International Journal of Mellitus 2 (2010) 169–174

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International Journal of Diabetes Mellitus

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Original Article High glucose-induced DNA-binding activities of nuclear factor of activated T cells 5 and carbohydrate response element binding protein to the myo- gene are inhibited by sorbinil in peripheral blood mononuclear cells from patients with mellitus and nephropathy ⇑ Bingmei Yang , Andrea Hodgkinson, Beverley A. Millward, Andrew G. Demaine

Molecular Medicine Research Group, Institute of Biomedical and Clinical Science, Peninsula Medical School, University of Plymouth, Plymouth PL6 8BU, United Kingdom article info abstract

Article history: Aims: To investigate whether high glucose induces myo-inositol oxygenase (MIOX) expression in periph- Received 12 May 2010 eral blood mononuclear cells through transcription factors, nuclear factor of activated T cells 5 (NFAT5) Accepted 28 August 2010 and carbohydrate response element binding protein (ChREBP), which may contribute to the pathogenesis of diabetic nephropathy. Methods: 34 patients with type 1 diabetes mellitus (20 with nephropathy, 14 without complications) and Keywords: 9 healthy controls were recruited in this study. Peripheral blood mononuclear cells were exposed to nor- Myo-inositol mal, high glucose conditions with/without an aldose reductase inhibitor (ARI), using electrophoretic Myo-inositol oxygenase mobility shift assays the DNA-binding activities of NFAT5 and ChREBP to corresponding sites in the pro- Type 1 diabetes mellitus Diabetic nephropathy moter region of MIOX gene were analysed. The protein levels and the activity of MIOX were mea- Aldose reductase sured. Aldose reductase inhibitors Results: DNA-binding activities of NFAT5 and ChREBP were increased under high glucose conditions and decreased in the presence of the ARI in all groups. In the presence of ARI, the DNA-binding activities of NFAT5 and ChREBP were significantly decreased by 41% (NFAT5: 0.91 ± 0.06 vs. 1.54 ± 0.12; p = 0.01) and 49% (ChREBP: 0.92 ± 0.08 vs. 1.81 ± 0.22; p = 0.001) compared with high glucose in patients with nephropathy. ARI treatment decreased the protein levels of MIOX under high glucose conditions in patients with nephropathy (0.81 + 0.19 vs. 1.3 + 0.04; p = 0.049). Summary/conclusions: There was a trend for increased binding activities of NFAT5 and ChREBP accompa- nied with increased protein levels under high glucose, particularly in patients with nephropathy. ARI treatment prevented these increases and this effect was more obvious in the patients with nephropathy compared to the uncomplicated subjects. Ó 2010 International Journal of Diabetes Mellitus. Published by Elsevier Ltd. All rights reserved.

1. Introduction cose to fructose via sorbitol. Under physiological conditions, most of the cellular glucose is phosphorylated into glucose 6-phosphate, It has been widely accepted that long-term exposure to high and only a minor portion is metabolised through the polyol path- blood glucose plays an important role in the development of dia- way. However, high glucose conditions may saturate the betic nephropathy [1]. High glucose, together with increased flux involved in the phosphorylation of glucose, and as a result, one- through the polyol pathway causes myo-inositol (MI) depletion third of intracellular glucose enters the polyol pathway, which which may have a role to play in the development of diabetic causes an accumulation of sorbitol within cells. Accumulation of nephropathy. However, the mechanism of MI depletion is still un- sorbitol is accompanied by a depletion of MI, and this alters clear. It has been proposed that myo-inositol oxygenase (MIOX) Na+/K+ ATPase activity and impairs phosphatidylinositol syntheses, plays a key role in causing the depletion of MI. which is a common precursor of important secondary signalling There are two enzymes in the polyol pathway, aldose reductase molecules. Several studies have shown that MI depletion is (AKR1B1) and sorbitol dehydrogenase. Together, they convert glu- associated with diabetic nephropathy, retinopathy, neuropathy and diabetic cataracts [2–7]. MIOX is the first and rate-limiting en- zyme in the MI metabolism pathway, which is the glucuronate– ⇑ Corresponding author. Address: Molecular Medicine Research Group, The John xylulose pathway and is the only pathway for MI catabolism. It Bull Building, Research Way, Peninsula Medical School, University of Plymouth, Plymouth PL6 8BU, United Kingdom. Tel.: +44 1752 437415; fax: +44 1752 517846. has been shown that an increase in MIOX enzyme activity is in E-mail addresses: [email protected], [email protected] (B. Yang). proportion to serum glucose concentrations [8]. Therefore, MIOX

