Preventive Effect of Molecular Iodine in Pancreatic Disorders from Hypothyroid Rabbits

Pancreatic alterations such as inflammation and insulin resistance accompany hypothyroidism. Molecular iodine (I2) exerts antioxidant and differentiation actions in several tissues, and the pancreas is an iodine-uptake tissue. We analyzed the effect of two oral I2 doses on pancreatic disorders in a model of hypothyroidism for 30 days. Adult female rabbits were divided into the following groups: control, moderate oral dose of I2 (0.2 mg/kg, M-I2), high oral dose of I2 (2.0 mg/kg, H-I2), oral dose of methimazole (MMI; 10 mg/kg), MMI + M-I2,, and MMI + H-I2. Moderate or high I2 supplementation did not modify circulating metabolites or pancreatic morphology. The MMI group showed reductions of circulating thyroxine (T4) and triiodothyronine (T3), moderate glucose increments, and significant increases in cholesterol and low-density lipoproteins. Acinar fibrosis, high insulin content, lipoperoxidation, and overexpression of GLUT4 were observed in the pancreas of this group. M-I2 supplementation normalized the T4 and cholesterol, but T3 remained low. Pancreatic alterations were prevented, and nuclear factor erythroid-2-related factor-2 (Nrf2), antioxidant enzymes, and peroxisome proliferator-activated receptor gamma (PPARG) maintained their basal values. In MMI + H-I2, hypothyroidism was avoided, but pancreatic alterations and low PPARG expression remained. In conclusion, M-I2 supplementation reestablishes thyronine synthesis and diminishes pancreatic alterations, possibly related to Nrf2 and PPARG activation.


Introduction
Preclinical and clinical studies have described a close relationship between thyroid status and pancreatic function [1,2].Thyroid hormones are critical for the normal development and function of the endocrine and exocrine pancreas during the neonatal period [3].In adults, thyroid hormones induce pancreatic acinar cell proliferation [4].Hypothyroidism is accompanied by pancreatic alterations such as pancreatitis, insulitis, insulin resistance, and type 2 diabetes mellitus (T2DM) [1,5,6].Pharmacological hypothyroidism modifies insulin secretion and the expression of GLUT4, hexokinase, and glucokinase [6][7][8][9][10].Previous studies have reported that methimazole-induced hypothyroidism in female rabbits promotes interstitial edema and degenerative changes in pancreatic acinar cells [11,12].Many of these alterations could be explained by the direct effect of thyroid hormones on differentiation target genes and their impact on the oxidative state.In the thyroid and other organs, the inflammation (edema, vascularization, and infiltration) observed during hypothyroidism has been explained by the low expression of antioxidant thyroid-dependent enzymes such as catalase (Cat) and glutathione peroxidase [8].
Moreover, some authors have postulated that the pancreatic redox equilibrium could be modulated by the local presence of iodine per se.Iodine may have an ancestral antioxidant function in all iodide-concentrating cells, from primitive algae to more recent vertebrates [13].In the chemical form of molecular iodine (I 2 ), its reductive capacity in vitro (by the ferric reducing antioxidant power assay) is ten times more efficient than ascorbic acid and 100 times more potent than potassium iodide, while in vivo, it binds reactive oxygen species, thereby neutralizing • OH radicals, resulting in less cellular oxidative damage [14].It has also been demonstrated that I 2 acts as a direct activator of the nuclear factor erythroid-2-related factor-2 (Nrf2) pathway, triggering the expression of several phase II protective antioxidant enzymes such as superoxide dismutase type 1 (Sod1) and Cat [15].In addition, I 2 can bind to arachidonic acid and activate the peroxisome proliferator-activated receptor type gamma (PPARG), prompting metabolic, antioxidant, and immuno-regulatory effects [16].Studies have only recently begun analyzing the mechanisms involved in the actions of iodine in the pancreas.Thus, in the spontaneous type 1 diabetes model (Bio-Breeding/Worcester rats, or BB rats), moderate iodine supplementation prevented the incidence of this pathology, decreasing insulitis [17].Similar results were described in the murine model of streptozotocin-induced pancreatitis, where inflammation processes and fibrosis activation by stellar pancreatic cells were prevented [18].Moreover, it is well established that B-cells exhibit a continuous expression of deiodinase type 3 (Dio3), which, in addition to regulating the active thyroid hormones, generates local concentrations of free iodine [19].The purpose of the present study was to evaluate oral I 2 supplementation in pancreatic disorders associated with hypothyroidism.

