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Endocrinology, doi:10.1210/en.2006-1358
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Endocrinology Vol. 148, No. 5 2198-2208
Copyright © 2007 by The Endocrine Society

Induction of Monocyte Chemoattractant Protein-1 Expression by Angiotensin II in the Pancreatic Islets and ß-Cells

Galina Chipitsyna, Qiaoke Gong, Chance F. Gray, Yasir Haroon, Erdinc Kamer and Hwyda A. Arafat

Department of Surgery, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Address all correspondence and requests for reprints to: Hwyda A. Arafat, M.D., Ph.D., Department of Surgery, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, Pennsylvania. E-mail: hwyda.arafat{at}jefferson.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Angiotensin II (AngII), the principal hormone of the renin-angiotensin system, is actively generated in the pancreas and has been suggested as a key mediator of inflammation. Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that plays an important role in the recruitment of mononuclear cells into the pancreatic islets. In this study, we investigated the potential molecular basis for the role of AngII in islet inflammation through studying its effect on MCP-1. AngII significantly increased the expression of MCP-1 mRNA and protein in the RINm5F ß-cell line and activated MCP-1 promoter. AngII-MCP-1 mRNA induction was inhibited by an AngII type 1 receptor antagonist but was unchanged by an AngII type 2 receptor antagonist. AngII-MCP-1 induction was inhibited by the tyrosine kinase inhibitor genistein, suggesting a MAPK signaling mechanism. AngII activated the phosphorylation of ERK1/2 but not p38 or c-Jun NH2-terminal MAPKs. Inhibition of ERK1/2 activation reduced the AngII-induced MCP-1 synthesis. In nonobese diabetic mice pancreata, the temporal pattern of angiotensin-converting enzyme expression correlated well with progression of insulitis and ß-cell destruction. Immunostaining of pancreatic serial sections show colocalization of angiotensin-converting enzyme with MCP-1 in ß-cells in the islets. In freshly isolated islets from normoglycemic mice, AngII alone and in combination with IL-1ß elicited an inflammatory response by stimulation of MCP-1. Our data suggest a positive autocrine/paracrine action for the local pancreatic AngII-generating system during insulitis and provide the first insight into an AngII-initiated signal transduction pathway that regulates MCP-1 as a possible inflammatory mechanism in the islets.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TYPE 1 DIABETES DEVELOPS as a consequence of autoimmunity, leading to ß-cell destruction (1). In the early stages of insulitis, activated macrophages and T cells are attracted to the islets and produce cytokines and free radicals, which contribute to ß-cell dysfunction and death (2, 3). The mechanisms regulating the attraction of monocytes and T cells to the islets remain to be clarified.

Monocyte chemoattractant protein-1 (MCP-1), a protein from the C-C chemokine subfamily is expressed in human and rodent islets (4, 5). A biologically active MCP-1 is released from the pancreatic islets and plays a relevant role in the clinical outcome of islet allografts in type 1 diabetic patients, as suggested by the observation that high MCP-1 secretion is negatively correlated with successful engraftment and long-lasting insulin independence (5). The temporal pattern of MCP-1 expression along with other C-C chemokines is well correlated with the progression of insulitis and ß-cell destruction in the islets of the nonobese diabetic (NOD) mice (4, 6). MCP-1 attracts monocytes (7), T cells (8), and natural killer cells (9) to the site of inflammation. Transgenic mice expressing MCP-1 under control of the insulin promoter develop an intense insulitis (10). Although these observations suggest that MCP-1 produced by cytokine-stimulated islet cells is involved in the recruitment and activation of immune cells during insulitis, the upstream effector molecules and the signaling cascades involved in its regulation in the islets have not been fully elucidated.

The circulating renin-angiotensin system (RAS) is well known to play important roles in the nervous, cardiovascular, and renal systems. The circulatory RAS cascade contains several key components, namely the precursor angiotensinogen, the critical Zn2+-dependent metallopeptidase, angiotensin I (AngI)-converting enzyme (ACE), AngI, and the bioactive octapeptide AngII as well as multiple G protein-coupled receptor subtypes including AngII type 1 receptor (AT1R) and AT2R (11). AT1R and AT2R belong to the heterotrimeric G protein-coupled receptor superfamily. Activation of AT1R leads to coupling with heterotrimeric G proteins and activation of phospholipase C-ß, receptor tyrosine kinases, and nonreceptor tyrosine kinases (23). AngII signaling through AT1R activates MAPK (24), activates nuclear factor-{kappa}B (NF-{kappa}B), and increases the expression of NF-{kappa}B-dependent genes (12). NF-{kappa}B triggers the expression of proinflammatory genes such as MCP-1 in the pancreatic islets and ß-cells (13). AT2R activation is associated with increased tyrosine phosphatase activity (14, 15).

