Biochemical and Molecular Roles of Nutrients

Plasma Lipids and Fatty Acid Synthase Activity Are Regulated by ShortChain Fructo-Oligosaccharides in Sucrose-Fed Insulin-Resistant Rats1,2 Nasrin Agheli,3 Morvarid Kabir, Sibilla Berni-Canani, Estelle Petitjean, Abdelghani Boussairi,* Jing Luo, Francis Bornet,† Gerard Slama4 and Salwa W. Rizkalla Department of Diabetes and INSERM U341, *Department of Biochemistry and Toxicology, Hoˆtel-Dieu Hospital, 75004 Paris, France and †Eridania Beghin-Say, Nutrition and Health Service, Vilvoorde, Belgium ABSTRACT The aim of this study was to evaluate the chronic effects of a short-chain fructo-oligosaccharide (FOS)-containing diet on plasma lipids and the activity of fatty acid synthase (FAS) in insulin-resistant rats. Normal male Sprague-Dawley rats, 5 wk old, were randomly assigned to two groups and fed either a sucrose-rich diet (S, 575 g sucrose /kg diet and 140 g lipids/kg diet) or a sucrose-rich diet supplemented with 10 g/100 g shortchain fructo-oligosaccharides (S/FOS). A third reference group (R) was fed a standard nonpurified diet (g/kg, 575 g starch, 50 g fat). After 3 wk the sucrose-fed rats (compared with the R group) were characterized by the following: 1) higher insulin responses after a glucose challenge (P õ 0.05); 2) heavier liver (P õ 0.001) and retroperitoneal adipose tissue (P õ 0.01); 3) hypertriglyceridemia (P õ 0.0001) and higher plasma free fatty acids (P õ 0.0001); and 4) higher fatty acid synthase activity in the liver but a low activity in the adipose tissue (P õ 0.001). The addition of FOS to the diet resulted in 11% lower liver weight than in the S group (P õ 0.05) and tended to result in lower adipose tissue weight (P õ 0.11). Plasma triglycerides and plasma free fatty acids were lower in S/FOSthan in S-fed rats (P õ 0.05). Chylomicrons / VLDL, and intermediate density lipoprotein (IDL) concentrations did not differ between groups, nor was plasma cholesterol influenced by diet. Hepatic FAS activity was lower in S/ FOS-fed rats than in the S-fed rats (P õ 0.05). In adipose tissue, however, this activity tended to be greater in rats fed S/FOS than in rats fed the S diet (P õ 0.07). In conclusion, in a rat model of diet-induced (57.5% sucrose and 14% lipids) insulin resistance, the addition of short-chain FOS prevented some lipid disorders, lowered fatty acid synthase activity in the liver and tended to raise this activity in the adipose tissue. Short-chain FOS, in addition to being a nondigestible sweetener with good bulking capacity, might be useful in the treatment of insulin resistance and hyperlipidemia. J. Nutr. 128: 1283–1288, 1998. KEY WORDS: • short-chain fructo-oligosaccharides • fatty acid synthase • sucrose • plasma lipids • rats

Lipid abnormalities associated with diabetes and/or insulinresistant states should be viewed as being of equal if not of greater importance than hyperglycemia itself. Insulin resistance enhances very low density lipoprotein (VLDL)5 secretion and might produce hypertriglyceridemia (Arrol et al. 1994), which is the most common lipid disorder in humans (Grundy 1984). This disorder is commonly related to abnormalities in lipoprotein composition, which in turn are atherogenic (Bierman 1992).

Treatment of diabetes by low energy diets, oral hypoglycemic agents and insulin therapy frequently is not sufficient for perfect control of lipid metabolism. A high fiber diet has been reported to reduce elevated plasma triglyceride (TG) levels in humans (Anderson 1980). Nondigestible oligosaccharides are a new category of low energy sweeteners that share many properties with fermentable dietary fibers. The short-chain fructo-oligosaccharides (FOS) belong to this class of food ingredients. They are not hydrolyzed by digestive enzymes such as intestinal disaccharidase and the pancreatic amylase (Tokunga et al. 1986). In the colon, FOS are fermented mainly by Bifidobacteria (Wang and Gibson 1993) and produce mainly lactate and short-chain fatty acids such as acetate, propionate and butyrate, which are utilized by the colonocytes (butyrate) or reach the liver by the portal vein (Dankert et al. 1981). Some investigators found that FOS decreased plasma lipids in normal rats (Delzenne et al. 1993, Fiodalsio et al. 1995, Oku et al. 1984). In healthy subjects, we found also that FOS decreased basal hepatic glucose production (Luo et al. 1996). FOS might influence glucose and lipid metabolism through the action of its end products, the short-chain fatty acids. This

