Nutr. Metabol. 18: 283 293 (1975)
An Investigation in Rats, into the Metabolic Consequences of Early Ingestion of Dietary Carbohydrates I. Macdonald, T. Rebella and Anne Keyser' Department o f Physiology, G u y ’s Hospital Medical School, London
Key Words. Triglyceride • Cholesterol • Liver-fatty acids - Serum-fatty acids • Adiposefatty acids • Glucose - Sucrose • Fructose • Rats
Abstract. Weanling rats were given diets containing a high proportion o f either glucose, sucrose or fructose until they reached 200 g weight, when they were either transferred to a control (chow) diet for 4 weeks or killed. The results were compared with rats receiving a control diet throughout. The triglyceride (TG ) and cholesterol levels in liver and in serum had returned to values similar to the control animals 4 weeks after being given the control diet. The fatty acid profile o f liver TG and adipose lipid tended to take longer to return to normal.
In some people normal intakes o f carbohydrate may aggravate disorders of lipid metabolism, and moreover, at least in the short term, intakes o f sucrose appear to emphasise pre-existing abnormalities o f the serum lipids (7). The aim o f the present experiments was to determine whether or not dif ferent dietary carbohydrates, taken by young animals, would cause metabolic changes which persisted or first appeared after sexual maturity. This question is clinically relevant since some children take large amounts o f sucrose (glucose and fructose) in confectionery and beverages.
1 Wc arc grateful to Beccham Products Ltd. for a research grant which enabled these studies to be carried out. We should also like to thank Dr. A. MacRae for his help.
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Received: March 17, 1975 ; accepted: May 12, 1975.
284
Macdonald/Rebello/Key ser Table I. Composition o f the experimental diets Parts by weight Glucose, sucrose or fructose Calcium caseinate (//-Methionine Dried yeast Salt mixture (see reference 2) Methyl cellulose Sunflower seed oil
70 21 0.5 4 3.5 3 1
12,000 III vitamin A . 1,200 IU vitamin D, 30 mg vitamin C/kg food
Methods Three groups o f 10 weanling male rats o f the Wistar strain were given a diet containing either glucose, sucrose or fructose as the main carbohydrate. The composition o f the diets is given in table 1. The animals were weighed weekly, and the food (presented ad libitum) taken from the feeding pot was estimated by weighing the pots each day. When each animal reached 200 g in body weight it was either killed (immediate group = I) or placed on a control (chow) diet for 4 weeks before being killed (delayed group = D). Three groups o f 6 animals were given a control (chow) diet throughout and the amount o f food eaten by this group was not measured. In the morning, 7 h prior to death, the food was removed from the cage and 5 h later, under light ether anaesthesia, each animal was given, by stomach tube, 5 uCi o f uniformally labelled 14C glucose, sucrose or fructose (with 0.5 g 'cold’ carbohydrate in 2 ml water). The carbohydrate was the same as that- which formed the major part o f the diet. Each o f the three radioactive carbohydrates was given to a group o f three rats which had been on a control (chow) diet. At post-mortem the liver was removed, blotted and weighed, and serum was obtained, as was a small piece o f peri-renal fat.
cation o f the Eolch method. The portion o f peri-renal fat was mixed with chloroform and. after filtration, the solvent was evaporated. Triglyceride and cholesterol estimation. Determination o f the concentration o f triglyc eride (TG) in the liver and in the serum was carried out using a modified semi-automated method (4). The radio-activity in the TG fraction was measured after separation by thin layer chromatography (10) from a known amount o f total lipid. With adipose tissue, a known weight of lipid was counted without prior separation, it being assumed that 90 99 % of the lipid extracted was TG (6). An automated method (8) was used for the cholesterol estimation. Fatty acid composition. Liver T G separated by thin-layer chromatography, was saponi fied with alcoholic KO H and the fatty acids were extracted and methylated. Analysis was
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A nalytical Methods Liver, serum and adipose tissue. After homogenising the liver the lipid was extracted by the method o f Folch et al. (5). The serum lipid was extracted using the Carlson (3) modifi
Consequences o f Dietary Carbohydrates in Rats
285
carried out by gas-liquid chromatography (G LC ) using a flame ionisation detector. The fatty acid profile o f the adipose tissue was also determined by G L C . Radio-activity. After the TG band on the thin-layer chromatograph plate (identified by iodine staining) had been scraped o ff, it was mixed with approximately 0.5 g o f ‘Cab-O-Sil’ (Koch-Light) and 6 ml o f scintillant. Counting was carried out in a Nuclear Enterprises 8102 with 93 % efficiency. Statistical comparisons were made using Student’s t test.