In addition to the circulating RAS, numerous tissues, including the pancreas (16, 17), possess their own AngII-generating systems that may finely tune specific functions via paracrine/autocrine actions (18, 19). The biological role of the local pancreatic AngII during the development of islet inflammation in type 1 diabetes has not been investigated.

In this study, we tested the hypothesis that AngII may contribute to islet inflammation through induction of MCP-1 in the islets and ß-cells. We analyzed its effect on MCP-1 gene transcription, synthesis, and protein production and the signaling mechanisms involved. We also examined the presence of the constitutive AngII-generating system in NOD mice pancreata through analyzing the temporal expression of the enzyme responsible for generation of AngII, ACE, in correlation with hyperglycemia and ß-cell destruction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
AngII was purchased from AnaSpec Inc., (San Jose, CA). Recombinant human IL-1ß was from R&D Systems (Minneapolis, MN). Losartan was from Merck (Whitehouse Station, NJ), and PD123319 was from Sigma Chemical Co. (St. Louis, MO). Rabbit polyclonal antibodies for total and phospho-ERK1/2 (Thr185/Tyr189), total c-jun NH2-terminal protein kinase (JNK) and phospho-JNK (Ser473), and total and phospho-p38 (Thr180/Tyr182) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Goat polyclonal IgG antibody for ACE was from Santa Cruz Biotechnology (Santa Cruz, CA), polyclonal goat antihuman MCP-1 was from R&D Systems, and rat-specific MCP-1 ELISA kit was from Assay Design (Ann Arbor, MI). Horseradish-peroxidase-conjugated donkey antigoat and antirabbit IgG were from Vector Laboratories Inc. (Burlingame, CA). Genistein and sodium orthovanadate were obtained from Sigma, and the MEK1/2 inhibitor U0126 was from Cell Signaling.

Cell culture
RIN, clone 5F (RINm5F), an insulinoma cell line derived from the NEDH rat islet cell tumor, was purchased from American Type Culture Collection (Manassas, VA) and grown at 37 C under a humidified, 5% CO2 atmosphere in RPMI 1640 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum and 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml amphotericin B. After overnight incubation in serum-free medium, the cells were treated with AngII (10–8 to 10–6 mol/liter) for 1–6 h, after which the cells and media were harvested. The cells were also treated with IL-1ß (0.1 ng/ml) as a positive control. To evaluate whether AngII binding to AT1R or AT2R results in induction of MCP-1 mRNA, cells were preincubated with AT1R antagonist losartan or AT2R antagonist PD123319 for 1 h, before stimulation by AngII. The concentrations of AngII and antagonists used in these experiments were selected based on dose-response studies.

ELISA
Rat MCP-1 concentration in the cell culture media was measured using rat-specific ELISA kit (Assay Design). Spectrophotometric evaluation of MCP-1 levels were made by Synergy HT multidetection microplate reader (BioTeck, Winooski, VT).

Transient transfection and MCP-1 promoter studies
The rat MCP-1 promoter –514(enh)luc (GenBank accession no. AF079313) in a luciferase expression vector pGL3 basic (Promega, Madison, WI) was kindly provided by Dr. Decio Eizirik, Free University Brussels, Belgium (13). Cells were seeded into 24-well culture plates (105). At ~80% confluence, they were cotransfected with the pGL3 vectors containing the rat luciferase-labeled MCP-1 promoter, and pGFP as transfection control, using TransFast reagent (Promega), as previously described (20). Two hours later, serum-containing medium was overlaid, and the cells were incubated for an additional 24 h. The cells were then incubated with serum-free medium for 18 h followed by addition of AngII (10–9 to 10–7 mol/liter). Luciferase activities were assayed with the Dual-Luciferase Reporter Assay System (Promega) in a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA). Transfection efficiency was normalized using the total protein concentration of the cell lysates. The results for AngII-treated cells are expressed as a fold induction of the luciferase activity of the same construct in control conditions, taking the control (no AngII added) value as 1.

Mice
Female NOD mice were purchased from Taconic Farm (Hudson, NY). The incidence of diabetes in our colony is about 50% at 20 wk of age in the females. The animals had free access to tap water and pelleted food. All animal studies were performed in accordance with guidelines set forth by the Animal Care Committee of Thomas Jefferson University. Fasting blood glucose (FBG) was monitored using a glucometer (Accu-Check; Roche, Indianapolis, IN). Mice with a reading of FBG more than 200 mg/dl on two consecutive occasions were diagnosed as being positive for type 1 diabetes. According to age and blood glucose level, animals were divided into three main groups (n = 5): 2–4 wk (normoglycemic), 13–15 wk (FBG 200–250 mg/dl), and 20–22 wk (FBG > 350 mg/dl).

