Nutrient Physiology, Metabolism, and Nutrient

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Chronic Intake of High-Fat and High-Sucrose Diets Differentially Affects Glucose Intolerance in Mice Maho Sumiyoshi,* Masahiro Sakanaka,* and Yoshiyuki Kimuray1 * Division of Functional Histology, Department of Integrated Basic Medical Science and yDivision of Biochemical Pharmacology, Department of Fundamental Medical Science, School of Medicine, Ehime University, Shitsukawa, Toon City, Ehime, 791-0295, Japan

KEY WORDS: high-fat high-sucrose glucose intolerance obesity lean

Insulin resistance is associated with a number of metabolic disorders such as obesity, hyperlipidemia, and hypertension. These factors can increase the risk of coronary heart disease (1,2). Both genetic and environmental factors contribute to the development of metabolic abnormalities. Diet represents one environmental factor that can influence a metabolic disorder. Several experimental studies demonstrated that the macronutrient composition of a diet is an important environmental determinant of the quality of insulin action (3–6). High-fat (HF)2 and high-sucrose (HS) intakes were shown to contribute to syndromes such as hyperlipidemia, glucose intolerance, hypertension, and atherosclerosis (7–9). Numerous studies showed that a HF and/or HS diet induces insulin resistance in rodents (10–14). The pathogenesis of insulin resistance caused by HF and/or HS is unclear. It was reported that excess circulating free fatty acids (FFA) and glucose may contribute to insulin resistance (15,16). 1

On the other hand, it was reported that adipocytokines such as leptin, adiponectin, tumor necrosis factor (TNF)-a, plasminogen activator inhibitor (PAI)-1, and IL-6 are secreted from adipose tissue. The alteration of lipid metabolism and the secretion of adipocytokines in adipose tissue may be critical for the development of lifestyle-related diseases such as obesity, hyperlipidemia, diabetes, hypertension, and atherosclerosis. Thus, it is important to clarify the mechanisms underlying the metabolic disorders that result from the long-term consumption of HF or HS diets. A HF diet causes obesity, and it seems likely that obesity contributes to insulin resistance (17). On the other hand, it is unclear whether insulin resistance due to longterm consumption of a HS diet is associated with obesity. The diabetes- and obesity-prone C57BL/6 mouse fed a HF diet is a good model of human obesity and type II diabetes (18–20). In this study, we examined the effects of consuming high-fat or high-sucrose diets for a long time (55 wk) on insulin resistance and lipid metabolic alterations in C57BL/6J mice.

To whom correspondence should be addressed. E-mail: [email protected].

ac.jp. 2 Abbreviations used: AUC, area under the curve; Cmax, maximal concentration; FAS, fatty acid synthase; FFA, free fatty acids; G6P, glucose-6-phosphate; G6Pase, glucose-6-phosphatase; HF, high-fat; HS, high-sucrose; HMG-CoA, 3-hydroxymethylglutaryl CoA; ITT, insulin tolerance test; LL, low-fat, low-sucrose; LPL, lipoprotein lipase; MCP-1, monocyte chemoattractant protein-1; OGTT, oral glucose tolerance test; Pi, inorganic phosphate; PPAR, peroxisome proliferatoractivated receptor; TC, total cholesterol; TG, triglyceride.

MATERIALS AND METHODS Materials. Cornstarch, casein, and the mineral and vitamin mixtures were purchased from Oriental Yeast. No-salt butter was provided by Yotsuba Nyugyou. The Triglyceride E-Test, Triglyceride-Test,

0022-3166/06 $8.00 Ó 2006 American Society for Nutrition. Manuscript received 6 September 2005. Initial review completed 10 October 2005. Revision accepted 19 December 2005. 582

Downloaded from jn.nutrition.org by guest on December 11, 2011

ABSTRACT Intakes of some macronutrients can comprise risk factors for life-style-related diseases such as obesity, hyperlipidemia, diabetes, hypertension, and atherosclerosis. In this study, we examined the effects in C57BL/6J mice of consuming excess fat or sucrose for a long period of time (55 wk). Another group of mice consumed a low-fat, low-sucrose (LL) diet. Mice fed the high-fat (HF) diet gained weight and developed hyperlipidemia and hyperleptinemia. At 25 wk, but not at 55 wk, hepatic glucose-6-phosphatase (G6Pase) activity of the mice fed the high sucrose (HS) diet was greater than that of mice fed the LL or HF diet. Those fed the HS diet were not obese and had greater hepatic lipogenic and gluconeogenic enzyme activities. The HF and HS diets resulted in different types of glucose intolerance. In an oral glucose tolerance test, mice fed the HF diet had a delay in the clearance of glucose compared with those fed the LL diet, perhaps due to the peripheral insulin resistance that resulted from higher levels of circulating free fatty acids. Feeding the HS diet for 55 wk induced hyperglycemia 10 min after oral glucose administration, although blood glucose declined rapidly after i.p. insulin injection. This finding suggests that the effects of chronic HS diet intake may be due to the reduction in early insulin secretion from pancreatic islets and the increase in sucrase activity in the small intestine. It is important to consider the effects of macronutrients in lean as well as obese mice to clarify the pathogenesis of the metabolic disorders. J. Nutr. 136: 582–587, 2006.

