Interleukin (IL)-1β, the sole proinflammatory cytokine released from pancreas-infiltrating macrophages, inhibits glucose-stimulated insulin secretion (GSIS), causing hyperglycemia in Cohen diabetes-sensitive (CDs) rats fed a diabetogenic-diet (CDs-HSD). Because IL-1β blockade is a potential therapeutic target in diabetes, we examined whether treating CDs rats with IL-1β antibody (IL-1βAb; 0.5 mg/kg body weight) could counteract the inhibition of GSIS and hyperglycemia. We found that daily IL-1βAb injections had a beneficial effect on glucose tolerance and insulin secretion in CDs-HSD rats. In the oral glucose tolerance test, IL-1βAb–treated CDs-HSD rats showed lower blood glucose concentrations (P < 0.001) and higher GSIS (P < 0.05) compared with nontreated CDs-HSD rats. IL-1βAb treatment also protected the exocrine pancreas; the number of infiltrating macrophages decreased by 70% (P < 0.01) and IL-1β expression decreased by 85% (P < 0.01). In parallel, a 50% reduction (P < 0.01) in the rate of apoptosis and in fat infiltration (P < 0.05) was noted in the exocrine parenchyma of IL-1βAb–treated CDs-HSD rats compared with nontreated CDs-HSD rats. Altogether, these data demonstrate that blocking IL-1β action by IL-1βAb counteracted β-cell dysfunction and glucose intolerance, supporting the notion that prevention of pancreas infiltration by macrophages producing IL-1β is of crucial importance for the preservation of β-cell function and prevention of diabetes.

Recent studies suggest that interleukin (IL)-1β is an important player in β-cell dysfunction and death in type 1 and type 2 diabetes mellitus (T2DM) (17). IL-1β has been shown to reduce glucose-stimulated insulin secretion (GSIS) in isolated islets (4,5). In vivo studies of patients with T2DM and animal models have shown evidence of elevated IL-1β expression in the pancreas, in insulin target tissues, as well as in the circulation (1,3,4,6,7). Moreover, IL-1β antibody (IL-1βAb) treatment has improved glycemic control in animals and humans (3,4,8,9), supporting the notion that efficient targeting of IL-1β with a specific monoclonal antibody may prevent hyperglycemia.

The Cohen diabetes-sensitive (CDs) rat is a genetic model of nutritionally induced diabetes that maintains normoglycemia on a regular diet (RD) but develops impaired GSIS and hyperglycemia when fed a high-sucrose, low-copper diet (HSD). In this animal model the development of hyperglycemia is associated with a mild pancreatic inflammation, that is, peri-islet infiltration with fat and macrophages expressing the proinflammatory cytokine IL-1β (6,7). In a recent in vitro study we demonstrated that IL-1β modulates GSIS by inhibiting the activity of the islet mitochondrial respiratory chain enzyme cytochrome c-oxidase (COX) (5). In the current study we analyzed the role of IL-1β in β-cell dysfunction by neutralizing IL-1β in vivo and examined whether treating CDs rats with a rat-specific IL-1βAb counteracts GSIS inhibition and hyperglycemia. The CDs rat is a unique animal model for this purpose because IL-1β is the sole cytokine expressed in the infiltrating macrophages surrounding the islets, which permits its role in β-cell dysfunction and disease manifestation to be studied without the confounding effect of other cytokines.

Animals and Diets

CDs rats are bred and maintained in the animal facility at the Hebrew University School of Medicine, Jerusalem, Israel. Rats were fed ad libitum an RD (Teklad, 2018; Harlan Laboratories) composed of 54% carbohydrate (ground whole wheat, ground alfalfa, and bran); 21% protein (skimmed milk powder); 6% fat; 5% salts, vitamins, and trace elements, including an adequate copper content (15 ppm); 7% humidity; and 7% ash. Custom-prepared HSD contained 72% sucrose; 18% vitamin-free casein; 5% salt mixture no. II USP (MP Biomedicals, Solon, OH); 4.5% butter; and 0.5% corn oil, vitamins, and copper (0.9 ppm) (10,11). Animal studies were approved by the institutional committee for animal use and care. Eight-week-old male CDs rats fed an RD were either switched to HSD for an additional period of 15 days or maintained on the RD for the same period. Body weight was monitored weekly.

Study Design

Preventive therapy was performed using a custom-made, rat-specific monoclonal antibody (Eurogentec, Liege, Belgium). This IL-1βAb contains the epitope of the SILK6 clone known to bind to the amino acid sequence 123–143 of IL-1β, a region crucial for the binding to its receptor. Thus this IL-1βAb effectively binds to the active site of the released cytokine IL-1β, thereby blocking its biological activity (12). CDs rats fed an HSD for 15 days were treated in parallel with saline or injections of IL-1βAb (0.5 mg/kg body weight, subcutaneously) for the first 5 days or the entire 15 days of HSD feeding. Normoglycemic CDs rats fed an RD for 15 days and treated with saline were studied as a normoglycemic reference group. In this study we used the dose range of IL-1βAb used in various human studies (1315).