The NOD mouse strain is derived from the ICR mouse strain. As nondiabetic prone controls, three groups of age-matched ICR mice (n = 3) were studied. Pancreata were isolated, cleaned from surrounding fat and lymph nodes, and fixed in neutral formalin, snap-frozen in liquid nitrogen, or incubated in RNAlater (Ambion, Austin, TX).

RNA extraction and semiquantitative PCR
Total RNA was isolated from pancreata, islets, or ß-cells with Trizol reagent (Life Technologies), according to the manufacturer’s protocol. RNAs were quantified and DNase digested, and cDNAs were prepared using ImProm-II Reverse Transcription System (Promega) and then subjected to semiquantitative PCR using Master Mix (Promega). The primers used were as follows: MCP-1 rat forward, 5'-TCTACAGAAGTGCTTGAGGTGGTTG-3', and reverse, 5'-CCTGTTGTTCACAGTTGCTGCC-3'; ACE mouse forward, 5'-TGAGAAAAGCACGGAGGTATCC-3', and reverse, 5'-AGAGTTTTGAAAGTTGCTCACATCA-3'; and MCP-1 mouse forward, 5'-GTGAAGCTTAGCTCTCTCTTCCTCCACCACCA-3', and reverse, 5'-CACGGATCCTTTACGGGACAACTTCACATTCAAA-3'.

Upstream and downstream primers that could anneal with the 3'-untranslated region of rat GAPDH were included in the PCR as an internal standard: forward, 5'-TCTACAGAAGTGCTTGAGGTGGTTG-3', and reverse, 5'-CCTGTTGTTCACAGTTGCTGCC-3'. The linear range of amplification for each set of primers was determined to ensure that we used a number of cycles in the linear range. Additional analysis was performed to ensure that the densitometry used provided a linear response. PCR products were electrophoresed on 2% agarose gels, and band intensities were quantified using Kodak Electrophoresis Documentation and Analysis System 290 (EDAS 290; Kodak, New Haven, CT).

Protein isolation and Western blot analysis
Pancreata were lysed in modified RIPA lysis buffer (21), and the protein concentrations in the supernatant were determined using the BCA protein assay reagent (Pierce, Rockford, IL). Equal protein concentrations (50 µg) were denatured in a gel loading buffer at 85 C for 5 min and then loaded onto 10% SDS-polyacrylamide slab gels and transferred to polyvinylidene difluoride membranes and incubated at 4 C overnight with goat polyclonal ACE antibody diluted in PBS/Tween 20 (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA). Proteins from ß-cells treated with AngII were blotted with anti-total and anti-phospho-ERK1/2 MAPK, anti-total and anti-phospho-p38 MAPK, and anti-total and anti-phospho-stress-activated protein kinase (anti-phospho-SAPK)/JNK (Cell Signaling Technology). Lysate from the same experiment was separated on two gels and probed with either phosphospecific or total antibody as control for sample variation in protein content. To avoid sample loading errors, ß-actin expression was determined in the blots to adjust and normalize the amount of sample loaded. The protein bands were visualized with enhanced chemiluminescence reagents (ECL Plus Western Blotting Detection System; Amersham Pharmacia Biotech, Piscataway, NJ) and analyzed, and intensity was quantified using Kodak Electrophoresis Documentation and Analysis System 290 (EDAS 290).

Immunohistochemistry
To localize ACE in the NOD mice pancreata and study its expression and relationship to MCP-1, formalin-fixed, paraffin-embedded tissue blocks were prepared from pancreata of diabetic and nondiabetic mice. Serial sections at 5 µm were stained with the following antibodies: a goat polyclonal antibody against ACE (Santa Cruz) (1:200) and a goat polyclonal antibody against human MCP-1 (R&D Systems) (1:200). To identify where these proteins are expressed in the islet cells, we used ready-to-use antibodies against mouse insulin, glucagon (BioGenex, San Ramon, CA), and somatostatin (Accurate Chemical, Westbury, NY) (1:200). A Vectastain Universal Elite ABC kit and diaminobenzidine chromogenic substrate (Vector Laboratories Inc.) were used according to the manufacturer’s protocol to visualize the tissue reaction to the antibodies. Insulin was visualized by alkaline phosphatase reaction (red), whereas diaminobenzidine was used to visualize the rest of the islet hormones in addition to ACE and MCP-1 (brown).