ALTERATION OF DIET-INDUCED GLUCOSE INTOLERANCE

TABLE 1 Composition of experimental diets LL

HS

HF

g/kg Milk fat Cornstarch Sucrose Casein Cellulose Mineral mix1 Choline chloride Vitamin mix1 Water Energy, MJ/kg

30.0 415.0 50.0 200.0 30.0 35.0 4.0 10.0 224.0 12.26

60.0 65.0 500.0 200.0 30.0 35.0 4.0 10.0 96.0 14.89

450.0 171.0 100.0 200.0 30.0 35.0 4.0 10.0 0.0 22.40

1 Mineral and vitamin mixtures (AIN-76) (21) were purchased from Oriental Yeast.

epididymal adipose tissue segments were stained with hematoxylin and eosin. The plates were photographed, and .100 adipose cells were randomly selected and their cell diameters measured.

Enzyme activities Lipoprotein lipase (LPL). LPL activity in muscle and epididymal adipose tissue was determined as described by Iverius et al. (24). Tissue was homogenized in 0.178 mol/L Tris buffer, pH 8.2, containing 0.25 mol/L sucrose, 2 g/L deoxycholate, and 50 mg/L heparin. After centrifugation at 12000 3 g, the supernatant was used for the assay. Lipolysis. Epididymal adipose tissue (50 mg) was added to HBSS (pH 7.4) containing 2.5% bovine serum albumin with or without norepinephrine (0.1 mmol/L; Wako Pure Chemical) and insulin (1 nmol/L; Wako Pure Chemical). After incubation at 378C for 30 min, the FFA released was determined by the method of Han et al. (25) using copper reagent. Glucose-6-phosphatase (G6Pase). The liver microsomal fraction was separated by the method of Daniele et al. (26) with slight modifications. Briefly, the liver was cut and homogenized immediately on ice in 10 mmol/L HEPES buffer (pH 7.2) containing 0.25 mol/L sucrose and protease inhibitor, using a Teflon glass homogenizer. The homogenate was centrifuged at 9000 3 g for 20 min at 48C; the supernatant was then further centrifuged at 105,000 3 g for 60 min at 48C. The microsomal pellet was suspended in homogenized buffer. The assay quantified the amount of inorganic phosphate (Pi) released from glucose-6-phosphate (G6P) as G6Pase activity. The microsomal fraction was added to 20 mmol/L Tris buffer (pH 7.4) containing 5 or 20 mmol/L G6P, and incubated at 378C for 10 min. The reaction was stopped by the addition of 8% trichloroacetic acid, and Pi was determined by the methods of Fiske and Subbarow (27). 3-Hydroxymethylglutaryl CoA (HMG-CoA) reductase. The liver microsomal fraction was separated by the method described above. The microsomal pellet was suspended in 0.1 mol/L phosphate buffer (pH 7.4), and added to a reaction mixture containing 0.128 mmol/L HMG-CoA (14C-HMG-CoA, 144 MBq/mmol), 1 mmol/L NADPH, 10 mmol/L dithiothreitol, and 10 mmol/L EDTA in 0.12 mol/L phosphate buffer (pH 7.4). The mevalonic acid released was separated on a silica gel 60 F254 TLC plate (Merck) (28). The plate was exposed to an imaging plate (Fuji film), and examined with an imaging analyzer BAS 1000 (Fuji film). Fatty acid synthase (FAS). The liver was homogenized on ice in 10 mmol/L HEPES buffer (pH 7.2) containing 0.25 mol/L sucrose and protease inhibitor, and centrifuged at 12,000 3 g for 10 min at 48C. The activity was determined by measuring the incorporation of 14 C-acetyl-CoA into fatty acids (29). The reaction was stopped by the addition of chloroform:methanol (2:1, v:v), and mixed using a vortex mixer. After centrifugation (1500 3 g; 10 min), the supernatant was removed by aspiration, and the residue was washed twice with water under acidic conditions. The residual radioactivity was quantified with a liquid scintillation counter. Sucrase. The mucosa of the small intestine was scraped off on ice and homogenized in PBS. After centrifugation at 600 3 g for 10 min at 48C, the supernatant was used for the assay (30). The glucose concentration was determined with a Glucose C-II Test Kit (Wako Pure Chemical). Immunoblotting. Muscle was homogenized in a solubilizing buffer [50 mmol/L HEPES buffer (pH 7.4) containing 5 mmol/L EDTA, 150 mmol/L NaCl, 1% TritonX-100 and protease inhibitor] and then centrifuged at 12,000 3 g for 15 min at 48C. The amount of protein in the supernatant was measured using a protein assay reagent (Bio-Rad). After denaturing at 998C, the protein was resolved by SDS-PAGE. The gel was transferred to polyvinylidene fluoride membrane, and the membrane was blocked with 5% skim milk. The membrane was incubated with anti-PPARa antibody (1:250). Immunoreactivity was visualized with alkaline phosphatase-conjugated goat anti-rabbit IgG (ICN Pharmaceuticals) and the ECF system (Amersham Biosciences). Statistical analysis. All values are expressed as means 6 SEM. Data were analyzed by 1-way, 2-way, or repeated-measures ANOVA. When the F-test was significant, means were compared using Fisher’s Protected LSD test with StatView (SAS Institute). Differences were considered significant at P , 0.05.