Results
Figure 1 shows the food consumption and body weight gain for all groups.During the first two weeks, the MMI treatment resulted in reduced food intake, which could be attributed to the sour taste of MMI.However, no significant changes were observed compared to the control group (Figure 1A).The body weight gain was similar between groups (Figure 1B).observed during hypothyroidism has been explained by the low expression of antioxidant thyroid-dependent enzymes such as catalase (Cat) and glutathione peroxidase [8].
Moreover, some authors have postulated that the pancreatic redox equilibrium could be modulated by the local presence of iodine per se.Iodine may have an ancestral antioxidant function in all iodide-concentrating cells, from primitive algae to more recent vertebrates [13].In the chemical form of molecular iodine (I2), its reductive capacity in vitro (by the ferric reducing antioxidant power assay) is ten times more efficient than ascorbic acid and 100 times more potent than potassium iodide, while in vivo, it binds reactive oxygen species, thereby neutralizing °OH radicals, resulting in less cellular oxidative damage [14].It has also been demonstrated that I2 acts as a direct activator of the nuclear factor erythroid-2-related factor-2 (Nrf2) pathway, triggering the expression of several phase II protective antioxidant enzymes such as superoxide dismutase type 1 (Sod1) and Cat [15].In addition, I2 can bind to arachidonic acid and activate the peroxisome proliferator-activated receptor type gamma (PPARG), prompting metabolic, antioxidant, and immunoregulatory effects [16].Studies have only recently begun analyzing the mechanisms involved in the actions of iodine in the pancreas.Thus, in the spontaneous type 1 diabetes model (Bio-Breeding/Worcester rats, or BB rats), moderate iodine supplementation prevented the incidence of this pathology, decreasing insulitis [17].Similar results were described in the murine model of streptozotocin-induced pancreatitis, where inflammation processes and fibrosis activation by stellar pancreatic cells were prevented [18].Moreover, it is well established that B-cells exhibit a continuous expression of deiodinase type 3 (Dio3), which, in addition to regulating the active thyroid hormones, generates local concentrations of free iodine [19].The purpose of the present study was to evaluate oral I2 supplementation in pancreatic disorders associated with hypothyroidism.
Table 2 shows the analysis of pancreatic islets.The study involved measuring 100-250 islets per group.Moderate or high I 2 doses did not impact the percentage of islets, regardless of their size or the mean length of each category.However, MMI-induced hypothyroidism (MMI group) caused a reduction in the number of cells in small islets, which suggests decreased cell proliferation.The moderate I 2 dose prevented the reduction, whereas the high dose did not.
Since the moderate or high dose of I 2 did not modify the circulating or pancreatic parameters, our subsequent analysis focused on these groups: Control, MMI, MM + M-I 2 , and MMI + H-I 2 .
Figure 3 shows the status of the pancreatic acinus.Compared with the control group, the MMI group increased the area covered by collagen deposits (14.8 + 0.05 vs. 2.4 + 0.2) and proteoglycan fibers (17.3 + 0.06 vs. 2.4 + 0.03) on interlobular septa (Figure 3A).Moreover, a significant increase was observed in the area covered by blood vessels (823 ± 38 vs. 292 ± 41) and in the number of immune cells in the blood vessels of islets (9.3 ± 1.5 vs. 4.6 ± 0.3; Figure 2B) in the MMI group compared with the control group.Moderate I 2 supplementation reduced collagen and proteoglycan deposits to basal levels, while high I 2 supplementation could not recover the acinar injury induced by MMI.However, this high I 2 dose maintained the basal amount of blood vessels and immune cell infiltration in islets.The image in Figure 4 depicts the immunoreactivity of insulin and PPARG and the expression of insulin, PPARG, and GLUT4 proteins in whole pancreas tissue.The MMI group had a higher expression of insulin (0.56 + 0.09 vs. 0.06 + 0.02) and GLUT4 (0.95 + 0.09 vs. 0.21 + 0.06) than the control group, indicating a possible alteration in insulin synthesis and delivery.This group also exhibited a decreased PPARG expression (1.0 + 0.02 vs. 2.8 + 0.05).Moderate I 2 concentrations prevented the increase in insulin and GLUT4 expression caused by hypothyroidism and retained the PPARG amount (2.1 + 0.07).However, the high I 2 dose did not prevent the rise in insulin (5.2 + 0.07) and GLUT4 (0.6 + 0.02) or the reduction of PPARG expression (0.9 + 0.05) caused by MMI treatment.