1 Supported by grants from Pierre and Marie Curie University and the National Institute of Health and Medical Research (INSERM). 2 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 USC section 1734 solely to indicate this fact. 3 Current address: National Nutrition and Food Technology Research Institute, 19666, Tehran, Iran. 4 To whom correspondence should be addressed. 5 Abbreviations used: F, fructose; FAS, fatty acid synthase; FOS, fructo-oligosaccharides; G, glucose; HDL, high density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; R, reference group; S, sucrose; S/FOS, sucrose-rich diet supplemented with 10 g/100 g fructo-oligosaccharides; TG, triglycerides; VLDL, very low density lipoprotein.

0022-3166/98 $3.00 q 1998 American Society for Nutritional Sciences. Manuscript received 16 October 1997. Initial review completed 15 December 1997. Revision accepted 7 April 1998.

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concept was strengthened in part by a recent study in our laboratory (Boillot et al. 1995) in which 3 wk of feeding rats a diet rich in propionate, one of the short-chain fatty acids, decreased plasma glucose concentrations. The lipogenic capacities of both liver and adipose tissue are essentially controlled by fatty acid synthase (FAS), a multiunit enzyme complex that catalyzes the synthesis of long-chain fatty acids from acetyl CoA and malonyl CoA (Wakil et al. 1983). Nutritional and hormonal factors regulate lipogenic enzyme activities and gene expression (Iritani 1992, Prip-Buus et al. 1995, Semenkovich et al. 1993). Carbohydrate feeding was found to increase the activity of these enzymes in the liver (Prip-Buus et al. 1995). However, the effect on FAS of an insoluble fermentable carbohydrate has not yet been studied. Therefore, in this work, we studied the influence of dietary FOS on the metabolism of plasma lipids and lipoproteins and on plasma short-chain fatty acid concentrations in insulinresistant rats. We also evaluated FAS activity in liver and adipose tissue. We chose a rat model of diet-induced insulin resistance, i.e., sucrose-fed rats. This is a different model than other forms of insulin resistance because hypertriglyceridemia and hyperinsulinemia are observed soon after initiating sucrose, but insulin resistance does not develop for several days. In the literature, studies using different models of sucrose-fed rats demonstrated either an impairment of insulin action (Boyd et al. 1990, Klimes et al. 1994, Storlien 1988) or even an increase in insulin sensitivity of peripheral tissue (Kergoat et al. 1987). This apparent discrepancy may be due to differences in the relative proportions of carbohydrates, fat and protein in the diets. It depends also on whether Wistar or Sprague-Dawley rats are studied. In this study, we used Sprague-Dawley rats fed a diet containing 575 g sucrose/kg diet and 140 g lipid/kg diet. Previously, we showed that 3 wk of sucrose feeding decreased glucose transport activity, and GLUT 4 proteins and gene expression in adipocytes (Fluteau-Nadler et al. 1996; Rizkalla, S. W., Berni-Canani, S., Kabir, M., Slama, G., unpublished results). This model is characterized by whole-body insulin resistance measured by an intraperitoneal glucose tolerance test (Fluteau-Nadler et al. 1996) as well as peripheral insulin resistance. MATERIALS AND METHODS Diets. The powdered diets (modified 210 diet) used were purchased from the UAR (Villemoisson-sur-Orge, France). The two experimental diets [sucrose (S) and S/FOS] were prepared from a basal sucrose diet as described in Table 1. The best way to prepare the two experimental diets with nearly equal energy values was to add 50 g sucrose to 950 g of the basal sucrose diet for the experimental sucrose diet (resulting energy value, 18.3 kJ/g) and to add 100 g FOS to 900 g of the basal sucrose diet for the S/FOS diet (energy value, 17.9 kJ/ g, assuming that the FOS energy value is 8.5 kJ/g). The FOS were supplied as ACTILIGHTR (which is a mixture of 2-, 3- and 4-linked fructose (F) moieties bound to a glucose (G) molecule as follows: GF2, 45%; GF3, 45%; GF4, 10%; 950 P, Be´ghin-Say, Neuilly sur Seine, France). This method of diet formulation resulted in slight variations in the composition of the two experimental diets. The S experimental diet contained (g/kg diet) 596 g sucrose, 133 g lipids and 200 g proteins, whereas the S/FOS diet contained 517 g sucrose, 100 g FOS, 127 g lipids and 189 g protein. Because the slight differences in the amount of sucrose in the experimental S and the S/FOS diets might induce different glycemic responses, we evaluated the acute effects of three oral carbohydrate loads (the quantity of carbohydrate in the experimental diets) on postprandial glycemia.

