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Abstract

Transplantation of islets isolated from deceased donor pancreata is an attractive method of β-cell replacement therapy for patients with type 1 diabetes (T1D). However, the loss of islet cell viability and function during the peritransplant period is a limiting factor to long-term islet engraftment. Activation of the isoenzyme PKCe may improve islet survival and function. The current study assesses the effects of PKCe activation on islet graft function in a syngeneic streptozotocin-induced diabetic mouse model. Islets were isolated from wild-type BALB/c mice preconditioned with either a PKCe activator (ψεRACK) or a TAT carrier control peptide. Islets were further treated with the same agents during isolation, purification, and incubation prior to transplantation. Two hundred seventy-five islet equivalents were transplanted under the kidney capsule of streptozotocin-induced diabetic BALB/c mice. Islet function was assessed by measurement of blood glucose levels every 3 days for 42 days after transplant and through an intraperitoneal glucose tolerance test (IPGTT). The time for return to euglycemia in mice transplanted with islets treated with ψεRACK was improved at 14 ± 6 days versus 21 ± 6 days with TAT-treated islets. The IPGTT showed a 50% reduction in the area under the curve associated with an improved insulin response in mice transplanted with ψεRACK-treated islets compared to TAT-treated islets. A preconditioning regimen using PKCe agonist before pancreatic recovery and during islet isolation improves islet graft function and resistance to high glucose stress after transplantation.

Keywords

Islet Transplantation Protein kinase C ε (PKCε)Diabetes

Introduction

Islet cell transplantation has clinical promise and is widely studied for its application in the treatment of type 1 diabetes (T1D) (23). However, islet cell demise during the 30-day peritransplantation period remains a major challenge to successful islet engraftment (8). Throughout the isolation and peritransplant period, islet cells are exposed to ischemia, oxidative stress, and cytokine injury, which may activate apoptotic pathways in the insulin-producing β-cell. Thus, inhibition of apoptotic pathways and subsequent activation of survival pathways throughout the isolation and peritransplant period may improve islet graft function and viability (2,3,19 –21).

Protein kinase C e (PKCe) is a member of the protein kinase C superfamily of serine/threonine kinases and is involved in the regulation of β-cell survival through the activation of Akt in the phosphoinositide 3 (PI3)-kinase-dependent pathway (5,6,18). Akt, a serine/ threonine kinase, inhibits B-cell CLL/lymphoma 2 (BCL2) family proapoptotic factors [caspase-9, BCL2-associated agonist of cell death (Bad), and BCL2-associated X protein (Bax)] (12) and suppresses the activation of c-Jun N-terminal kinase (JNK) (26), which is involved in both immune- and stress-mediated β-cell apoptosis (20). Akt-mediated JNK inhibition also improves insulin sensitivity and glucose tolerance in a type 2 diabetic mouse model (11). In addition to reducing islet cell death (14), PKCe activation may influence islet cell function. The translocation of PKCe from the cytosol to the plasma membrane is associated with the β-cell insulin response to glucose (29). Previous studies have linked PKCe activation with enhanced islet cell function in vitro (22), and conversely, inhibition and downregulation of PKCe have each shown to reduce insulin secretory function (10,25). In a previous in vitro study, we found that preconditioning of donor mice with a PKCe activator, ψ∊ receptor for activated C kinase (ψ∊RACK), led to a significant reduction in islet cell death during isolation (13). In the current investigation, PKCe activation by ψ∊RACK was evaluated to improve islet graft viability and function in a syngeneic diabetic mouse transplant model.

Materials and Methods

Animals

Fourteen-week-old BALB/c female mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). All mice were housed in the Stanford Blood Center animal facility. All animal procedures were approved by Stanford University's Institutional Animal Care and Use Committee (IACUC).

Peptide Synthesis

The PKCe selective agonist peptide, ψ∊RACK, was conjugated to the transactivator of transcription_47–57 (TAT_47–57) carrier peptide for transmembrane delivery as previously described (4). The TAT_47–57 peptide was used as a control peptide. Both peptides were synthesized by American Peptide (Sunnyvale, CA, USA).

