J Nutr Sci Vitaminol, 65, 45–51, 2019

Alteration of Metabolism by a High-Fat Diet Is Associated with Plasma Transaminase Activities and Glucose Intolerance in Rats

Reika Yoshitsugu, Keidai Kikuchi, Hitoshi Iwaya, Nobuyuki Fujii, Shota Hori, Dong Geun Lee and Satoshi Ishizuka*

Laboratory of Nutritional Biochemistry, Research Group of Bioscience and Chemistry, Division of Fundamental Agriscience Research, Research Faculty of Agriculture, Hokkaido University, Sapporo 060–8589, Japan (Received July 3, 2018)

Summary Ingestion of a high-fat (HF) diet is known to enhance bile acid (BA) secretion, but precise information about the BA molecular species is lacking, especially information on the conjugated BAs in enterohepatic circulation. As cholesterol is the precursor of BAs, we analyzed alterations of the entire BA metabolic pathway in response to a HF diet without the addition of cholesterol and BA in the diet. Additionally, we evaluated the relationships between BA metabolism and some disorders, such as plasma transaminase activities and glucose intolerance induced by the HF diet. Acclimated WKAH/HkmSlc male rats (3 wk old) were divided into two groups fed a control or the HF diet for 22 wk. Fasting blood glu- cose was measured during the experimental period, and an intraperitoneal glucose tolerance test was performed at week 21. As a result, ingestion of the HF diet selectively increased the concentration of taurocholic acid in the bile and small intestinal contents as well as deoxycholic acid in the large intestinal contents and feces. These results indicated a selec- tive increase of 12a-hydroxylated BA concentrations in response to the HF diet. Moreover, fecal 12a-hydroxylated BA concentration was positively correlated with cumulative energy intake, visceral adipose tissue weight, and glucose intolerance. The present study suggests that fecal 12a-hydroxylated BA is a non-invasive marker that can detect the early phase of glucose intolerance. Key Words bile acids, high-fat diet, energy intake, liver damage, glucose tolerance

The expansion of noncommunicable diseases (NCDs) this instrumental analysis enables us to accurately eval- has increased, and overweight and unhealthy diets are uate BA metabolism, including conjugated BAs. factors associated with the incidence of disease (1). During the development of instrumental analysis, Consumption of an energy-dense diet is considered to information on BA metabolism has gradually been be related to NCDs. Actually, obesity (2) and various revealed. For example, BAs undergo deconjugation disorders (3), such as insulin resistance and dyslipid- and/or 7a-dehydroxylation by some intestinal bacte- emia, related to energy metabolism are observed in ani- ria (8). Aging increases the ratio of (CA) to mal models fed a high-fat diet. Many researchers have (CDCA) in rodents (9). The CA/ attempted to reveal the underlying mechanisms in the CDCA ratio in primary BAs in humans has not been development of these disorders, but these efforts are still systematically investigated. However, there are some underway. reports showing the BA composition in bile, serum, Ingestion of a high-fat diet promotes bile acid (BA) or feces, which enabled us to calculate the CA/CDCA secretion as shown by fecal excretion of secondary BAs ratio of 0.3–0.5 in infants until 72 h after birth (10), in animal and human studies (4, 5). In these studies, of ~0.5 in children ages 12–59 mo (11), and of .1.1 BAs were primarily analyzed by gas chromatography in adults 24–41 y of age (12). Taurocholic acid (TCA) (GC) and GC/mass spectrometry (MS). In this method, is the taurine-conjugated form, and deoxycholic acid deconjugation and derivatization steps of the extracted (DCA) is the 7a-dehydroxylated form of CA (13). Both BAs are necessary before analysis with GC/MS, which TCA and DCA possess an a-hydroxyl group in the ste- makes it difficult to distinguish unconjugated and con- roid ring. In rodents, there is an additional BA synthetic jugated BAs in the BA profile. Those preparatory steps pathway from CDCA to a- and b-muricholic acids in are not required in current methods with liquid chro- the liver (14). Considering the diversity of primary BA matography (LC)/MS (6, 7), and the whole BA profile synthesis, it is appropriate to use the term “proportion can be analyzed at the same time. The development of of 12a-hydroxylated BAs (12aBAs)” as a more general expression rather than the CA/CDCA ratio. * To whom correspondence should be addressed. BA metabolism is closely related to several disorders, E-mail: [email protected] such as carcinogenesis in the liver and intestine (15),