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TABLE 1 Composition of basal sucrose diet Component

Amount g/kg

Sucrose Vegetable and animal fat1 Casein Mineral mix2 Vitamin mix3 Cellulose

575 140 210 45 10 20

1 Supplied in g/kg diet: corn oil 28, peanut oil 28 and lard 84. 2 Supplied in g/kg (or mg/kg as indicated) diet: phosphorus, 3.425;

calcium, 4.5; potassium, 2.7; sodium, 1.8; magnesium, 0.45; manganeac, 0.04; iron, 0.14; copper, 5.63 mg; zinc, 0.02; cobalt, 0.04 mg; and iodine, 0.22 mg. 3 As described previously (Luo et al. 1992).

Acute study Test loads. Three carbohydrate loads were administered to normal rats; they consisted of 2 g sucrose (representing the quantity of sucrose in the experimental S diet), 1.7 g sucrose (quantity of sucrose alone in the S/FOS diet) or 1.7 g sucrose and 0.3 g FOS (quantities of sucrose / FOS in the S/FOS diet). Nondiabetic male SpragueDawley rats (body weight, 200 g; Elevage Janvier, Le Genest St-Isle, France) were used. Rats were placed in individual polypropylene cages for sedentary animals under a 12-h light:dark cycle at 247C. Rats were trained for 2 wk as follows: each morning a small amount of powdered commercial diet containing (g/kg diet) 575 g carbohydrate, 55 g lipids and 225 g protein (semipurified diet #210, UAR) was introduced into the cage and removed after 15 min. Rats then had free access to diet from 1400 to 2000 h and were food deprived overnight. By the end of the training period, rats were accustomed to eating a given amount of food within 15 min. After 2 wks, the three carbohydrate oral load tests were performed at 800 h on rats that had been food deprived overnight. Rats were randomly assigned to three groups, each receiving one of three test loads. Pentobarbital anesthesia was administered only to rats that consumed all of the offered food. Blood samples were taken from the tip of the tail after 30, 60, 90, 120, 150 and 180 min to measure plasma glucose levels. Fasting plasma glucose levels (0 min) were determined on a separate day under similar conditions. Approval for the use of laboratory animals was given by the French Ministry of Agriculture. The protocol complied with the NIH guidelines (NRC 1985).

Chronic study Animals and diets. Male Sprague-Dawley rats (Elevage Janvier), 5 wk old, were housed in grill-bottomed metal wire cages. The rats were randomly assigned to two groups. Each group received for 3 wk one of the two experimental diets: 1) the experimental S diet (n Å 12), or 2) the S/FOS diet (n Å 7). A third group of 12 rats was used as a reference (R). This group was fed a nonpurified standard powdered diet (no. 210, UAR) containing (g/kg diet) 575 g starch, 50 g lipids and 235 g casein. The experiment was designed to include seven rats per group. However, to further characterize the sucrosefed insulin-resistant rats, the number of rats was increased to 12 in the reference and the sucrose-fed groups to confirm high plasma glucose, insulin and lipid levels as well as insulin resistance and high hepatic FAS activity. The additional rats were studied concurrently with the initial seven rats and were used to evaluate other parameters in the sucrose-fed rats. Because there was no significant difference (in body weight or food intake) between the initial seven rats per group and the additional five rats for the S and the R groups, the 12 rats were grouped together. Food intake was adjusted to equalize energy intake in the two

