Biosci. Biotechnol. Biochem., 77 (11), 2305–2308, 2013 Note Inhibition of Postprandial Hyperglycemia by Either an Insulin-Dependent or -Independent Drug Reduces the Expression of Related to Inflammation in Peripheral Leukocytes of OLETF Rats

y Chihiro IMAI,1 Tomomi HARAZAKI,1 Seiya INOUE,1 Kazuki MOCHIZUKI,1;2 and Toshinao GODA1;

1Laboratory of Nutritional Physiology, Graduate School of Nutritional and Environmental Sciences, The University of Shizuoka, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan 2Research Branch of Food and Nutritional Sciences, Department of Local Produce and Food Sciences, Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi 400-8510, Japan

Received June 10, 2013; Accepted July 24, 2013; Online Publication, November 7, 2013 [doi:10.1271/bbb.130451]

Treatment with the dipeptidyl peptidase-4 inhibitor, already been reported that insulin resistance and hyper- anagliptin, or with the -glucosidase inhibitor, miglitol, glycemia in patients with IGT and type 2 diabetes were reduced the oral sucrose load-inducible expression of associated with elevated plasma levels of IL-1, IL-6, interleukin (IL)-1 , IL-18, - , IL-12, IL-18 and TNF- .5–10) These inflamma- S100a8, S100a9, S100a11, IL-1R2, IL-1Rn and tumor tory cytokines activate macrophages/monocytes and necrosis factor receptor 2 genes in peripheral leukocytes induce the infiltration of macrophages into many tissues, of Otsuka Long-Evans Tokushima fatty (OLETF) rats resulting in the development and progression of arterio- at the stage of impaired glucose tolerance. Inhibiting sclerosis, insulin resistance, of islet -cells, postprandial hyperglycemia reduced the expression of nephropathy and other complications.8) One of our genes related to inflammation in peripheral leukocytes previous studies has shown that intermittent treatment of OLETF rats. with miglitol, an inhibitor of -glucosidases that participate in carbohydrate digestion in the small Key words: postprandial hyperglycemia; inflammatory intestine,11) reduced the expression of such inflammatory cytokine; peripheral leukocytes; -glucosi- cytokine genes as IL-1, IL-18 and TNF- in peripheral dase inhibitor; dipeptidyl peptidase-4 (DPP- leukocytes of low-dose streptozotocin (STZ)-treated rats 4) inhibitor with intermittent postprandial hyperglycemia.12) Such dipeptidyl peptidase-4 (DPP-4) inhibitors, which reduce Many recent studies have demonstrated that patients postprandial hyperglycemia by enhancing gut hormone with type 2 diabetes had a higher subsequent incidence incretins, as glucagon-like peptide-1 (GLP-1) and of such arteriosclerosis-related problems as cardiovas- glucose-dependent insulinotropic polypeptide (GIP) cular disease (CVD). In particular, the development of have recently been developed. These DPP-4 inhibitors CVD in subjects with impaired glucose tolerance (IGT) promote insulin secretion by enhancing the incretin as well as type 2 diabetic patients was related to the concentration in the blood through the inhibition of elevation of postprandial hyperglycemia. Epidemiolog- incretin breakdown. It is most likely that reducing ical studies like the DECODE and Funagata trials have postprandial hyperglycemia by DPP-4 inhibitors would reported that IGT, but not impaired fasting glucose, was reduce the risk of CVD by repressing inflammatory strongly and positively associated with the subsequent cytokine expression in peripheral leukocytes. We exam- incidence of CVD.1,2) In addition, the STOP-NIDDM ined in this study whether either the DPP-4 inhibitor, study on patients with IGT has demonstrated that anagliptin, or miglitol would reduce the expression of inhibiting postprandial hyperglycemia by the -glucosi- the inflammation-related genes in peripheral leukocytes dase inhibitor, acarbose, reduced the development of of Otsuka Long-Evans Tokushima fatty (OLETF) rats, type 2 diabetes and CVD.3,4) The inhibition of post- which exhibit obesity in addition to the late onset of prandial hyperglycemia in patients with IGT and type 2 chronic and slowly progressive hyperinsulinemia, hyper- diabetes is therefore important for suppressing associ- glycemia and hyperlipidemia caused by overeating,13) at ated complications, in particular CVD. Recent studies the stage of IGT. have shown that such inflammatory cytokines as Twenty-four male OLETF rats at 4 weeks of age were interleukin (IL)-1, IL-6, IL-12, IL-18 and tumor obtained from the Otsuka GEN Research Institute necrosis factor (TNF)- secreted from activated leuko- (Tokushima, Japan). The rats were raised in individual cytes, including neutrophils, monocytes and macro- cages and maintained at a constant temperature phages, were linked to diabetic complications. It has (23 2 C) and humidity (55 5%) under a 12-h

