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Pathogenesis of obesity by food restriction in OLETF rats-increased intestinal monoacylglycerol acyltransferase activities may be a crucial factor
Diabetes Research and Clinical Practice 57 (2002) 75 – 82 www.elsevier.com/locate/diabres
Pathogenesis of obesity by food restriction in OLETF rats-increased intestinal monoacylglycerol acyltransferase activities may be a crucial factor Yang Luan a,b, Tsukasa Hirashima a, Zhi-Wei Man a, Min-Wei Wang b, Kazuya Kawano a,*, Takumi Sumida a a
Biological Research Institute, Otsuka Pharmaceutical Co. Ltd., 463 -10 kagasuno Kawauchi-cho, Tokushima 771 -0192, Japan b Shenyang Pharmaceutical Uni6ersity, Shenyang, People’s Republic of China Received 20 September 2001; received in revised form 28 January 2002; accepted 18 February 2002
Abstract Obesity was considered to be one of the causes of non-insulin-dependent diabetes mellitus (NIDDM). However, the mechanism responsible for obesity has not yet been fully elucidated. In this study, we first examined the relationship between food intake amount and obesity in a NIDDM model animal, and then we focused on triacylglycerol (TG) synthetase activity, which play important roles in hypertriglyceridemia (HTG) associated with obesity. Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model of NIDDM, characterized by obesity, HTG and insulin resistance. In this study, OLETF rats were allocated to a food-satiated group (satiated) or food-restricted group (to eliminate the effects of hyperphagia on obesity, amount of daily food intake was the same as that in their control strain Long-Evans Tokushima Otsuka (LETO) rats). Changes in body weight, body fat, intraabdominal fat weight, and TG content in liver were measured and biochemical blood tests and activity assay of TG synthetase (monoacylglycerol acyltransferase (MGAT) and diacylglycerol acyltransferase (DGAT)) were performed. Results: (1) The body weight in the restricted OLETF rats was significantly decreased to 71.7% of that in the satiated OLETF rats, which was almost the same value as that in the LETO rats. However, body fat and intraabdominal fat weight were significantly increased in restricted OLETF rats and satiated OLETF rats compared with LETO rats. (2) Plasma TG, insulin, glucose, leptin and hepatic TG content were significantly higher in OLETF rats than the values in LETO rats. (3) MGAT activity in the small intestine from both satiated and restricted OLETF rats was significantly higher than that in LETO rats. DGAT activity in OLETF rats was not significantly different from that in LETO rats. In conclusion, the body fat weight and plasma TG were still significantly accelerated in OLETF rats at the same food intake as LETO rats. MGAT activity in the small intestine from OLETF rats was also significantly higher than those of LETO rats. Therefore, high MGAT activity in the small intestine may play an important role in HTG and obesity, subsequently hastening the development of NIDDM in OLETF rats. © 2002 Elsevier Science Ireland Ltd. All rights reserved.
* Corresponding author. Tel.: + 81-88-665-2126. E-mail address: k – [email protected] (K. Kawano). 0168-8227/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0168-8227(02)00026-8
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Keywords: NIDDM; MGAT activity; OLETF rat; Obesity
1. Introduction In recent years, although the precise etiology of NIDDM (non insulin-dependent diabetes mellitus) has not been clarified, a substantial number of studies have suggested that obesity is one of the crucial factors leading to this syndrome [1 –3]. TG accumulation in muscle might cause insulin resistance in skeletal muscle [5]. Hypertriglyceridemia (HTG) caused a high portal FFA concentration that led to insulin resistance in the liver [6– 8]. TG stored in islets is potentially toxic to cells, impairs b-cell function and subsequently inhibits insulin secretion [4,27]. Otsuka Long-Evans Tokushima Fatty (OLETF) rats display HTG at an early age, develop insulin resistance and subsequently glucose intolerance in adulthood. These characteristic features closely resemble those of human type II diabetes, suggesting that the OLETF rat is a useful animal model for analyzing the pathogenetic mechanism(s) of NIDDM with obesity. The control strain Long-Evans Tokushima Otsuka (LETO) rats, established from the same colony of Long-Evans rats from Charles River Canada (St. Constant, Quebec, Canada), is normal and never develops diabetes [23]. Using this model animal, in earlier studies we demonstrated that HTG resulted in significant TG stores in the islets and subsequently inhibited glucose-induced insulin secretion, at least in part, via reduced glucokinase activity in the islets, suggesting that fat droplets in islets may play an important role in hastening the development of NIDDM in this model [4]. Hyperphagia has been reported in OLETF rats [9]. In a previous study, we demonstrated that 30% food restriction in OLETF rats markedly ameliorated TG accumulation in tissues, thereby improved diabetes [10,11]. However, body fat, and intraabdominal fat in 30% food restricted OLETF rats were still significantly increased compared with LETO rats although body weight was similar [12]. These
results suggested that there may be some other causes of obesity besides hyperphagia [12]. However, as the amount of food intake in the two groups was different, it was difficult to compare OLETF rats with LETO rats to find other factors causing obesity. We report, here a study using in vivo approaches in OLETF and LETO rats, and we focused our attention on TG biosynthesis to better understand the mechanism responsible for obesity in NIDDM. To eliminate the effects of hyperphagia, daily food intake of the restricted group was the same amount as that in the LETO group. The study involved (1) examination of sequential measurement of body weight, body fat weight and visceral fat weight, (2) blood tests for plasma TG, insulin, glucose, leptin and hepatic TG content, and (3) enzyme activity assay for MGAT and DGAT, the key enzyme in TG biosynthesis.