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experimental groups (S and S/FOS) by pair-feeding; body weight was measured weekly. The pair-feeding was performed as follows. Food intake was measured every day; at the end of wk 1, the FOS-fed rats consumed less energy than the S-fed rats. From the beginning of wk 2 to the end of wk 3, the S-fed rats were offered each day the amount of diet that equaled the energy intake of the FOS-fed rats on the previous day. At the end of the experimental period, the rats were decapitated in the fed state. Blood was collected and plasma was separated and stored at 0207C for glucose, insulin and lipid measurements. Plasma lipoproteins were measured immediately using fresh plasma samples. Samples of the liver and the retroperitoneal adipose tissue were removed and homogenized immediately to determine FAS activity. Separation of plasma lipoproteins. Plasma (2 mL) was adjusted to 1.21 kg/L with solid KBr and ultracentrifuged on a density gradient (rotor SW1 Beckman, 120,000 1 g, 24 h at 157C) (Mathe´ et al. 1991). The different lipoproteins were separated as follows: chylomicrons / VLDL (d õ 1.006 kg/L), intermediate density lipoprotein (IDL) (d Å 1.006–1.019 kg/L), low density lipoprotein (LDL; d Å 1.019– 1.063 kg/L), high density lipoprotein (HDL1) / HDL2 (d Å 1.063– 1.12 kg/L) and HDL3 (d Å 1.12–1.21 kg/L). Determination of plasma lipids, glucose and insulin. TG, phospholipids and total and free cholesterol were assayed in total plasma and in each lipoprotein fraction by using the enzymatic Pap 1000 kit for assay of TG (Bio-Merieux, Marcy, l’Etoile, France), enzymatic Pap 150 kit for assay of phospholipids (Bio-Merieux) and Chod Pap kit (Boehringer Mannheim, Meylan, France) for assay of total and free cholesterol. Esterified cholesterol was calculated as the difference between total and free cholesterol, and cholesterol ester as esterified cholesterol value 1 1.67. Total free fatty acids were measured in plasma by using the enzymatic NEFA Kit (Biolyon, Dardilly, France). Total proteins were determined in each lipoprotein fraction (BCA Protein Assay Reagent A, Pierce, IL). Plasma glucose was determined by using a glucose oxidase method (Glucose Analyzer 2, Beckman, Fullerton, CA); plasma insulin was also measured (Insulin RIA kits, Pasteur, Paris, France). Determination of short-chain fatty acid concentrations. Concentrations of acetate, butyrate and propionate in plasma were determined by gas chromatography (Boussairi et al. 1995). Determination of FAS activity. Liver and adipose tissue samples were immediately homogenized in ice-cold 0.25 mol/L sucrose, 1 mmol/L dithiothreitol and 1 mmol/L EDTA, pH 7.4; cytosolic fractions were obtained by centrifugation at 100,000 x g for 1 h at 47C. The FAS activity in liver and retroperitoneal adipose tissue was measured immediately in duplicate by measuring malonyl Co A–dependent oxidation of NADPH at 377C (Halestrap and Denton 1973). One unit of enzyme activity represents 1 mmol of NADPH oxidized per minute at 377C. Protein concentrations were measured by using bovine serum albumin as standard (Bradford 1976).The results were expressed both as mU/g of adipose tissue and as mU/mg protein.

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FIGURE 2 Areas under the glycemic and insulin response curves (AUC) after an intraperitoneal glucose challenge in rats consuming a sucrose (S) or a nonpurified reference (R) diet. Values are means { SEM, n Å 7. *Difference between R and S, P õ 0.05.

