of Proinsulin Processing Enzymes in Beta Cells of Non-diabetic and Type 2 Diabetic Humans

Abstract

Although there is evidence indicating transcriptional and functional heterogeneity in human beta cells, it is unclear whether this heterogeneity extends to the expression level of the enzymes that process proinsulin to insulin in beta cells. To address this question, the expression levels of prohormone convertases (PC) 1/3, proprotein convertase 2 (PC2), and carboxypeptidase E (CPE) were determined in immune-stained sections of human pancreas. In non-diabetic donors, the level of proprotein convertase 1/3 (PC1/3) expression varied among beta cells of each islet but the average per islet was similar for all islets of each donor. Although the average PC1/3 expression of all islets examined per sample was unique for each pancreas, donors had similar levels of proinsulin/insulin expression. PC2 expression in beta cells showed less pronounced inter- and intraislet variation while CPE levels were fairly constant. The relationship between PC1/3 and PC2 expression levels was variable among different donors. Type 2 diabetes had an uneven effect on the expression levels of all three enzymes as they decrease only in some islets in a section. These findings suggest the presence of intraislet, but not interislet, variation in the expression of the proinsulin processing enzymes in non-diabetic subjects and a heterogeneous effect of type 2 diabetes on enzyme expression in islets.

Introduction

The increase in insulin synthesis plays a key role in the initial compensatory mechanisms to obesogenic stimuli and the development of insulin resistance. Type 2 diabetes (T2D) only develops when the beta cell response fails to restore normoglycemia and eventually leads to the development of glucotoxicity, lipotoxicity, endoplasmic reticulum (ER), oxidative stress, and defects in insulin secretion. The effects of stressors that lead to this metabolic dysfunction have been reviewed elsewhere.^1–3

The hormone is translated as preproinsulin and cleaved into proinsulin in the ER.⁴ The maturation of proinsulin into insulin occurs in the secretory granules and is mediated by the prohormone convertases (PC) 1/3, proprotein convertase 2 (PC2), and carboxypeptidase E (CPE).^5–8 Proinsulin segments are generally allocated as A-chain, B-chain, and C-peptide. Studies in rodent islets established that proinsulin is cleaved first by proprotein convertase 1/3 (PC1/3) at the B-C junction, which is then further processed by CPE. After this step, PC2 cleaves at the A-C junction and then processed by CPE.^3,9 The final products of proinsulin processing include insulin and C-peptide (reviewed by Rhodes³). Analysis of proinsulin maturation in mice lacking either enzyme confirmed the sequence of proinsulin cleavage by the processing enzymes.^7,10,11

There is now substantial evidence supporting the presence of phenotypic and functional heterogeneity in beta cells.^12–14 The availability of human tissues allowed us to explore whether human beta cells differ in the expression of the proinsulin processing enzymes. This analysis indicated that PC1/3 expression varied significantly between individual beta cells of an islet, whereas the estimated level of enzyme expression per islet was similar for all islets of each non-diabetic donor. Moreover, the evidence suggested the existence of a unique PC1/3 level for each donor. In spite of this difference, donors with either high or low PC1/3 level had similar expression levels of proinsulin/insulin. PC1/3 was also expressed by somatostatin (delta) cells, but in contrast to beta cells, the expression level of the convertase in delta cells was similar in islets of all non-diabetic donors. The intensity of PC2 in beta cells also varied, but to a lesser extent than PC1/3 while level of CPE was similar in human islets of all tissues. Similar interindividual variation in PC2 and PC1/3 was found between some T2D donors. However, a noted characteristic of islets of T2D donors was the presence a significant heterogeneity in enzyme levels between islets in the same pancreatic section, indicating that islets are differentially affected by the metabolic insult. These observations suggest that expression of the convertases, and particularly of PC1/3, in human islets is regulated by multiple factors and result in intraislet and interindividual heterogeneity.

Materials and Methods

Source of Human Tissues

Pancreata of the two groups of donors analyzed were provided by network of Pancreatic Organ Donors (nPOD). List of donors is indicated in Tables 1 and 2. Tissue sections were obtained from the tail of the pancreas.

Table 1.

List of Donor Controls.