Antibody specificities were validated with nonimmune isotype serum. Negative control sections, where the primary or secondary antibodies were omitted, were also prepared.

Islet isolation and treatment
Islets were isolated from normoglycemic 4- to 6-wk-old female NOD mice as previously described (20). Briefly, 20 ml cold Hanks’ buffer/type IV collagenase solution was infused into the bile duct. The inflated pancreas was cleaned from the surrounding fat and lymph nodes, minced, and digested in a shaker-type water bath at 37 C. Islets were recognized and handpicked under the stereomicroscope after their staining with dithiazone. Islets were aliquoted and cultured in RPMI medium containing 5 mmol/liter glucose and supplemented with 10 mmol/liter HEPES, 1% L-glutamine, and penicillin/streptomycin. Islets were allowed to equilibrate overnight before their treatment. AngII (10–7 mol/liter) with or without IL-1ß (0.1 ng/ml) in the presence or absence of losartan (100 µM) was added for 3 h after which the islets and media were harvested.

Statistical analysis
All experiments were performed four to six times. Data were analyzed for statistical significance by ANOVA with post hoc Student’s t test analysis. These analyses were performed with the assistance of a computer program (JMP 5 Software; SAS, Cary, NC). Differences were considered significant at P ≤ 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
AngII induces MCP-1 mRNA accumulation and secretion of MCP-1 in cultured ß-cells
To investigate whether AngII can directly increase MCP-1 mRNA accumulation in ß-cells, serum-starved RINm5F cells were treated with or without AngII. Cells were treated also with IL-1ß (0.1 ng/ml) as a positive control. By RT-PCR, there was little MCP-1 mRNA expression in unstimulated ß-cells (control). AngII induced an increase in MCP-1 mRNA accumulation between 10–8 and 10–7mol/liter. The increase in MCP-1 mRNA levels could be detected after 3 h of AngII stimulation and returned to baseline at 6 h after AngII treatment (Fig. 1AGo). To examine whether the increase in MCP-1 mRNA levels in response to AngII is associated with MCP-1 production, MCP-1 protein levels in the media were determined by ELISA. Extracellular MCP-1 protein concentration increased markedly from 0.05 to 4.4 and 6.6 ng/ml after 6 and 12 h of AngII stimulation in RINm5F cells, respectively (Fig. 1BGo).


Figure 1
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  		FIG. 1. AngII induces MCP-1 mRNA accumulation and secretion of MCP-1 in cultured ß-cells. A, RINmF5 cells were grown for 24 h in serum-starved medium and then treated with AngII (10–8 to 10–6 mol/liter) for 3 and 6 h. PCR analysis of MCP-1 mRNA transcripts revealed the presence of a very small amount of MCP-1 mRNA in control untreated cells. The response of MCP-1 mRNA in AngII shows a visible up-regulation of MCP-1 mRNA levels at 3 h at an AngII concentration of 10–7 mol/liter. Levels were reduced at 6 h (300- and 109-bp bands correspond to the amplified MCP-1 and GAPDH, respectively). The MCP-1 mRNA contents are expressed as ODs corrected for GAPDH. Values are expressed as mean ± SEM of three experiments. *, P < 0.05 vs. control levels. B, MCP-1 protein in culture media was measured using mouse-specific ELISA kit. Levels of MCP-1 showed significant up-regulation with AngII and IL-1ß after 6 and 12 h of stimulation. *, P < 0.005 vs. control untreated cells. C, AngII-induced MCP-1 mRNA expression in RINm5F cells is blocked by AT1R antagonist. Cells were preincubated for 1 h with AT1R antagonist losartan or the AT2R PD123319 before addition of AngII (10–7 mol/liter) for 3 h. MCP-1 and GAPDH mRNA content was analyzed by RT-PCR. The MCP-1 mRNA content is expressed as ODs corrected for GAPDH (300- and 109-bp bands correspond to the amplified MCP-1 and GAPDH, respectively). Values are expressed as mean ± SEM of three experiments. *, P < 0.05 vs. cells treated with AngII alone using one-way repeated ANOVA with subsequent all-pairwise comparison procedure by Student’s t test.

 
AngII-induced MCP-1 mRNA expression in RINm5F cells is blocked by AT1R antagonist
To determine the receptor that mediates the AngII-induced MCP-1 gene expression in ß-cells, the AT1R blocker losartan and the AT2R blocker PD123319 were added for 1 h before addition of AngII to the cells. Losartan at 100 nM prevented the increase in MCP-1 by AngII. Losartan had no effect on the IL-1ß-induced mRNA expression. Pretreatment of the cells with PD123319 had no effect on AngII-induced MCP-1 mRNA expression (Fig. 1CGo). These data suggest that the induction of MCP-1 gene expression by AngII is mediated through AT1R in RINm5F ß-cells.