Islet Cell Isolation and Experimental Design

Donor mice were treated with either ψ∊RACK or TAT-carrier peptide (20 nmol/L) through a 0.2 ml intraperitoneal (IP) injection, 45 min prior to pancreatic harvest (Fig. 1). Untreated donor mice also served as controls. Animals were anesthetized, and the abdomen was opened for exposure of the pancreas. Collagenase type V (Sigma-Aldrich, St Louis, MO, USA) at 0.7 mg/ml in a buffer solution (Perfusion Solution; Cellgro-Mediatech, Inc., Herndon, VA, USA) was injected through the main bile duct. The distended pancreas was removed and incubated in the enzyme solution at 37°C for 15 min with vigorous agitation. Islet purification was obtained through centrifugation on Euro-Ficoll (Cellgro-Mediatech, Inc.) gradients containing either 1 mmol/L of ψ∊RACK peptide, TAT control peptide, or no peptide. Islets were then handpicked under an inverted microscope, plated in Roswell Park Memorial Institute (RPMI) 1640 medium (VWR, Radnor, PA, USA) supplemented with 10% fetal bovine serum (Gemini BioProducts, West Sacramento, CA, USA), 2 mmol/L l-glutamine (Sigma-Aldrich), 100 μg/ml penicillin, 100 μg/ml streptomycin, 1 mmol/L hydroxyethyl piperazineethanesulfonic acid (HEPES), and 0.25 μg/ml fungizone (all Invitrogen, Carlsbad, CA, USA) and incubated with either 1 μM of ψ∊RACK peptide, TAT control peptide, or no peptide at 37°C in 5% CO₂ for 2 h prior to transplantation. Isolations for control islets pretreated with TAT or no peptide were run in parallel with the ψ∊RACK-treated islets used in the experimental group.

Figure 1.

ψ∊RACK/TAT administration and assessment protocol. Abbreviations: ψ∊RACK, ψ∊ receptor for activated C kinase; TAT, transactivator of transcription; IP, intraperitoneal; IPGTT, IP glucose tolerance test.

Islet Cell Transplantation

In preparation for transplantation, BALB/c female mice weighing 20–25 g were rendered diabetic by an IP injection of streptozotocin (STZ; Sigma-Aldrich) [110 mg/kg in 0.1 M sodium citrate buffer (Sigma-Aldrich)] repeated after 48 h. Diabetes was confirmed by two consecutive blood glucose level readings greater than 250 mg/dl using an automated glucometer (Roche Diagnostics Mississauga, ON, Canada) on tail vein blood.

Approximately 275 islet equivalents (IEQs) were transplanted under the left kidney capsule (24) of STZ-induced diabetic mice divided into three groups receiving 1) ψ∊RACK-treated islets (n = 6), 2) TAT-treated islets (n = 7), or 3) untreated islets (n = 3). Posttransplantation, blood glucose levels were measured in fed mice every third day for 42 days to detect a return to normoglycemia (euglycemia), defined as less than 200 mg/dl.

Intraperitoneal Glucose Tolerance Test

At 4 weeks post-islet transplantation, recipient mice were fasted overnight (16 h) and injected IP with d-glucose solution (2 g/kg; Sigma-Aldrich). Blood glucose levels were monitored on tail vein blood at 0, 10, 20, 30, 45, 60, 90, and 120 min following glucose injection. Relative area under the curve (AUC) was calculated by subtracting baseline blood glucose levels from those measured postinjection using GraphPad Prism software (La Jolla, CA, USA).

Nephrectomy

At 42 days posttransplant, the kidney containing the islet graft was resected through a midline incision and processed for insulin graft content analysis and immunohistochemistry. Blood glucose levels were then monitored daily for return to hyperglycemia following islet graft removal.

Insulin Content of the Kidney

The whole islet graft-containing kidneys were homogenized with a dounce (Sigma-Aldrich) in 10 ml cold acid ethanol [1.5% volume 1 N HCl (Sigma-Aldrich) in 75% ethanol (Fisher Scientific, Pittsburgh, PA, USA)] and centrifuged at 800 × g at 4°C for 5 min. The samples were incubated for 12 h at 4°C. The supernatant was pH adjusted to 8.5 with ammonium hydroxide (Sigma-Aldrich) and stored at −20°C. Insulin levels were measured using a mouse insulin enzyme-linked immunosorbent assay (ELISA) kit per manufacturer's protocol (Mercodia AB, Uppsala, Sweden) and normalized to total protein content measured by micro bicinchoninic acid (BCA) protein assay (Thermo Scientific, Rockford, IL, USA).