Number	Age	Sex	Body Mass Index	C-Peptide Levels
6012	68	F	23.7	2.97
6013	65	M	24.2	2.8
6017	59	F	24.8	9.89
6020	60	M	29.8	2.82
6022	75	M	30.6	4.99
6060	24	M	32.7	13.63
6102	45	F	35.1	0.55
6126	25	M	25.1	0.88
6165	45	F	25	4.45
6168	58	M	25.2	—
6179	20	F	20.7	27.4
6234	20	F	25.6	6.89
6254	38	M	30.5	6.43
6288	55	M	37.7	12.96
6290	58	M	22.5	7.46
6295	47	F	30.4	10.9
6335	18	M	23.6	8.85
8003	50	M	26.6	—

Table 2.

List of Donors With Type 2 Diabetes.

Number	Age	Sex	Body Mass Index	C-Peptide Levels	Duration (in Years)	Cause of Death
6127	44.8	F	30.4	0.08	10	brain hemorrhage
6139	37.2	F	45.4	0.6	1.5	CVD
6221	61	F	33.7	3.05	4	DKA
6249	45	F	32.3	4.17	15	CVD
6280	47	M	28.1	3.71	10	CVD
6300	67	M	23.5	3.19	10	CVD

Abbreviations: CVD, cardiovascular disease; DKA, diabetic ketoacidosis.

Immunohistochemistry

Sections were processed for immunostaining according to the protocols recommended by nPOD with some modifications. Sections were deparaffinized and sequentially incubated in two 5-min rinse in 0.1 M Tris saline (TS), 30 min incubation at 95C to 100C in a retrievagen solution (pH 6), followed by an 1 hr cool off period at room temperature. Then, slides were washed for 5-min in two changes of 1× TS, incubated for 30 min in a blocking solution (TS + 3.3% lamb serum (Invitrogen, Carlsbad, CA) + 0.3% Triton X-100 (Sigma-Aldrich, St Louis, MO), followed by two 5-min washes in 1× TS and incubation overnight in primary antisera diluted in 1× TS + 0.1% lamb serum. The following day, sections were washed twice in 1× TS, incubated in secondary antibodies for 2 hr in the dark, followed by two 5-min washes in TS and cover-slipped using mounting medium (Vector labs).

Source of Antisera

PC1/3: Abcam (Cambridge, MA), Clone #3532

Western blot analysis indicates that the antibody binds to the 92-kDa (precursor), 88-kDa, 83-kDa, and 66-kDa active forms of the enzyme^15,16 and to a 101-kDa molecule.¹⁶ Incubation of the antibody with the peptide used for immunization eliminates staining in glucagon-like peptide-1 (GLP-1)-expressing mouse intestinal L cells.¹⁶ PC1/3 antisera were also kindly provided by Dr I. Lindberg (University of Maryland) from a stock prepared by the D. F. Steiner’s laboratory (University of Chicago; #R-20).^17,18 This antibody binds to the 87-kDa and 68-kDa forms of the enzyme. In human tissues, the antibody specifically labels neuroendocrine cells.¹⁷ Both antibodies to PC1/3 tested positive for GLP-1⁺ intestinal cells (Supplementary Fig. 1A and B). PC2: antibodies tested were from Cell Signaling (Danvers, MA, #14013S) and D. F. Steiner (University of Chicago; Prep#4).^17,18 Antibodies from both sources recognize the processed forms (69 kDa and 72 kDa) of the enzyme. Insulin: guinea pig antisera (Linco, END Millipore, Burlington, MA). This antibody is a “pan-insulin” antibody, similar to those used by others,¹⁹ that recognizes not only insulin but also its precursors. Monoclonal antisera to glucagon: Sigma-Aldrich (St Louis, MO), clone K79bB10. Monoclonal antisera to somatostatin: Accurate Chemical Co, Westbury NY; clone #YMC1020. Monoclonal antisera to GLP-1 were purchased from Abcam (Cambridge, MA). This antibody is reported by the manufacturer to be specific for the amidated (active) form of the incretin. Antibody dilutions: For human tissues, the antisera to PC2 (Cell Signaling) was used at 1:200 dilution. All other primary antisera were used at a 1:500 dilution. Each staining was repeated at least 2 times per tissue sample.