2. Materials and methods
2.1. Animals and composition of experimental groups Male OLETF rats and their control counterpart LETO rats were maintained in our animal facilities under specific pathogen-free conditions and supplied with CRF-1 solid food (protein 23.1, carbohydrate 53.5, and fat 5.9%; Oriental Yeast, Tokyo, Japan) plus tap water ad libitum. Thirty LETO rats were used in this study; 20 were used in the initial study to determine the food intake from 5 weeks of age, and the remaining animals (10) were used to study TG accumulation and TG synthatase activities as a normal control (LETO group, n= 10). OLETF rats were randomly allocated to two groups: the first group maintained on food satiation (satiated OLETF group, n= 5), the second restricted group given the save daily amount of food as LETO rats from 5 weeks of age (restricted
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OLETF group, n= 9). Experiments were performed from 5 to 21 weeks of age. The animals were fasted for 16 h before each experiment.
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method from other portions of the liver, [15] and was measured by the method described above.
2.5. Preparation of subcellular fractions 2.2. Measurement of body weight, body fat and blood tests Daily food intake of satiated OLETF rats was measured every-day. Body weight was measured once per week. Body fat was analyzed at 20 weeks old. Rats were anesthetized with pentobarbital (50 mg/kg, i.p.) and inserted into a small-animal body composition analyzer (EM-SCAN model SA-2, EM-SCAN Inc., Springfield, IL USA) to estimate total body fat content. Blood tests were performed at 20 weeks of age. Using blood collected from the caudal artery of rats fasted for 16 h, plasma levels of TG, glucose, insulin and leptin were measured. Plasma levels of TG were determined using Lipidos Liquids (Ono Pharmaceutical, Osaka, Japan). Plasma glucose levels were measured using a Glucose B-Test Wako Kit (Wako Pure Chemical Industries, Osaka, Japan). Plasma insulin was determined using an enzymelinked immunosorbent assay (ELISA) Insulin Kit (Morinaga Biochemical Industries, Japan). Plasma leptin levels were measured using a rat leptin kit (Morinaga Biochemical Industries, Japan).
2.3. Oral glucose tolerance test (OGTT) OGTT was performed at 20 weeks of age. Glucose (2 g/kg) was orally administered to rats fasted for 16 h, and then blood was collected from the caudal artery at 0, 30, 60, 90 and 120 min. Plasma glucose levels were measured by the method described above.
2.4. Measurement of intraabdominal fat weight and TG content in hepatic tissues At the end of the experiment (21 weeks of age), animals were euthanized under anesthesia, and the intraabdominal fat (fat in the epididymis, mesentery, and retroperitoneum) were removed and measured. TG was extracted by the Folch
After removal the of liver and small intestine segments (the first 15 cm) and retroperitoneal adipose from euthanized rats, the small intestine segments were cleaned with several volumes of normal saline, and the tissues were then frozen immediately in liquid nitrogen and stored at − 80 °C until use. The subcellular fractions were prepared according to the methods described elsewhere [16] [17]. Briefly, the tissues (retroperitoneal adipose, liver and small intestine) were rinsed in medium (50 mM Tris –HCl, 140 mM KCl, 0.1 mM EDTA, pH 7.4), and the intestinal mucosa was scraped with tweezers. Liver and adipose tissue were cut into small tissue fragments with scissors. These fragments were suspended in six volume of cold medium and homogenized with a Polytron PT 3000. The homogenates were centrifuged at 15000× g for 15 min at 4 °C, the obtained supernatant was ultracentrifuged at 100000× g for 60 min at 4 °C. After carefully removing the supernatant, the pellets were then washed with the medium and centrifuged under the same conditions. The resultant pellets were suspended in buffer (175 mM Tris–HCl-8 mM MgCl2-0.1% BSA, pH 7.4) and homogenized with ten strokes in a glass homogenizer. The obtained microsomes were frozen at − 80 °C until use. The concentration of microsomal proteins was determined with a Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA).
2.6. Assay of enzyme acti6ities 2.6.1. Monoacylglycerol acyltransferase (MGAT) The reaction mixture (final volume, 0.2 ml) contained 175 mM Tris–HCl buffer, pH 7.5, 8 mM MgCl2, 0.1% serum albumin (BSA), 0.25% Triton X-100, 25 mM [14C]palmitoyl CoA(0.04 mCi) (specific radioactivity, 55 mCi/mmol, Amersham Pharmacia Biotech UK), 150 mM 2monoolein (Sigma Chemicals, St. Louis, MO. dispersed in 99% acetone). The reaction was
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started by addition of microsomal proteins. Incubation was carried out at 37 °C. The reaction was linear for 6 min with 30 g of microsomal proteins [18] [19] [20]. The reaction was terminated by addition of 1.5 ml of stop solution [chloroform/ methanol (1:1)]. Then the contents were mixed with 0.25 ml distilled water, and centrifuged at 2500 rpm for 2 min. After centrifugation, 650 ml of the chloroform layer was taken, and dried under N2. The dry lipids were dissolved in 20 ml of chloroform, and aliquots of 10 ml were used as samples separated on thin layer plates coated with Silica Gel G 60 A (Clifton, NJ, USA). The separation of lipids was performed with the solvent system heptane/diethylether/acetic acid (60/40/4, v/v/v), di- and triacylglycerol (TG) levels were taken as measures of enzyme activity. The radioactivity was measured in a RLG system (BAS 2000 and imaging plates BAStation, Fuji Photo Film Ltd. Tokyo, Japan).