45 46 Yoshitsugu R et al. type 2 diabetes (16) and cardiovascular diseases (17). Table 1. Diet composition. However, fundamental information on BA metabolism on a high-fat diet is still unclear despite the attention C HF that has been paid to the relationship between BAs and the development of NCDs. In this study, we assessed BA g/kg diet 1 metabolism in rats fed a high-fat diet and explored the Dextrin 529.5 299.5 Casein2 200.0 200.0 relationship between BA metabolism and several disor- Sucrose3 100.0 100.0 ders observed with high-fat diet consumption. Soybean oil4 70.0 70.0 MATERIALS AND METHODS Lard — 230.0 Cellulose5 50.0 50.0 Animals. The study was approved by the Institu- Mineral mixture6 35.0 35.0 tional Animal Care and Use Committee of National Vitamin mixture7 10.0 10.0 Corporation Hokkaido University (approved number: Choline bitartrate8 2.5 2.5 14-0026), and all animals were maintained in accor- l-Cystine8 3.0 3.0 dance with the Hokkaido University Manual for Imple- 1 TK-16 (Matsutani Chemical Industry Co., Ltd., Hyogo, menting Animal Experimentation. Wistar King A Hok- Japan). kaido male rats (3-wk-old NBRP Rat No: 0154) (WKAH/ 2 NZMP Acid Casein (Fonterra Co-Operative Group Lim- HkmSlc, Japan SLC, Inc., Shizuoka, Japan), an inbred ited, Auckland, New Zealand). strain of Wistar rats, were housed in an air-conditioned 3 Nippon Beet Sugar Manufacturing Co., Ltd., Tokyo, room at 2262˚C with 5565% humidity, and the light Japan period was from 8:00 to 20:00. The rats were housed 4 J-Oil Mills, Inc., Tokyo, Japan. individually in wire-bottomed cages and allowed ad libi- 5 Crystalline cellulose (Ceolus PH-102, Asahi Kasei tum access to diet and water. The rats were acclimatized Chemicals Corp., Tokyo, Japan). 6 with a control diet (C) based on the AIN-93G formulation AIN-93G mineral mixture (18). 7 AIN-93 vitamin mixture (18). (Table 1) (18) for 2 wk. The acclimatized rats (n520) 8 Wako Pure Chemical Industries, Ltd., Osaka, Japan. were then divided into two groups and were fed either a C (n510) or a high-fat diet (HF) (n510) (Table 1) for another 22 wk. Tail vein blood was collected every 2 wk from week 1 to week 15 and at week 21 during the test - (5b-cholanic acid-3a,6a,7b-triol, period after food deprivation for 16 h to measure fasting MCA), CDCA (5b-cholanic acid-3a,7a-diol), DCA plasma glucose concentration. Additionally, feces were (5b-cholanic acid-3a,12a-diol), hyocholic acid (5b- collected every 2 wk from week 2 to week 22 to measure cholanic acid-3a,6a,7a-triol, HCA), hyodeoxycholic fecal BA compositions. At week 22, bile juice was col- acid (5b-cholanic acid-3a,6a-diol, HDCA), ursodeoxy- lected through a polyvinyl catheter (SV-35; id 0.5 mm, cholic acid (5b-cholanic acid-3a,7a-diol, UDCA), litho- od 0.9 mm; Natsume Seisakusyo, Tokyo, Japan) inserted cholic acid (5b-cholanic