1877-5934/$ - see front matter Ó 2010 International Journal of Diabetes Mellitus. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijdm.2010.08.005 170 B. Yang et al. / International Journal of Diabetes Mellitus 2 (2010) 169–174 may be responsible for the MI depletion found in diabetic was always associated with retinopathy. Retinopathy was defined complications. as more than five dots or blots per eye; hard or soft exudates, new MIOX is predominantly expressed in the kidney, nerves and li- vessels, or fluorescein angiographic evidence of maculopathy or ver [9–12]. The promoter region of both the human and murine previous laser treatment for pre-proliferative or proliferative reti- MIOX genes contain osmotic response element(s) (OREs) and car- nopathy; and maculopathy or vitreous haemorrhage. Fundoscope bohydrate response element(s) (ChREs). The binding activities of was performed by both a diabetologist and an ophthalmologist. the nuclear factor of activated T cells 5 (NFAT5) and carbohydrate response element binding protein (ChREBP) to OREs and ChREs, respectively, significantly increase under high glucose conditions 2.2. Cell isolation and cultures [8,13]. High glucose and osmolytes significantly increase the tran- scriptional activity of MIOX by in vitro assays. Further- Peripheral venous blood samples (20 ml) were collected into 5% more, increased MIOX expression has been shown in the kidneys EDTA Vacutainers (Becton Dickinson, UK). The peripheral blood of diabetic mice [8]. In preliminary experiments, we have shown mononuclear cells (PBMCs) were separated by using Histopaque that human peripheral blood mononuclear cells (PBMCs) express (Sigma, Dorset, UK) and grown in RPMI 1640 supplemented with the MIOX gene by a direct sequencing of reverse transcriptase D-glucose at a concentration of 5.5 mmol/l, 10% calf serum and polymerase chain reaction (RT-PCR) products and Western blot- 2 mmol/l L-glutamine, 100 units/ml penicillin G sodium and ting. In related studies, we have shown that NFAT5 is involved in 100 mg/ml streptomycin sulfate with PHA-P at a concentration of the regulation of AKR1B1 under high glucose conditions [14] and 5 lg/ml in a 37 °C incubator with a controlled, humidified atmo- in the presence of an aldose reductase inhibitor (ARI), the binding sphere of 95% air/5% CO2. The cells were divided into three groups activities of nuclear factor kappa B (NFjB) [15] and NFAT5 (unpub- in 200 ml-flasks. Group 1 (normal conditions-NG): cells were cul- lished data) to OREs in the promoter of the AKR1B1 gene were sup- tured in the above medium (D-glucose at a final concentration of pressed. Therefore, our hypotheses are that high glucose increases 5.5 mmol/l). Group 2 (high glucose conditions-HG): 19.5 mmol/l MIOX protein levels and activity which might be regulated through extra D-glucose was added into above-mentioned media (D-glucose increased binding activities of NFAT5 to OREs and ChREBP to ChREs at a final concentration of 25 mmol/l). Group 3 (aldose reductase in the promoter region of the MIOX gene. These binding activities inhibitor conditions (ARI) with HG): sorbinil (at a final concentra- may be different in the PBMCs from patients with nephropathy, tion of 10 lmol/l), was added 3 h later after 19.5 mmol/l extra D- compared to those without complications. Inhibition of AKR1B1 glucose was added to the media. All cells were incubated for may suppress MIOX protein levels and activity through reducing 5 days. At the end of the incubation time, cells were harvested the production of sorbitol through the polyol pathway. and nuclear and cytoplasmic proteins were extracted as below. To the best