Total Cholesterol E-Test, and NEFA C-Test Kits were purchased from Wako Pure Chemical. The ELISA Insulin and ELISA Leptin Kits were purchased from Morinaga. The Adiponectin ELISA Kit was from Otsuka Pharmaceutical. The JE/MCP-1 ELISA Kit was purchased from R&D systems. Anti-peroxisome proliferator-activated receptor (PPAR)-a antibody was obtained from Santa Cruz Biotechnology. Human recombinant insulin was purchased from Novo Nordisk. 3-Hydroxy-3-methyl [3-14C] glutaryl-CoA and [1-14C] acetyl-CoA were obtained from Amersham Biosciences UK. [9,10-3H(N)]-triolein and [1-14C]-palmitic acid were from Perkin Elmer Life Sciences. Other chemicals were of reagent grade. Animals and diets. Male C57BL/6J mice (4 wk old) were obtained from Japan SLC and housed in a room with a 12-h light:dark cycle and controlled for temperature and humidity. They were fed a standard laboratory diet (8 g water, 51.3 g crude carbohydrate, 24.6 g crude protein, 5.6 g crude lipid, 3.1 g crude fiber, 6.4 g mineral mixture, and 1 g vitamin mixture/100 g diet; Oriental Yeast) for 1 wk to adapt to the lighting conditions. After this period of adaptation, they were given free access to water and the experimental diets (Table 1) for 55 wk. The diets were a low-fat low-sucrose (LL) diet (3% fat, 5% sucrose, wt/wt), a HF diet (45% fat, wt/wt), and a HS diet (50% sucrose, wt/wt). Body weight and food intake were recorded weekly. Because both the LL and HS diets contained water, intake of these diets was corrected using the dry weight of the diet. The energy intakes of the mice fed the diets were constant throughout the experiment [kJ/(moused): LL, 54.50 6 0.53; HS, 52.67 6 0.55; HF, 47.89 6 0.49]. Mice were treated according to the ethical guidelines of the Animal Center, School of Medicine, Ehime University. The Animal Studies Committee of Ehime University approved the experimental protocol. Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). The OGTT was carried out at wk 6 and every 5 wk beginning at wk 10. After at least 4 h of food deprivation, glucose (556 mmol/mouse) was administered orally to the mice. The ITT was conducted at wk 5, 15, and 45. After 4 h of food deprivation, insulin (5.6 nmol/kg) was injected i.p. Blood samples were taken from the tail at the indicated times and blood glucose concentrations were measured using GLUCOCARDä (GT-1640, Arkray). In the OGTT at wk 40, the plasma was separated and insulin was measured using the ELISA kit. Plasma measurements. At wk 55, blood was obtained by venous puncture from mice anesthetized with NEMBUTALÒ (Dainippon Pharmaceutical). Plasma was separated by centrifugation (2000 3 g; 15 min) and frozen at 2208C. Triglyceride (TG), total cholesterol (TC), and FFA concentrations in the plasma at wk 55 were determined using the respective test kits; insulin, leptin, adiponectin, and monocyte chemoattractant protein 1 (MCP-1) concentrations in plasma were measured using the respective ELISA kit. Liver lipid concentrations. TG and TC concentrations in liver were measured by the methods of Fletcher (22) and Zak et al. (23). Histological examination. Epididymal adipose tissue was fixed in 10% (v/v) buffered formalin, embedded in paraffin, and sectioned. The

583

SUMIYOSHI ET AL.

584

RESULTS

TABLE 2 Body, adipose tissue and liver weights, and liver lipid concentrations in mice fed the LL, HS, and HF diets for 55 wk1 LL

HS b

40.3 6 1.9

54.5 6 1.8a

1.57 6 0.34b 0.40 6 0.05b 0.70 6 0.26b

Adipose tissue 1.45 6 0.28b 0.53 6 0.09b 0.86 6 0.27b

3.53 6 0.22a 1.05 6 0.08a 5.97 6 0.39a

3.43 6 0.57 190.74 6 48.69b 41.44 6 9.01

Liver 3.01 6 0.40 191.55 6 29.50b 32.47 6 5.04

3.46 6 0.34 295.73 6 21.26a 38.71 6 2.87

Body weight, g

40.7 6 1.5

Epididymal, g Mesenteric, g Subcutaneous, g Liver weight, g TG, mmol/g TC, mmol/g

HF b

1 Values are means 6 SEM, n ¼ 6. Data were analyzed by 1-way ANOVA; differences among means were analyzed using Fisher’s protected Least Significant Difference multiple test. Means in a row with superscripts without a common letter differ, P , 0.05.