acid-3a-ol, LCA), hyocholic into the common bile-pancreatic duct under anesthe- acid (5b-cholanic acid-3a,6a,7a,-triol, HCA), TCA (5b- sia with sodium pentobarbital (Somnopentyl, 50 mg/ cholanic acid-3a,7a,12a-triol-N-(2-sulphoethyl)-amide), kg body weight, Kyoritsu Seiyaku Corporation, Tokyo, tauro-a-muricholic acid (5b-cholanic acid-3a,6b,7a- Japan). Immediately after the bile collection for 10 min, triol-N-(2-sulphoethyl)-amide, TaMCA), tauro-b-muri­ aortic blood was collected from the aorta abdominalis cholic acid (5b-cholanic acid-3a,6b,7b-triol-N-(2-sulpho- of the rats. An anticoagulant agent (heparin, 50 U/mL ­ethyl)-amide, TbMCA), tauro--muricholic acid (5b- blood, Nakarai Tesque, Inc., Kyoto, Japan) and a prote- cholanic acid-3a,6a,7b-triol-N-(2-sulphoethyl)-amide, ase inhibitor (aprotinin, 500 kIU/mL blood, Wako Pure TMCA), taurochenodeoxycholic acid (5b-cholanic acid- Chemical Industries, Ltd., Osaka, Japan) were added 3a,7a-diol-N-(2-sulphoethyl)-amide, TCDCA), tauro- to the collected blood, and the plasma was separated. deoxycholic acid (5a-cholanic acid-3a,12a-diol-N-(2- The rats were killed by exsanguination thereafter. The sulphoethyl)-amide, TDCA), taurohyodeoxycholic acid liver, visceral adipose tissues, and intestinal contents (5b-cholanic acid-3a,6a-diol-N-(2-sulphoethyl)-amide, (jejunum, ileum, cecum, and colon) were collected and THDCA), taurolithocholic acid (5b-cholanic acid-3a-ol- weighed. Feces were collected through the last day of N-(2-sulphoethyl)-amide, TLCA), glycocholic acid (5b- the experimental period. cholanic acid-3a,7a,12a-triol-N-(carboxymethyl)-amide, BA analysis. The levels of each BA in the bile, intesti- GCA), glycochenodeoxycholic acid (5b-cholanic acid- nal contents, and feces were extracted and measured as 3a,7a-diol-N-(carboxymethyl)-amide, GCDCA), gly- previously reported (6, 7). The individual BA concentra- codeoxycholic acid (5b-cholanic acid-3a,12a-diol-N- tions were measured with nordeoxycholic acid (23-nor- (car­boxymethyl)-amide, GDCA), glycohyodeoxycholic acid 5b-cholanic acid-3a,12a-diol) as the internal standard. (5b-cholanic acid-3a,6a-diol-N-(carboxymethyl)-amide, The BAs measured in this experiment were as follows: GHDCA), glycoursodeoxycholic acid (5b-cholanic acid- CA (5b-cholanic acid-3a,7a,12a-triol), ursocholic acid 3a,7b-diol-N-(carboxymethyl)-amide, GUDCA), glycol­itho- (5b-cholanic acid-3a,7b,12a-triol, UCA), a-muricholic cholic acid (5a-cholanic acid-3a-ol-N-(carboxymethyl)- acid (5b-cholanic acid-3a,6b,7a-triol, aMCA), b-muri- amide, GLCA), 7-oxo-deoxycholic acid (5b-cholanic acid- cholic acid (5b-cholanic acid-3a,6b,7b-triol, bMCA), 3a,12a-diol-7-one, 7oDCA), 7-oxo-lithocholic acid (5b- Bile Acids in High-Fat Diet-Induced Glucose Intolerance 47 cholanic acid-3a-ol-7-one, 7oLCA), and 12-oxo-litho- stasis model assessment of insulin