of our knowledge, there has been no study that has investigated the factors involved in the regulation of the MIOX 2.3. Extraction of nuclear protein and cytoplasmic proteins gene and enzyme activity in patients with type 1 diabetes mellitus (T1D) and nephropathy. Therefore, the aims of this study were to Cells were collected and re-suspended in 100 ll of buffer A investigate the regulation of MIOX expression under high glucose (10 lmol/l HEPES, pH 7.9, 1.5 mmol/l MgCl2, 0.5 mmol/l dithio- conditions in the PBMCs from patients with T1D, with or without threitol (DTT), 0.2% NP-40, 100 mmol/l 4-(2-aminoethyl)-bez- nephropathy. enesulfonyl fluoride (AEBSF), 18.4 mg/ml sodium orthovanadate, 42 mg/ml sodium fluoride and 2.2 mg/ml aprotonin) and held on 2. Materials and methods ice for 15 min. The resulting cell lysate was then centrifuged at 13,000 rpm for 10 min. The supernatant containing cytoplasmic 2.1. Subjects proteins was transferred into a fresh tube and stored at À80 °C for Western blotting and MIOX activity assays. The nuclear pellets The following Caucasoid subjects were included in this study: were re-suspended in 50 ll of buffer C (20 mmol/l HEPES pH 7.9, 34 patients with T1D and 9 ethnically matched healthy controls. 25% glycerol, 0.42 mol/l NaCl, 1.5 mmol/l MgCl2, 0.5 mmol/l DTT, All patients with T1D as defined by The Expert Committee On 0.2 mmol/l EDTA, 100 mmol/l AEBSF, 18.4 mg/ml sodium ortho- The Diagnosis And Classification Of Diabetes Mellitus [16] had at- vanadate, 42 mg/ml sodium fluoride and 2.2 mg/ml aprotonin), tended the Diabetes Clinic, Derriford Hospital, Plymouth. The study and incubated on ice for 10 min. After centrifugation at was approved by the Local Research Ethical Committee, and in- 13,000 rpm for 10 min the supernatant containing the nuclear pro- formed consent was obtained from all subjects. The criteria for dia- tein was transferred into a fresh tube and stored at À80 °C until use betic microvascular complications have been published previously in the electrophoretic mobility shift assay (EMSA). The concentra- [17]. tions of both nuclear and cytoplasmic proteins were determined using a CoomassieÒ Plus Protein Assay kit (Peribo Science Ltd., Chest, UK). 2.1.1. Uncomplicated Patients (n = 14) have been diagnosed with T1D for at least 20 years but remain free of retinopathy (fewer than five dots or 2.4. Electrophoretic mobility shift assay blots per fundus), proteinuria (urine Albustix negative on at least three consecutive occasions over 12 months) and neuropathy NFAT5 and ChREBP probes with consensus sequence to the OER (overt neuropathy was defined if there was any clinical evidence and ChRE motifs CCTCCTCCAGGAAAGCCTTTACCCTCC and GAG- of peripheral or autonomic neuropathy). CACGTGACCTACCCGTGTTG GGACACGTGAGG [8,13] of the MIOX gene were labeled with [a-32P] deoxy-ATP by T4 polynucleotide ki- 2.1.2. Diabetic nephropaths nase (Amersham Pharmacia Biotech, Buckinghamshire, UK). The la- Patients (n = 20) have had T1D for at least 8 years with persis- beled probes along with the gel-binding buffer were incubated tent proteinuria (urine Albustix positive on at least three consecu- with 25 lg of nuclear proteins at room temperature for 20 min. tive occasions over 12 months or three consecutive total urinary The binding mixtures were resolved by electrophoresis on a 4% protein excretion rates >0.5 g/24 h) in the absence of hematuria non-denaturing polyacrylamide gel at 100 V for 3–4 h. The gel or infection on midstream urine samples. Diabetic nephropathy was exposed to X-Omax photographic paper. B. Yang et al. / International Journal of Diabetes Mellitus 2 (2010) 169–174 171