Characterization of Proconvertase Antibodies: PC1/3

Preliminary testing of both PC31 antibodies in human pancreas revealed differences in PC1/3 expression in islets from the same donors. Thus, although the level of expression of PC1/3 was similar in islets from the some donors (Supplementary Fig. 1C and D), they differed markedly in other samples (Supplementary Fig. 1E and F). In addition, the (Steiner) PC1/3 antisera revealed the presence of PC31⁺IN⁻ cells (Supplementary Fig. 1E) that were rarely visualized with the Abcam antibody (Supplementary Fig. 1D and F). In this study, the expression of only the PC1/3 antisera from D. F. Steiner’s laboratory was examined.

PC2

It was found that the PC2 antibody from Cell Signaling and from D. F. Steiner’s laboratory had different specificity for the convertase expressed by either alpha or beta cells. The cell signaling antibody had low sensitivity for the enzyme in beta cells but high for alpha cells (Supplementary Fig. 1G) while the PC2 from D. F. Steiner’s laboratory was equally sensitive for the enzyme in both cell types (Supplementary Fig. 1H). In this study, PC2 expression was examined using the PC2 antibody from D. F. Steiner’s. Unfortunately, this antibody was in short supply, which limited the scope of the analysis.

Secondary Antibodies

Alexa Fluor 488 and 564 antimouse and antirabbit IgGs were purchased from Molecular Probes (Eugene, OR). For double-label experiments, Alexa Fluor dyes that fluoresced at different wavelengths and conjugated to secondary antibodies produced in different species were used. Secondary antibodies produced in one species (i.e., donkey) directed against primary antibodies of guinea pig, mouse, or rabbit origin often resulted in nonspecific binding and were avoided. All secondary antisera were highly cross-absorbed and were used at a 1:200 dilution. Same batch of secondary antiserum was used in all experiments to prevent alterations in baseline staining of control tissues. To control for nonspecific binding of secondary antibodies, a primary antisera (i.e., insulin) was incubated with secondary antibodies produced in different hosts. No inappropriate binding was found in all combinations tested. Similarly, no label was obtained when sections were incubated with only secondary antibodies.

Confocal Microscopy

Stained sections were analyzed using a Leica SP5 confocal microscope. We performed sequential scanning of the samples to prevent cross talk of the fluorophores.

Measurement of Total Fluorescence (TF)

TF was measured using Image J, a program provided by the National Institutes of Health (NIH). Measurements were performed according to instructions provided in theolb.readthedocs.io/en/latest/imaging/measuring-cell-fluorescence-using-imagej.html. With this program, the area of interest is selected and the corrected TF in that area was calculated according to the following formula: TF= fluorescence intensity (FI) / islet area. As islets had different areas, area values were normalized to a value of 10. Sections from non-diabetic and T2D donors used for measurement of TF were processed together for staining; a histological section from non-diabetic donor was included for comparison; no significant difference was found between TF values of sections of control pancreas stained at different times. All sections were examined using the same settings of the confocal microscope to eliminate bias in the evaluation of enzyme expression.

To measure the FI of PC1/3, PC2, and CPE in beta cells, the slides used for quantitation were processed for immunostaining for both insulin and the enzyme, which were visualized with secondary antibodies that fluoresced at different wavelengths. Using image J, the islet area containing double-labeled cells was delineated, thus excluding the non-insulin cells (not double-labeled). Then, the insulin fluorescence (usually red) was eliminated and the FI corresponding to CPE, PC2, or PC1/3 (green) in the islet area was determined. This approach is illustrated in Supplementary Fig. 2. Then, an average of the FI of individual areas of each islet, such as that illustrated in Supplementary Fig. 2, was calculated. It should be noted that while the drawing tools from Image J offer a greater flexibility in outlining the areas of interest than those used in this figure, the delineated areas cannot be saved for a demonstration. The drawing in Supplementary Fig. 2 was done with Photoshop and is included only to demonstrate the approach.

Statistical Methods

Data were analyzed using a Student’s t-test to compare values from two groups. To compare more than two groups, a mixed linear model was applied to the scores, which were power-transformed to reduce skew. The only fixed effect was intercept; subject ID was introduced as a random factor. A chi-square test of zero variance is reported. Also reported are best linear unbiased estimates (BLUEs) of subject-specific means (back-transformed to the original metric), with 95% confidence intervals. Pairwise tests of differences in means were conducted among subjects. Differences were considered statistically significance at p<0.05.

Study Approval

Human tissues had Institutional Review Board exempt status.

