Trigonelline Ameliorates Oxidative Stress in Type 2 Diabetic Goto-Kakizaki Rats

Abstract

Previously, we showed that trigonelline (TRG) exerts antidiabetic effects in type 2 diabetic Goto-Kakizaki (GK) rats and also lowers blood and liver thiobarbituric acid reactive substances and urinary 8-hydroxydeoxyguanosine when compared with those levels in GK control rats without TRG. These results suggested that TRG also mitigates oxidative stress, which accelerates diabetes. In this study, the mechanisms of TRG prevention of oxidative stress were determined by measuring erythrocyte and liver antioxidant enzyme activities, and expressions of genes associated with reactive oxygen species production, and carbohydrate and lipid metabolisms by DNA microarray. Erythrocyte and liver glutathione peroxidase, and liver catalase activities in the GK rats fed with TRG were significantly lower than those of the GK control rats. TRG downregulated the gene expressions involved with NADPH oxidase and mitochondrial electron transfer system when compared with those of the GK control group. These results suggested that mitigation of diabetes by TRG is mediated by its ameliorating effects on oxidative stress.

Introduction

I t is recognized that reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide tend to be produced in patients suffering from hypertension, diabetes, and obesity.^1
–3 It is reported that ROS are produced along with the progression of diabetes, and that their production is caused by nicotinamide adenine dinucleotide phosphate (NADPH)–oxidase,⁴ xanthine oxidase,⁵ and the mitochondrial electron transfer system.⁶ For example, tumor necrosis factor (TNF)α and advanced glycation end-products (AGE) are known to accelerate the production of ROS via NADPH oxidase.^7
–9 ROS produced through these processes oxidize DNA, proteins, and lipids, causing impairment of cellular functions. Ookawara et al. reported that the antioxidant enzyme, superoxide dismutase (SOD) produced ROS when glycation continuously progressed in hyperglycemia, and the activity of this enzyme was decreased with increased ROS.¹⁰ It is also known that oxidation of pancreatic β cells by ROS causes a decrease in insulin secretion,¹¹ and that ROS induce insulin resistance by impairing insulin signaling. Treatment with antioxidant enzymes, such SOD or catalase (CAT), is reported to be useful for normalizing these defects.¹²

Trigonelline (TRG) is one of the alkaloids having a pyridine ring, and is found in some foods, like coffee¹³ or pumpkin.¹⁴ It has been known that TRG is excreted as methyl-2-pyridone-5-carboxlic acid and TRG is found in urine after its oral administration.^15,16 TRG is reported to improve cognitive function^17,18 and auditory neuropathy,^19,20 and exhibits anticancer actions.²¹ It is also reported that TRG improves glucose tolerance in men with obesity;²² however, its mechanism is undefined. Previously, we reported that TRG has an antidiabetic effect in type 2 diabetic Goto-Kakizaki (GK) rats and that feeding of TRG resulted in decreased serum levels of insulin and TNFα, as well as decreased levels of liver lipids (total cholesterol, triglyceride [TG], and free fatty acid).¹⁴ It has also been reported that blood and liver thiobarbituric acid reactive substances (TBARS), and urinary 8-hydroxydeoxyguanosine (8-OHdG) levels in the GK rats fed with TRG were significantly lower, suggesting that TRG might be useful for improving oxidative stress. As TRG does not have strong antioxidative activity,^21,23 it is very interesting to determine the reasons why TRG can prevent the oxidation of DNA and lipids in vivo. The aim of this study was to determine the mechanism of TRG for ameliorating oxidative stress in diabetic GK rats.