2.5. Western blotting 3. Results

All of the cytoplasmic proteins from each individual within each Clinical characteristics of patients with T1D with or without group was pooled together and 50 lg of this was loaded onto a 7.5 diabetic nephropathy are shown in Table 1. There were no differ- or 10% precast SDS–PAGE (BIO-RAD Laboratory Ltd., Hempstead, ences in age, gender, age at onset of diabetes, duration of diabetes, UK), electrophoresed for 2–3 h at 100 V and then transferred to Haemoglobin A1c (Hb1Ac) and plasma glucose levels between the nitrocellular membrane (Amersham, USA) overnight. Next, the two groups. Estimated glomerular filtration rate (eGFR) was signif- membrane was blocked with 5% non-fat milk and 0.05% Tween icantly lower in patients with nephropathy compared with uncom- 20-PBS for 1 h at room temperature. Immunoblotting was per- plicated subjects (59.4 ± 4.5 vs. 74.5 ± 3.2; p = 0.021). formed with a primary goat antibody against human MIOX (Insight The DNA-binding activities of NFAT5 to the ORE and ChREBP to Biotechnology Ltd., Wembley, UK) in a 1:500 dilution. Secondary the ChRE motifs in PBMCs exposed to either normal glucose, high antibody against goat IgG of horse-radish peroxidase-conjugated glucose or, ARI with high glucose conditions, from nephropaths, was used in 1:5000 dilution (Sigma, UK). A chemiluminescence uncomplicated and normal control subjects are shown in Fig. 1a kit (Pierce, UK) and Kodak X-Omax film (Amersham, UK) were used and b. DNA-binding activities of NFAT5 and ChREBP to their motifs to detect the amount of protein. In order to have equal amounts of increased under high glucose conditions in all study groups; how- proteins loaded in wells, a-tubulin levels were measured using a ever, these increases did not reach statistical significance. ARI mouse antibody against human a-tubulin in 1:5000 dilution (Sig- treatment significantly decreased the DNA-binding activities of ma, UK) and a secondary antibody against mouse IgG of horse-rad- ish peroxidase-conjugated in 1:5000 dilution (Sigma, UK). DNA-binding activities of NFAT5 and ChREBP and protein levels Table 1 of MIOX were analysed and quantified using a phosphoimager Clinical characteristics of patients with type 1 diabetes mellitus and normal controls. (BIO-RAD, UK) with multi-analyst software. All results were ex- pressed as means of fold increase, due to high glucose treatment, Nephropaths Uncomplicated Normal controls (n = 20) (n = 14) (n =9) calculated by dividing the density in high glucose-treated cells by the density in untreated cells, or as a mean fold decrease due Male:Female 6:14 4:10 4:5 Age (years) 49.6 ± 13.9 48.1 ± 14.9 (19– 31.5 ± 5.14 (26– to ARI treatment, calculated by dividing the density in high glucose (30–82) 74) 41) with ARI-treated cells by density in high glucose-treated cells. Age at onset of 12.3 ± 8.8 (1– 15.2 ± 9.4) (2– – diabetes (years) 35) 38) Duration of diabetes 34.9 ± 8.1 34.3 ± 9.4) (20– – (years) (21–49) 53) 2.6. MIOX activity assay Plasma glucose 12.7 ± 0.5 11.0 ± 0.4 – (mmol/L) Pooled cytoplasmic proteins from each subject group were used HbA1c (%) 7.9 ± 0.1 8.4 ± 0.1 – to perform MIOX enzyme activity by following the methods used eGFR (mls/min/ 59.4 ± 4.5* 74.5 ± 3.2 – 2 by Prabhu’s group [13]. Two-hundred and fifty microgram of cyto- 1.73 m ) plasmic proteins was mixed with 50 mM sodium acetate buffer Data are means ± SE (ranges) in years. Levels of plasma, Haemoglobin A1c (HbA1c) (pH 6.0), 2 mM L-cysteine, 1 mM ferrous ammonium sulfate and and estimated glomerular filtration rate (eGFR) are expressed as means + SE in 2 with 60 mM of MI or without MI as a negative control and paral- mmol/l, % and mls/min/1.73 m , respectively. Diabetic nephropaths, uncomplicated and normal controls were defined in Section 2.1. leled tubes with D-glucuronic acid as positive controls. The mixture * Vs. the uncomplicated, p = 0.02. then was incubated at 30 °C for 1 hour and the reaction was termi- nated by the addition of 100 ll of 25% trichloroacetic acid. The products, D-glucuronic acid in the supernatant was determined by orcinol reagent [13,18,19]. Briefly, 200 mg of orcinol were dis- solved in 81.4 ml of concentrated HCl and then 2 ml of 2% ferric a Nephropath Uncomplicated Normal control chloride was added in. The final solution was made up to 100 ml with H2O. This solution must be made fresh. Two millilitre of the orcinol reagent was then added into the reaction tubes, which then NFAT5 were incubated at 100 °C in a water bath for 30 min. After they were cooled down to room temperature, 200 ll was transferred into 96-well-microplates in triplicates and D-glucuronic acid was measured on a spectrophotometer (GENios, Tecan, UK) at a wave- length of 650 nm. The MIOX enzyme activity was normalized by NG HG ARI+HG NG HG ARI+HG NG HG ARI+HG the activity without MI in the incubation reaction tubes. MIOX en- zyme activities were expressed as mean of fold increase due to b Nephropath Uncomplicated Normal control high glucose treatment, calculated by dividing the optical density (OD) value in high glucose-treated cells by the OD value in un- ChREBP treated cells or as mean of fold decrease due to ARI treatment, cal- culated by dividing the OD value in high glucose with ARI-treated cells by the OD value in high glucose-treated cells.