Discussion
The present study confirms that hypothyroidism injures pancreatic physiology and that the oxidative stress observed in this condition could be related to altered signaling in the Nrf2/Keap1/ARE pathway with deficient expression of antioxidant enzymes such as Sod1 and Cat, as has been previously suggested [7,8].The evident attenuation of some pancreatic damage observed in animals supplemented with moderate doses of I 2 could be partly explained by the re-establishment of synthesis and circulating levels of thyroid hormone.These data are consistent with previous work in which we demonstrated that iodide transporters such as NaI-symporter (NIS) or Pendrin do not uptake I 2 , but the thyroid gland can uptake this form of iodine through a facilitating mechanism [20].This agrees with data found in a family with a specific inactivated NIS mutation, but the consumption of Laminaria algae, which contain different chemical forms of iodine (including I 2 ), attenuated the hypothyroidism syndrome associated with this alteration [21].Our results indicate that this oxidized chemical form of iodine does not require thyroid peroxidase, which is inhibited by MMI, to bind thyroglobulin-generating T4 and T3.The present work also shows that moderate I 2 supplementation partially reestablished euthyroid pancreatic status by preventing the decrease in Dio1 and maintaining the high expression of Sod1 and Cat; however, it could not normalize Dio3.Nevertheless, the moderate dose of I 2 was adequate to prevent circulating lipid alterations (cholesterol and TAG).Indeed, the normalized values of sCD163, a biomarker of macrophage activation in various inflammatory diseases (e.g., macrophage activation syndrome and sepsis), and during the development of T2DM [22,23] are consistent with the prevention observed in almost all pancreatic damage such as fibrosis, collagen, and immune infiltrations.In addition, these normalized values restored pancreatic functionality by decreasing insulin storage and GLUT4 overexpression.This glucose transporter has been related to the energy required for glucagon and insulin synthesis [24].
Although we cannot separate the effect of thyroid hormones reestablished from the moderated I 2 dose, several reports have described the direct actions of iodine per se.Hypercholesterolemia was reduced in overweight women with iodine supplementation [25].Moderate iodine diets improve the lipid profile in mice, increasing LDLR and scavenger receptor class B type-1 in the liver [26].In addition, part of the effects observed with moderate I 2 supplementation could be explained by the activation of PPARG receptors.I 2 supplementation is accompanied by 6-iodolactone (6-IL) formation in the mammary gland.6-IL is an iodolipid derived from arachidonic acid and an agonist ligand of PPARG [27].It has been proposed that the improvement in glucose homeostasis observed with thiazolidinediones, PPARG agonists, could be related to enhanced B-cell function.The activation of PPARG in B-cells involves the induction of anti-inflammatory mechanisms [28], the reduction of oxidative stress [29], and the inhibition of amyloid formation [30].
Moreover, the prevention of inflammation and fibrosis in the pancreatic acini observed in the MMI + M-I 2 group could be associated with the anti-inflammatory actions of I 2 .In vitro, I 2 promotes the release of anti-inflammatory cytokines such as interferon-gamma, interleukin 6 (IL6), IL10, and IL8-CXCL8 in normal lymphocytes [31].The protection against fibrosis formation could also be explained by the activation of PPARG by stimulating phosphatase and tensin homolog expression, which decreases the TGFB1 and PI3K/Akt pathways [32].It has been described that pancreatic stellate cell (PSc) activation promotes the failure of B-cell function and increases fibrosis [33].Natural compounds with antioxidant properties, such as resveratrol and curcumin, can inhibit the activation of these cells and diminish the production of reactive oxygen species and collagen in vitro [34,35] and in mice with cerulein-induced chronic pancreatitis [36].
Hypothyroidism increases the expression of GLUT4 and insulin.Considering that glucose is the most crucial factor in regulating the architecture of islets, the moderate dose of I 2 restored the effects found in the MMI group.It has been described that B-cell mass and islets increase during the first stage of T2DM.This increase is accompanied by greater insulin production [37,38] or excessive synthesis of misfolded proinsulin [39].A new formation of islets has been observed in the hypothyroid pancreas [10].Two mechanisms are involved in new islet formation: 1) the replication of preexisting B-cells and neogenesis [40] or 2) A-cell phenotype modification to B-cells through the activation of Arx and Dnmt1 genes to regenerate islet function [41].In this sense, in our previous studies using the murine model of streptozotocin-induced pancreatitis, I 2 supplementation prevented the increase in the number of A-cells, thereby decreasing the inflammation and canceling the fibrosis activation by PSc [18].We also found that I 2 modulates the cell cycle and participates in the transdifferentiation of the cell population through PPARG activation in cancerous mammary cells [16].Lipotoxicity has been proposed as a mechanism of B-cell failure, mainly through ceramides and oxidative lipid production, promoting alterations in the mitochondria and nucleus [42,43].In this regard, I 2 supplementation inhibits lipid peroxidation in both normal and cancer cells [14,18], and we observed the same effect in the present work with both I 2 concentrations, indicating a sustained antioxidant effect.
On the other hand, the combination of MMI and high doses of I 2 maintained a euthyroid state, but it was accompanied by differential serum and pancreatic alterations that were not observed with I 2 alone (H-I 2 group).The MMI + H-I 2 group showed almost all circulating parameters in the normal range, including elevated HDL-C and lower triglycerides and VLDL-C, indicating a favorable lipid metabolism.However, the high concentration of I 2 was unable to prevent the increased amount of collagen and proteoglycans in the interlobular septa of pancreatic tissue, as well as the infiltration of immune cells, even with the increased response on antioxidant signaling (Nrf2, Sod1, and Cat), suggesting that these pancreatic damages could be related to the extrathyroidal effects of MMI + H-I 2 .Several recent reports have indicated that MMI treatment could be accompanied by pancreatic injury, and one was administered with elevated concentrations of iodine [44].The mechanism involved in this combination is unknown, but the inhibition of PPARG expression could explain this unexpected result.Studies that directly correlate MMI exposure with the inhibition of PPARG actions do not exist, but it has been described that low PPARG expression increases PSc proliferation and activation, generating high fibrosis and collagen deposits [42].
These results show that I 2 supplementation at moderate doses prevents some metabolic and pancreatic alterations associated with hypothyroidism.The anti-inflammatory and lipid modulation effects could be related to antioxidant Nrf2 mechanisms and PPARG activation.The therapeutic effect of I 2 in chronic pancreatic diseases related to inflammation is currently being analyzed.