Materials and Methods

Animal care

Male Wistar and GK (GK/Slc) rats (8 weeks old) were purchased from Clea Japan, Inc. (Tokyo, Japan). Wistar rats were used as normal rats without diabetes. After acclimatizing for 4 days, the Wistar and GK rats were assigned to two groups (Wistar; each five rats per group, GK; six rats per group), respectively, individually housed in stainless steel cages with wire netting at the bottom. The rats were fed on either the basal diet (control group) or the basal diet containing 0.056% (0.406 mmol) TRG (TRG group) for 43 days. This amount of dietary TRG is the same as that in the previous study.¹⁴ The basal diet was composed of casein (15%), α-cornstarch:sucrose 2:1 (v/v) mixture (70.5%), corn oil (5%), AIN-93G-MX mineral mixture (3.5%), and AIN-93-VX vitamin mixture (1%). TRG was added to the diet in place of an equal amount of the α-cornstarch:sucrose mixture. TRG was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All animals were maintained in a room with a 12-h light–12-h dark cycle at 22°C±2°C and 40%–60% humidity. The diet and water were given ad libitum. The rats were scarified after 10 h of fasting on the last day of the feeding period of the experimental diet. Blood was collected by cardiac puncture from rats anesthetized with Nembutal (50 mg/mL; Dainippon Pharmaceutical Co., Osaka, Japan). The liver was dissected out and stored at −80°C until needed for analyses. The rats were cared for at all times according to the institutional guidelines of Yamagata University.

Antioxidant enzyme activities

Preparation of erythrocyte and liver homogenates

Blood (0.1 mL) mixed with 0.9% NaCl (1.9 mL) was centrifuged at 1000 g for 5 min. The erythrocytes obtained as precipitates were washed thrice with ice-cold saline and suspended in 2.9 mL deionized water. Contents of hemoglobin in the erythrocyte lysate were measured using the Wako Hemoglobin B-test kit (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

Liver (0.6 g) was homogenized with a five-fold-volume of potassium phosphate buffer (0.1 M pH 7.4, added with 1 mM ethylenediaminetetraacetic acid) and two-fold KCl (2.3%) in a glass-type homogenizer, followed by centrifugation at 1000 g for 10 min at 4°C. After sonication of the supernatants for 2 min, the mixture was recentrifuged at 10,000 g for 20 min at 4°C. The supernatants were used for the measurement of the SOD, CAT, and glutathione peroxidase (GPx) activities. The protein content was measured by the method described by Lowry et al. ²⁴

Enzyme activities

SOD activity was measured by the xanthine/xanthine oxidase method.²⁵ One unit of this enzyme activity was defined as the amount of enzyme required to inhibit 50% of the standard level at 560 nm. CAT activity was determined by the method of Chance and Meahly, measuring the decrease in the absorbance at 240 nm due to decomposition of the hydrogen peroxide.²⁶ GPx activity was determined from the decrease in the NADPH due to the reaction.^27,28 One unit of CAT and GPx activity was defined as the amount of enzyme required to decompose 1 mmol hydrogen peroxide or oxidize 1 mmol NADPH per minute.

Measurement of reduced form glutathione and oxidized form glutathione levels

A homogenate for measuring the liver reduced form glutathione (GSH) and oxidized form glutathione (GSSG) was prepared by homogenizing the liver with a 10-fold volume of ice-cold 0.4 N perchloric acid. The supernatant, obtained by centrifuging the homogenate at 13,500 g for 5 min under 4°C, was filtered through a 0.2-μ m filter. GSH and GSSG contents in the filtrate were determined by an ion-pairing reverse-phase high-performance liquid chromatography coupled to a coulometric detector according to the method described by Harvey et al.²⁹

DNA microarray

The DNA microarray analysis was carried out in the GK rats. The RNA was isolated from the liver using the RNeasy Mini kit (Qiagen N.V., Hilden, Germany), and its quantity and quality were assessed spectrophotometrically using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). The isolated RNA was converted to cDNA using the WT Sence Target Labeling kit (NuGen Technologies, Inc., San Carlos, CA, USA).