Glucose Tolerance

Rats fasted overnight underwent an oral glucose tolerance test (OGTT) performed on day 16 following 15 days of HSD feeding. Blood glucose concentration was assessed before (t = 0) and 30, 60, 90, and 120 min after gavage administration of glucose (350 mg glucose/100 g body weight). Blood glucose was measured using a glucometer (Elite; Bayer, Leverkusen, Germany) and serum insulin with an ELISA assay (Mercodia AB, Uppsala, Sweden).

Collection of Tissues

Rats were killed on day 16 immediately after the OGTT; the pancreas and spleen were dissected, cleaned of external fat, and weighed, as described previously (6,7,11). Results were compared with tissues from CDs rats fed an RD or HSD for 15 days.

Immunohistochemistry and Morphological Evaluation

The pancreases of the different experimental groups were dissected and fixed either in buffered 4% paraformaldehyde for immunohistochemistry or in a mixture of 2% paraformaldehyde and 2% glutaraldehyde for quantification of adipocytes containing fat vacuoles (6,7). Immunohistochemistry used the avidin biotin complex or a double immunofluorescence method to identify β-cells, CD68 macrophages, and IL-1β expression using the following primary antibodies: insulin (A565; DAKO, Germany), CD68 macrophages (clone ED1), IL-1β (AAR15G, clone Silk6 [12]), interferon (IFN)-γ (MCA1301), tumor necrosis factor (TNF)-α (AAR33), and MCP1 (AAR31; Serotec, Düsseldorf, Germany) (6,7), IL-2 (AF-502-NA; R&D Systems, Wiesbaden, Germany), and IL-6 (500-P73G; PeproTech, Hamburg, Germany). Paraffin and semithin sections were examined with a BX61 light-microscope (Olympus, Hamburg, Germany). Areas of acinar cells and adipocytes were determined morphometrically, and the proportion of adipose tissue was expressed as a percentage of the total pancreas area measured on the same section (6,7). The number of macrophages and IL-1β expression were quantified in four different areas per section in four different animals per treatment group (6,7). Apoptosis in exocrine and endocrine pancreatic parenchyma was quantified by TUNEL staining (Roche, Mannheim, Germany).

Statistics

Data are represented as mean ± SEM. Comparisons between the different treatment groups and the RD control groups were analyzed by two-way ANOVA and the Bonferroni posttest to compare multiple groups with the SigmaStat program (Jandel Corp., San Rafael, CA).

Glucose Tolerance Test and Pancreas Organ Weight

CDs rats fed an HSD for 15 days and treated in parallel with IL-1βAb for 5 days demonstrated a tendency toward lower blood glucose, while 15 days of IL-1βAb treatment revealed a significant reduction in blood glucose followed by a significantly higher insulin response (P < 0.05; Fig. 1A and B) compared with CDs rats fed an HSD for 15 days. The insulinogenic index (insulin/glucose) demonstrated an increased ability of CDs rats treated with IL-1βAb for 15 days to secrete insulin in response to glucose both 30 and 60 min after glucose administration (P < 0.01 and P < 0.05, respectively, vs. control; Fig. 1C). In fact, glucose and insulin concentrations, as well as the insulinogenic index, were comparable with those observed in normoglycemic CDs rats fed an RD (Fig. 1A–C). IL-1βAb treatment prevented the reduction in pancreas weight that occurred in hyperglycemic CDs rats. Pancreas weight of these rats was, therefore, greater than of nontreated CDs rats fed an HSD for 15 days and comparable with that of normoglycemic CDs rats fed an RD (Fig. 1D). No differences in body weight (data not shown) were observed between the different study groups.

The glucose area under the curve of CDs rats treated with IL-1βAb was significantly lower (P < 0.01) compared with that of nontreated CDs rats fed an HSD for 15 days and was comparable with that of normoglycemic CDs rats fed an RD (Fig. 1E). Insulin area under the curve (Fig. 1F) was significantly higher (P < 0.05) in IL-1βAb–treated rats compared with nontreated CDs rats fed an HSD for 15 days and was comparable with that of normoglycemic CDs rats fed an RD, further confirming the effectiveness of the IL-1βAb treatment.

Morphological Analysis of Pancreatic Parenchyma in IL-1βAb–Treated CDs Rats Fed an HSD Compared With Untreated CDs Fed an HSD or RD

Pancreatic β-cells of IL-1βAb–treated or untreated CDs rats fed an HSD demonstrated comparable dense insulin immunoreactivity (Fig. 2A and B). The percentage of apoptosis measured in acinar cells of the exocrine pancreatic parenchyma was significantly lower in IL-1βAb–treated rats compared with control untreated CDs rats fed an HSD (Fig. 2C and D and Table 1). The total number of macrophages infiltrating the exocrine pancreas and IL-1β expression in these macrophages, as well as the percentage of adipose tissue in the exocrine parenchyma, were all significantly reduced after 15 days of IL-1βAb treatment compared with untreated HSD-fed control CDs rats (P < 0.01 and P < 0.05, respectively; Fig. 2E–J and Table 1). Shorter duration of IL-1βAb treatment during the first 5 days of the 15-day HSD feeding showed a significant (P < 0.05) albeit weaker protective effect on these parameters compared with 15 days of IL-1βAb treatment (Table 1). Normoglycemic CDs rats fed an RD displayed a small number of macrophages that did not express IL-1β and a small amount of adipose tissue in their exocrine parenchyma (Table 1).