Animals
Adult chinchilla-breed virgin female rabbits (Oryctolagus cuniculus) of 8-9 months were housed under controlled temperature (20 ± 2 • C) conditions and a 16:8 h light: dark cycle.Animals were provided pellet food (120 g/day) and water ad libitum.Rabbits were randomly assigned to the following experimental groups: control (control; n = 6), moderate oral (drinking water) dose of I 2 (0.2 mg/kg; M-I 2 ; n = 3), a high oral dose of I 2 (2.0 mg/kg; H-I 2 ; n = 4); oral dose of methimazole (10 mg/kg; MMI; n = 6), MMI + M-I 2 (n = 6), and MMI + H-I 2 (n = 6).The body weight of the females was measured before and at the end of treatments.After four weeks of treatment, the rabbits were anesthetized with sodium pentobarbital (90 mg/kg, i.p.) and euthanized with an overdose of the same anesthetic.The Ethics Committee at Universidad Autónoma de Tlaxcala approved this experimental design following the guidelines of Mexican Law for the Production, Care, and Use of Laboratory Animals.Immediately after death, the left lobe of the pancreas was collected, histologically processed, embedded in Paraplast X-TRA (Sigma-Aldrich, St Louis, MO, USA), and longitudinally cut at a thickness of 5 µm using a microtome (Thermo Scientific, Model Finesse 325, Waltham, MA, USA).The right lobe of the pancreas was frozen at −80 • C for biochemical measures.