2.6.2. Diacylglycerol acyltransferase (DGAT) The reaction mixture (final volume, 0.2 ml) contained 175 mM Tris– HCl buffer, pH 7.5, 8 mM MgCl2, 0.1% serum albumin (BSA), 0.25% Triton X-100, 100 mM [14C] palmitoyl CoA (0.05 mCi), 1.2 mM 1,2-diolein (Sigma Chemicals dispersed in 99% acetone). The reaction was started by addition of ml of microsomal protein (120 mg). The reaction was linear for 20 min at room temperature and was terminated by the addition of 1.5 ml of stop solution [chloroform/methanol (1:1)]. Then, the contents were mixed with 0.25 ml of 0.5 N NaOH for lipid extraction, and centrifuged at 2500 rpm for
2 min [21,22]. After centrifugation, 650 ml of the chloroform layer was taken, and dried under N2. The dry lipids were dissolved in 20 ml of chloroform, and aliquots of 10 ml were used as samples separated on thin layer plates coated with Silica Gel G 60 A. Separation was performed with the solvent system petroleum ether/diethylether/acetic acid (80/20/2, v/v/v). The produced lipids were assayed according to the method described for MGAT.
2.7. Statistical analysis Values a expressed as means 9 S.E. For the body weight, body fat, intraabdominal fat, hepatic TG content, plasma TG, glucose, insulin and leptin levels, mean values were compared by t-test between the restricted OLETF group and LETO control group and between the restricted OLETF group and satiated OLETF group. In OGTT, at each time point, the results were assessed by t-test between the groups as described above. For enzyme activity, mean values were compared by t-test between the restricted OLETF group and LETO control group and between the satiated OLETF group and LETO control group. In these analyses, a PB0.05 was used as the criterion for statistical significance.
3. Results General characters of OLETF and LETO rats. Table 1 shows differences in body weight, body fat,
Table 1 General characteristics of LETO and OLETF Characteristic
Restricted OLETF (n = 9)
LETO (n = 10)
Satiated OLETF (n =5)
Body weight (g) Body fat (% of body weight) Intraabdominal fat (% of body weight) Plasma TG (mg/dl) Hepatic TG content (mg/g) Plasma glucose (mg/dl) Plasma insulin (pg/ml) Plasma leptin (pg/ml)
430.791.0+++ 11.6390.27***+++ 5.8490.1**++ 47.5 9 1.8*+++ 16.3290.47*+++ 127.49 0.7***+++ 1110.09 30.2**+++ 5501.0 9233.3***+++
424.2 9 0.91 7.14 9 0.14 3.55 9 0.04 34.1 9 0.5 11.26 9 0.31 114.7 90.4 651.0 9 21.20 1662.4 949.24
600.8 9 6.76 18.58 9 0.52 8.77 9 0.18 120.4 96.66 28.78 90.49 155.4 92.20 3563.0 9 174.0 14 903.1 9778.2
Values are the means 9 S.E.; after overnight fasting, blood was withdrawn from the vena cava. *PB0.05, **PB0.01, ***PB0.001, 6 LETO. +PB0.05, ++PB0.01, +++PB0.001, 6 satiated OLETF.
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Fig. 1. Changes in food intake in the satiated OLETF group (", n =5) from 6 to 21 weeks of age, restricted OLETF group (, n =9) and LETO group (, n =10) from 5 to 21 weeks of age. Values are the means 9 S.E.
intraabdominal fat, hepatic TG content, and plasma TG, glucose, insulin and, leptin levels in the fasting state in OLETF and LETO rats at 20–21 weeks of age. Although their body weights were similar, restricted OLETF rats showed significantly increased body fat (% of body weight) and intraabdominal fat (% of body weight), with values of 1.6 —(PB 0.01) and 1.7-fold (P B 0.001) greater than those of LETO rats, respectively. Body weight, body fat and intraabdominal fat were significantly reduced in restricted OLETF rats compared with satiated OLETF rats at 21 weeks old (P B 0.01). Hepatic TG content, plasma TG, glucose, insulin and leptin levels in restricted OLETF rats were significantly higher than those in LETO rats, although they were evidently improved compared with satiated OLETF rats. Hepatic TG content in restricted OLETF rats was 1.5-fold higher than that in LETO rats. Fig. 1 illustrates sequential changes in food intake in each experimental group during the study. The food intake in restricted OLETF rats was the same as that in LETO rats, and the average value was about 70% of that in satiated OLETF rats. Fig. 2 shows the differences in intraabdominal fat (% of body weight) in each experimental group at 21 weeks of age. A sharp accumulation of retroperitoneal fat was observed in restricted OLETF rats at the end of the experimental period. Oral glucose tolerance test Changes in plasma glucose during OGTT in each experimen-
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Fig. 2. Differences in intra-abdominal intra-abdominal fat, including retroperitoneal ( ), epididymal( ) and mesenteric (b) fats weights (% of body weight) in restricted OLETF group (n =9), satiated OLETF group (n =5) and LETO group (n = 10) at 21 weeks of age. Values are means 9S.E. **PB0.01, (t-test) 6 restricted OLETF group.
tal group at 20 weeks of age are illustrated in Fig. 3. OGTT score was evidently ameliorated in foodrestricted OLETF rats compared with satiated OLETF rats, but then still had significantly higher
Fig. 3. Plasma glucose responses in the OGTT in the satiated OLETF group (, n = 5) and restricted OLETF group ( , n = 9) and LETO group ( , n = 10) at 20 weeks of age. Rats were fasted for 16 h before the test. Values are means 9S.E. *PB0.05, **PB 0.01, ***PB 0.001 (t-test) 6 LETO group. +++ PB 0.001, 6 satiated OLETF group.