Additional expression of the proinflammatory markers analyzed (the chemokine MCP1 and the cytokines IL-2, IL-6, TNF-α, and IFN-γ) were not expressed to a significant extent in the infiltrating macrophages.

Novel treatments for diabetes, which specifically target the preservation of β-cell function, have been the focus of recent studies (14,8,13,16). In our study the deleterious effects of a diabetogenic HSD, causing β-cell dysfunction in a CDs rat model (57,11), were counteracted using a rat-specific IL-1β monoclonal antibody. This antibody also protected the exocrine pancreas from the deleterious effects induced by the HSD. Acinar cell apoptosis, infiltration of fat, and the total number of peri-islet macrophages, as well as the percentage of those expressing IL-1β (57,11), were all markedly lower in IL-1βAb–treated CDs rats. Therefore the current observations showing that specific IL-1βAbs are able to fully counteract the deleterious process triggered exclusively by IL-1β provide convincing evidence linking IL-1β toxicity and diabetes development in this animal model. Notably, the IL-1βAb dose selected in our study was comparable with the dose that successfully counteracted the deleterious effects of IL-1β on glucose homeostasis and β-cell function in diabetic patients (14,15). The extent of protection provided by IL-1βAb was associated with the duration of the treatment, demonstrating pronounced protection when IL-1βAb was injected every day during the 15 days of HSD feeding and a weaker effect when IL-1βAb was given during only the first 5 days of HSD feeding. As shown in our previous studies (57,11,14,15) and confirmed in the current study, IL-1β is the sole proinflammatory cytokine expressed by macrophages infiltrating the pancreas. An expression of other proinflammatory markers such as the chemokine MCP1 or the cytokines IL-2, IL-6, TNF-α, and IFN-γ could not be detected in the infiltrating macrophages. This latter observation varies from the Goto-Kakizaki rat model of T2DM, in which expression of these additional cytokines and chemokines also was observed (1719). This confirms that in the CDs rat model, IL-1β toxicity is apparently the crucial and exclusive mediator of GSIS inhibition and hyperglycemia. On the other hand, the observation that blocking IL-1β action reduced both IL-1β expression, as well as the number of infiltrating macrophages, successfully preventing both inflammation and β-cell dysfunction, is in agreement with studies performed using the Goto-Kakizaki rat as well as in diet-induced obese hyperglycemic mice. In these studies IL-1βAb treatment improved inflammation, glucose metabolism, and β-cell function (3,9,1720). Thus the cumulative evidence from the literature and our current observations reinforce the important role that IL-1β plays in mediating β-cell toxicity in T2DM (14,15).

The association to diabetic patients was mentioned in preclinical studies reporting increased IL-1β production, reduced insulin secretion, and β-cell dysfunction (1,8,14,15,19). The precise mechanism inducing increased IL-1β production in T2DM, however, is not fully elucidated. In our recent in vivo studies we demonstrated reduced activity of the islet mitochondrial respiratory chain enzyme COX and GSIS in association with peri-islet infiltration of macrophages expressing IL-1β (6,7). We also documented reduced COX activity concurrent with GSIS inhibition in isolated islets of CDs rats exposed in vitro to IL-1β (5). We therefore hypothesize that the IL-1β secreted by peri-islet–infiltrating macrophages triggers a vicious sequence of events causing β-cell dysfunction (57). In the current study the anti–IL-1β treatment probably breaks this vicious sequence of events, decreasing both the number and the activity of the infiltrating macrophages, thus maintaining the integrity of the exocrine parenchyma as well as preventing β-cell dysfunction. This indicates that IL-1β neutralization is sufficient to prevent β-cell failure, thereby guaranteeing the success of the preventive therapy.

In conclusion, our study supports the concept that inflammation in close vicinity to islets may have a detrimental effect on the ability of β-cells to secret insulin in response to glucose stimulation (14,15). Moreover, it illustrates the complexity of the interrelationships between inflammation, β-cell dysfunction, and diabetes development and supports the notion that preventing pancreatic inflammation is important to preserve β-cell function and avoid diabetes development.

Funding. This work was supported by a grant for a joint research project by the Ministry for Science and Culture of Lower Saxony, Hannover, Germany.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. G.A.-H. researched data and wrote, reviewed, and edited the manuscript. A.J. designed the study, researched data, and wrote the manuscript. S.L. and I.R. designed the study, interpreted data, and reviewed and edited the manuscript. S.W.-Z. designed the study, researched data, and wrote, reviewed, and edited the manuscript. S.W.-Z. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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