NG HG ARI+HG NG HG ARI+HG NG HG ARI+HG 2.7. Statistical analysis Fig. 1. NFAT5: nuclear factor of activated T cell 5; ChREBP: carbohydrate response Data were analysed through SPSS. t-test, or one-way analysis of element binding protein; NG: cells were cultured under normal conditions (D-glucose at a final concentration of 5.5 mmol/l). HG: cells were cultured under variance (ANOVA) was used to test DNA-binding activities, MIOX high glucose conditions (D-glucose at a final concentration of 25 mmol/l). ARI + HG: protein level, and MIOX enzyme activity between different groups. cells were cultured under high glucose conditions with aldose reductase inhibitor, A value of p < 0.05 was considered to be statistically significant. sorbinil (at a final concentration of 10 lmol/l). 172 B. Yang et al. / International Journal of Diabetes Mellitus 2 (2010) 169–174

Table 2 DNA-binding activities of NFAT5 and ChREBP to the ORE and ChRE in the promoter of the MIOX gene; MIOX protein levels and MIOX enzyme activity in patients with T1D with diabetic nephropathy or uncomplicated and normal controls.

Stress Nephropaths (n = 20) Uncomplicated (n = 14) Normal controls (n =9) DNA binding ChREBP to ChRE HG/NG 1.81 ± 0.22 (n = 11)* 1.30 ± 0.15 (n = 8) 1.41 ± 0.18 (n =8) ARI + HG/HG 0.92 ± 0.08 (n = 11) 0.92 ± 0.15 (n = 8) 0.95 ± 0.21 (n=8) NFAT5 to ORE HG/NG 1.54 ± 0.12 (n = 13)# 1.20 ± 0.10 (n = 8) 1.24 ± 0.10 (n =9) ARI+HG/HG 0.91 ± 0.06 (n = 13) 1.13 ± 0.24 (n = 8) 0.90 ± 0.06 (n =9) MIOX proteins HG/NG 1.3 ± 0.04 [n =6]§ 1.03 ± 0.04 [n = 4] 1.16 ± 0.56 [n =3] ARI + HG/HG 0.81 ± 0.19 [n = 6] 0.85 ± 0.05 [n = 4] 0.73 ± 0.12 [n =3] MIOX activity HG/NG 1.1 ± 0.4 [n = 3] 1.1 ± 0.1 [n = 3] 0.9 ± 0.1 [n =3] ARI + HG/HG 0.8 ± 0.1 [n = 3] 1.0 ± 0.4 [n = 3] 1.2 ± 0.3 [n =3]