resistance (HOMA- cholic acid (5b-cholanic acid-3a-ol-12-one, 12oLCA). IR) (21) was calculated by using the data in week 21. All BAs, with the exception of UCA, were purchased Statistics. The significance of differences between from Steraloids, Inc. (Newport, RI, USA); UCA was the control and HF groups was determined using Stu- obtained from Toronto Research Chemicals (Toronto, dent’s t-test. For correlation analysis, linear regression Ontario, Canada). The 12aBAs in this study are as fol- was analyzed by using the least squares method. A p lows: CA, UCA, DCA, TCA, TDCA, GCA, GDCA, 7oDCA value ,0.05 was considered to be significant. JMP ver- and 12oLCA. Among these BAs, 12oLCA possesses an sion 12.2.0 (SAS Institute Inc., Cary, NC, USA) was used oxo group at position 12 on the steroid ring and is not a for the statistical analyses. genuine 12aBA, but we included 12oLCA in the 12aBA RESULTS family in the present study because this molecule can originate from one of the 12aBAs, DCA (19). The HF diet reduced diet consumption, probably due Blood aminotransferase activities, glucose, and insulin. to its high energy density (Table 2). In contrast, total Blood plasma was stored at 280˚C until it was used in energy intake in the test period was significantly higher the analyses. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using a Transaminase CII-test Wako (Wako). Plasma glu- Table 2. Food intake, growth, and organ weights. cose and insulin concentrations were analyzed using a Glucose CII-test kit (Wako) and a Rat Insulin ELISA kit C HF (AKRIN-010T) (Shibayagi Co. Ltd., Shibukawa, Japan), respectively. Cumulative food intake Intraperitoneal glucose tolerance test (IPGTT). We per- (g) 2,417635 1,970627* formed an IPGTT (20) at week 21 during the experimen- (kcal) 9,6436125 10,1576138* tal period. The rats were fasted for 16 h, and then, the Final body weight (g) 427.566.4 471.8611.4* blood was collected from the tail vein. A glucose solu- Organ weight (g/100 g body weight) tion was injected intraperitoneally (1 g/kg), and blood Liver 3.1060.06 2.8260.05* samples were collected from the tail vein at the indicated Visceral fat 7.1860.12 8.4360.27* time points after glucose injection. The blood samples Mesenteric adipose tissue 1.6360.07 1.5860.04 were collected into tubes containing heparin (final con- Epididymal adipose tissue 2.1160.11 1.9460.07* centration 50 IU/mL; Ajinomoto Co., Inc., Tokyo, Japan) Retroperitoneal adipose 2.9560.06 4.4760.28* tissue and aprotinin (final concentration 500 Kallikrein inhib- Perirenal adipose tissue 0.4960.02 0.4360.02* itor units/mL; Wako). The plasma was separated by cen- trifugation at 2,500 3g for 10 min at 4˚C and stored at * Significant different from the values in C (Student’s 280˚C until analysis. The relative value of the homeo- t-test, p,0.05, n510).