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Fig. 4B shows differences in DGAT activities in OLETF and LETO rats. DGAT activities in intestinal, adipose and liver tissue showed no significant differences between OLETF and LETO rats. 4. Discussion
Fig. 4. Differences in MGAT activities in the small intestine (A) and DGAT activities (B) in liver, intestine and adipose tissues of restricted OLETF group ( , n= 9), LETO group ( , n= 10) and satiated OLETF group ( , n= 5) at 21 weeks of age. Values are the means 9 S.E. *PB 0.05, **PB0.01, (t-test) 6 LETO group. The specific activity of MGAT in different tissues was calculated on the basis of radioactivity in the DG fraction plus 1/2 of the radioactivity present in the TG fraction.
plasma levels of glucose than LETO rats at 30 and 60 min (PB 0.001). The sum of plasma glucose during the OGTT in restricted OLETF rats was about 1.12-fold (838.61936.76 mg/dl) higher than the value in LETO rats (747.70922.99 mg/ dl). Enzyme activities Fig. 4A shows differences in MGAT activity in the intestine in OLETF and LETO rats. MGAT activity was significantly increased in OLETF rats compared with LETO rats; the value in restricted OLETF rats was 2.7fold (PB 0.001) higher and that in satiated OLETF rats was 2.2-fold (P B 0.01) higher than that in LETO. No significant change in the MGAT activity was noted between satiated and restricted OLETF rats.
OLETF rats resemble human obese patients with NIDDM. There have been some reports about lipid production in OLETF rats, for example, hepatic acyl-coenzyme A synthetase (ACS) and microsomal triglyceride transfer protein (MTP) messenger RNAs were shown to be expressed at high levels in these animals [13]. However, it has been reported that OLETF rats show hyperphagia, a major factor responsible for obesity [9]. Satiated rats were used in these previous study, it will be difficult to determine whether these results were affected by hyperphagia. In the present study, to eliminate the effects of hyperphagia, OLETF rats were given the same amount of food daily as LETO rats. We found that body fat weight was still significantly increased in restricted OLETF rats, indicating that there may be some other factor(s) causing obesity in tissues besides hyperphagia, such as differences in enzyme activity or genetic factors. As the food intake was the same in restricted OLETF and LETO groups, it was possible to make comparisons between the two groups to find abnormalities in lipid metabolism in OLETF rats. In our previous study, 30% food-restricted OLETF rats showed no abnormalities in the activity of lipoprotein lipase (LPL) compared with LETO rats, [14] suggesting that the clearance of TG-rich lipoprotein is not delayed. In the present study, we wonder whether TG be overproduced in OLETF rats. We then focused our attention on the roles of two key enzymes in TG biosynthesis: MGAT and DGAT. MGAT uniquely catalyzes extraneous lipid to TG in the intestinal mucosa, and DGAT is believed to control the one common step of synthesis of triglycerides in all tissues. Results showed that MGAT activities in OLETF rats were significantly higher than those in LETO rats. On the other hand, no significant difference in the DGAT activity was noted between OLETF rats and LETO rats.
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The small intestine is the main tissue involved in TG synthesis. Although it is possible that increased MGAT activity may cause overproduction of TG, it has not been confirmed in vivo. Assuming that the following effects occur in vivo. The same amount of food were taken in restricted OLETF and LETO groups, which means the same amount of substrates were digested by pancreatic lipase to monoacylglycerol and FFAs, the increased MGAT activity in OLETF rats might increase the production of diacylglycerol, and then cause the overproduction of TG, although DGAT activity is normal. But the relationship between the increased MGAT activity and the increased body fat and HTG would be studied more in the future. Morphological studies showed that both the small intestinal wet weight and surface area were 30% larger in OLETF rats than in LETO rats, [24,29] suggesting that not only activity but also quantity of MGAT might be increased in OLETF rats, and both may be involved in the increased TG synthesis. Previous reports suggested that the small intestine plays a key role in the pathogenesis of TG accumulation in tissues and hyperlipidemia in diabetes, [25,26] the results of the present study were consistent with this hypothesis. Intestinal MGAT resides mainly in the mucosal fraction and this activity differs between segments to other, with the jejunum being the most active [28]. In this study, we isolated the mucosal microsomal fraction only from jejunum (the first 15 cm of the small intestine) to measure the enzyme activities. On the other hand, body weight, body fat, index of blood test and OGTT test in restricted OLETF were markedly decreased compared with satiated OLETF, indicating that diet therapy is useful in ameliorating abnormalities in lipid and glucose metabolism of diabetes by improving insulin resistance and b-cell function. Enzyme activity assay showed that MGAT activity in restricted and satiated OLETF rats were similar and both of them were significantly higher than that of LETO rats, whereas DGAT activity showed no significant difference between the satiated and restricted groups in statistics, although there is a tendency that DGAT activity in satiated OLETF rats was
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higher than that of restricted OLETF rats and LETO rats. In this study, even though restricted OLETF rats were given the same amount of food daily as LETO, body fat weight and plasma insulin, glucose and leptin were increased compared with LETO rats, suggesting that the restricted OLETF rats will be a useful model in which to study obesity and its relationship with NIDDM. The present study represents the first indication that increased MGAT activity might play a key role in HTG and obesity. There have been previous reports concerning MGAT activity in Zucker rats [16] and streptozotocin-induced diabetic rats [17], but to date there have seen no reports in human subjects with obesity. If the same abnormality occurs in human subjects as OLETF rats, MGAT inhibitor might become a promising triglyceride lowing drug, we expect that the results of the present study will contribute to elucidation of human HTG and obesity, subsequently in those individuals with NIDDM. In conclusion, increased small intestinal MGAT activities accelerated triglyceride synthesis, and thus might be a causative factor leading to HTG and obesity and be involved in the development of diabetes in OLETF rats.