Intraperitoneal glucose tolerance test. To characterize the sucrose-fed insulin-resistant rats, an intraperitoneal glucose tolerance test was performed in rats consuming the S diet for 3 wk compared with the R group (n Å 7 rats/group). On the morning of the experiment, food was removed at 0800 h. At 1400 h, rats were anesthetized with pentobarbital. Blood samples were taken from the tip of the tail (time 0). A glucose challenge (0.2 g/100 g body weight) was given intraperitoneally, and other blood samples were taken at 15 and 50 min. Statistical analysis. In the acute study, plasma glucose areas under the curves after the three test loads were compared by one-way AVOVA. In the chronic study, unpaired Student’s t test was used to compare rats fed sucrose and the reference group. Another comparison was made between the two experimental groups (S and S/FOS). All analyses were conducted with Statview 512/ Software program (Brain Power, Calabasas, CA). Data are expressed as means { SEM. Differences were considered significant when P õ 0.05. FIGURE 1 Acute glycemic responses in rats after a test meal containing 1.7 g sucrose, 2 g sucrose or a mixture of 1.7 g sucrose and 0.3 g fructo-oligosaccharides (FOS). Values are means { SEM, n Å 7.

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RESULTS Test loads. Plasma glucose responses after the three test loads did not differ (Fig. 1).

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TABLE 2 Characteristics of rats fed a diet containing sucrose (S) or fructo-oligosaccharide (S/FOS) for 3 wk1 Body weight

Diet

Retroperitoneal adipose tissue weight

Liver weight

Plasma glucose

Plasma insulin

mmol/L

pmol/L

7.8 { 0.2 7.1 { 0.2*

534 { 78 468 { 36

g 318 { 6 301 { 5

S S/FOS

13.5 { 0.4 12.0 { 0.4*

2.8 { 0.3 2.1 { 0.2

1 Values are means { SEM, n Å 7. * Different than S, P õ 0.05.

Characteristics of the sucrose-fed compared with reference rats. Plasma glucose (7.7 { 0.2 vs. 6.0 { 0.2 mmol/L, P õ 0.0001) and insulin (492 { 30 vs. 186 { 12 pmol/L, P õ 0.001) in food-deprived S-fed rats were higher than in the R group. After an intraperitoneal glucose charge, the areas under the insulin response curves were higher in the S-fed rats than in the R rats (P õ 0.05), whereas those under the glucose response curves did not differ (Fig. 2). The S-fed rats had greater liver (P õ 0.001) and retroperitoneal adipose tissue weights (P õ 0.01) than R rats. S-fed rats had also higher plasma TG (3.42 { 0.18 vs. 1.82 { 0.18 mmol/L, P õ 0.0001) and free fatty acid (0.97 { 0.06 mmol/L, P õ 0.05) concentrations compared with the R group. Moreover, FAS activity (n Å 12) was higher in liver (93 { 9 vs. 53 { 6 mU/mg protein, P õ 0.001) and lower in adipose tissue (497 { 95 vs. 1422 { 170 mU/mg protein, P õ 0.001) of S-fed rats than in the R group. Chronic study. Energy intake in the pair-fed rats consuming the S and S/FOS diets was 391 kJ/d at the end of the experimental period, which resulted in similar body weights (Table 2). Liver weight was 11% lower (P õ 0.05) and adipose tissue weight tended to be lower (25%, P õ 0.11) in S/FOScompared with S-fed rats. Plasma free fatty acids (P õ 0.05) and plasma TG (P õ 0.05) were lower in S/FOS- than Sfed rats. Plasma phospholipids, total and free cholesterol and cholesterol esters were not influenced by the S/FOS diet (Table 3). There were no significant differences in the plasma concentrations of different lipoproteins after 3 wk of consuming either S or S/FOS. The levels of LDL, HDL1 / HDL2 and HDL3 did not differ. There was no difference in the ratio between cholesterol esters and TG, phospholipids or proteins. Neither TG/total lipoprotein nor core lipid/surface in the different lipoproteins differed between groups (data not shown). Plasma glucose was lower (P õ 0.05) in rats consuming the S/FOS diet than in those consuming the S diet (Table 2). Plasma insulin did not differ between the two groups. Rats fed