y To whom correspondence should be addressed. Tel: +81-54-264-5533; Fax: +81-54-264-5565; E-mail: [email protected] Abbreviations: ANCOVA, analysis of covariance; ANOVA, analysis of variance; CVD, cardiovascular disease; DPP-4, dipeptidyl peptidase-4; GIP, glucose-dependent insulinotropic polypeptide; GLP-1, glucagon-like peptide-1; HPRT, hypoxanthine-guanine phosphoribosyltransferase; IGT, impaired glucose tolerance; IL, interleukin; IL-1R, interleukin 1 receptor; IL-1Rn, interleukin 1 receptor antagonist; OLETF, Otsuka Long-Evans Tokushima fatty; RAGE, receptor for advanced glycation endproduct; TNF-, tumor necrosis factor-; TNFR, tumor necrosis factor receptor; SEM, standard error of the mean; STZ, streptozotocin 2306 C. IMAI et al. Table 1. Sequences of Oligonucleotide Primers Used for Real-Time RT-PCR

Target mRNA Sequence 50-TGTGATGAAAGACGGCACAC-30 Interleukin 1 beta (IL-1) (#78) 50-CTTCTTCTTTGGGTATTGTTTGG-30 50-CAACGAATCCCAGACCAGAC-30 Interleukin 18 (IL-18) (#76) 50-ACATCCTTCCATCCTTCACAG-30 50-GTCTACTGAACTTCGGGGTGA-30 Tumor necrosis factor alpha (TNF-) (#63) 50-ATGAGAGGGAGCCCATTTG-30 50-GCCACAAGGAGTAACAGAGCTT-30 S100 calcium binding A8 (S100a8) (#26) 50-TGACGACTTTATTCTGTAGACATATCC-30 50-AAGGACTTGCCAAATTTTCTGA-30 S100 calcium binding protein A9 (S100a9) (#85) 50-GGACAGTTGATTGTCCTGGTTT-30 50-CGACCGCATGATGAAGAAG-30 S100 calcium binding protein A11 (S100a11) (#44) 50-AAGCCACCAATAAGGTTGAGAA-30 50-GCAAGGTACACGAATGCAGA-30 Interleukin-1 receptor type 2 (IL-1R2) (#116) 50-CACACCAACTTCCAGAGTGC-30 50-TCTGGAGATGACACCAAGCTC-30 Interleukin-1 receptor antagonist (IL-1Rn) (#25) 50-GCGCTTGTCTTCTTCTTTGTTC-30 50-GAGGCCCAAGGGTCTCAG-30 Tumor necrosis factor receptor 2 (TNFR2) (#1) 50-GCTGCCATGGGAAGAATC-30 50-AGTCAACGGGGGACATAAAA-30 Hypoxanthine-guanine phosphoribosyltransferase (HPRT) (#22) 50-CTTCAACAATCAAGACGTTCTTTC-30

The numbers in parentheses indicate ID of a universal probe (Roche Diagnostics, Tokyo, Japan) used to detect the signals of each .

light/dark cycle (lights on: 07:00–19:00). After being 350 fed a standard MF laboratory diet (Oriental Yeast, 300 a Tokyo, Japan) for 10 d, all the rats consumed a 40% a a 250 sucrose solution ad libitum from a water bottle during ab ab b the period of 6–10 weeks of age. Four weeks after 200 starting administration of the sucrose solution, the rats b b b were given regular water and fed an experimental high- 150 Control carbohydrate rodent diet until 13 weeks of age. The diet 100 was based on the recommendations of the American Blood glucose (mg/dL) Miglitol Anagliptin Institute of Nutrition (AIN)14) and contained 20% (w/w) 50