Acknowledgements We are grateful to Dr Kenji Shima, and Dr Masamichi Kuwajima, University of Tokushima, for providing valuable advice and guidance during these experiments.
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Pathogenesis of obesity by food restriction in OLETF rats-increased intestinal monoacylglycerol acyltransferase activities may be a crucial factor Yang Luan a,b, Tsukasa Hirashima a, Zhi-Wei Man a, Min-Wei Wang b, Kazuya Kawano a,*, Takumi Sumida a a
Biological Research Institute, Otsuka Pharmaceutical Co. Ltd., 463 -10 kagasuno Kawauchi-cho, Tokushima 771 -0192, Japan b Shenyang Pharmaceutical Uni6ersity, Shenyang, People’s Republic of China Received 20 September 2001; received in revised form 28 January 2002; accepted 18 February 2002
Abstract Obesity was considered to be one of the causes of non-insulin-dependent diabetes mellitus (NIDDM). However, the mechanism responsible for obesity has not yet been fully elucidated. In this study, we first examined the relationship between food intake amount and obesity in a NIDDM model animal, and then we focused on triacylglycerol (TG) synthetase activity, which play important roles in hypertriglyceridemia (HTG) associated with obesity. Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model of NIDDM, characterized by obesity, HTG and insulin resistance. In this study, OLETF rats were allocated to a food-satiated group (satiated) or food-restricted group (to eliminate the effects of hyperphagia on obesity, amount of daily food intake was the same as that in their control strain Long-Evans Tokushima Otsuka (LETO) rats). Changes in body weight, body fat, intraabdominal fat weight, and TG content in liver were measured and biochemical blood tests and activity assay of TG synthetase (monoacylglycerol acyltransferase (MGAT) and diacylglycerol acyltransferase (DGAT)) were performed. Results: (1) The body weight in the restricted OLETF rats was significantly decreased to 71.7% of that in the satiated OLETF rats, which was almost the same value as that in the LETO rats. However, body fat and intraabdominal fat weight were significantly increased in restricted OLETF rats and satiated OLETF rats compared with LETO rats. (2) Plasma TG, insulin, glucose, leptin and hepatic TG content were significantly higher in OLETF rats than the values in LETO rats. (3) MGAT activity in the small intestine from both satiated and restricted OLETF rats was significantly higher than that in LETO rats. DGAT activity in OLETF rats was not significantly different from that in LETO rats. In conclusion, the body fat weight and plasma TG were still significantly accelerated in OLETF rats at the same food intake as LETO rats. MGAT activity in the small intestine from OLETF rats was also significantly higher than those of LETO rats. Therefore, high MGAT activity in the small intestine may play an important role in HTG and obesity, subsequently hastening the development of NIDDM in OLETF rats. © 2002 Elsevier Science Ireland Ltd. All rights reserved.
* Corresponding author. Tel.: + 81-88-665-2126. E-mail address: k – [email protected] (K. Kawano). 0168-8227/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0168-8227(02)00026-8
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Keywords: NIDDM; MGAT activity; OLETF rat; Obesity
1. Introduction In recent years, although the precise etiology of NIDDM (non insulin-dependent diabetes mellitus) has not been clarified, a substantial number of studies have suggested that obesity is one of the crucial factors leading to this syndrome [1 –3]. TG accumulation in muscle might cause insulin resistance in skeletal muscle [5]. Hypertriglyceridemia (HTG) caused a high portal FFA concentration that led to insulin resistance in the liver [6– 8]. TG stored in islets is potentially toxic to cells, impairs b-cell function and subsequently inhibits insulin secretion [4,27]. Otsuka Long-Evans Tokushima Fatty (OLETF) rats display HTG at an early age, develop insulin resistance and subsequently glucose intolerance in adulthood. These characteristic features closely resemble those of human type II diabetes, suggesting that the OLETF rat is a useful animal model for analyzing the pathogenetic mechanism(s) of NIDDM with obesity. The control strain Long-Evans Tokushima Otsuka (LETO) rats, established from the same colony of Long-Evans rats from Charles River Canada (St. Constant, Quebec, Canada), is normal and never develops diabetes [23]. Using this model animal, in earlier studies we demonstrated that HTG resulted in significant TG stores in the islets and subsequently inhibited glucose-induced insulin secretion, at least in part, via reduced glucokinase activity in the islets, suggesting that fat droplets in islets may play an important role in hastening the development of NIDDM in this model [4]. Hyperphagia has been reported in OLETF rats [9]. In a previous study, we demonstrated that 30% food restriction in OLETF rats markedly ameliorated TG accumulation in tissues, thereby improved diabetes [10,11]. However, body fat, and intraabdominal fat in 30% food restricted OLETF rats were still significantly increased compared with LETO rats although body weight was similar [12]. These
results suggested that there may be some other causes of obesity besides hyperphagia [12]. However, as the amount of food intake in the two groups was different, it was difficult to compare OLETF rats with LETO rats to find other factors causing obesity. We report, here a study using in vivo approaches in OLETF and LETO rats, and we focused our attention on TG biosynthesis to better understand the mechanism responsible for obesity in NIDDM. To eliminate the effects of hyperphagia, daily food intake of the restricted group was the same amount as that in the LETO group. The study involved (1) examination of sequential measurement of body weight, body fat weight and visceral fat weight, (2) blood tests for plasma TG, insulin, glucose, leptin and hepatic TG content, and (3) enzyme activity assay for MGAT and DGAT, the key enzyme in TG biosynthesis.