AngII induces MCP-1 promoter activity in RINm5F cells
RINm5F cells were transfected with rat MCP-1 promoter/luciferase gene construct. After 24 h of transfection, the cells were incubated with AngII (10–7 mol/liter) for 1 or 2 h, after which the luciferase activity in the cell lysates was measured. Significant increase in MCP-1 promoter activation is seen after incubation with AngII (Fig. 2Go). These data show that MCP-1 promoter responds directly to AngII.


Figure 2
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  		FIG. 2. AngII induces MCP-1 promoter activity in RINm5F cells. After 24 h of transfection, the cells were incubated with AngII (10–7 mol/liter) at different times. After incubation, the luciferase activity in the cell lysates was measured. AngII causes a time-dependent increase in MCP-1 promoter activity. Relative luciferase activity was calculated after deduction of the activity levels with pGL3 vector alone. Results represent mean ± SEM of triplicate determinations. All experiments were repeated at least three times to confirm the reproducibility of the observations.

 
AngII-induced MCP-1 gene expression requires tyrosine kinase activity
AngII signals through AT1R and induces protein tyrosine phosphorylation in many cell types (22). We investigated whether a similar signaling pathway mediates AngII-induced MCP-1 gene expression in RINm5F cells. To determine whether an increase in tyrosine phosphorylation induces MCP-1 mRNA accumulation, RINm5F cells were exposed to the protein tyrosine phosphatase inhibitor sodium orthovanadate (50–200 µM) for 3 h. Sodium orthovanadate induced an increase in MCP-1 mRNA accumulation in RINm5F at 50 µM with a maximal effect at 200 µM (Fig. 3AGo). This suggests that increased protein tyrosine phosphorylation can induce MCP-1 mRNA accumulation. To determine the involvement of protein kinase in AngII-induced MCP-1 expression, RINm5F cells were pretreated with or without the specific tyrosine kinase inhibitor genistein (60 µM) for 30 min. Genistein alone had no effect on basal MCP-1 mRNA levels (Fig. 3BGo). AngII-induced MCP-1 mRNA accumulation was completely inhibited by genistein (Fig. 3BGo).


Figure 3
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  		FIG. 3. AngII-induced MCP-1 gene expression requires both tyrosine kinase and ERK1/2 MAPK activity. A, The protein tyrosine phosphatase inhibitor sodium orthovanadate induces MCP-1 mRNA accumulation in RINm5F cells. Cells were treated with sodium orthovanadate (50–200 µM) for 2 h. MCP-1 mRNA levels were determined by RT-PCR. *, P < 0.05; #, P < 0.02 vs. control untreated cells using one-way repeated ANOVA with subsequent all-pairwise comparison procedure by Student’s t test. B, Tyrosine kinase inhibitor genistein blocks AngII-induced MCP-1 mRNA accumulation in RINm5F cells. Cells were pretreated with genistein for 1 h (60 µM) and then exposed to AngII (10–7 mol/liter) for 3 h. MCP-1 mRNA levels were determined by RT-PCR. Three independent experiments showed similar results. *, P < 0.05 vs. AngII-treated cells using one-way repeated ANOVA with subsequent all-pairwise comparison procedure by Student’s t test.

 
AngII-induced MCP-1 gene expression requires ERK1/2 MAPK activity
AngII signaling through AT1R activates members of the MAPK family, ERK1/2, p38 MAPK, and JNK/SAPK, in different tissues (23, 24). We tested whether AngII-induced MCP-1 mRNA expression involves activation of MAPK in RINm5F cells. After washing, the cells were lysed, and 40-µg protein aliquots were subjected to Western blot analysis. We probed the blots with antibodies specific for phosphorylated ERK1/2 (Thr183/Tyr185), phosphorylated p38 (Thr180/Tyr182), and phosphorylated JNK/SAPK (Thr183/Tyr185).

AngII increased ERK1/2 phosphorylation within 5 min of treatment (Fig. 4AGo) but not JNK or p38 (data not shown). Preincubation of the cells with MEK1/2 selective inhibitor U0126 abolished the AngII-induced MCP-1 gene expression (Fig. 4BGo). This suggests that specific activation of ERK1/2 kinase may play an important role in AngII-induced MCP-1 expression in RINm5F cells.