Thyroid Hormones and Metabolic and Inflammatory Variables
Blood samples were obtained by cardiac puncture from rabbits fasted for 12 h at the end of the experiment.The serum concentration of total thyroxine (T4) and total triiodothyronine (T3) were measured using ELISA (International Immuno-Diagnostics, Foster City, CA, USA).Glucose, total cholesterol (TC), and triacylglycerol (TAG) were measured using standard enzymatic methods (ELITech, Puteaux, France).High-density lipoprotein cholesterol (HDL-C) was measured by a precipitating method (ELITech, Puteaux, France).The concentrations of low and very low-density lipoproteins (LDL-C and VLDL-C) were calculated from the concentration of TAG using the Friedewald equations: [8].Serum soluble CD163 protein was measured by Western blot using goat antibody (1:200 dilution, sc-18794, Santa Cruz Biotechnology, Dallas, TX, USA) and donkey anti-goat antibody-HRP (1:3000 dilution; sc-2020, Santa Cruz Biotechnology).The protein preparation was performed according to the Western blot method described below and normalized by ~90% of the total protein content (Ponceau).

Acinar and Islet Morphology
Morphometric analysis of islets was performed using an optical microscope at 4× (Zeiss Axio Imager A1).Pancreas samples were stained with either Masson's trichrome to identify the presence of collagen or Periodic Acid-Schiff (PAS) to analyze proteoglycans and quantify the proportion (%) from three random regions (40×) from each animal using the ImageJ 1.47 program.Samples stained with PAS were counterstained with Mayer's hematoxylin to evaluate the morphometry of islets, blood vessels, and proteoglycans.Images from each pancreas were reconstructed.A random selection of these reconstructions permitted us to measure the cross-sectional area and count the number of cells for different islets in photographs at 40×.Islets were classified as small (<4000 µm 2 ), medium (4000-7000 µm 2 ), and large (>7000 µm 2 ) [11]).Moreover, in three selected areas per animal, photographed at 40×, we evaluated the area covered by intra-acinar collagen and proteoglycan and blood vessels and immune cells in islets [12].

Lipids and Peroxidation in the Pancreas
The pancreas sample (25 mg) was homogenized in ice-cold Tris buffer (20 mM, pH 7.4) and centrifuged at 3000 rpm for 10 min at 4 • C. The supernatant was collected and immediately tested with the lipid peroxidation microplate assay (Oxford Medical Research, Inc., San Louis, MO, USA).The kit uses the thiobarbituric acid reaction, and lipoperoxidation is expressed as micromoles of malondialdehyde (MDA) per microgram of protein of the pancreas.

Statistical Analyses
Statistical analyses were performed with GraphPad Prism v6.01 (GraphPad Software, La Jolla, CA, USA).The Kolmogorov-Smirnov test was used to check the normality of distribution.Results were expressed as mean ± SD.One-way ANOVA and Tukey's post hoc test, or Kruskal-Wallis's test, were used to determine significant differences between groups (p < 0.05).

Conclusions
These results show that I 2 supplementation at moderate doses prevents some metabolic and pancreatic alterations associated with hypothyroidism.The anti-inflammatory and lipid modulation effects could be related to antioxidant Nrf2 mechanisms and PPARG activation.