Body weight and adipose tissue. The weight of the mice increased during the 55-wk experiment (data not shown). The final body weight of mice fed the HF diet was significantly greater than that of mice fed the LL or HS diet (Table 2). Body fat weights were significantly greater in mice fed the HF diet than in the other groups (Table 2). The adipocyte diameter in epididymal adipose tissue was larger in mice fed the HF diet than in the other 2 groups (the mean cell diameter for 100 cells, LL: 77.74 6 5.78 mm, HS: 78.31 6 5.14 mm, HF: 123.81 6 2.90 mm), and the adipocyte size distribution was shifted toward larger cells in the HF diet-fed mice (Fig. 1 A, B). Plasma and liver lipids. The plasma TG and TC concentrations of mice fed the HF diet were significantly greater than in those fed the LL or HS diets (Table 3). The plasma FFA concentration was significantly greater in mice fed the HF diet than in those fed the HS diet and greater in both of those groups than in mice fed the LL diet (Table 3). Liver TG concentrations were greater in mice fed the HF diet than in those fed the LL or HS diets, whereas cholesterol concentration did not differ among the groups (Table 2). Leptin, adiponectin, and MCP-1. Plasma leptin concentrations were significantly elevated in mice fed the HF diet compared with those fed the LL or HS diets. However, the concentration of adiponectin did not differ among the 3 groups (Table 3). The plasma MCP-1 levels of mice fed the HF diet tended to be greater than in the other 2 groups (P , 0.08, Table 3). Lipolysis in adipose tissue. Basal lipolysis did not differ among the 3 groups (mmol FFA released/g epididymal adipose tissue) LL: 2.32 6 0.50, HS: 2.79 6 0.45, HF: 1.98 6 0.24. The basal lipolysis per fat pad in mice fed the HF diet was greater than that in mice fed the LL or HS diet. Therefore, at least in mice fed the HF diet, the plasma FFA level may depend on the volume of adipose tissue as a source of FFA. On the other hand, lipolysis in mice fed the LL diet and HS diet was significantly increased by the addition of norepinephrine, but it was not increased by adding norepinephrine to the adipose tissue of mice fed the HF diet. Norepinephrine-induced lipolysis in all 3 groups was not affected by insulin (Fig. 2A). Lipogenesis in liver. FAS activity in the livers of mice fed the LL and HS diets was greater than that in HF diet-fed mice (Fig. 2B). The activity of HMG-CoA reductase, one of the key enzymes of cholesterol synthesis, tended to be lower in mice fed

the HF diet than in those fed the LL (P 5 0.059) and HS diets (P 5 0.112) (Fig. 2C). PPARa protein and LPL activity in skeletal muscle. TG concentrations in femoral muscle were reduced in mice fed the HS diet (0.38 6 0.06 mmol/ g protein), compared with those fed the LL diet (0.55 6 0.05 mmol/mg protein) or HF diet (0.56 6 0.06 mmol/mg protein; P , 0.05). Muscle LPL activity was lower in mice fed the HF (0.25 6 0.02 mmol FFA released /mg protein) and HS diets (0.28 6 0.03 mmol FFA released /mg protein) compared with that of mice fed the LL diet (0.42 6 0.04 mmol FFA released /mg protein; P , 0.05). The amount of PPARa protein in muscle was greater in mice fed the LL diet than in those fed the HS diet or HF diet (data not shown). OGTT and ITT. In the OGTT, the concentration of blood glucose of mice fed the LL and HS diets increased to a maximum at 10 min after the oral administration of glucose, and then declined to the basal value (Fig. 3A). At wk 10, the plasma glucose concentration at 10 min was higher in mice fed the HS diet than in those fed the HF or LL diets. On the other hand, the glucose levels of mice fed the HF diet were significantly higher at 20, 30, and 60 min after the oral administration of glucose than those of mice fed the LL or HS diet, and the glucose levels at 10 to 60 min in mice fed the HF diet were maintained. The glucose concentrations at 10, 20, 30, and 60 min after the oral administration of glucose did not differ in this group. Thus, glucose clearance in mice fed the HF diet at wk 10 was impaired compared with that in mice fed the LL and HS diets (Fig. 3A). At wk 30 and 50, the concentration of glucose in the blood increased to a maximum at 10 min in all groups. The glucose levels of HS diet-fed mice at 10 min after administration were higher than those of mice fed the LL and HF diets. On the other hand, the blood glucose levels at 20, 30, or 60 min in the HF diet-fed mice were less (P , 0.05) than the maximal glucose levels at 10 min, at wk 30 and/or 50. Thus, at wk 30 and 55, the clearance of glucose from the blood after oral administration was apparently improved in the mice fed the HF diet (Fig. 3A). The different types of impaired glucose tolerance were caused by feeding the HS diet and the HF diet for a long time. In the ITT, the blood glucose level decreased rapidly with insulin injections (Fig. 3B). The blood glucose levels after insulin injection did not differ among the 3 diet-fed groups at wk 5. However, the blood glucose levels at 40 to 120 min after insulin injection in the HF diet-fed mice were higher than those


585

FIGURE 1 Adipocyte size (A) and frequency (B) in epididymal adipose tissue in mice fed the LL, HS, and HF diets for 55 wk. (A) Original magnification X100; bar ¼ 100 mm.