the S/FOS diet tended to have greater acetate concentrations (19%) than rats fed S (P Å 0.15). Propionate and butyrate were not affected by diet (Table 4). FAS activity in the liver was lower (P õ 0.05) in S/FOS- than in the S-fed rats. In adipose tissue, this activity tended to be greater (P Å 0.07) in the S/FOS- than in the S-fed rats (Table 5). DISCUSSION Several biological and epidemiologic studies have implicated lipid disorders and diabetes in the etiology of atherosclerosis and cardiovascular diseases (Carlson and Bottinger 1985, Stamler et al. 1993). In hyperlipedemic patients, a high fiber diet lowers plasma lipids (Story 1985, Ullrich 1987). In diabetic subjects, amelioration of high plasma lipids and glucose control were reported after a diet rich in fibers (Riccardi et al. 1984, Rivellese 1980). In this study, the dietary model (57.5% sucrose / 14% lipids) of insulin-resistant rats was characterized by hypertriglyceridemia due to increased VLDL-TG, and high free fatty acid levels. High plasma TG and VLDL-TG are major markers of the metabolic syndrome in which glucose intolerance and hyperinsulinemia increase the risk of coronary and cardiovascular diseases (Grundy and Vega 1992, Reaven 1988, Welin et al. 1991). It has been argued that high circulating free fatty acids, caused by an increase in hepatic TG synthesis, promote hyperglycemia and contribute to insulin resistance in different organs and tissues (Randle 1963). The latter hypothesis is supported by two recent studies in which 3 wk of consuming a sucrose diet resulted in decreased glucose transport activity, and GLUT 4 proteins and mRNA in adipocytes of Sprague-Dawley rats (Fluteau-Nadler et al 1996, and our unpublished data, Rizkalla, S. W., Berni-Canani, S., Kabir, M., Slama, G.). The addition of 10% FOS to the experimental sucrose diet lowered both plasma free fatty acids and plasma TG. The improvement of some lipid disorders by FOS might ameliorate hyperglycemia and insulin resistance. The consumption of FOS for 3 wk, however, had no effect on either

TABLE 3 Plasma lipids in rats fed a diet containing sucrose (S) or fructo-oligosaccharide (S/FOS) for 3 wk1 TG2

Diet

TC

FC

CE

PL

FFA

2.50 { 0.23 2.50 { 0.20

2.80 { 0.12 2.70 { 0.10

0.97 { 0.06 0.75 { 0.07*

mmol/L 3.00 { 0.16 2.10 { 0.21*

S S/FOS

2.59 { 0.15 2.66 { 0.10

1.00 { 0.07 0.95 { 0.10

1 Values are means { SEM, n Å 7. * Different than S, P õ 0.05. 2 TG, triglycerides; TC, total cholesterol; FC, free cholesterol; CE, cholesterol esters (esterified cholesterol / fatty acids); PL, phospholipids; FFA,

free fatty acids.

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TABLE 4 Plasma short-chain fatty acid concentrations in rats fed a diet containing sucrose (S) or fructo-oligosaccharide (S/FOS) for 3 wk1 Diet

Acetate

Butyrate

Propionate

mmol/L S S/FOS

911 { 36 1115 { 40

11.8 { 0.2 11.9 { 0.6

24.7 { 1.0 24.1 { 0.8

1 Values are means { SEM, n Å 7.

plasma cholesterol or plasma phospholipids. In normal rats, Fiordaliso et al. (1995) showed that FOS could reduce total cholesterol and phospholipid levels when consumed for longer periods (16 wk) than the duration of this study (3 wk). The ratios between the different constituents of VLDL, however, did not differ in the S- and S/FOS-fed rats. This is consistent with the concept that VLDL composition could not be changed by moderate variations in plasma TG (Agheli et al. 1992). Short-chain FOS, in this study, modified not only some plasma lipids but also FAS activity, one of the major enzymes implicated in lipogenesis (Wakil et al. 1983). In liver, the activity of this enzyme was increased by sucrose feeding and normalized by FOS supplementation. A causal relationship seemed to exist between hepatic FAS activity and plasma TG concentrations. Both were increased by sucrose feeding and lowered by FOS supplementation. These results are consistent with those of Goodridge (1987) who demonstrated in rats that a high carbohydrate diet increases FAS activity and lipogenesis in the liver. In adipose tissue, however, this regulation was the opposite, i.e., there was a decrease with sucrose feeding and a tendency to increase with the addition of FOS. These results were confirmed by an increase in the quantity of FAS mRNA in the adipose tissue of S/FOS-fed rats compared with S-fed rats (a preliminary result with two Northern blots, Ageli, N., Rizkalla, S. W., Boillot, J., Slama, G.). The regulation found in adipose tissue might be simply a feedback mechanism or a compensatory response to the variations in plasma lipid levels. Shillabeer et al. (1992) showed that adipose tissue FAS mRNA levels demonstrated near reciprocity with hepatic levels. They suggested that the regulation of FAS mRNA levels in adipose tissue may depend on substrate availability rather than on plasma hormonal concentrations, and thus may differ from hepatic regulation. These authors suggested that adipose tissue FAS levels are constitutively high unless suppressed by exogenous substrate availability. This reciprocal regulation of the lipogenic capacity of liver and adipose tissue was found