2. Materials and methods
2.1. Animals and composition of experimental groups Male OLETF rats and their control counterpart LETO rats were maintained in our animal facilities under specific pathogen-free conditions and supplied with CRF-1 solid food (protein 23.1, carbohydrate 53.5, and fat 5.9%; Oriental Yeast, Tokyo, Japan) plus tap water ad libitum. Thirty LETO rats were used in this study; 20 were used in the initial study to determine the food intake from 5 weeks of age, and the remaining animals (10) were used to study TG accumulation and TG synthatase activities as a normal control (LETO group, n= 10). OLETF rats were randomly allocated to two groups: the first group maintained on food satiation (satiated OLETF group, n= 5), the second restricted group given the save daily amount of food as LETO rats from 5 weeks of age (restricted
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OLETF group, n= 9). Experiments were performed from 5 to 21 weeks of age. The animals were fasted for 16 h before each experiment.
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method from other portions of the liver, [15] and was measured by the method described above.
2.5. Preparation of subcellular fractions 2.2. Measurement of body weight, body fat and blood tests Daily food intake of satiated OLETF rats was measured every-day. Body weight was measured once per week. Body fat was analyzed at 20 weeks old. Rats were anesthetized with pentobarbital (50 mg/kg, i.p.) and inserted into a small-animal body composition analyzer (EM-SCAN model SA-2, EM-SCAN Inc., Springfield, IL USA) to estimate total body fat content. Blood tests were performed at 20 weeks of age. Using blood collected from the caudal artery of rats fasted for 16 h, plasma levels of TG, glucose, insulin and leptin were measured. Plasma levels of TG were determined using Lipidos Liquids (Ono Pharmaceutical, Osaka, Japan). Plasma glucose levels were measured using a Glucose B-Test Wako Kit (Wako Pure Chemical Industries, Osaka, Japan). Plasma insulin was determined using an enzymelinked immunosorbent assay (ELISA) Insulin Kit (Morinaga Biochemical Industries, Japan). Plasma leptin levels were measured using a rat leptin kit (Morinaga Biochemical Industries, Japan).
2.3. Oral glucose tolerance test (OGTT) OGTT was performed at 20 weeks of age. Glucose (2 g/kg) was orally administered to rats fasted for 16 h, and then blood was collected from the caudal artery at 0, 30, 60, 90 and 120 min. Plasma glucose levels were measured by the method described above.
2.4. Measurement of intraabdominal fat weight and TG content in hepatic tissues At the end of the experiment (21 weeks of age), animals were euthanized under anesthesia, and the intraabdominal fat (fat in the epididymis, mesentery, and retroperitoneum) were removed and measured. TG was extracted by the Folch
After removal the of liver and small intestine segments (the first 15 cm) and retroperitoneal adipose from euthanized rats, the small intestine segments were cleaned with several volumes of normal saline, and the tissues were then frozen immediately in liquid nitrogen and stored at − 80 °C until use. The subcellular fractions were prepared according to the methods described elsewhere [16] [17]. Briefly, the tissues (retroperitoneal adipose, liver and small intestine) were rinsed in medium (50 mM Tris –HCl, 140 mM KCl, 0.1 mM EDTA, pH 7.4), and the intestinal mucosa was scraped with tweezers. Liver and adipose tissue were cut into small tissue fragments with scissors. These fragments were suspended in six volume of cold medium and homogenized with a Polytron PT 3000. The homogenates were centrifuged at 15000× g for 15 min at 4 °C, the obtained supernatant was ultracentrifuged at 100000× g for 60 min at 4 °C. After carefully removing the supernatant, the pellets were then washed with the medium and centrifuged under the same conditions. The resultant pellets were suspended in buffer (175 mM Tris–HCl-8 mM MgCl2-0.1% BSA, pH 7.4) and homogenized with ten strokes in a glass homogenizer. The obtained microsomes were frozen at − 80 °C until use. The concentration of microsomal proteins was determined with a Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA, USA).
2.6. Assay of enzyme acti6ities 2.6.1. Monoacylglycerol acyltransferase (MGAT) The reaction mixture (final volume, 0.2 ml) contained 175 mM Tris–HCl buffer, pH 7.5, 8 mM MgCl2, 0.1% serum albumin (BSA), 0.25% Triton X-100, 25 mM [14C]palmitoyl CoA(0.04 mCi) (specific radioactivity, 55 mCi/mmol, Amersham Pharmacia Biotech UK), 150 mM 2monoolein (Sigma Chemicals, St. Louis, MO. dispersed in 99% acetone). The reaction was
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started by addition of microsomal proteins. Incubation was carried out at 37 °C. The reaction was linear for 6 min with 30 g of microsomal proteins [18] [19] [20]. The reaction was terminated by addition of 1.5 ml of stop solution [chloroform/ methanol (1:1)]. Then the contents were mixed with 0.25 ml distilled water, and centrifuged at 2500 rpm for 2 min. After centrifugation, 650 ml of the chloroform layer was taken, and dried under N2. The dry lipids were dissolved in 20 ml of chloroform, and aliquots of 10 ml were used as samples separated on thin layer plates coated with Silica Gel G 60 A (Clifton, NJ, USA). The separation of lipids was performed with the solvent system heptane/diethylether/acetic acid (60/40/4, v/v/v), di- and triacylglycerol (TG) levels were taken as measures of enzyme activity. The radioactivity was measured in a RLG system (BAS 2000 and imaging plates BAStation, Fuji Photo Film Ltd. Tokyo, Japan).