fraction from the liver did not differ among mice fed the 3 diets for 55 wk (data not shown). At wk 25, the G6Pase activity of the mice fed the HS diet was significantly greater than that of mice fed the LL or HF diet (Table 5). DISCUSSION

TABLE 3 Plasma glucose, lipid, insulin, leptin, adiponectin, and MCP-1 concentrations in mice fed the LL, HS, and HF diets for 55 wk1 LL

FIGURE 2 Lipolysis in epididymal adipose tissue (A), hepatic FAS (B) and HMG-CoA reductase activities (C) in mice fed the LL, HS, and HF diets for 55 wk. Norepinephrine (0.1 mmol/L)-induced lipolysis in epididymal adipose tissue was measured with or without insulin (1 nmol/L). Lipolytic activity is expressed as a percentage of the basal activity. Values are mean 6 SEM, n ¼ 6. Data were analyzed by 2-way ANOVA (A) or 1-way ANOVA (B and C); differences among means were analyzed using Fisher’s protected Least Significant Difference multiple test. Means without a common letter differ, P , 0.05.

HS

HF

Glucose, mmol/L 6.84 6 0.97 5.50 6 0.46 8.25 6 1.25 TG, mmol/L 0.38 6 0.05b 0.53 6 0.07b 1.40 6 0.16a b b TC, mmol/L 3.20 6 0.55 2.41 6 0.40 6.48 6 0.52a FFA, mmol/L 0.29 6 0.03c 0.61 6 0.11b 0.94 6 0.12a b c Insulin, nmol/L 0.65 6 0.17 0.23 6 0.07 1.07 6 0.16a Leptin, mg/L 28.14 6 12.83b 42.97 6 15.82b 108.94 6 8.47a Adiponectin, mg/L 15.84 6 1.78 18.40 6 2.22 19.29 6 1.68 MCP-1, ng/L 94.24 6 16.57 87.14 6 17.70 134.10 6 8.49 1 Values are means 6 SEM, n ¼ 6. Data were analyzed by 1-way ANOVA; differences among means were analyzed using Fisher’s protected Least Significant Difference multiple test. Means in a row with superscripts without a common letter differ, P , 0.05.

White adipose tissue is now recognized as a secretary organ, secreting adipocytokines such as leptin, adiponectin, TNF-a, and PAI-1. PPARg induces adipocytes to differentiate, and leptin and adiponectin are involved in the metabolism of glucose and lipids. In the present study, we found that longterm feeding of a HF diet to mice caused obesity, with increases in fat volume, fat size (Fig.1 and Table 2), and PPARg protein levels (data not shown). Insulin did not affect epididymal adipose tissue lipolytic activity (Fig. 2A). Other features of obese mice included elevated TG and PPARa concentrations in muscle, reduced muscle LPL activity, increased hepatic TG concentrations, increased plasma FFA, insulin, and leptin concentrations, and glucose intolerance after an ITT. Although circulating FFA upregulate the secretion of insulin from the pancreas (31), the increase in FFA induces peripheral insulin resistance (15,32). It was reported that the accumulation of TG and the reduction of PPARa expressions in muscle may cause insulin resistance (33). In the present study, the muscle TG


in LL and HS diet-fed mice at wk 15 and 40. The blood glucose levels at 40 to 120 min after insulin injection in the HS diet-fed mice were lower than those in mice fed the LL and HF diets at wk 15 and 40, although a higher glucose level at 10 min was maintained in the OGTT. In the OGTT at wk 45, plasma insulin levels were significantly higher in mice fed the HF diet than in those fed the LL diet (Cmax and area under the curve [AUC] 0–1h); plasma glucose concentrations did not differ between mice fed the LL and HF diets (Table 4). Otherwise, glucose levels were significantly higher in mice fed the HS diet than in those fed the LL diet (Cmax and AUC 0–1 h); insulin levels did not differ between mice fed the LL and HS diets. Thus, the impaired glucose tolerance of mice fed the HF diet is attributed to peripheral insulin resistance, which was apparently improved by compensative hyperinsulinemia. On the other hand, the decline in glucose levels after insulin injection in the HS diet-fed mice was greater than that in the LL diet-fed mice. Insulin secretion at the time of the OGTT was lower in mice fed the HS diet even though their blood glucose level was higher compared with mice fed the LL diet. Intestinal sucrase activity and hepatic glucogenesis. The small intestinal sucrase activities in mice fed the LL [5.67 6 0.51 mmol glucose/(mg protein0.5 h)] and HS diets [6.71 6 0.78 mmol glucose/(mg protein0.5 h)] were greater than in those fed the HF diet [3.44 6 0.15 mmol glucose/(mg protein0.5 h), P , 0.05]. The G6Pase activity of the microsomal

SUMIYOSHI ET AL.