Results

In Non-diabetic Donors, PC1/3 Expression in Beta Cells Reveals Intra- but not Interislet Variation

Previous analysis of PC1/3 expression in human islets^20,21 reported that beta cells expressed the convertase. However, these studies left unresolved whether the convertase was expressed at similar levels by all beta cells of each islet, by most islets in sections from each individual pancreas, and/or by islets from different non-diabetic donors.

Initial analysis was aimed to determine whether islets from different donors express similar levels of PC1/3. Sections of pancreas from 18 non-diabetic donors were stained for insulin and PC1/3 and the FI of PC1/3 measured in five islets/donor. Representative islets from each donor are illustrated in Fig. 1. The comparison of the TF (Fig. 2A) suggested the presence of significant variation between donors.

Figure 1.

Interindividual variation in PC1/3 expression. Photomicrographs illustrate expression of PC1/3 in representative islets of pancreas of 18 donor controls. Islets were double-labeled for insulin (red) and PC1/3 (green). Beta cells with high level of PC1/3 are yellow while cells with lower level of the convertase appear red/orange. Note the variation in enzyme expression between different islets. Bar: 20 µm. Abbreviation: PC1/3, proprotein convertase 1/3.

Figure 2.

Measurement of PC1/3 total fluorescence in islets of controls. (A) Graph of total fluorescence of islets illustrated in Fig. 1. Values represent the average fluorescence of five islets per donor. Note the presence of interindividual variation in PC1/3 expression. Donor identification number is indicated in the x-axis. (B) Histogram compares the total fluorescence in islets from four donor control; donor identification number is indicated in the x-axis. Values represent the mean ± standard error of the mean of 25 to 40 islets/donor. There was highly significant intersubject variance, p<0.001. Pairwise comparison of BLUEs among subjects indicated that every subject differed from all other subjects with p<0.01. (C) Total fluorescence of proinsulin/insulin is similar in islets of two donor controls. Staining intensity was measured in islets of donors that had significant difference in PC1/3 levels (28–30 islets/donor). Comparison of expression levels of PC3/1 (B) and insulin (C) indicate that variations in PC1/3 level are not due to differences in tissue processing. Abbreviations: PC1/3, proprotein convertase 1/3; BLUEs, best linear unbiased estimates; a.u., arbitrary unit; NS, not significant.

To further test for the presence of interindividual differences in PC1/3 levels, tissue sections from four non-diabetic donors (#8003, #6017; #6290; #6335) were double-labeled for insulin and PC1/3, and the TF was determined (25–40 islets per donor). It was found that, in each donor, most islets display similar levels of PC1/3 expression. Measurement of the TF confirmed that islets from each donor have similar values and support the presence of interindividual differences in PC1/3 expression (Fig. 2B). To ascertain whether this variation was due to technical differences in the processing of the tissues, the level of proinsulin/insulin was determined in islets from two of the tissue samples with significant difference in the level of PC1/3 (#8003 and #6335). This analysis revealed that islets from both samples had similar level of insulin/proinsulin between donors (Fig. 2C). Future analysis will seek to determine whether the interindividual differences in PC1/3 levels are correlated with variations in the concentration of mature insulin.

Of interest is the presence of different levels of PC1/3 in beta cells of the individual islets. This trait is illustrated in Fig. 3, which shows representative islets from the tissue samples evaluated for PC1/3 levels in Fig. 2B. Similar intraislet heterogeneity was found in islets of all other donors examined. In contrast to beta cells, islets contain PC1/3⁺IN⁻ cells expressing similar staining intensity. This observation suggests that different signals regulate PC1/3 expression in the two cell types.

Figure 3.

Intraislet variation in PC1/3 expression in beta cells of donors control. (A) Shown are representative islets of donors indicated in Fig. 2B double-labeled for insulin (red) and PC1/3 (green). Note that level of PC1/3 varies between beta cells of the same islet irrespectively of total fluorescence level for the islet. Bar: 15 µm. Abbreviation: PC1/3, proprotein convertase 1/3.

Pancreas of some non-diabetic donors contained islets with areas that were devoid of endocrine cells (Fig. 4A and B). In some instances, these empty spaces were filled with a stained precipitate (Fig. 4C). In other cases, only the trace of an islet remained (Fig. 4D). These figures exemplify what probably are different stages in beta cell death and suggest that this process is a normal, albeit infrequent, occurrence in normoglycemic donors. Unfortunately, efforts to ascertain whether these “empty” areas contain amyloid deposits, which is characteristic of T2D,²² were unsuccessful due to technical difficulties. Taken together, these observations indicate that levels of PC1/3 in islets on non-diabetic donors differ between beta cells of the same islet and between islets from different donors, but they are fairly homogeneous among islets of the same donor.