Abbreviations: NFAT5, nuclear factor of activated T cell 5; ChREBP, carbohydrate response element binding protein; MIOX, myo-inositol oxygenase; ORE, osmotic response element; ChRE, carbohydrate response element. NG: cells were cultured under normal conditions (D-glucose at a final concentration of 5.5 mmol/l). HG: cells were cultured under high glucose conditions (D-glucose at a final concentration of 25 mmol/l). ARI + HG: cells were cultured under high glucose conditions with aldose reductase inhibitor, sorbinil (at a final concentration of 10 lmol/l). The density value was defined as 1 under NG conditions. Data are expressed as means of fold changes ± SE under HG compared to NG conditions (HG/NG) or under the ARI with HG conditions compared to HG conditions without ARI (ARI + HG/HG) in peripheral blood mononuclear cells from subjects with diabetic nephropathy or uncomplicated and normal controls. Sample sizes in the gel shift assay are shown within parentheses. The subjects in the electrophoretic mobility shift assays were overlapped. Pooled cytoplasmic proteins from each subject group were used to perform Western blotting for measuring levels of MIOX protein and an assay for determining MIOX enzyme activity. Results are shown herein from 3 to 6 independent measurements (square brackets). * Vs. nephropaths under ARI + HG conditions (p = 0.001). # Vs. nephropaths under ARI + HG conditions (p = 0.01). § Vs. nephropaths under ARI + HG conditions (p = 0.049).

Nephropath Uncomplicated Normal control

MIOX

α-tubulin NG HG ARI+HG NG HG ARI+HG NG HG ARI+HG

Fig. 2. MIOX: myo-inositol oxygenase; NG: cells were cultured under normal conditions (D-glucose at a final concentration of 5.5 mmol/l). HG: cells were cultured under high glucose conditions (D-glucose at a final concentration of 25 mmol/l). ARI + HG: cells were cultured under high glucose conditions with aldose reductase inhibitor, sorbinil (at a final concentration of 10 lmol/l).