Fig. 1. Changes in BA profiles in the feces of the rats fed the C and HF diets. The BA concentration was calculated from the BA compositional data shown in total BAs, 12aBAs, primary BAs, and secondary BAs. Values are shown as the mean with the SE (n510). Open circles, C; filled circles, HF. Asterisks indicate significant differences compared to C (p,0.05). 48 Yoshitsugu R et al.

Fig. 2. BA compositions in bile, intestinal contents, and feces of the rats fed the C and HF diets at the end of the experi- mental period. Twenty-nine molecular species of BA were determined in all the samples, and the major molecular species are shown. Values are shown as the mean with the SE (n510). Open bars, C; filled bars, HF. Asterisks indicate significant differences compared to C (p,0.05).

in the HF-fed rats than in the control rats. Consistent with the energy intake, body weight in the HF-fed rats was greater than that in the C-fed rats, along with an increased weight in visceral adipose tissues. The relative liver weight was lower in the HF-fed rats, but the actual values of the liver weight were almost identical in both groups (13.3960.42 in C, 13.4160.43 in HF). We determined changes in BA excretion in the feces of the rats fed the C or HF diet (Fig. 1). HF diet con- sumption enhanced total and secondary BA excretions Fig. 3. Changes in aminotransferase activities in the from week 10, but an increase in the concentration blood plasma of the rats fed the C and HF diets. Blood of 12aBAs could be detected much earlier in week 4. was taken from the tail vain after 16 h of food depri- These levels were almost stable during the last half of vation at the indicated time points, and aspartate and the experimental period. In contrast, there was no clear alanine aminotransferase activities were measured in the separated plasma. Values are shown as the mean alteration in the primary BA excretion. Next, we ana- with the SE (n510). Open circles, C; filled circles, HF. lyzed BA composition in bile juice, intestinal contents, Asterisks indicate significant differences compared to C and feces at the end of the experimental period (Fig. 2). (p,0.05). TCA was the major component of BAs in the bile and small intestine (jejunal and ileal contents), especially in the HF-fed rats. The concentration was approximately than that of DCA in the control rats. An increase in the 10–20 mm in the C group and 25–40 mm in the HF 12oLCA concentration was also observed in the colon group at these sites. The concentration of BAs was much and feces. The concentration of MCA was nearly the lower in the large intestine (cecal and colonic contents). same as that of DCA, especially in the large intestinal The most abundant BA in the large intestine and feces contents and feces of the rats fed the HF diet. of the HF-fed rats was DCA, whereas the concentration Significantly higher values in the HF group were of MCAs (sum of bMCA and MCA) appeared greater detected for both AST and ALT from week 5 (Fig. 3). The Bile Acids in High-Fat Diet-Induced Glucose Intolerance 49 time point was almost the same (in weeks 11 and 12) and in humans considering the relationship between when the difference in 12aBAs was observed. The value BA metabolism and gastrointestinal carcinogenesis. The was continuously increased throughout the experimen- researchers observed an increase in bile CA concentra- tal period in the rats fed the HF diet. There was a sig- tion as well as fecal DCA concentration in the rats fed a nificant increase in the highest glucose concentration high-fat and high-sucrose diet, suggesting an increase in the IPGTT performed at week 21 (Fig. 4). Likewise, in BA secretion by a high-fat diet. However, as a conse- an increase in insulin secretion was observed at 30 min quence of the technical limitations at that time, there in the HF-fed rats in response to the glucose injection. was no information on conjugated BAs in the profiles. Similar changes were found in the glucose AUC as well The bile CA in the rat study (4) should be mainly TCA, as the DAUC (Table 3). There was no difference in the which was confirmed in the present study. Additionally, HOMA-IR, although a decrease was observed in the fast- we observed a selective increase in bile TCA concentra- ing glucose of the HF-fed rats. We analyzed the relation- tion by the HF diet. TCA possesses high water solubility, ships among energy intake, glucose tolerance, and fecal and the critical micellar concentration is approximately 12aBA concentration (Fig. 5). The total energy intake 8–12 mm (22). The present study suggests that the bile was significantly correlated with glucose tolerance. TCA concentration in the control is sufficient to form Interestingly, there was a significant correlation between emulsions and contributes to lipid digestion as well as total energy intake and fecal 12aBA concentration. absorption. The TCA concentration in the HF-fed rats Moreover, there was a positive correlation between fecal might increase in response to the addition of high levels 12aBA concentration and glucose DAUC in the IPGTT. of dietary lipids. Secreted BAs in the duodenum are reabsorbed by DISCUSSION an apical sodium-dependent BA transporter (ASBT) at Pioneering studies performed by Reddy and colleagues the ileal epithelial cells (23). The present study demon- (4, 5, 12) demonstrated an increase in BA secretion fol- strated a higher proportion of TCA than MCAs in the lowing HF diet consumption in experimental animals small intestinal contents and in the portal blood of the HF-fed rats despite comparable proportions of 12aBAs and non-12aBAs in the large intestine and feces. TCA might be highly available in the small intestine because

Table 3. Parameters in IPGTT in rats fed C or HF at week 21.