2.6.2. Diacylglycerol acyltransferase (DGAT) The reaction mixture (final volume, 0.2 ml) contained 175 mM Tris– HCl buffer, pH 7.5, 8 mM MgCl2, 0.1% serum albumin (BSA), 0.25% Triton X-100, 100 mM [14C] palmitoyl CoA (0.05 mCi), 1.2 mM 1,2-diolein (Sigma Chemicals dispersed in 99% acetone). The reaction was started by addition of ml of microsomal protein (120 mg). The reaction was linear for 20 min at room temperature and was terminated by the addition of 1.5 ml of stop solution [chloroform/methanol (1:1)]. Then, the contents were mixed with 0.25 ml of 0.5 N NaOH for lipid extraction, and centrifuged at 2500 rpm for
2 min [21,22]. After centrifugation, 650 ml of the chloroform layer was taken, and dried under N2. The dry lipids were dissolved in 20 ml of chloroform, and aliquots of 10 ml were used as samples separated on thin layer plates coated with Silica Gel G 60 A. Separation was performed with the solvent system petroleum ether/diethylether/acetic acid (80/20/2, v/v/v). The produced lipids were assayed according to the method described for MGAT.
2.7. Statistical analysis Values a expressed as means 9 S.E. For the body weight, body fat, intraabdominal fat, hepatic TG content, plasma TG, glucose, insulin and leptin levels, mean values were compared by t-test between the restricted OLETF group and LETO control group and between the restricted OLETF group and satiated OLETF group. In OGTT, at each time point, the results were assessed by t-test between the groups as described above. For enzyme activity, mean values were compared by t-test between the restricted OLETF group and LETO control group and between the satiated OLETF group and LETO control group. In these analyses, a PB0.05 was used as the criterion for statistical significance.
3. Results General characters of OLETF and LETO rats. Table 1 shows differences in body weight, body fat,
Table 1 General characteristics of LETO and OLETF Characteristic
Restricted OLETF (n = 9)
LETO (n = 10)
Satiated OLETF (n =5)
Body weight (g) Body fat (% of body weight) Intraabdominal fat (% of body weight) Plasma TG (mg/dl) Hepatic TG content (mg/g) Plasma glucose (mg/dl) Plasma insulin (pg/ml) Plasma leptin (pg/ml)
430.791.0+++ 11.6390.27***+++ 5.8490.1**++ 47.5 9 1.8*+++ 16.3290.47*+++ 127.49 0.7***+++ 1110.09 30.2**+++ 5501.0 9233.3***+++
424.2 9 0.91 7.14 9 0.14 3.55 9 0.04 34.1 9 0.5 11.26 9 0.31 114.7 90.4 651.0 9 21.20 1662.4 949.24
600.8 9 6.76 18.58 9 0.52 8.77 9 0.18 120.4 96.66 28.78 90.49 155.4 92.20 3563.0 9 174.0 14 903.1 9778.2
Values are the means 9 S.E.; after overnight fasting, blood was withdrawn from the vena cava. *PB0.05, **PB0.01, ***PB0.001, 6 LETO. +PB0.05, ++PB0.01, +++PB0.001, 6 satiated OLETF.
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Fig. 1. Changes in food intake in the satiated OLETF group (", n =5) from 6 to 21 weeks of age, restricted OLETF group (, n =9) and LETO group (, n =10) from 5 to 21 weeks of age. Values are the means 9 S.E.
intraabdominal fat, hepatic TG content, and plasma TG, glucose, insulin and, leptin levels in the fasting state in OLETF and LETO rats at 20–21 weeks of age. Although their body weights were similar, restricted OLETF rats showed significantly increased body fat (% of body weight) and intraabdominal fat (% of body weight), with values of 1.6 —(PB 0.01) and 1.7-fold (P B 0.001) greater than those of LETO rats, respectively. Body weight, body fat and intraabdominal fat were significantly reduced in restricted OLETF rats compared with satiated OLETF rats at 21 weeks old (P B 0.01). Hepatic TG content, plasma TG, glucose, insulin and leptin levels in restricted OLETF rats were significantly higher than those in LETO rats, although they were evidently improved compared with satiated OLETF rats. Hepatic TG content in restricted OLETF rats was 1.5-fold higher than that in LETO rats. Fig. 1 illustrates sequential changes in food intake in each experimental group during the study. The food intake in restricted OLETF rats was the same as that in LETO rats, and the average value was about 70% of that in satiated OLETF rats. Fig. 2 shows the differences in intraabdominal fat (% of body weight) in each experimental group at 21 weeks of age. A sharp accumulation of retroperitoneal fat was observed in restricted OLETF rats at the end of the experimental period. Oral glucose tolerance test Changes in plasma glucose during OGTT in each experimen-
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Fig. 2. Differences in intra-abdominal intra-abdominal fat, including retroperitoneal ( ), epididymal( ) and mesenteric (b) fats weights (% of body weight) in restricted OLETF group (n =9), satiated OLETF group (n =5) and LETO group (n = 10) at 21 weeks of age. Values are means 9S.E. **PB0.01, (t-test) 6 restricted OLETF group.
tal group at 20 weeks of age are illustrated in Fig. 3. OGTT score was evidently ameliorated in foodrestricted OLETF rats compared with satiated OLETF rats, but then still had significantly higher
Fig. 3. Plasma glucose responses in the OGTT in the satiated OLETF group (, n = 5) and restricted OLETF group ( , n = 9) and LETO group ( , n = 10) at 20 weeks of age. Rats were fasted for 16 h before the test. Values are means 9S.E. *PB0.05, **PB 0.01, ***PB 0.001 (t-test) 6 LETO group. +++ PB 0.001, 6 satiated OLETF group.