The cDNA was hybridized onto GeneChip^® Array (Rat Gene 1.0 ST Array, 27,342 genes Affymetrix, Inc., Santa Clara, CA, USA) for 16 h at 45°C. GeneChip was washed and scanned by the GeneChip 3000 scanner. Using data analysis system GeneChip Operating Software, the acquired sample was confirmed as array image data. The magnitude of gene expression was converted into the txt file extracted as numerical value with GeneChip data analysis Expression Console^™ software (Affymetrix). The standardized data were analyzed by algorithm Robust Multichip Analysis set in Expression Console. Three rat livers in the control and TRG groups of GK rats were analyzed. The genes that showed more than 1.5-fold change in their expression compared with the control group were considered as genes affected by feeding of TRG.

The genes that showed the changes in their expression were annotated based on NetAffx (www.affymetrix.com). The classification of pathway for interesting genes was performed using Uniprot and KEGG database. Abbreviations of genes in DNA Microarray are shown as gene symbols.

Real-time reverse transcription–polymerase chain reaction

Quantification of mRNA was also performed on the mRNA levels by real-time polymerase chain reaction (PCR). The confirmed genes are as follows: glucose-6-phoshatase (gene symbol: G6pc), angiopoietin-like 4 (Angptl4), cytochrome P450, and subfamily 51 (Cyp51). Levels of these genes were relative values to the housekeeping gene β-actin (Actb). Primers were purchased from Sigma-Aldrich Co.

G6pc: sense 5′-CTACCTTGCGGCTCACTTTC-3′, antisense 5′-ATCCAAGTGCG AAACCAAAC-3′; Angptl4: sense 5′-CAGAACAGCAAGATCCAGCA-3′, antisense, 5′-CCTCTTTCCCCTCGAAGTCT-3′; Cyp51: sense 5′-GTGCCAAATGCAGTTTTCCT-3′, antisense 5′-TCAGACAGCGCTTCAAACAC-3′; Actb: sense 5′-ACCCACACTGTGCCCATCTA-3′, antisense 5′-CGTCACACTTCATGATG-3′.

Reactions were carried out with StepOne^™ Real-Time PCR System (Applied Biosystems, Inc., Foster City, CA, USA) using the SYBR Green Qiagen One-Step RT-PCR kit (Qiagen N.V.). The program profile was 95°C for 30 sec and 45 cycles of denaturation for 5 sec at 95°C, and annealing for 15 sec at 55°C and extension for 15 sec at 72°C.

Statistical analysis

Each value is given as the mean±standard error of the mean. Significant differences between the two groups were determined by Welch's t-test and P<.05 was considered as statistically significant. In the case of DNA microarray data, the values with P<.01 were considered statistically significant. The false discovery rate (FDR) was calculated according to Benjamini and Hochberg,³⁰ and the threshold of the FDR was set at 5%.

RESULTS

There were no differences in erythrocyte and liver SOD, CAT, and GPx activities when compared between the control and TRG groups in the Wistar rats (Table 1). Although liver SOD activity of the GK rats did not differ significantly between the control and TRG groups, the erythrocyte and liver CAT, and GPx activities in the TRG group either exhibited a tendency to be lower or were significantly lower compared with the control group (Table 1). The liver GSH levels did not differ between the control and TRG group in the GK rats, but the GSH/GSSG ratios in the TRG group were higher than in the control group (Table 1).

Table 1.

Effects of Trigonelline on Erythrocyte and Liver Antioxidant Enzymes Activities, and Liver Glutathione Levels

	Dietary group
	Wistar (n=5)		GK (n=6)
Antioxidant enzyme activity	Control	TRG	Control	TRG
Erythrocyte
CAT (U/mg of Hb)	3.90±0.89	4.11±0.32	7.96±1.21	6.70±0.38^†
GPx (U/mg of Hb)	23.1±2.2	20.8±3.9	34.0±3.3	24.8±2.0^*
Liver
SOD (U/mg of protein)	1.04±0.59	1.21±0.99	0.931±0.061	1.06±0.09
CAT (U/mg of protein)	63.8±1.4	61.5±2.7	76.0±1.1	50.5±6.3^*
GPx (U/mg of protein)	0.921±0.071	0.901±0.105	1.15±0.05	0.751±0.087^*
Liver glutathione level
GSH (mmol/kg of liver)	—	—	4.27±0.11	4.16±0.09
GSSG (mmol/kg of liver)	—	—	0.0592±0.0123	0.0239±0.0054^*
GSH/GSSG ratio	—	—	82.4±18.1	214±41^*

Values are means±standard error of the mean.