0 casein, 27.8% corn starch, 27.8% sucrose, 9.5% corn oil, 0 15 30 60 120 180 5% lard, 3.5% AIN93 vitamin mix, 1% AIN93G mineral Time after sucrose loading (min) mix, 5% cellulose, 0.3% L-cysteine and 0.25% choline bitartrate. After confirming that the rats exhibited a Fig. 1. Changes in the Blood Glucose Concentration Following Oral sufficient level of IGT, the animals were assigned to one Sucrose Loading with or without Miglitol or Anagliptin to OLETF Rats. of three groups (n ¼ 8) between which there were no The symbols and bars represent the mean SEM (n ¼ 8). significant differences in the 14-h fasted plasma glucose a{bValues not sharing a common superscript are significantly concentrations ([mean standard error of the mean different from each other at each time point (p < 0:05). (SEM)], 142:7 1:8 mg/dL), HbA1c (4:8 0:1%) and body weight (453:1 1:2 g). At 13 weeks of age, all rats to the guidelines of the Animal Usage Committee of underwent oral sucrose loading (2 g/kg of body weight the University of Shizuoka. Plasma glucose was deter- as a 40% sucrose solution) either without treatment mined by using a Glucose C-II-test kit (Wako Pure (control group), with the miglitol -glucosidase inhibitor Chemical Industries, Osaka, Japan). RNA extraction and (0.2 mg/mL), or with the anagliptin DPP-4 inhibitor real-time RT-PCR by the universal probe library (Roche (3 mg/kg of body weight). Miglitol was included in the Molecular Biochemicals, Tokyo, Japan) were performed sucrose solution, while a dose of 0.6 mg/mL of as previously described.12) The PCR primer sequences anagliptin was orally administered 30 min before the for the rat peripheral leukocytes are listed in Table 1. sucrose administration. Both anagliptin and miglitol All data are expressed as the mean SEM. The were provided by Sanwa Kagaku Kenkyusho Co. (Mie, significance of differences between groups was deter- Japan). Blood samples were collected from the tail vein mined by Tukey’s test, based on a one-way analysis of by using a capillary containing sodium fluoride and variance (ANOVA, Fig. 1) or based on a paired two- heparin-lithium (Terumo Co., Tokyo, Japan) immedi- way analysis of covariance (ANCOVA, Fig. 2). A level ately prior to (0 min) and 15, 30, 60, 90, 120 and of p < 0:05 was considered to indicate statistical 180 min after sucrose administration for glucose meas- significance. urement, and immediately prior to and 180 min after The blood glucose concentration immediately prior to sucrose administration for RNA extraction. The exper- the sucrose loading was approximately 190 mg/dL in all imental procedures used in the present study conformed groups, and was markedly increased in the control group Postprandial Hyperglycemia and Inflammation 2307

a IL-1β b IL-18 c TNF-α 20 1 4 a ** a 3 15 * a ** 10 0.5 2 b b b 5 b 1 b b 0 0 0 d S100a8 e S100a9 f S100a11 10 3 40 a ** a a ** 30 2 ** 5 20 ab ab b 1 b 10 b b

Relative mRNA levels Relative 0 0 0 g IL-1R2 h IL-1Rn i TNFR2 0.25 a 3 0.004 ** a ** * 0.2 0.003 2 0.15 b b 0.002 0.1 1 b 0.001 0.05 b

0 0 0 ControlMiglitolAnagliptin Control Miglitol Anagliptin Control Miglitol Anagliptin