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also in rats fed fructose; lipogenesis was low in adipose tissue but increased in the liver (Chevalier et al. 1972). The mechanism of action of such a nondigestible, but fermentable polysaccharide on lipid metabolism is not known. First, the fermentation end products, the short-chain fatty acids, could be involved. Plasma acetate tended to be greater in rats fed the S/FOS diet. There were no differences in propionate or butyrate levels. These short-chain fatty acids reach the portal blood and enter into the liver (Dankert et al. 1981). This step clears the propionate and butyrate presented but only 50–75% of the acetate. The uncleared acetate then enters the peripheral circulation and reaches the peripheral tissues, mainly the muscles (Roberfroid et al. 1993). In this study, we found that plasma short-chain fatty acids were composed of 97% acetate, 2% propionate and 1% butyrate. In the cecum of rats fed a diet containing FOS, Roberfroid et al (1993) found that the short-chain fatty acids were comprised of 83% acetate, 13% propionate and 4% butyrate. The difference between the amounts of propionate and butyrate in the plasma and in the cecum suggest that they might be metabolized elsewhere, perhaps in the liver or other organs, before reaching the plasma. Second, the slight change in the amount of sucrose in the experimental groups (S and S/FOS) might have a role in the observed results. In this study, a slight variation in the sucrose concentration of the diet had no effect on postprandial glycemic responses. Thus, the above hypothesis is unlikely. Finally, differences in plasma glucose and insulin levels at the end of the experimental period might play an important role in the observed results. Three weeks of FOS supplementation decreased plasma glucose levels of rats that were not food deprived. Furthermore, it was clearly demonstrated in vitro (Semenkovich et al. 1993) that in Hep G2 cells, the addition of glucose increased three- to fivefold the FAS activity and mRNA mass. Similarly, Prip-Buus et al. (1995) showed that the glucose metabolism is directly involved in the regulation of FAS gene expression in the liver, and that the effect of hormones is due in part to their capacity to induce glucose phosphorylation. In vivo (Iritani et al. 1992), a high carbohydrate diet initiated the transcription and also increased the mRNA concentrations of lipogenic enzymes. Thus, decreased plasma glucose in association with the trend toward increased plasma acetate after FOS supplementation might be implicated in the normalization of FAS activity, especially in the liver. We conclude that in sucrose-fed insulin-resistant rats, FOS supplementation for 3 wk lowered both plasma TG and plasma free fatty acids. It normalized FAS activity in the liver and tended to increase this activity in the adipose tissue. Fructooligosaccharides may be of interest from a preventive and therapeutic point of view for the pathogenesis related to hyperlipoproteinemia and might be used as a low energy sweetener in diabetic or insulin-resistant subjects.

TABLE 5 Fatty acid synthase activity in the liver and retroperitoneal adipose tissue of rats fed a diet containing sucrose (S) or supplemented with fructo-oligosaccharide (S/FOS) diet for 3 wk1 Diet

Liver

Retroperitoneal adipose tissue

mU/mg protein

mU/g tissue

mU/mg protein

mU/g tissue

93 { 9 63 { 8*

2181 { 215 1394 { 109*

497 { 95a 835 { 143

883 { 188 1282 { 146

S S/FOS

1 Values are means { SEM, n Å 7, except for the hepatic FAS activity of sucrose-fed rats, n Å 12. * Different from S, P õ 0.05.

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ACKNOWLEGMENTS We thank A. Quignard-Boulange´ (INSERM U465), F. Guyon (Biochemistry and Toxicology Department) and B. Guy Grand (Nutrition Department) for the opportunity to perform measurements in their laboratories.

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