Significant differences between the control and TRG groups in each Wistar or GK rats were determined by Welch's t-test: ^* P<.05, ^† P<.1 compared with GK control rats.

GK, Goto-Kakizaki; TGR, trigonelline; CAT, catalase; GPx, glutathione peroxidase; SOD, superoxide dismutase; GSH, reduced form glutathione; GSSG, oxidized form glutathione.

To examine the expression of genes related to carbohydrate and lipid metabolisms, antioxidant enzymes, and ROS (superoxide anion) generation, DNA microarray analysis was carried out in the GK rats. When compared with the control group, 58 genes were upregulated and 229 genes were downregulated in the TRG group (Supplementary Tables S1 and S2; Supplementary Data are available online at www.liebertpub.com/jmf). Gene expressions of antioxidant enzymes (gene symbols: Cat, Gpx1, Gsr, and Gstk1) were downregulated in the TRG group (Table 2). The activities of CAT and GPx were lowered in the GK TRG group. Additionally, gene expressions involved with NADPH oxidase (Cybb, Nox4) and mitochondrial electron transfer system subunit (Ndufs1, Ndufs8, Ndufa9, Ndufa10, Ndufb6, Cox7a2, and Cox8a) were also observed to be downregulated by feeding TRG (Table 2).

Table 2.

Expression of Genes Concerned with Antioxidantive Enzymes and Superoxide Anion Generation

Function	Gene symbol	Gene title	Fold change ^a
Antioxidantive enzyme	Cat	Catalase	0.65
	Gpx1	Glutathione peroxidase 1	0.60
	Gsr	Glutathione reductase	0.58
	Gstk1	Glutathione S-transferase kappa1	0.64
NADPH oxidase	Cybb	Cytochrome b-245, beta polypeptide	0.59
	Nox4	NADPH oxidase 4	0.62
Mitochondria electron transfer system	Ndufs1	NADH dehydrogenase (ubiquinone) Fe-S protein 1	0.53
	Ndufs8	NADH dehydrogenase (ubiquinone) Fe-S protein 8	0.66
	Ndufa9	NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9	0.45
	Ndufa10	NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 10	0.46
	Ndufb6	NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6	0.33
	Cox7a2	Cytochrome c oxidase, subunit VIIa2	0.65
	Cox8a	Cytochrome c oxidase, subunit VIIIa	0.65

Expression of genes concerned with antioxidative enzymes and superoxide anion generation was compared between GK rats given the diet with or without TRG. Values are means, n=3 independent experiments.

Relative value to control group.

Genes associated with glycolysis (Pdhb, Pklr, Pfkfb1, and Gck), gluconeogenesis (G6pc, Slc37a4), and glucose uptake (Slc2a2) were downregulated by feeding TRG (Table 3). Genes in TCA cycle (Pdk4, Acly), genes of enzymes involved in metabolism of starch and sucrose (Mgam, Ugp2, and Amy1a), and genes of enzymes concerned with the insulin signal pathway (Mknk2, Gys2, Pik3r1, Phkg2, Ppp1cb, Foxo1, Kras, and Ppp1r3c) showed changes when animals were fed with TRG. Overall lipid metabolism was downregulated, as seen by the reduced expression of genes associated with cholesterol biosynthesis (Lss, Hmgcr, Sqle, Fdft1, Cyp51, and Sc4mol), fatty acid metabolism (Acsl5, Acacb, Acaca, and Srebf1), and genes concerned with peroxisome proliferator-activated receptor (PPAR) α signaling (Fads2, Ep300, Ppara, Angptl4, Cyp27a1, Pnpla2, Cyp7a1, and Cpt1a; Table 4). In addition, the TNFα receptor gene (Tnfrsf1a) was also downregulated by feeding of TRG. Expressions of the main genes concerned with carbohydrates and lipid metabolisms are shown in Supplementary Figures S1 and S2.