Figure 4.

PC1/3 expression in islets with apparent beta cell loss. Photomicrograph A to D illustrates islets from donor control #6179 immunostained for insulin and PC1/3. Images in A and B illustrate islets displaying significant levels of both molecules; these islets were located near the islet illustrated in C, which contains a stained precipitate. Note that while islets in Fig. 4A and B contain regions devoid of beta cells, these areas lack a visible precipitate. Fig. D illustrates an islet “remnant” that was found in the same section of pancreas. Bar: 20 µm. Abbreviation: PC1/3, proprotein convertase 1/3.

PC1/3 was expressed not only by insulin cells, but also by a population of cells that were IN negative. It was initially hypothesized that these were cells containing GLP-1, which is cleaved from the proglucagon precursor by PC1/3.²³ Recent reports suggest that GLP-1 is produced not only by intestinal L cells, but also by cells present in islets.²⁴ To test whether the PC1/3⁺IN⁻ cells are GLP-1⁺, sections of pancreas from non-diabetic and diabetic donors were processed for immunostaining. As illustrated in Supplementary Fig. 3A and B, PC1/3⁺ cells in islets of euglycemic (A) or diabetic donors (B) did not stain for GLP-1. Further technical tests are required to visualize the form of PC1/3 present in GLP-1⁺ cells. Finally, costaining of sections of human pancreas for PC1/3 and somatostatin revealed that the PC1/3⁺ cells were delta cells (Supplementary Fig. 3C and D). In contrast, in rodents, prosomatostatin is processed mainly by PC2 into somatostatin.^10,25

Donors With T2D Display Intraislet Variation in PC1/3 Expression

To determine the effect of diabetes on PC1/3 expression levels, sections of pancreas from donors with T2D (#6221, #6280, #6300, #6249; Table 2) that were double-labeled for insulin and PC3/1 were examined by confocal microscopy. Because pancreata from the T2D group contain fewer islets, only approximately 15 islets/donor were examined. The determination of the TF indicated the presence of islets with high, medium, and low levels of PC1/3 in islets of donor (Fig. 5D) #6221, suggesting a gradual loss of this enzyme in insulin/proinsulin positive cells. Representative islets are illustrated in Fig. 5A to C. Similar variability in the level of PC1/3 expression was found in islets of donor #6280 (Fig. 5E). On the contrary, PC1/3 levels in islets of the other two donors examined were either generally low (Fig. 6A; donor #6300) or high (Fig. 6B; donor #6249).

Figure 5.

Heterogeneous decrease of PC1/3 in islets of T2D donors. (A–C) Representative islets from T2D donor #6221 immunostained for insulin (red) and PC1/3 (green) illustrate the presence, in the same section of pancreas, of islets with high (A), medium (B), and low (C) level of the convertase. Also note the persistence of insulin/proinsulin staining after loss of PC1/3 expression. Bar: 40 µm. (D and E) Graphs indicate total fluorescence of PC1/3 in islets of T2D donor #6221 and #6280, respectively. Note the presence of significant interislet variation in fluorescence intensity. Islets with high, medium, and low fluorescence intensities are indicated with black, green, and red lines, respectively. Abbreviations: PC1/3, proprotein convertase 1/3; a.u., arbitrary unit.

Figure 6.

Intraindividual variation in PC1/3 expression is reduced in some T2D donors. Graphs in A and B illustrate the fluorescence intensity for PC1/3 in islets of donor #6300 and #6249, respectively. Sections were immunostained for insulin and PC1/3 and the fluorescence calculated as indicated in methods. Note that although there is some variation in total fluorescence between islets, most islets of each donor have similar values. Bar at a.u. = 150 µm is included to facilitate the comparison. Abbreviations: PC1/3, proprotein convertase 1/3; T2D, type 2 diabetes; a.u., arbitrary unit.

Taken together, these findings suggest that the effect of the metabolic dysfunction on convertase expression varies between islets of the same donor and may also differ between donors.