586

TABLE 5 Liver G6Pase activity in mice fed the LL, HS, and HF diets for 25 wk1 LL

HS

HF

Pi, mmol/mg protein G6P, 5 mmol/L G6P, 20 mmol/L

1.24 6 0.05b 2.50 6 0.13b

1.45 6 0.08a 3.17 6 0.18a

0.68 6 0.03c 1.51 6 0.05c

1 Values are means 6 SEM, n ¼ 7. Data were analyzed by 1-way ANOVA; differences among means were analyzed using Fisher’s protected Least Significant Difference multiple test. Means at same G6P concentration without a common letter differ, P , 0.05.

concentrations in the HF and LL diet-fed mice were greater than those of the HS diet-fed mice. Furthermore, muscle PPARa expression in the HF and HS diet-fed mice was lower than that in the LL diet-fed mice. These findings suggest that the insulin resistance caused by feeding the HF diet for a long time may be due to the decrease in PPARa expression and the accumulation of TG in the muscle. Although the HS diet-fed mice had potent insulin sensitivity in the ITT, HS diet-fed mice demonstrated glucose intolerance in the OGTT. The glucose intolerance after feeding the HS diet cannot be explained by the reduction in muscle PPARa expression. Further studies are warranted to clarify the relation between PPARa expression and lipid metabolism in the muscle of HS diet-fed mice. Yamuuchi et al. (34) reported that adiponectin stimulated glucose utilization and FFA oxidation by activation of the 59AMP-activated protein kinase. However, the concentration of adiponectin did not differ among the groups (Table 3). Arita et al. (35) reported that levels of adiponectin were reduced in obese individuals and that the reduction was inversely correTABLE 4 Plasma insulin and glucose levels during an OGTT in mice fed the LL, HS, and HF, diets for 45 wk1

Cmax (10 min) Glucose, mmol/L Insulin, nmol/L AUC (0–1 h) Glucose, (mmolh)/L Insulin, (nmolh)/L

LL

HS

HF

14.34 6 1.26

24.33 6 0.88a

16.94 6 1.06b

2.32 6 0.29b 10.69 6 1.18b 1.09 6 0.14b

3.14 6 0.52ab 13.29 6 0.45a 1.20 6 0.21ab

3.92 6 0.47a 11.53 6 0.46ab 1.66 6 0.16a

1 Values are mean 6 SEM, n ¼ 6. Data were analyzed by 1-way ANOVA; differences among means were analyzed using Fisher’s protected Least Significant Difference multiple test. Means in a row with superscripts without a common letter differ, P , 0.05.


FIGURE 3 Plasma glucose concentrations of mice fed the LL, HS, and HF diets at OGTT in wk 10, 30, and 55 (A), and at ITT during wk 5, 15, and 40 (B). Values are mean 6 SEM, n ¼ 6. Data were analyzed by repeated-measures ANOVA; differences among means were analyzed using Fisher’s protected Least Significant Difference multiple test. Means at the same time without a common letter differ, P , 0.05.

lated with an increase in visceral fat volume; on the other hand, it was reported that there was no relation between the plasma adiponectin level and obesity (36,37). In the present study, the adiponectin level, glucose intolerance, and visceral adipose tissue weight were not interrelated. The visceral fat weight of female C57BL/6J mice fed a HF diet for 70 wk was much greater than that of male mice; conversely, adiponectin levels were lower in the female mice than in the male mice (unpublished data). Insulin is a lipogenic hormone, acting to increase the expressions and activities of acetyl CoA carboxylase, FAS, malic enzyme, and HMG-CoA reductase (38). Recently, Najjar et al. (39) reported that acute insulin did not significantly reduce hepatic FAS activity in refed obese hyperinsulinemic (ob/ ob) mice compared with normoinsulinemic mice, and suggested that this is consistent with the blunted ability of insulin to activate insulin receptors in chronic hyperinsulinemia. We found that hepatic FAS activity in mice fed the LL and HS diets was elevated compared with those fed the HF diet (Fig. 2B), but the FAS activity of HF diet-fed mice with hyperinsulinemia was not affected compared with the mice fed a laboratory diet (data not shown). Moreover, the HMG-CoA reductase in the HF dietfed mice was lower than that in the LL and HS diet-fed mice (Fig. 2C) or laboratory diet-fed mice (data not shown). These findings also show that the HF diet causes insulin resistance of liver lipid metabolism. Therefore, it seems likely that the elevated plasma FFA levels in mice fed the HF diet may be associated with fat volume and basal lipolysis in white adipose tissue and the accumulation of TG in muscle and liver; consequently, the mice may develop insulin resistance and hyperinsulinemia (Table 3, 4, Fig. 2A). In addition, because hyperleptinemia was reported to suppress second-phase insulin secretion (40), we suggest that glucose intolerance in mice fed the HF diet in OGTT may be caused by the regulation of insulin secretion from the pancreas through higher levels of circulating FFA and leptin. Although atherosclerosis was not induced, a slight accumulation of lipids in the main artery occurred in mice fed the HF diet (data not shown). It was shown recently that MCP-1 is responsible for the macrophage accumulation in atherogenesis (41–43). Obesity is associated with the accumulation of macrophages and the overexpression of MCP-1 in adipose tissue (44,45). Plasma MCP-1 levels in HF diet-fed mice tend to be greater than those in LL or HS diet-fed mice (P , 0.08, Table 3). On the basis of these results in HF diet-fed mice, we suggest that the incidence of atherosclerosis may arise together with obesity by feeding a HF diet. It is of great interest that glucose intolerance differed between mice fed the HS and HF diets for a long period time (Fig. 3). In the OGTT, the Cmax of blood glucose (at 10 min) was higher in the mice fed the HS diet than in those fed the LL or HF diets. Thus, the blood glucose clearance in the HS dietfed mice was impaired 10 min after OGTT (Fig. 3A), but the