Methods for Immunostain

After nephrectomy, kidney samples were fixed in 10% formalin (Fisher Scientific). The fixed samples were embedded in paraffin, cut into 5-μm-thick sections, and mounted on glass slides (Thermo Scientific, Rockford, IL, USA). Samples prepared for histological analysis were stained with hematoxylin and eosin (Richard-Allen Scientific, Kalamazoo, MI, USA). Insulin granules in parallel samples were detected with immunoperoxidase staining as previously described (17). Controls were performed to exclude nonspecific staining.

In Vitro Glucose-Stimulated Insulin Secretion

Aliquots of 35 IEQs from each group (untreated or treated with either ψ∊RACK or TAT peptide) were washed for 5 min with low (2.5 mmol/L) glucose in Krebs-Ringer solution [0.1% bovine serum albumin (BSA), 25 mmol/L HEPES, 115 mmol/L NaCl, 24 mmol/L NAHCO₃, 5 mmol/L KCl, 1 mM MgCl₂, 2.5 mM CaCl₂; all Sigma-Aldrich] and underwent a 60-min static incubation with low (2.5 mmol/L) followed by high (28 mmol/L) glucose concentrations in Krebs-Ringer solution. Insulin secretion was measured after each incubation using the mouse ELISA kit (Mercodia AB). Values are reported as a stimulation index (SI) defined as the ratio of insulin (μg/L) secreted with high glucose incubation to insulin (μg/L) secreted with low glucose incubation (n≥5 for all groups).

Statistical Analysis

Results were expressed as mean ± SEM. Unpaired Student's t test or one-way ANOVA with Bonferroni post hoc analysis, as appropriate, was employed using GraphPad Prism software. A value of p < 0.05 was considered significant.

Results

Blood Glucose Normalization After Islet Transplantation

The posttransplantation blood glucose levels of ψ∊RACK-treated islet recipients were compared to untreated islet (Fig. 2A) and to TAT-treated islet recipient mice (Fig. 2B) and showed no statistical significant difference. Return to euglycemia was reached at 14 ± 6 days for mice transplanted with ψ∊RACK-treated islets (n = 6), at 21 ± 6 days for mice transplanted with TAT-treated islets (n = 7), and at 24 ± 11 days for control mice transplanted with untreated islets (n = 3) (p > 0.05). By 42 days posttransplant, 100% of mice transplanted with ψ∊RACK-treated islets, 71.4% of mice transplanted with TAT-treated islets, and 66.7% of mice transplanted with untreated islets had returned to euglycemia (Fig. 2C).

Figure 2.

Time for return to euglycemia. Blood glucose measurements in fed mouse recipients of (A) ψ∊RACK-treated islets (circle, n = 6) versus TAT-treated islets (square, n = 7) and (B) ψ∊RACK-treated islets (circle, n = 6) versus untreated islets (triangle, n = 3); vertical dotted line signifies graft removal; data are expressed as mean ± SEM. Mean body weight of mice at baseline was 22 g. (C) Percentage of mice returning to euglycemia over time for recipients of ψ∊RACK-treated islets (solid line, n = 6), TAT-treated islets (dashed line, n = 7), or untreated islets (dotted line, n = 3). Return to euglycemia was reached at 14 ± 6 days for recipients of ψ∊RACK-treated islets, at 21 ± 6 days for recipients of TAT-treated islets, and at 24 ± 11 days for recipients of untreated control islets. No statistically significant differences among groups are shown.

ψ∊RACK Treatment Enhances Glucose Tolerance and Islet Secretory Function

At 4 weeks posttransplantation, the IP glucose tolerance test (IPGTT) showed improved insulin response to high glucose in mice transplanted with ψ∊RACK-treated islets at 60 and 90 min when compared to mice transplanted with TAT-treated islets, and at 10, 60, and 90 min when compared to mice transplanted with untreated islets, no significant statistical difference was observed (p > 0.05) (Fig. 3A). The IPGTT area under the curve was reduced by 50% for mice transplanted with ψ∊RACK-treated islets compared to control mice transplanted with untreated islets (p < 0.05) (Fig. 3B).

Figure 3.