Intra- and Interislet Variation of PC2 Expression

Due to the limited supply of the PC2 antibody, pancreas of only two normoglycemic (#6290 and #6335) and two diabetic donors (#6127 and # 6139) were examined. Analysis of pancreas of non-diabetic donors that were double-labeled for insulin and PC2 indicated the intensity of PC2 staining in beta cells was similar in islets from each donor. Representative islets are illustrated in Fig. 7A to C. It was also determined that the average of PC2 fluorescence was statistically different between the two non-diabetic donors examined (Fig. 7D). Interestingly, islets of donors may differ in the levels of PC2 and PC1/3. This is illustrated in Fig. 8, which shows islets from individual donors with either different (A, B) or similar (C, D) levels of each convertase.

Figure 7.

Intraindividual variation in PC1/3 expression is reduced in some T2D donors. Photomicrographs illustrate representative islets of donor #6290 (A, B) and donor 6179 (C) stained for PC2 (green) and insulin (red). IN⁺PC2⁺ cells are yellow. Note that variation in PC2 expression level among beta cells is less pronounced than with PC1/3. Bar: 20 µm. Insert in A shows, in high magnification, the area indicated with an arrow in that figure. This insert illustrates the overlap in distribution of both markers and the presence of non-beta cells expressing the convertase. (D) Graph indicates the total fluorescence (in a.u.) of islets examined. Note the presence of statistical significant difference between the two donors control. Abbreviations: PC1/3, proprotein convertase 1/3; PC2, proprotein convertase 2; IN⁺, insulin+; a.u., arbitrary unit.

Figure 8.

Non-coordinated variation of PC2 and PC1/3 expression in islets of control donors. Figure 8A and B illustrates islets of donor #6290 stained for insulin and either (A) PC2 or (B) PC1/3, respectively. Figure 8C and D illustrate islets of donor #6179 immunostained for insulin and either (C) PC2 or (D) PC1/3, respectively. Note that islets of donor #6290 have high PC2 and low PC1/3 levels, whereas donor #6179 has similar level of both enzymes. Bar: 30 µm. Abbreviations: PC2, proprotein convertase 2; PC1/3, proprotein convertase 1/3.

The level of PC2 levels was examined in sections from pancreas of T2D donors. Representative islets are illustrated in Fig. 9A to C and the measurement of the TF in Fig. 9D (donors #6139) and 9E (donor #6127). This analysis revealed differences between islets in the level of the convertase, although expression of the enzyme persisted even in islets with few beta cells. These observations indicate that the effect of T2D on PC2 content in islets is related to the degree of beta cell dysfunction. In addition, they indicate that the cells lose the convertase but retain insulin/proinsulin expression.

Figure 9.

PC2 expression in islets of T2D donors. Photomicrographs A to C show islets from T2D donor #6139 immunostained for insulin (red) and PC2 (green). Note that beta cells in islets with abnormal morphology, such as those shown in Fig. 7A, maintain PC2 expression. Insert in Fig. 7A is a high magnification image of the area indicated with an arrow illustrating double-label IN⁺PC2⁺ cells and IN⁻PC2⁺ cells. Bar for Fig. 9A to C: 25 µm. (D and E) Graphs indicate the total fluorescence (in a.u.) of islets from donors #6139 and #6127. Although there is variation in the values of enzyme levels, this disparity is less pronounced than that found in islets stained for PC1/3. Abbreviations: PC2, proprotein convertase 2; IN⁺, insulin+; T2D, type 2 diabetes; PC1/3, proprotein convertase 1/3; a.u., arbitrary unit.

The Homogeneous Expression of CPE by Beta Cells Is Altered by T2D

The level of CPE expression was determined in pancreatic islets from four non-diabetic donors (#8003, #6288, #6179, #6335) and four diabetic donors (#6127, #6280, #6221, #6300). Representative islets are illustrated in Fig. 10A and B, respectively. The level of staining was similar in both groups as confirmed by the absence of statistical significance between the respective total (Fig. 10D) fluorescence. Islets of T2D also contain islets expressing insulin and low CPE levels (Fig. 10C); these were not included in the calculation of the TF of T2D islets.

Figure 10.