Figure 4
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  		FIG. 4. AngII-induced MCP-1 gene expression requires ERK1/2 MAPK activity. A, Time-dependent activation of ERK1/2 MAPK signaling pathway by AngII in RINm5F cells. Representative Western blot probed with phospho-antibody against the activated from of ERK1/2 showing increased phosphorylation of ERK1/2 after incubation of AngII (10–7 mol/liter) for 5–60 min. Blots were stripped and developed with anti-total ERK1/2 and ß-actin as controls for equal protein loading. B, Effect of MEK1/2 inhibitor U0126 on AngII-induced increase in MCP-1 mRNA. Cells were pretreated with the inhibitor (10–30 µM) for 10 min before incubation with AngII (10–7 mol/liter) for 3 h. Data represent three independent experiments. *, P < 0.05 vs. AngII-treated cells using one-way repeated ANOVA with subsequent all-pairwise comparison procedure by Student’s t test.

 
NOD pancreatic AngII-generating system expression correlates with hyperglycemia and progression to severe diabetes
We then investigated the in vivo protein levels of ACE as an indirect assessment of AngII generation in the NOD mice pancreata, in correlation with the establishment and/or progression of insulitis. The intrapancreatic levels of ACE in three groups of female NOD mice (prediabetic, 2–4 wk; diabetic FBG 200–250 mg/dl, 13–15 wk; and diabetic FBG >350 mg/dl, 20–22 wk) were analyzed by Western blotting. The mice were compared with age-matched non-diabetic-prone ICR mice. Age-blood glucose-dependent analyses showed that ACE protein is detectable in the female NOD mice at 2 wk of age before the onset of insulitis (Fig. 5AGo). At 13–15 wk of age, and with onset of hyperglycemia, the expression of ACE protein in NOD pancreata is increased about 2-fold. With the establishment of hyperglycemia, ACE levels continue to rise to reach approximately 3.5-fold prediabetic levels.


Figure 5
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  		FIG. 5. Expression of an AngII-generating system in NOD mice pancreata. A, Expression of ACE protein. Representative Western immunoblot of protein extracts from NOD mice pancreata between 2 and 22 wk of age. Pancreatic ACE protein is expressed as one band at approximately 190 kDa. An increase in ACE expression is seen in the 13- to 15-wk diabetic group that became significant in the severely diabetic group (20–22 wk). Blots were stripped and reprobed with actin antibody to control for loading errors. Average densitometry values of the samples were multiplied to obtain the arbitrary levels. Data are means ± SEM of n = 5 in each group. *, P < 0.05 vs. noromoglycemic mice (2–4 wk). B, ACE mRNA expression. PCR analysis of ACE mRNA transcripts revealed the presence of a considerable amount of ACE mRNA in normoglycemic pancreata. Visible up-regulation of ACE mRNA levels is detected in the second group and became significant in the older diabetic group (85- and 109-bp bands correspond to the amplified ACE and GAPDH, respectively). The ACE mRNA contents are expressed as ODs corrected for GAPDH. Data are means ± SEM of n = 5 in each group. *, P < 0.05, #, P < 0.02 vs noromoglycemic mice (2–4 wk).

 
ACE mRNA levels showed the same pattern seen with the protein (Fig. 5BGo). Interestingly, in ICR mice, ACE protein levels (Fig. 6AGo) were generally higher than in NOD mice, and the expression pattern was reversed, where young ICR mice had high levels of ACE and older mice expressed low ACE levels. ACE mRNA followed the same pattern (Fig. 6BGo). These results demonstrate the importance of the NOD genetic background in the control of the patterns of ACE expression and show that the progression to destructive insulitis in NOD mice is associated with higher intrapancreatic ACE mRNA and protein levels.


Figure 6
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  		FIG. 6. A, Representative Western immunoblot of protein extracts from ICR mice pancreata between 2 and 22 wk of age. Pancreatic ACE protein is expressed as one band at approximately 190 kDa. High levels of ACE expression seen in the 2- to 4- and 13- to 15-wk groups are decreased in the older mice at 20–122 wk. Blots were stripped and reprobed with actin antibody to control for loading errors. Average densitometry values of the samples were multiplied to obtain the arbitrary levels. Data are means ± SEM of n = 3 in each group. *, P < 0.05 vs. 2- to 4-wk group. B, PCR analysis of ACE mRNA transcripts revealed the presence of a considerable amount of ACE mRNA in ICR mice pancreata. Visible down-regulation of ACE mRNA levels is detected in the oldest group (85- and 109-bp bands correspond to the amplified ACE and GAPDH, respectively). The ACE mRNA contents are expressed as ODs corrected for GAPDH. Data are means ± SEM of n = 3 in each group. *, P < 0.05 vs. 2- to 4-wk group.