Insulin secretory function in response to glucose challenge improved in recipients of ψ∊RACK-treated islets. (A) IP glucose tolerance 4 weeks after islet transplantation in mouse recipients of ψ∊RACK-treated islets (circle, n = 6), TAT-treated islets (square, n = 7), and untreated islets (triangle, n = 3). Data are expressed as mean ± SEM. (B) Area under the curve (AUC) of IP glucose tolerance test 4 weeks after islet transplantation in mouse recipients of ψ∊RACK-treated islets (n = 6), TAT-treated islets (n = 7), and untreated islets (n = 3). Data are expressed as mean ± SEM. *p < 0.05 for ψ∊RACK-treated versus untreated.

Effect of ψ∊RACK Preconditioning on In Vitro Glucose-Stimulated Insulin Secretion

The SI for ψ∊RACK-treated islets was improved but not significantly compared to TAT-treated islets (p = 0.6002) and untreated islets (p = 0.2888) (Fig. 4). ψ∊RACK-treated islets showed a mean SI of 3.102 ± 0.7831 compared to 2.097 ± 0.3165 for TAT-treated islets and 1.398 ± 0.9130 for untreated islets.

Figure 4.

In vitro insulin response to glucose challenge. The in vitro insulin response to glucose challenge is measured by the stimulation index, insulin release ratio at high/low glucose, of ψ∊RACK-treated islets, TAT-treated islets, and untreated islets (n≥5 for all groups). Data are expressed as mean ± SEM. No statistically significant differences among groups are shown.

Effect of PKC∊ Activation on Graft Insulin Content and Islet Morphology

Islet graft-bearing kidneys were removed at 42 days posttransplantation, homogenized, and analyzed for total insulin content measured by ELISA. Total insulin content was similar in ψ∊RACK-treated islet grafts compared to TAT-treated islet grafts (Fig. 5A). Immunohistochemistry analysis on a representative kidney graft sample confirmed staining of insulin in the cytoplasm of ψ∊RACK- versus TAT-treated islet grafts (Fig. 5B–D).

Figure 5.

Insulin content of kidney grafts (A) containing ψ∊RACK-treated islets (n = 5) and TAT-treated islets (n = 2) at 4 weeks after islet transplantation, normalized to total bicinchoninic acid (BCA) protein content. Data are expressed as mean ± SEM. (B) Immunohistochemistry with insulin staining (black arrow) of a representative sample of ψ∊RACK-treated islets versus (C) control TAT-treated islets transplanted under the kidney capsule at 4 weeks after transplantation with 40× magnification, and (D) immunohistochemistry negative control for insulin.

Discussion

This study investigated the effects of the ψ∊RACK preconditioning regimen (ψ∊RACK treatment of both mouse pancreatic donors and freshly isolated islets) in a syngeneic mouse islet transplant model. The time from transplant to diabetes reversal was improved in the ψ∊RACK-treated group, although the posttransplant blood glucose levels of the ψ∊RACK-treated and TAT-treated groups were similar. On the other hand, the ψ∊RACK pretransplant conditioning regimen improved the insulin response to high glucose stress posttransplantation, as demonstrated by enhanced IPGTT performance with a 50% reduction in the area under the curve in ψ∊RACK-treated islet grafts compared to untreated islet grafts (Fig. 3B). PKCe plays a role in glucose-stimulated insulin secretion, and several studies have shown that direct inhibition of PKCe translocation leads to reduced glucose-stimulated insulin secretion (1,28). ψ∊RACK-treated islets challenged in vitro with high versus low glucose concentration showed a moderately increased SI (p = 0.2888, ψ∊RACK-treated vs. untreated); this observation may be due to increased β-cell sensitivity in vitro to low glucose as well as high glucose stimulation in the ψ∊RACK-treated group. Conversely, the greater improvement in islet function seen in vivo suggests that environmental factors expressed under physiological conditions may be needed for optimal effect of ψ∊RACK preconditioning on islet function.