Table 3.

Expression of Genes Concerned with Carbohydrate Metabolism

Function	Gene symbol	Gene name	Fold change ^a
Glycolysis	Pdhb	Pyruvate dehydrogenase (lipoamide) beta	0.59
	Pklr	Pyruvate kinase, liver, and RBC	0.50
	Pfkfb1	6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 1	0.44
	Gck	Glucokinase	0.33
Gluconeogenesis	G6pc	Glucose-6-phosphatase, catalytic subunit	0.50
	Slc37a4	Solute carrier family 37 (glucose-6-phosphate transporter), member 4	0.48
Glucose uptake	Slc2a2	Solute carrier family 2 (facilitated glucose transporter), member 2 (GLUT2)	0.50
TCA cycle	Pdk4	Pyruvate dehydrogenase kinase, isozyme 4 (negative regulation)	2.58
	Acly	ATP citrate lyase	0.45
Pentose-phosphate pathway	G6pd	Glucose-6-phosphate dehydrogenase	0.49
	H6pd	Hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase)	0.43
Amino sugar and nucleotide sugar metabolism	Nans	N-acetylneuraminic acid synthase	0.50
Starch and sucrose metabolism	Mgam	Maltase-glucoamylase	0.49
	Ugp2	UDP-glucose pyrophosphorylase 2	0.49
	Amy1a	Amylase, alpha 1A (salivary)	0.47
Glycan metabolismbiosynthesis	Man2a1	Mannosidase, alpha, class 2A, member 1	0.49
	Mgat2	Mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase	0.48
	Fuca2	Fucosidase, alpha-L-2, plasma	0.48
Heparan sulfate biosynthesis	Ext1	Exostoses (multiple) 1	0.47
Insulin signal pathway	Ppp1r3b	Protein phosphatase 1, regulatory (inhibitor) subunit 3B	0.58
	Mknk2	MAP kinase-interacting serine/threonine kinase 2	0.56
	Gys2	Glycogen synthase 2	0.50
	Pik3r1	Phosphoinositide-3-kinase, regulatory subunit 1 (alpha)	0.50
	Phkg2	Phosphorylase kinase, gamma 2 (testis)	0.47
	Ppp1cb	Protein phosphatase 1, catalytic subunit, beta isoform	0.44
	Foxo1	Forkhead box O1	0.42
	Kras	v-ki-ras2 Kirsten rat sarcoma vial oncogene homolog	0.42
	Ppp1r3c	Protein phosphatase 1, regulatory (inhibitor) subunit 3C	0.31
Inositol phosphate metabolism	Pten	Phosphatase and tensin homolog	0.48

Expression of genes involved in carbohydrate metabolism was compared between GK rats given the diet with or without TRG. Values are means, n=3 independent experiments.

Relative value to control group.

Table 4.