 
ACE distribution in the diabetic pancreas and colocalization with MCP-1 in ß-cells
Clear ACE and MCP-1 immunoreactivity could be seen in the islets cells of the diabetic pancreata (Fig. 7Go). Staining of serial sections with islet antibodies revealed the colocalization of ACE with MCP-1 in insulin- and somatostatin-positive cells and to a lesser extent in the glucagon-positive cells. The close proximity between an AngII-generating system and MCP-1 suggests a potential paracrine/autocrine interaction in the pancreatic islets and ß-cells. So, next we investigated the relationship between AngII and MCP-1 in the pancreatic islets. We also added IL-1ß, which induces the expression of MCP-1 in the pancreatic islets (13), as a positive control.


Figure 7
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  		FIG. 7. Colocalization of ACE and MCP-1 in NOD mice islets. To identify which cells in the islets express ACE and MCP-1, paraffin-embedded NOD pancreatic serial sections were stained with antibodies against insulin, glucagon, and somatostatin. It appears that most islet cells express ACE and MCP-1. Negative control samples where the primary antibody was omitted did not show nonspecific reaction. Original magnification, x200.

 
AngII alone and in combination with IL-1ß elicit an inflammatory response in the islets by stimulation of MCP-1
MCP-1 mRNA was analyzed in normoglycemic NOD mice islets exposed to AngII (10–7 mol/liter) with or without pretreatment with IL-1ß (0.1 ng/liter). AngII significantly induced MCP-1 mRNA in the pancreatic islets, an effect that was potentiated by IL-1ß. Losartan, an AT1R antagonist, blocked the AngII-mediated induction of MCP-1 but not the IL-1ß-mediated induction (Fig. 8Go). These data demonstrate the contribution of AngII as a proinflammatory molecule in the islets.


Figure 8
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  		FIG. 8. AngII mediates an AT1R-depednet MCP-1 induction in NOD mice islets. Islets were preincubated for 1 h with AT1R antagonist losartan before addition of AngII (10–7 mol/liter) or IL-1ß. MCP-1 and GAPDH mRNA content were analyzed by RT-PCR. The MCP-1 mRNA contents are expressed as ODs corrected for GAPDH (300- and 109-bp bands correspond to the amplified MCP-1 and GAPDH, respectively). Values are expressed as mean ± SEM of three experiments. *, P < 0.05 vs. untreated islets; #, P < 0.05 vs. cells treated with AngII alone using one-way repeated ANOVA with subsequent all-pairwise comparison procedure by Student’s t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The molecular and cellular mechanisms that contribute to monocytes and T cells infiltrating into the islets as a key initial step in the development of insulitis are poorly understood. MCP-1 is a peptide chemokine that plays an important role in monocyte and T cell recruitment to the islets (4, 7, 13). Previous studies have shown that the temporal pattern of MCP-1 expression correlates with the progression of insulitis and ß-cell destruction in NOD mice (4, 6), and when MCP-1 was overexpressed in the islets of transgenic mice, it induced mononuclear cell infiltration (10). However, only few studies investigated the upstream effector molecules and the signaling cascades involved in regulation of MCP-1 in the islets and ß-cells. In the present study, we introduce AngII as a novel trigger for MCP-1 expression in the pancreatic islets and ß-cells and propose a previously undescribed role for AngII in islet inflammation in type 1 diabetes.

We show here that AngII is a potent stimulator of MCP-1 expression in the pancreatic islets and ß-cells. AngII induced MCP-1 accumulation rapidly in the pancreatic islets and with significant magnitude. In ß-cells, dose-response studies demonstrated a significant induction of MCP-1 mRNA and protein levels at a physiological concentration of AngII (10–7 mol/liter). The maximal effect concentration at 10–7 mol/liter is similar to other AngII actions that have been reported (25, 26).

We also show that AngII-induced MCP-1 gene expression occurs through an AT1R-mediated mechanism and through induction of its promoter activity, as demonstrated by our promoter studies (Fig. 3Go). Addition of losartan, an AT1R antagonist, prevented the AngII-MCP-1 production, an effect that was not observed when PD123319, an AT2R antagonist, was added. AT1R belongs to the heterotrimeric G protein-coupled receptor superfamily. Because increased the expression of AT1R mRNA in vascular smooth muscles has been shown to result in elevation of the functional response to AngII (27), we assume that AngII itself could have a regulatory effect on AT1R in ß-cells, which could consequently contribute to AngII-MCP-1 induction. Indeed, AngII addition to ß-cells results in a concomitant increase in AT1R mRNA expression (unpublished observation), confirming observations from previous studies in renal proximal tubules (29). Thus, it is possible that AngII induces MCP-1 transcripts in the pancreatic islets and ß-cells directly through acting on its promoter and indirectly through AT1R up-regulation.