blood glucose concentration in the HS diet-fed mice was rapidly lowered by the ITT (Fig. 3B). The small intestinal sucrase activity was increased by feeding the HS diet for a long time. Furthermore, hepatic G6Pase activity was increased by feeding the HS diet for 25 wk, but its activity in mice fed that diet for 55 wk was not affected. Therefore, these findings suggest that the glucose intolerance in HS diet-fed mice is not associated with the hepatic gluconeogenesis, but rather may be due to a reduction in early insulin secretion from pancreatic islets and an increase in sucrase activity in the small intestine. Further studies are warranted to clarify the mechanisms (e.g., glucose-stimulated insulin release, glucose transporter expression in islets, and pancreatic injury). Thus, we found that the excess consumption of fat and sucrose diets induced 2 different types of glucose intolerance not only in obese but also in lean mice. It is important to consider the effects of macronutrients lean as well as obese mice to clarify the pathogenesis of metabolic disorders. Further study is warranted to clarify the mechanisms of insulin resistance in both obese and lean animals fed the HF or HS diets for a long time.

1. Plutzky J. Emerging concepts in metabolic abnormalities associated with coronary artery disease. Curr Opin Cardiol. 2000;15:416–21. 2. Reaven GM. Role of insulin resistance on human disease. Diabetes. 1988;37:1595–607. 3. Axen KV, Dikeakos A, Sclafani A. High dietary fat promotes Syndrome X in nonobese rats. J Nutr. 2003;133:2244–9. 4. Bessesen DH. The role of carbohydrates in insulin resistance. J Nutr. 2001;131:2782S–6. 5. Harris RB, Kor H. Insulin sensitivity is rapidly reversed in rat by dietary fat from 40% to 30% of energy. J Nutr. 1992;122:1811–22. 6. Storlien LH, Kraegen EW, Jenkins AB, Chisholm DJ. Effects of sucrose vs starch diets on in vivo insulin action, thermogenesis, and obesity in rats. Am J Clin Nutr. 1988;47:420–7. 7. Dobrian AD, Davies MJ, Prewitt RL, Lauterio TJ. Development of hypertension in a rat model of diet-induced obesity. Hypertension. 2000;35: 1009–15. 8. Mann JI. Diet and risk of coronary heart diseases and type2 diabetes. Lancet. 2002;360:783–9. 9. Fried SK, Rao SP. Sugar, hypertriglyceridemia, and cardiovascular disease. Am J Clin Nutr. 2003;78:873S–80. 10. Ahren B, Scheurink AJ. Marked hyperleptinemia after high-fat diet associated with severe glucose intolerance in mice. Eur J Endocrinol. 1998;139: 461–7. 11. Brenner RR, Rimoldi OJ, Lombardo YB, Gonzalez MS, Bernasconi AM, Chicco A, Basabe JC. Desaturase activities in rat model of insulin resistance induced by a sucrose-rich diet. Lipids. 2003;38:733–42. 12. Muurling M, Jong MC, Mensink RP, Hornstra G, Dahlmans VEH, Pijl H, Voshol PJ, Havekes LM. A low-fat diet has a higher potential than restriction to improve high-fat diet-induced insulin resistance in mice. Metabolism. 2002;51: 695–701. 13. Pagliassotti MJ, Prach P, Koppenhafer T, Pan DA. Changes in insulin action, triglycerides, and lipid composition during sucrose feeding in rats. Am J Physiol Regul Integr Comp Physiol. 1996;271:R1319–26. 14. Ryu MH, Cha YS. The effects of high-fat or high-sucrose diet on serum lipid profiles, hepatic acyl-CoA synthetase, carnitine palmitoyltransferase-I, and the acetyl-CoA carboxylase mRNA levels in rats. J Biochem Mol Biol. 2003;36:312–8. 15. Arner P. Insulin resistance in type 2 diabetes: role of fatty acid. Diabetes Metab Res Rev. 2002;18:S5–9. 16. Tomas E, Lin YS, Dagher Z, Saha A, Luo Z, Ido Y, Ruderman NB. Hyperglycemia and insulin resistance: possible mechanisms. Ann N Y Acad Sci. 2002;967:43–51. 17. Kahn BB, Flier JS. Obesity and insulin resistance. J Clin Invest. 2000;106: 473–81.