The ψ∊RACK preconditioning enhancing islet response to high glucose supports findings by Mendez et al. showing that PKCe activation plays a role in the insulin secretory process (16). PKCe activation is directly involved in the signaling cascade of glucose-stimulated insulin production by enhancing conversion of proinsulin to mature insulin I and II (9). Moreover, a recent study by Warwar et al. showed that PKCe regulates biosynthetic pathways of insulin production by affecting early steps in the glucose-dependent processing of proinsulin (27). The enhanced insulin response to high glucose observed by PKCe activation with ψ∊RACK may be through the same mechanism of modulating glucose-triggered insulin conversion. In addition, PKCe aids in the transport of newly synthesized proinsulin from the endoplasmic reticulum to the trans-Golgi network (TGN) (9). Indeed, PKCe's anchoring protein, β coatomer protein 1 (β′COP1), localizes PKCe to the endoplasmic reticulum and is involved in shuttling proinsulin to the TGN (9). This enhanced proinsulin trafficking provides a second mechanism to explain the heightened insulin response to glucose stimulation observed in the ψ∊RACK-treated islet group. Lastly, PKCe activity plays a role in glucose-triggered insulin secretion by interacting with inositol hexakisphosphate (InsP₆), a protein that recruits secretory granules to the site of insulin exocytosis (9).

The involvement of PKCe in proinsulin conversion and insulin secretion, but not in insulin synthesis, may also explain the similarity in graft insulin levels among study groups. PKCe plays a role in insulin processing and secretion primarily in response to high glucose concentration, whereas the total graft insulin content compared between ψ∊RACK-treated and control grafts was measured at physiologic normoglycemic conditions (Fig. 5A).

PKCe is a possible therapeutic target in the apoptosis pathway, and its activation was also evaluated as a strategy to interrupt this damaging process during the islet peritransplant period. Our group previously reported increased islet viability (88%) at 1 h postisolation following ψ∊RACK preconditioning compared to 65% with TAT control peptide (13). As well, apoptosis (evaluated by measurement of DNA–histone complexes) was significantly reduced in vitro in the ψ∊RACK-treated islets compared to TAT-treated control islets for the first 40 h following isolation (13).

The current in vivo study shows an improvement in the rate of diabetes reversal in the ψ∊RACK-treated islet recipients suggesting that there is engraftment of a higher amount of functioning β-cell mass, although islets' expression of markers of apoptosis was not analyzed in vivo posttransplantation. Future studies will assess islet graft expression of apoptosis markers with additional immunohistochemistry staining of graft tissue harvested at consecutive time points posttransplantation.

As the focus of this study was on preconditioning treatment, we did not evaluate ψ∊RACK treatment of the recipient posttransplantation. But other studies evaluated treatments targeting islet apoptosis-associated caspases to protect islet survival posttransplantation (7,15). The current protocol differs as it treats islet cells prior to pancreatic harvest and during isolation. Future studies will compare ψ∊RACK preconditioning alone to protocols with PKCe agonist preconditioning and posttransplantation treatment of the mouse recipient.

Conclusion

This study shows that PKCe activation by a ψ∊RACK preconditioning regimen improves the rate of diabetes reversal and enhances islet metabolic function in vivo after transplantation in a diabetic mouse host. ψ∊RACK preconditioning regimen during pancreatic harvest and isolation improves resistance to high glucose stress after transplantation in a diabetic mouse transplant model. Future studies will evaluate the efficacy of a ψ∊RACK treatment in the recipient as well and validate the current results using a larger animal model before consideration for clinical use.

Footnotes

Acknowledgments

We thankfully acknowledge the funding from Kai Pharmaceutical, now Amgen (Thousand Oaks, CA, USA), and we are grateful to Dr. Daria Mochly Rosen and her laboratory for guidance in the experimental design. This work was also funded in part by the Maria and George Erdi Foundation. We acknowledge Alan Le for his contribution to the islet cell graft preparation for analysis. The authors declare no conflicts of interest.

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A Preconditioning Regimen with a PKCε Activator Improves Islet Graft Function in a Mouse Transplant Model

Abstract

Keywords

Introduction

Materials and Methods

Animals

Peptide Synthesis

Islet Cell Isolation and Experimental Design

Islet Cell Transplantation

Intraperitoneal Glucose Tolerance Test

Nephrectomy

Insulin Content of the Kidney

Methods for Immunostain

In Vitro Glucose-Stimulated Insulin Secretion

Statistical Analysis

Results

Blood Glucose Normalization After Islet Transplantation

ψ∊RACK Treatment Enhances Glucose Tolerance and Islet Secretory Function

Effect of ψ∊RACK Preconditioning on In Vitro Glucose-Stimulated Insulin Secretion

Effect of PKC∊ Activation on Graft Insulin Content and Islet Morphology

Discussion

Conclusion

Footnotes

Acknowledgments

References