CPE expression is similar in islet cells of control and T2D donors. Photomicrographs illustrate representative islets of controls (A) and T2D (B, C) donors immunostained for CPE (green) and insulin (red). Note that the expression of the enzyme is similar in controls and T2D groups. (C) illustrates an islet from a T2D sample with few beta cells and low CPE levels. Bar: 20 µm. (D) Histogram document that the total fluorescence of islets of control and T2D, measured as indicated in methods, is similar (10 islets/donor, 4 donors/group). Islets similar to that in C were not included in the calculation of the FI. Abbreviations: CPE, carboxypeptidase E; T2D, type 2 diabetes; FI, fluorescence intensity; a.u., arbitrary unit; NS, not significant.

Loss of Proinsulin Converting Enzymes Is not Correlated With the Appearance of Bihormonal Cells

Recent evidence suggests that the differentiated state of mature beta cells can express poly-hormonal traits in response to genetic manipulations, in vitro culture, or metabolic stress.^26–28 Those findings raised the possibility that beta cells of T2D donors that have lost the expression of the proconvertases become bi-hormonal cells. To test this possibility, sections of pancreas from non-diabetic and T2D donors that were immunostained for insulin and either glucagon or somatostatin were compared. As expected, IN⁺ cells of non-diabetic donors did not coexpress glucagon or somatostatin (Fig. 11A and C). Analysis of islets from T2D donors indicate that IN⁺ cells did not stain for glucagon (Fig. 11B and D) or somatostatin (Fig. 11E), indicating that the cells did not acquired a mixed beta–non-beta cell phenotype. The lack of agreement between these results and those reported by others may be due to different approaches used in the analysis (in vivo vs. in vitro) or to distinct properties of rodent and human islet cells.

Figure 11.

In T2D, insulin cells do not express non-beta cell hormones. Photomicrographs illustrate islets of control (A, C) and T2D donors (B, D, E). Islets in A and B were stained for insulin and glucagon; islets in C, D, and E were immunostained for insulin and somatostatin. Note the absence, in both staining combinations, of “yellow” cells, which would result from the coexpression of two hormones. Bar: 15 µm. Abbreviation: T2D, type 2 diabetes.

Discussion

It is generally believed that the correct pathway of proinsulin processing is determined, in part, by the affinity of the convertases to cleave specific sites of the precursor molecule. Based on their enzymatic specificity, it is likely that proinsulin is first cleaved by PC1/3 and that the product of this cleavage is processed by PC2.²⁹ It has also been proposed that the presence of a higher concentration of PC1/3 than³⁰ PC2 also plays a role in the correct processing of proinsulin in beta cells.^7,31 If the relative enzyme concentrations were the only determinants of correct proinsulin processing, it would be anticipated that each of them would be present at similar levels in all beta cells. The aim of this study was to ascertain whether there is homogeneity in the expression level of each of the three insulin processing enzymes and to determine how this attribute is affected by T2D.

The findings indicate the presence of considerable differences in PC1/3 expression between beta cells of the same islet in non-diabetic donors. This observation suggests that functional heterogeneity of beta cells, such as distinct responsiveness to glucose,³² is correlated with changes in the expression levels of PC1/3. However, the value of PC1/3 level per islet, measured as TF, was similar for all islets of each individual donors, and its average was distinctive for each donor. The presence of interindividual variation in PC1/3 expression may be related to the differences in the process of fixation of the tissues. While this could explain the low levels of PC1/3 and PC2 expression in some donors, other donors with low levels of PC1/3 had significant expression of either PC2 or CPE. These observations suggest that the differences in average PC1/3 levels in islets from different non-diabetic donors are not due to a fixation artifact. An alternate possibility is that PC1/3 expression in humans is affected by multiple signals that become dysregulated during the death process, affecting enzyme levels.