C HF Fig. 4. Changes in glucose and insulin concentrations in the blood plasma taken from the tail vein of the Fasting glucose (mg/dL) 92.363.6 80.062.4* rats fed the C and HF diets during IPGTT at week 21. Glucose AUC (mg/dL·h) 242.967.3 276.866.2* After collection of tail vein blood in fasting conditions Glucose DAUC (mg/dL·h) 58.368.7 124.368.6* (16 h food deprivation) at time 0, a glucose solution Fasting insulin (nm) 0.6960.14 0.8060.12 was intraperitoneally injected (1 g/kg rats), and tail Insulin AUC (nm·h) 1.6760.14 2.3260.24 vein blood was collected at the indicated time points to Insulin DAUC (nm·h) 0.6060.11 0.7260.07 measure glucose and insulin concentrations. Values are Relative HOMA-IR 1.0060.14 1.3860.12 shown as the mean with the SE (n510). Open circles, C; filled circles, HF. Asterisks indicate significant differ- * Significant different from the values in C (Student’s ences compared to C (p,0.05). t-test, p,0.05, n510).

Fig. 5. Correlations among total energy intake, fecal 12aBA concentration, and glucose tolerance of the rats fed the C and HF diets. Total energy intake was calculated from diet consumption throughout the experimental period and the energy density of each diet. Glucose DAUC was calculated from the data shown in Fig. 4. The fecal 12aBA concentration was calculated from the data in week 22 as shown in Fig. 2. Individual data are shown as circles (open circles, n510 for C; filled circles, n510 for HF). 50 Yoshitsugu R et al. the absorption rate of TCA by ASBT is relatively high present study, but no difference was found until week compared to CA (13, 24), which results in increased 15. Later, we observed a significant difference in fasting TCA concentration in organs related to enterohepatic glucose at week 21, but the value in the HF-fed rats was circulation. These results suggest that TCA is preferen- lower than that in the control rats. There was no differ- tially used to support lipid absorption even in rodents ence in the HOMA-IR, but significantly higher values in that synthesize substantial amounts of MCAs. There the AUC of the plasma glucose levels were found in the are two pathways for BA synthesis: the classical (neu- HF-fed rats in the IPGTT. Food deprivation is necessary tral) pathway and alternative (acidic) pathway (13, 14); for the glucose tolerance test to obtain a steep altera- Cyp8b1 synthesizes 12aBAs in the classical pathway, tion of blood glucose levels during IPGTT. However, a and Cyp27a1 synthesizes non-12aBAs in the alterna- small effect of dietary intervention on glucose tolerance tive pathway. Therefore, the present study suggests that might disappear following frequent food deprivation. an HF diet activates the classical pathway rather than One example in this study might be some of the insulin- the alternative pathway in liver BA synthesis. related parameters in the IPGTT. Notably, the fasting In this study, we observed an increase in plasma AST condition of a treatment with a weak effect, especially and ALT activities at week 5 when the fecal 12aBA con- on glucose regulation, should be carefully determined in centration was already elevated in the HF-fed rats. The terms of duration and frequency in the experiment to increased TCA is used as the precursor of DCA in the obtain accurate results. large intestine. Various 12aBAs, such as CA and DCA, Dietary cholesterol is the precursor of BAs (13), and are involved in the increase of these transaminase activ- a high-cholesterol diet may hide the intact BA metabo- ities (25). Especially, DCA provokes DNA damage and lism in response to an increase in energy consumption. cell death via induction of reactive oxygen and nitrogen In this study, we selected a HF diet without the addition species (26). The changes in the transaminase activities of dietary cholesterol or BAs to elucidate sterol metabo- of the present study were consistent with these obser- lism on an HF diet. The present study demonstrated that vations. The increase in DCA, the 7a-dehydroxylated BA metabolism reflects energy consumption status as product of CA in the 12aBAs, absorbed in the distal well as glucose intolerance in the body. In conclusion, digestive tract is a possible factor to increase plasma this study suggests that 12aBA is a reliable marker to transaminases via cell death of hepatocytes. Addition- evaluate the onset of glucose tolerance rather than fast- ally, there was an interesting correlation between the ing glucose, fasting insulin, and HOMA-IR. fecal 12aBAs and glucose intolerance. Interestingly, the influence of the HF diet was on the glucose-related Acknowledgments parameters but not on the insulin-related parameters This work was supported by JSPS KAKENHI Grant (Table 3). 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