NFAT5 to the ORE motif by 41% (1.54 ± 0.12 vs. 0.91 ± 0.06, through both competitive (its stereochemical similarity between p = 0.01) and ChREBP to the ChRE motif by 49% (1.81 ± 0.22 vs. D-glucose and MI) and non-competitive mechanisms (sorbitol 0.92 ± 0.08, p = 0.001) in the nephropaths (Table 2). However, the accumulation as a result of increased flux of polyol pathway) fold decrease due to ARI in the binding activities of NFAT5 and [20]. Evidence supporting the non-competitive mechanism is the ChREBP were not significantly different in DN compared to the fact that the ARI significantly ameliorated the decrease in MI up- uncomplicated group (NFAT5: 0.91 ± 0.06 vs. 1.13 ± 0.24, p > 0.05; take. This suggests a close link between MI depletion and the pol- ChREBP: 0.92 ± 0.08 vs. 0.92 ± 0.15; p > 0.05) (Table 2). Further- yol pathway. Recent studies on the MIOX gene structure provided more, in the presence of ARI, the protein levels of MIOX were de- another possible mechanism by which MI levels are regulated in creased in patients with nephropathy and this decrease just the cells. MIOX catalysis is the first committed step in the glucuro- reached statistical significance (1.3 + 0.04 vs. 0.81 + 0.19; nate–xylulose pathway, which is the only pathway for MI metabo- p = 0.049). Again, the fold decrease in the MIOX protein levels lism. As the expression of the MIOX gene is ORE and ChRE- was not significant between patients with nephropathy and the dependent, its expression levels may be regulated by NFAT5 and uncomplicated group (0.81 + 0.19 vs. 0.85 + 0.05; p > 0.05) (Table ChREBP under high glucose conditions [8]. The glucuronate–xylu- 2, Fig. 2). Furthermore, the DNA-binding activities of NFAT5 and lose pathway can also provide xylulose 5-phosphate, a cellular sen- ChREBP are correlated within individuals (data not shown), when sor that activates ChREBP during hyperglycaemia [21]. Therefore, there is an increase in DNA binding of NFAT5 there is also in- high glucose may not only directly cause increased DNA-binding creased DNA binding of ChREBP. activity of ChREBP to ChRE, but also the end product, xylulose 5- MIOX enzyme activity was assessed by an orcinol reagent. phosphate, provides a positive feedback loop on MIOX expression. There was no significant difference of MIOX enzymatic activity un- In this study, we have demonstrated that high glucose induced the der different conditions and between all study groups (Table 2). binding activities of NFAT5 and ChREBP to ORE and ChRE in the MIOX gene, respectively, although, these increases were not statis- tically significant. This trend may suggest that high glucose is in- 4. Discussion volved in the regulation of MIOX gene expression. However, these increases were much higher in patients with nephropathy, There is considerable evidence that altered inositol metabolism and were also accompanied by an increase in MIOX protein levels. (MI depletion) due to hyperglycaemia is associated with microvas- These results are similar to our previous studies, which have cular complications in both T1D and T2D [2–7]. A decreased uptake shown that the DNA-binding activity of NFAT5/NFjB to ORE/jB rate of MI under high glucose conditions has been suggested to motif in the promoter region of AKR1B1 [14,15] is increased in contribute to MI depletion, seen in diabetes and its complications PBMCs from patients with T1D and nephropathy, compared to B. Yang et al. / International Journal of Diabetes Mellitus 2 (2010) 169–174 173 the uncomplicated group under high glucose conditions. Further- obvious in the patients with nephropathy, compared to the uncom- more, our previous studies have shown that mRNA and protein lev- plicated subjects. Our results suggested that NFAT5 and ChREBP els of AKR1B1 and sorbitol dehydrogenase were increased in may be involved in the regulation of MIOX expression under high PBMCs from patients with T1D and nephropathy [14,22]. These re- glucose conditions in patients with T1D with nephropathy and that sults clearly indicated that the response to uptake and metabolism sorbitol may be involved in the process of DNA binding of NFAT5 of D-glucose is different for patients with T1D and with or without and ChREBP in the MIOX promoter. Using ARIs may slow down, nephropathy. The exact mechanisms have still to be elucidated and or prevent the development of diabetic nephropathy by inhibiting explored, but genetic associations such as AKR1B1 or glucose both the polyol and glucuronate–xylulose pathways to decrease transporter 1 (GLUT1) gene polymorphisms, may contribute to the production of sorbitol and to restore the MI levels within cells, the differences in gene expression [23,24]. Interestingly, there are respectively. single nucleotide polymorphisms close to the OREs in the MIOX gene. This could be another possible factor affecting MIOX expres- Acknowledgements sion levels through altered DNA-binding