0 min 180 min

Fig. 2. Quantitative RT-PCR Analysis of Inflammation-Related Genes in the Peripheral Leukocytes of OLETF Rats Subjected to Oral Sucrose Loading with or without Miglitol or Anagliptin. Values represent the mean SEM of 5–8 animals. Values not sharing a common letter are significantly different from one another among the treatment groups after 180 min (as determined by Tukey’s test based on repeated two-way ANCOVA). Asterisks denote significant differences (Tukey’s test based on repeated two-way ANCOVA) compared with the 0 min value ( p < 0:01, p < 0:05). by the sucrose loading, with a maximum level of to the pathogenesis of atherosclerosis by acting as 275 13 mg/dL being attained 30 min after sucrose cytokine/chemokine-like factors via binding to the loading. The blood glucose concentration in the animals receptor for advanced glycation end-products (RAGE) given the sucrose solution with miglitol was signifi- on leukocytes.15,16) IL-1R2 and TNFR2 are the respec- cantly lower than that in the control group after 15 and tive receptors for IL-1 and TNF-. The results of this 30 min (p < 0:05). The blood glucose concentration in present study suggest that postprandial hyperglycemia the rats given the sucrose solution with anagliptin was was associated with the development and progression of significantly lower than that in the control group after 30 CVD by enhancing the expression of inflammation- and 60 min (p < 0:05) (Fig. 1). The mRNA levels of related genes in peripheral leukocytes of an animal genes including IL-1, IL-18, TNF-, S100a8, S100a9, model of obesity and insulin resistance. As shown in our S100a11, interleukin-1 receptor 2 (IL-1R2), interleukin previous study, intermittent sucrose loading enhanced 1 receptor antagonist (IL-1Rn) and tumor necrosis factor postprandial hyperglycemia and the expression of IL-1 receptor 2 (TNFR2) in the peripheral leukocytes were and TNF- genes in peripheral leukocytes of low-dose significantly higher after 180 min than the basal levels in STZ-treated rats, this being a model of reduced insulin the control group, but not in the miglitol or anagliptin secretion capacity.12) Combining the results of the groups. mRNA of interleukin-1 receptor 1 (IL-1R1), previous study with those of the current study, post- tumor necrosis factor receptor 1 (TNFR1) and interleu- prandial hyperglycemia induced the expression of IL-1 kin 18 receptor 1 (IL18R1) did not change among the and TNF- genes in peripheral leukocytes of an animal groups (data not shown). At 180 min after sucrose model with either insulin secretion deficiency or insulin loading, the mRNA levels of the IL-1, IL-18, TNF-, resistance. It should be noted that IL-1Rn was induced S100a11, IL-1R2 and IL-1Rn genes were significantly by a single oral sucrose load in peripheral leukocytes. lower in both drug groups than in the control group, with Since IL-1Rn is an antagonist for IL-1, it is possible the mRNA level of the S100a8 gene being significantly that a single oral sucrose load induced the anti- lower in the miglitol group than in the control group. inflammation step as a negative feedback system. It The mRNA level of the S100a9 gene was also should be examined whether immediate sucrose loading significantly lower in the anagliptin group than in the alters the IL-1/IL-1Rn mRNA ratio in peripheral control group (Fig. 2). leukocytes of OLETF rats at the earlier and later stages We have demonstrated in this study that a single oral of type 2 diabetes. In addition, it should be noted that the sucrose load administered to OLETF rats not only induction of IL-1, TNF- and IL-18 mRNAs by a induced postprandial hyperglycemia, but also enhanced single oral sucrose load was greater in OLETF rats used the expression of such inflammation-related genes as IL- in this study than in STZ-induced moderate postprandial 1, IL-18, TNF-, S100 proteins, IL-1R2, IL-1Rn and hyperglycemic rats in a previous study,12) although the TNFR2. It has been reported that S100 proteins, postprandial hyperglycemic levels without drugs were including S100a8 and S100a9, were intimately related similar (the glucose level in OLETF rats after 30 min 2308 C. IMAI et al. was 274:6 13:0 mg/dL; that in STZ rats was In conclusion, this study has demonstrated that acute 240:1 6:4 mg/mL). STZ-treated rats are known as a postprandial hyperglycemia led to enhanced expression model of insulin secretion deficiency by inducing the of the genes related to inflammation in peripheral apoptosis of pancreatic -cells. On the other hand, leukocytes, and that inhibiting postprandial hyperglyce- OLETF rats are a model that exhibits obesity, in addition mia by either anagliptin or miglitol reduced the to the late onset of chronic and slowly progressive expression of these genes. hyperinsulinemia, hyperglycemia and hyperlipidemia caused by overeating.13) Recent studies have demon- Acknowledgments strated that a metabolic abnormality with obesity and lipid abnormality, which is frequently called the meta- This work was supported by grant-aid