Our data show that MCP-1 promoter was induced significantly as early as after 1 h of stimulation. The implications of this acute response to AngII could be critical in conditions where pancreatic AngII generation is increased. Additional studies are required to analyze the AngII-specific cis-elements on the MCP-1 promoter and whether or not the AngII-induced MCP-1 up-regulation is mediated through NF-{kappa}B, which is a primary transcription factor necessary for triggering the expression of MCP-1 in ß-cells (13). Studies in this regard are currently ongoing in our lab.

MAPKs encoded by the ERK genes are a family of serine/threonine protein kinases activated as early responses to a variety of stimuli involved in cell growth, transformation, and differentiation (30). They are also involved in the activation of AP-1 and NF-{kappa}B (31). Two isoforms of ERK referred to as p44 (ERK1) and p42 (ERK2), are activated by phosphorylation of threonine and tyrosine residues by MAPK kinase (MEK) (32, 33). AngII rapidly activates MAPKs, particularly ERK1 and ERK2, in vascular smooth muscle cells (30, 34) and in pancreatic cancer cells (12). Using a selective inhibitor for MEK activation, U0126, we demonstrated that AngII-induced MCP-1 mRNA occurs through a MEK-sensitive mechanism (Fig. 4BGo). AngII had no effect within this time period on the phosphorylation of either p38 or SAPK/JNK. Additional studies are now required to fully delineate the specific signaling pathway by which AngII ultimately modulates MCP-1 synthesis in the islets.

No data have been reported previously concerning the regulation of AngII generation during islet inflammation in type 1 diabetes. In the present study, we demonstrate for the first time the presence of an active AngII-generating system in the pancreata of NOD mice. As early as 2 wk of age, ACE mRNA and protein were detectable in the nondiabetic pancreata. Development of insulitis and progression to hyperglycemia and ß-cell destruction correlated well with increased expression levels of ACE, suggesting that AngII generation is an active ongoing process with diabetes advancement. We also show that ACE is constitutively expressed in most of the islet cells, colocalizing with MCP-1, suggesting an endogenous autocrine/paracrine interaction.

Several studies have shown that the use of angiotensin blockade therapies could improve islet function and glucose intolerance in type 2 diabetes patients (35) and in animal models (36). The mechanisms mediating these effects are not clearly understood. However, adding ACE inhibitors in vitro to the islets leads to their protection against glucotoxicity and oxidative stress (37). AngII itself affects the islet short-term insulin release (38), but the impact of the long-term effects of AngII on the islet functions are yet to be determined.

The novel data from our study add new information about the role of the local pancreatic RAS as a source of islet inflammation and suggest that targeting AngII could be used as a novel therapeutic strategy to reduce MCP-1 levels in the islets and inhibit ß-cell-directed immunity in type 1 diabetes. Studies in this regard are currently ongoing in our lab.

Our study demonstrates that AngII elicits an inflammatory response in the islets and ß-cells by stimulation of MCP-1 production through an AT1R-ERK1/2-dependent mechanism. We also show that hyperglycemia and progression to diabetes correlate with up-regulation of ACE, the enzyme responsible for production of AngII. It is not clear from this study that AngII regulates MCP-1 in vivo, and our data from the NOD mice remain associative. However, the existence of AngII as a potential endogenous trigger for MCP-1 in the islets is unique and could provide a novel target for prevention of monocyte recruitment into the islets during the early stages of insulitis in type 1 diabetes.


   Footnotes
 
This work was supported by grants from the American Diabetes Association, Diabetes Transplant Fund, and Diabetes Action Research and Education Foundation. C.F.G. is supported by the Department of Surgery, Thomas Jefferson University.

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 15, 2007

Abbreviations: ACE, Angiotensin-converting enzyme; AngI, angiotensin I; AT1R, AngII type 1 receptor; FBG, fasting blood glucose; JNK, c-jun NH2-terminal protein kinase; MCP-1, monocyte chemoattractant protein-1; MEK, MAPK kinase; NF-{kappa}B, nuclear factor-{kappa}B; NOD, nonobese diabetic; RAS, renin-angiotensin system; SAPK, stress-activated protein kinase.

Received October 5, 2006.

Accepted for publication February 6, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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