activity. A recent study conducted in a renal tubule cell line [25] showed an increase in Grant support: This work was supported by Diabetes UK. MIOX mRNA and protein levels in the kidney of rats with diabetic We would like to thank all staff that work at Diabetes Research nephropathy, whilst also demonstrating that in vitro high glucose Network in Plymouth Campus, UK for organising and collecting pa- significantly increased MIOX secretion in rat renal tubular epithe- tient samples. We would like to thank Dr. Peter Oates (Department lial cells, suggesting that hyperglycaemia may be a direct cause of of Cardiovascular and Metabolic Diseases, Pfizer Global Research the MIOX increase in the kidney. and Development, Groton, CT 06340 USA) for providing aldose In diabetic complications, increased glucose levels are associ- reductase inhibitor: sorbinil. ated with high sorbitol accumulation in the kidney, nerve, retina, and lens followed by a depletion of MI [2,3,5]. The MI metabolic pathway is a source of , which would contribute to the in- References crease in AKR1B1 expression during hyperglycaemia. 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ARIs have Sorbitol, myo-inositol, and rod outer segment phagocytosis in cultured hRPE been shown to improve MI uptake under high glucose conditions. cells exposed to glucose. In vitro model of myo-inositol depletion hypothesis of This suggests that the depletion of MI results from a glucose-in- diabetic complications. Diabetes 1991;40:1335–45. [6] Lin LR, Reddy VN, Giblin FJ, Kador PF, Kinoshita H. Polyol accumulation in duced decrease in MI uptake, which is associated with increased cultured human lens epithelial cells. Exp Eye Res 1991;52:93–100. AKR1B1 enzyme activity [26,27]. Our findings, together with those [7] Arner RJ, Prabhu KS, Krishnan V, Johnson MC, Reddy CC. Expression of myo- reported in cell lines and human neutrophils [28] strongly suggest inositol oxygenase in tissue susceptible to diabetic complications. Biochem that the increased expression and activity of AKR1B1 is intricately Biophys Res Commun 2006;339:816–20. 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Biochem Biophys Res Commun 2004;324:1386–92. raise inositol levels in diabetes, and may thus help counteract [11] Yang Q, Dixit B, Wada J, Tian Y, Wallner EI, et al. Identification of a renal- hyperglycaemia. specific oxido-reductase in newborn diabetic mice. Proc Natl Acad Sci USA The absence of any significant detectable changes in MIOX en- 2000;97:9896–901. [12] Danesh FR, Wada J, Wallner EI, Sahai A, Srivastva SK, Kanwar YS. Gene zyme activity in our subjects is disappointing, and may be due to regulation of aldose-aldehyde- and a renal specific oxido reductase (RSOR) in a number of reasons including the assay itself. Most assays done the pathobiology of diabetes mellitus. Curr Med Chem 2003;10:1399–406. by other groups have been on homogeneous MIOX enzyme ob- [13] Prabhu KS, Arner RJ, Vunta H, Reddy CC. Up-regulation of human myo-inositol oxygenase by hyperosmotic stress in renal proximal tubular epithelial cells. J tained from a centrifuged homogenate of rat or hog kidneys. There Biol Chem 2005;280:19895–901. is no publication to our knowledge that describes the MIOX assay [14] Yang B, Hodgkinson AD, Oates P, Kwon HM, Millward BA, Demaine AG. in PBMCs. Although, our RT-PCR (data not shown) and Western Elevated activity of transcription factor nuclear factor of activated T-cells 5 blotting demonstrated that PBMCs expressed high levels of MIOX, (NFAT5) and diabetic nephropathy. Diabetes 2006;55:1450–5. [15] Yang B, Hodgkinson AD, Oates P, Millward BA, Demaine AG. High glucose MIOX enzyme activity may not be very high in these cases. On the induction of DNA binding activity of the transcription factor NFjB in patients other hand, the assay may not be sensitive and specific enough to with diabetic nephropathy. Biochim Biophys Acta 2008;1782:295–302. detect MIOX activity in the PBMCs extracted from our subjects pre- [16] The Expert Committee on the diagnosis and classification of diabetes mellitus: Report of the Expert Committee on the diagnosis and classification of diabetes pared in our way. mellitus, Diabetes Care 2003;26:S5–20. In summary, we have shown a trend for high glucose-increased [17] Heesom AE, Hibberd ML, Millward BA, Demaine AG. Polymorphism in the 5’- binding activities of NFAT5 and ChREBP accompanied with in- end of the aldose reductase gene is strongly associated with the development of diabetic nephropathy in type 1 diabetes. Diabetes 1997;46:287–91. creased protein levels, particularly in patients with nephropathy. [18] Reddy CC, Swan JS, Hamilton GA. Myo-inositol oxygenase from hog kidney. I. ARI treatment prevented these increases, and this effect was more purification and characterization of the oxygenase and of an enzyme complex 174 B. 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