Expression of Genes Concerned with Lipid Metabolism by Dietary Trigonelline

Function	Gene symbol	Gene name	Fold change ^a
Cholesterol biosynthesis	Lss	Lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase)	0.51
	Hmgcr	3-Hydroxy-3-methylglutaryl-coenzyme A reductase	0.50
	Sqle	Squalene epoxidase	0.50
	Fdft1	Farnesyl diphosphate farnesyl transferase 1	0.46
	Cyp51	Cytochrome P450, subfamily 51	0.42
	Sc4mol	Sterol-C4-methyl oxidase-like	0.39
Lipoprotein metabolism	Lcat	Lecithin cholesterol acyltransferase	0.36
Sphingolipid metabolism	Smpd1	Sphingomyelin phoaphodiesterase 1, acid lysosomal	0.50
	Lass2	LAG1 homolog, ceramide synthase 2	0.48
Glycerophospholipid metabolism	Chpt1	Choline phosphotransferase 1	0.41
Steroid hormone biosynthesis	Cyp17a1	Cytochrome P450, family 17, subfamily a, polypeptide 1	0.49
Bile acid biosynthesis	Hsd3b7	Hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 7	0.61
	Slc27a5	Solute carrier family 27 (fatty acid transporter), member 5	0.50
Fatty acid metabolism	Acsl5	Acyl-CoA synthetase long-chain family member 5	0.61
	Acacb	Acetyl-coenzyme A carboxylase beta	0.49
	Acaca	Acetyl-coenzyme A carboxylase alpha	0.48
	Srebf1	Sterol regulatory element binding transcription factor 1	0.48
PPAR alpha signaling	Fads2	Fatty acid desaturase 2	0.49
	Ep300	E1A binding protein p300	0.48
	Ppara	Peroxisome proliferator activated receptor alpha	0.48
	Angptl4	Angiopoietin-like 4	0.47
	Cyp27a1	Cytochrome P450, family 27, subfamily a, polypeptide 1	0.45
	Pnpla2	Patatin-like phosholipase domain containg 2	0.41
	Cyp7a1	Cytochrome P450, family 7, subfamily a, polypeptide 1	0.19
	Cpt1a	Carnitine palmitoyltransferase 1a, liver	0.50
Glycosylphosphatidylinositol (GPI)-anchor biosynthesis	Pigm	Phoshatidylinositol glycan anchor biosynthesis, class M	0.50
	Pign	Phosphatidylinositol glycan, class N	0.43
	Pigw	Phosphatidylinositol glycan anchor biosynthesis, class W	0.38
Calcium signaling pathway/renin-angiotensin system	Agtr1a	Angiotensin II receptor, type 1a	0.50
TNF signaling	Tnfrsf1a	Tumor necrosis factor receptor superfamily, member 1a	0.40

Expression of genes concerned with lipid metabolism was compared between GK rats given the diet with or without TRG. Values are means, n=3 independent experiments.

Relative value to control group.

To confirm downregulation of the above-mentioned genes by dietary TRG, the expression patterns of three genes (G6pc, Angptl4, and Cyp51) were determined by real-time PCR. The expression levels of the three genes in the TRG group were significantly lower than those in the control group, showing agreements with the results obtained by the microarray analysis (Fig. 1).

FIG. 1.

Effect of feeding trigonelline (TRG) on the gene expression levels of glucose-6-phosphatase (G6pc), angiopoietin-like 4 (Angptl4), and cytochrome P450 subfamily 51 (Cyp51) in GK rats. Normalization was performed with respect to β-actin. Values are means±standard error of the means (n=4). *P<.05, **P<.01 versus control (CON) group. GK, Goto-Kakizaki.

Discussion

We recently demonstrated that serum insulin and TNFα, liver lipid and TBARS, and urinary 8-OHdG levels in the TRG group of the GK rat were significantly lower than those in the control group without TRG.¹⁴ However, action mechanisms for these phenomena were not examined precisely in the previous article. This study was conducted to investigate the mechanisms for ameliorating diabetes and oxidative stress by TRG.

Although antioxidant enzyme activities in the Wistar rats did not differ between the control and TRG groups, those of GK rats showed a significant difference between the control and TRG groups. This result might be due to that ROS production prone to occur in the diabetes in GK rats. ROS production in the GK control group is thought to be greater than in the GK TRG group because of the higher levels of TBARS and 8-OHdG shown in the previous study. In the GK control group, there was an increase in the antioxidative enzyme activities with increases in ROS. One reason may be because their enzymes, as proteins, were not yet damaged by ROS. It was reported that CAT activity was higher in diabetic rats because of increased hydrogen peroxide.³¹ In this study, the lower CAT and GPx activities in the GK TRG group may indicate that feeding of TRG suppressed oxidative stress. Furthermore, the higher GSH/GSSG ratio in the GK TRG group may also explain that TRG-fed animals are under less oxidative stress than the control animals. However, the absolute amount of GSSG in the GK control and GK TRG groups was very little compared with te absolute amount of GSH. Thus, lower activity of antioxidant enzymes in the GK TRG group might be due to other causes and not due to a decrease in the amount of GSH. Since the antioxidant activity of TRG in vivo is not strong,^21,23 it is thought that regulation of antioxidant enzymes by TRG is involved with the greater suppression of oxidation. To examine the mechanism of TRG more precisely, the gene expressions were analyzed by DNA microarray.