Comparison of islets from diabetic donors indicated the presence of significant difference in PC1/3 expression in islets of two of the four donors examined. In these donors, as in the non-diabetic group, the levels of PC1/3 varied significantly between beta cells of each islet. In addition, most insulin cells of some islets showed a gradual decrease and even loss of convertase expression. The presence of intraislet heterogeneity in the expression of the proprotein convertases in response to an abnormal environment is intriguing. Studies in rodents revealed the presence of a subgroup of highly perfused islets,³³ which, in T2D, could increase the exposure of those islets to abnormal metabolic signals. The persistence of this difference in human pancreas may explain the phenotypic diversity of the islets in these two donors with T2D. Alternatively, the phenotypic differences between islets may reflect differences in islet age. While little is known about the process of islet formation in humans, there is evidence suggesting a slow process of beta cell renewal throughout life,³⁴ which would result in the presence of islets of different chronological age coexisting in the same pancreas. It has been reported that insulin resistance induces the appearance of aging markers in islets,³⁵ which would accelerate islet senescence in the islet population. If so, islets expressing low enzyme levels and with evidence of cell death would be those islets that had reached senescence. However, enzyme levels were similar in islets of the other two donors of the T2D group as, in those cases, all islets contain either high levels of PC1/3 in one donor or low levels of the enzyme in the other donor. Perhaps there are intrinsic differences between donors in their response to hyperglycemia and T2D. It may also be speculated that the slices of pancreas examined represent variations between different sections, and that the pancreas contain areas with normal or abnormal islets.

There is scant information regarding the regulation of PC1/3 level and activity. In rodents, the expression levels of PC1/3 and insulin are coordinately regulated by glucose,³⁶ and the decrease in insulin content with hyperglycemia^37,38 is correlated with a reduction in the level of the convertase.³⁶ Insulin content in beta cells also decreases in human islets in vitro.³⁹ If there were a direct relationship between insulin and PC1/3 levels in humans as in rodents, there would be a concomitant decrease in the level of PC1/3 in T2D. The observation that the level of PC1/3 mRNA is lower in human islets from T2D than in normoglycemic donors⁴⁰ supports this possibility.

PC1/3 is synthesized as an inactive precursor, and two major autocatalytic events occur first within the ER, and then at the secretory vesicles that generate a fully active 66-kDa form.⁴¹ Genome-wide association analysis of a normoglycemic population indicated that common PCSK1 variants were implicated in abnormal proinsulin levels and increased levels of proinsulin following glucose stimulation.^40,42 The variants found in the genetic analysis were associated with alterations in amino acid sequence of the protein that may affect its subcellular targeting in the beta cell and/or stability.^40,42,43 Further studies are required to extend these observations to the analysis of PSCK1 variants on proinsulin maturation.

PC1/3 is not only involved in the processing of islet proinsulin to insulin, but also in the maturation of other pro-peptides in the central nervous system and periphery.^44,45 In contrast to the common PCSK1 alleles discussed above, the rare PC1/3 deficiency in humans is characterized by obesity and acute malabsorption due to alterations in the processing of enteric hormones.⁴⁶ In contrast, the phenotypes of mice with ablation of PCSK1 show multiple endocrine abnormalities including growth retardation due to a defect in pro-growth hormone-releasing hormone (GHRH) processing.^11,15 The reason for this difference between species remains unknown. A mutation in the murine gene, termed PC1 ^N222d/N222d, displays a phenotype similar to that in humans as it does not affect growth but it results in defects in proinsulin processing due to decreased activity and expression level of the enzyme.⁴⁷ In spite of the defect in proinsulin processing, mice carrying this mutation remain normoglycemic due to a large increase in the beta cell mass.⁴⁷ It is conceivable that variant(s) similar to PC1 ^N222d/N222d will cause diabetes in humans, which lack a proliferative compensatory response.

The results reported here also indicate that the level of PC2 expression varied between different beta cells in islets cells of non-diabetic donor, probably reflecting the heterogeneity encountered in islet cells. However, the values of TF indicated similar levels of PC2 among all islets within each donor, although it differed between donors. TF levels of the two convertases also allowed determining whether they increase or decrease in synchrony in euglycemic donors. This comparison revealed that the relationship between the expression level of PC1/3 and PC2 is variable, suggesting that they are independently regulated. This finding supports the proposition that the precise sequence of proinsulin processing, depends, like other prohormones,⁴⁸ on other factors in addition to the concentration of each of the enzymes.

In summary, comparison of proconvertase expression in beta cells of non-diabetic and diabetic human donors indicates that the disease has a variable effect on the three proinsulin processing enzymes. Although some islets of T2D donors retain the expression of all three enzymes, their expression is extinguished in beta cells of islets with significant cell loss. An important caveat of this study is that these conclusions are based on the analysis of a very small number of human islets and may not reflect the characteristics of normal beta cells or response of most islets to the functional insults caused by diabetes. However, it is certain that further analysis of the effect of T2D on proconvertase levels in human beta cells will provide a better understanding of the nature of the beta cell functional alterations that lead to a disease state.