for young bolic syndrome, was more closely associated with the scientists (22680054) and for scientific research development of CVD than was a metabolic abnormality (23300276) from the Ministry of Education, Culture, without obesity and lipid abnormality.17,18) The response Sports, Science and Technology of Japan, and the to postprandial hyperglycemia of the inflammatory Global COE program, the Center of Excellence for cytokine and S100s genes in peripheral leukocytes Innovation in Human Health Sciences of the Ministry of may therefore be enhanced by the obesity and lipid Education, Culture, Sports, Science and Technology of abnormality. Japan. Interestingly, treating with either an insulin-depend- ent drug (anagliptin) or an insulin-independent drug References (miglitol) reduced the oral-sucrose-inducible expression of the genes encoding inflammatory cytokines, S100 1) Tominaga M, Eguchi H, Manaka H, Igarashi K, Kato T, and proteins and receptors for IL-1 and TNF- in periph- Sekikawa A, Diabetes Care, 22, 920–924 (1999). eral leukocytes. These results strongly suggest that 2) DECODE Study Group tEDEG, Arch. Intern. Med., 161, 397– 405 (2001). either an insulin-dependent or insulin-independent drug 3) Chiasson JL, Josse RG, Gomis R, Hanefeld M, Karasik A, and would enable suppression of the postprandial induction Laakso M, Lancet, 359, 2072–2077 (2002). of these inflammation-related genes in peripheral leu- 4) Zeymer U, Schwarzmaier-D’assie A, Petzinna D, and Chiasson kocytes. Our previous study has demonstrated that a JL, Eur. J. Cardiovasc. Prev. Rehabil., 11, 412–415 (2004). miglitol treatment for 20 d reduced the expression of 5) Spranger J, Kroke A, Mohlig M, Hoffmann K, Bergmann MM, IL-1, TNF- and S100 proteins in the peripheral Ristow M, Boeing H, and Pfeiffer AF, Diabetes, 52, 812–817 leukocytes of STZ hyperglycemic rats.19) Furthermore, (2003). 6) Kim ES, Im JA, Kim KC, Park JH, Suh SH et al., Obesity the miglitol treatment for 40 weeks from the pre-diabetic (Silver Spring), 15, 3023–3030 (2007). stage (5-week-old) repressed the TNF- mRNA level in 7) Bruun JM, Stallknecht B, Helge JW, and Richelsen B, Eur. J. peripheral severe leukocytes.20) Our previous studies Endocrinol., 157, 465–471 (2007). have demonstrated in humans that reducing postprandial 8) Shoelson SE, Lee J, and Goldfine AB, J. Clin. Invest., 116, hyperglycemia by miglitol in Japanese type 2 diabetic 1793–1801 (2006). patients for 3 months reduced the level of circulating 9) Hui H, Dotta F, Di Mario U, and Perfetti R, J. Cell. Physiol., 200 TNF- protein and the mRNA level in peripheral , 177–200 (2004). 21) 10) Jorns A, Gunther A, Hedrich HJ, Wedekind D, Tiedge M, and leukocytes. Whether a single dose of miglitol or Lenzen S, Diabetes, 54, 2041–2052 (2005). anagliptin to OLETF rats at the earlier and later stages of 11) Van de Laar FA, Lucassen PL, Akkermans RP, Van de Lisdonk type 2 diabetes and whether long-term treatment with EH, Rutten GE, and Van Weel C, Cochrane Database Syst. miglitol or anagliptin can suppress the levels of mRNA Rev., CD003639 (2005). in peripheral leukocytes and protein in the serum of 12) Tanaka Y, Mochizuki K, Fukaya N, Shimada M, and Goda T, inflammation-related genes in the peripheral leukocytes Br. J. Nutr., 102, 221–225 (2009). 13) Moran TH and Bi S, Philos. Trans. R. Soc. B Biol. Sci., 361, of OLETF rats needs to be examined. 1211–1218 (2006). It should be noted that insulin-dependent drugs, 14) Reeves PG, J. Nutr., 127, S838–S841 (1997). including DPP-4 inhibitors, could induce obesity, be- 15) Ehlermann P, Eggers K, Bierhaus A, Most P, Weichenhan D, cause insulin stimulates fatty acid synthesis. Indeed, a Greten J, Nawroth PP, Katus HA, and Remppis A, Cardiovasc. previous study has demonstrated that the -glucosidase Diabetol., 5, 6 (2006). inhibitor, acarbose, reduced body weight, whereas the 16) Foell D, Wittkowski H, Vogl T, and Roth J, J. Leukoc. Biol., 81, DPP-4 inhibitor, vildagliptin, had little effect.22) How- 28–37 (2007). 17) Wajchenberg BL, Endocr. Rev., 21, 697–738 (2000). ever, unlike -glucosidase inhibitors, DPP-4 inhibitors 18) Perrini S, Leonardini A, Laviola L, and Giorgino F, Arch. can suppress between-meal hyperglycemia. The com- Physiol. Biochem., 114, 277–286 (2008). bined use of an -glucosidase inhibitor and a DPP-4 19) Fukaya N, Mochizuki K, Shimada M, and Goda T, Nutrition, inhibitor could therefore reduce postprandial and be- 25, 657–667 (2009). tween-meal hyperglycemia and inhibit obesity. Further 20) Mochizuki K, Fukaya N, Tanaka Y, Fuchigami M, and Goda T, studies on animal models and type 2 diabetic patients Metabolism, 60, 1560–1565 (2011). should examine whether co-treating with an -glucosi- 21) Osonoi T, Saito M, Mochizuki K, Fukaya N, Muramatsu T, Inoue S, Fuchigami M, and Goda T, Metabolism, 59, 1816– dase inhibitor and a DPP-4 inhibitor for a longer period 1822 (2010). of time could improve glycemic control and reduce body 22) Pan C, Yang W, Barona JP, Wang Y, Niggli M, Mohideen P, weight. and Foley JE, Diabet. Med., 25, 435–441 (2008).