DNA microarray in the GK rats indicated that feeding of TRG downregulated the expression of genes involved with glucose uptake, glycolysis, gluconeogenesis, cholesterol biosynthesis, and fatty acid metabolism. Fasting blood glucose levels in the control and TRG groups were not significantly different in the previous study. However, expressions of genes involved with glycolysis and gluconeogenesis in the TRG group were changed in this study. Serum insulin levels in the TRG group were lower than that in the control group, indicating that feeding of TRG may be effective to prevent insulin resistance. Lower serum insulin levels in the TRG group were related to the downregulation of PI3K genes (Table 3). The lower serum TNFα level in the TRG group was related to the gene expression of Tnfrsf1a downregulation (Table 4). It is reported that the inhibition of tyrosine phosphorylation in insulin receptor substance by TNFα is the cause of increased insulin levels.³² Feeding of TRG might mitigate insulin resistance by regulation of phosphorylation. Lower liver lipid levels in the TRG group might be achieved by suppressing the expressions of the lipogenic genes, because downregulation of genes associated with cholesterol biosynthesis and fatty acid metabolism was observed in the microarray. Since it is known that higher levels of serum fatty acid induce gene expression of Cpt1 through PPARα,³³ lower levels of fatty acid in rats fed TRG, which was reported previously by us,¹⁴ might be due to the downregulation of gene expression of Ppara and Cpt1a.

As it is known that TNFα activates cytosolic NADPH oxidase to produce ROS,³⁴ and also induces ROS production in mitochondria via ceramide-dependent signaling pathways involved with the electron transport chain,³⁵ the lower serum TNFα level induced by TRG might be closely connected with inhibition of ROS production and related to the decrease in oxidative stress. In addition, it is also known that NADPH oxidase is activated by various stimuli, such as insulin,³⁶ AGE,⁹ and accumulated fat.³ Our previous study showed that serum insulin levels and serum and liver TG levels were decreased by feeding of TRG in the GK rats.¹⁴ Furthermore, hemoglobin A1c levels in the TRG group were lower compared with the control group, suggesting that AGE levels in the TRG group were lower compared with the control group. Feeding of TRG may suppress oxidative stress by inhibiting the formation of TNFα and AGE, and slowing fat accumulation, resulting in the suppression of ROS formation. Downregulation of the gene expressions involved with NADPH oxidase and electron transport chain in the TRG-fed rats may support that TRG suppresses the formation of ROS (Fig. 2).

FIG. 2.

A model illustrating effect of feeding TRG on antioxidant enzymes and glutathione. ▼, Lowering of gene expression;↓, lowering of enzyme activity or level; ⊥, inhibition.

In this report, it was demonstrated that expressions of genes associated with carbohydrate and lipid metabolisms, NADPH oxidation, and mitochondrial electron transfer were downregulated by feeding TRG in the GK rats. In parallel with these phenomena, decreases in the antioxidant enzyme (SOD, CAT, and GPx) activities were also observed. These results suggest that suppression of the generation of active oxygen species through suppression of formation of TNFα, glycation of hemoglobin, and accumulation of lipids, which may be closely related to the downregulation of gene expression of NADPH oxidase and mitochondrial electron transfer system, by dietary TRG may be the mechanism of the mitigation of diabetes in GK rats.

Footnotes

Acknowledgments

O.Y. designed and conducted the research, analyzed data, and wrote the article. A.T. analyzed real-time reverse transcription–PCR. K.I. had primary responsibility for the final content.

Author Disclosure Statement

There is no ethical problem or conflict of interest with regard to this manuscript.

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