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Fig. 4B shows differences in DGAT activities in OLETF and LETO rats. DGAT activities in intestinal, adipose and liver tissue showed no significant differences between OLETF and LETO rats. 4. Discussion
Fig. 4. Differences in MGAT activities in the small intestine (A) and DGAT activities (B) in liver, intestine and adipose tissues of restricted OLETF group ( , n= 9), LETO group ( , n= 10) and satiated OLETF group ( , n= 5) at 21 weeks of age. Values are the means 9 S.E. *PB 0.05, **PB0.01, (t-test) 6 LETO group. The specific activity of MGAT in different tissues was calculated on the basis of radioactivity in the DG fraction plus 1/2 of the radioactivity present in the TG fraction.
plasma levels of glucose than LETO rats at 30 and 60 min (PB 0.001). The sum of plasma glucose during the OGTT in restricted OLETF rats was about 1.12-fold (838.61936.76 mg/dl) higher than the value in LETO rats (747.70922.99 mg/ dl). Enzyme activities Fig. 4A shows differences in MGAT activity in the intestine in OLETF and LETO rats. MGAT activity was significantly increased in OLETF rats compared with LETO rats; the value in restricted OLETF rats was 2.7fold (PB 0.001) higher and that in satiated OLETF rats was 2.2-fold (P B 0.01) higher than that in LETO. No significant change in the MGAT activity was noted between satiated and restricted OLETF rats.
OLETF rats resemble human obese patients with NIDDM. There have been some reports about lipid production in OLETF rats, for example, hepatic acyl-coenzyme A synthetase (ACS) and microsomal triglyceride transfer protein (MTP) messenger RNAs were shown to be expressed at high levels in these animals [13]. However, it has been reported that OLETF rats show hyperphagia, a major factor responsible for obesity [9]. Satiated rats were used in these previous study, it will be difficult to determine whether these results were affected by hyperphagia. In the present study, to eliminate the effects of hyperphagia, OLETF rats were given the same amount of food daily as LETO rats. We found that body fat weight was still significantly increased in restricted OLETF rats, indicating that there may be some other factor(s) causing obesity in tissues besides hyperphagia, such as differences in enzyme activity or genetic factors. As the food intake was the same in restricted OLETF and LETO groups, it was possible to make comparisons between the two groups to find abnormalities in lipid metabolism in OLETF rats. In our previous study, 30% food-restricted OLETF rats showed no abnormalities in the activity of lipoprotein lipase (LPL) compared with LETO rats, [14] suggesting that the clearance of TG-rich lipoprotein is not delayed. In the present study, we wonder whether TG be overproduced in OLETF rats. We then focused our attention on the roles of two key enzymes in TG biosynthesis: MGAT and DGAT. MGAT uniquely catalyzes extraneous lipid to TG in the intestinal mucosa, and DGAT is believed to control the one common step of synthesis of triglycerides in all tissues. Results showed that MGAT activities in OLETF rats were significantly higher than those in LETO rats. On the other hand, no significant difference in the DGAT activity was noted between OLETF rats and LETO rats.
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The small intestine is the main tissue involved in TG synthesis. Although it is possible that increased MGAT activity may cause overproduction of TG, it has not been confirmed in vivo. Assuming that the following effects occur in vivo. The same amount of food were taken in restricted OLETF and LETO groups, which means the same amount of substrates were digested by pancreatic lipase to monoacylglycerol and FFAs, the increased MGAT activity in OLETF rats might increase the production of diacylglycerol, and then cause the overproduction of TG, although DGAT activity is normal. But the relationship between the increased MGAT activity and the increased body fat and HTG would be studied more in the future. Morphological studies showed that both the small intestinal wet weight and surface area were 30% larger in OLETF rats than in LETO rats, [24,29] suggesting that not only activity but also quantity of MGAT might be increased in OLETF rats, and both may be involved in the increased TG synthesis. Previous reports suggested that the small intestine plays a key role in the pathogenesis of TG accumulation in tissues and hyperlipidemia in diabetes, [25,26] the results of the present study were consistent with this hypothesis. Intestinal MGAT resides mainly in the mucosal fraction and this activity differs between segments to other, with the jejunum being the most active [28]. In this study, we isolated the mucosal microsomal fraction only from jejunum (the first 15 cm of the small intestine) to measure the enzyme activities. On the other hand, body weight, body fat, index of blood test and OGTT test in restricted OLETF were markedly decreased compared with satiated OLETF, indicating that diet therapy is useful in ameliorating abnormalities in lipid and glucose metabolism of diabetes by improving insulin resistance and b-cell function. Enzyme activity assay showed that MGAT activity in restricted and satiated OLETF rats were similar and both of them were significantly higher than that of LETO rats, whereas DGAT activity showed no significant difference between the satiated and restricted groups in statistics, although there is a tendency that DGAT activity in satiated OLETF rats was
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higher than that of restricted OLETF rats and LETO rats. In this study, even though restricted OLETF rats were given the same amount of food daily as LETO, body fat weight and plasma insulin, glucose and leptin were increased compared with LETO rats, suggesting that the restricted OLETF rats will be a useful model in which to study obesity and its relationship with NIDDM. The present study represents the first indication that increased MGAT activity might play a key role in HTG and obesity. There have been previous reports concerning MGAT activity in Zucker rats [16] and streptozotocin-induced diabetic rats [17], but to date there have seen no reports in human subjects with obesity. If the same abnormality occurs in human subjects as OLETF rats, MGAT inhibitor might become a promising triglyceride lowing drug, we expect that the results of the present study will contribute to elucidation of human HTG and obesity, subsequently in those individuals with NIDDM. In conclusion, increased small intestinal MGAT activities accelerated triglyceride synthesis, and thus might be a causative factor leading to HTG and obesity and be involved in the development of diabetes in OLETF rats.
Acknowledgements We are grateful to Dr Kenji Shima, and Dr Masamichi Kuwajima, University of Tokushima, for providing valuable advice and guidance during these experiments.
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