18. Harte RA, Kirk EA, Rosenfeld ME, LeBoeuf RC. Initiation of hyperinsulinemia and hyperleptinemia is diet dependent in C57BL/6 mice. Horm Metab Res. 1999;31:570–5. 19. Surwit RS, Kuhn CM, Cochrane C, McCubbin JA, Feinglos MN. Dietinduced typeII diabetes in C57BL/6J mice. Diabetes. 1988;37:1163–7. 20. Surwit RS, Feinglos MN, Rodin J, Sutherland A, Petro AE, Opara EC, Kuhn CM, Rebuffe-Scrive M. Differential effects of fat and sucrose on development of obesity and diabetes in C57BL/6J and A/j mice. Metabolism. 1995;44:645–51. 21. American Institute of Nutrition. Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J Nutr. 1977;107:1340–8. 22. Fletcher MJ. A colorimetric method for estimating serum triglycerides. Clin Chim Acta. 1968;22:393–7. 23. Zak B, Dickman RC, White EG, Cherney PJ. Rapid estimation of free and total cholesterol. Am J Clin Pathol. 1954;24:1307–15. 24. Iverius PH, Ostrund-Lindqvist AM. Preparation, characterization, and measurement of lipoprotein lipase. Methods Enzymol. 1986;129:691–704. 25. Han LK, Kimura Y, Okuda H. Reduction in fat storage during chitinchitosan treatment in mice fed a high-fat diet. Int J Obes. 1999;23:174–9. 26. Daniele N, Rajas F, Payrastre B, Mauco G, Zitoun C, Mithieux G. Phosphatidylinositol 3-kinase translocates onto liver endoplasmic reticulum and may account for the inhibition of glucose-6-phosphatase during refeeding. J Biol Chem. 1999;274:3597–601. 27. Fiske CH, Subbarow Y. The colorimetric determination of phosphorus. J Biol Chem. 1925;66:375–400. 28. Shapiro DJ, Imblum RL, Rodwell VW. Thin-layer chromatographic assay for HMG-CoA reductase and mevalonic acid. Anal Biochem. 1969;31:383–90. 29. Tijburg LB, Maquedano A, Bijleveld C, Guzman M, Geelen MJH. Effects of ethanol feeding on hepatic lipid synthesis. Arch Biochem Biophys. 1988;267: 568–79. 30. Dahlqvist A. Method for assay of intestinal disaccharidases. Anal Biochem. 1964;57:18–25. 31. Itoh Y, Kawamata Y, Harada M, Kobayash M, Fujii R, Fukusumi S, Ogi K, Hosoya M, Tanaka Y, et al. Free fatty acids regulate insulin secretion from pancreatic b cells through GPR40. Nature. 2003;422:173–6. 32. Griffin ME, Marcucci MJ, Cline GW, Bell K, Barucci N, Lee D, Goodyear LJ, Kraegen EW, White MF, Shulman GI. Free fatty acid-induced insulin resistance is associated with activation of protein kinase Cu and alternations in the insulin signaling cascade. Diabetes. 1999;48:1270–4. 33. Hegarty BD, Furler SM, Ye J, Cooney GJ. Kraegen EW. The role of intramuscular lipid in insulin resistance. Acta Physiol Scand. 2003;178:373–83. 34. Yamauchi T, Kamon J, Minokoshi Y, Ito Y, Wakai H, Uchida S, Yamashita S, Noda M, Kita S, et al. Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase. Nat Med. 2002;8:1288–95. 35. Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, Hotta K, Shimomura I, Nakamura T, et al. Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity. Biochem Biophys Res Commun. 1999;257:79–83. 36. Baratta R, Amato S, Degano C, Farina MG, Patane´ G, Vigneri R, Frittitta L. Adiponectin relationship with lipid metabolism is independent of body fat mass: evidence from both cross-sectional and intervention studies. J Clin Endocrinol Metab. 2004;89:2665–71. 37. Hoffstedt J, Arvidsson E, Sjo¨lin E, Wa˚hleń K, Arner P. Adipose tissue adiponectin production and adiponectin serum concentration in human obesity and insulin resistance. J Clin Endocrinol Metab. 2004;89:1391–6. 38. Nepokroeff CM, Lakshmanan MR, Ness GC, Dugan RE, Porter JW. Hormonal regulation of the diurnal rhythm of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in rat liver by insulin, glucagons, cyclic AMP and hydrocortisone. Arch Biochem Biophys. 1974;160:387–93. 39. Najjar SM, Yang Y, Fernstro¨m MA, Lee SA, DeAngelis AM, Rijaily GAA, Al-Share QY, Dai T, Miller TA, et al. Insulin acutely decreases hepatic fatty acid synthase activity. Cell Metab. 2005;2:43–53. 40. Ookuma M, Ookuma K, York DA. Effects of leptin on insulin secretion from isolated rat pancreatic islets. Diabetes. 1998;47:219–23. 41. Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in CCR2/mice reveals a role for chemokines in the initiation of atherosclerosis. Nature. 1998;394:894–7. 42. Egashira K. Molecular mechanisms mediating inflammation in vascular disease. Hypertension. 2003;41:834–41. 43. Kusano KF, Nakamura K, Kusano H, Nishii N, Banba K, Ikeda T, Hashimoto K, Yamamoto M, Fujio H, et al. Significance of the level of monocyte chemoattractant protein-1 in human atherosclerosis. Circ J. 2004; 68: 671–6. 44. Sartipy P, Losktoff DJ. Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci U S A. 2003;100:7265–70. 45. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest. 2003;112:1796–808.


LITERATURE CITED

587