Results F ood Intake and Weight Gain It took 28 days for the first animals to reach 200 g body weight, and only the data up to this time are considered for the estimation of food intake and weight gain (The longest time to reach 200 g was 61 days). The mean food consumption (excluding controls) was greatest for those with glucose as the dietary carbohydrate and least for those eating sucrose (fig. 1). The mean intake of 13.2 g/day/animal (SE ± 0.91) on the glucose diet was significantly greater than the mean sucrose intake (9.5 g/day/animal, SE ± 0.61; p = 0.005-0.001). Despite this there was no significant difference between the groups in mean body weight at 28 days (fig. 1). No group had diarrhoea and it is not possible to state the reason for the similar weight gain despite the difference in food intake.
T riglyceride Total amount (table II). The amount of liver TG was significantly greater in the fructose group, compared with each o f the other three groups at the end o f the period on a high carbohydrate diet. In the group o f animals on the fructose diet there was a significant fall in total TG 4 weeks after returning to the control diet. Specific activity, dpm/mg TG (table II). The fructose-I group had a signifi cantly greater TG-specific activity than either the glucose-I or sucrose-I groups. There was a reduction in the specific activity after the animals had returned to a control diet, a reduction which was significant in the sucrose and fructose groups (p < 0.025). Am ount incorporated, dpm/total TG (fig. 2). The amount o f radio-activity
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Liver Wet Weight and Cholesterol (table II) The wet weight was significantly greater in the animals which were returned to a control diet for 4 weeks before being killed. There was a tendency for the total* amount o f cholesterol in the liver to be greater in the D group. The amount o f cholesterol in the fructose-I group was significantly greater than in the compa rable control group.
286
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Fig. 1. Mean body weight (g) o f the rats during the first 28 days on the diets, together with the mean food intake (and SE). Values in parentheses indicate the number o f animals.
d p m / t o t a l TG
X 1,000 120
J
100
80
60
40
T
20
T
_x
—
G
s 1
....... F
± 3 G
S
F
0
Fig. 2. Mean amount (with SE) o f 14C incorporated in liver T G . G = Glucose; S = sucrose; F = fructose; 1= immediate group; D = delayed g ro u p .--------= Mean o f three controls.
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0
Consequences o f Dietary Carbohydrates in Rats
287
Table II. Mean values for the amount and specific activity o f T O , amount o f choles terol and liver weight in animals killed while on the high carbohydrate diet (immediate) and 4 weeks after returning to a chow diet (delayed) Diet
T G , mg
X
SE
39 30 45 87*
6.4 5.3 4.5
54 45 44 43
TG-specific activity, dpm/mg
Choles terol, mg
Liver weight, g
X
SE
X
SE
X
SE
1.2
169.6 62.5 147.9
13* 16 15* 18*
1 .1
632 455* 1,263*
9.9 7.7 8.5 9.4
0.39 0.47 0.59 o.so
7.1 8.3 5.6 3.9
20
1.4
270 246 270
115.3 46.0 106.2
17
1.6
22
1.6
23
1.7
11.9 10.5 11.1 12.4
0.65 0.71 0.43 0.71
Immediate animals Control Glucose Sucrose Fructose
1.4
1.0 1.0
Delayed animals Control Glucose Sucrose Fructose
* p < 0.025 when compared with delayed group.
Table III. Mean values o f the serum T G and cholesterol concentrations, together with the specific activity o f the T G , in rats killed while on a high carbohydrate diet (im mediate) and 4 weeks after returning to a control diet (delayed) 1 --------------------------------
Diet
TG-specific activity, dpm/mg
T G . mg
Cholesterol, mg/100 ml
X
SE
X
SE
X
SE
91 64 96 117
8.2 12.1 9.7 27.3
456* 565 2.033*
77.6 110.1 642.2
58 63 63 79
2.9 7.6 3.9 8.9
116 97 95 141
15.5 18.0 12.1 23.2
238 368 572
9.0 17.8 143.6
67 65 58 60
2.6 6.4 4.3 3.3
Immediate animals Control Glucose Sucrose Fructose
Control Glucose Sucrose Fructose
* p < 0.025 when compared with delayed group.
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Delayed animals
288
Macdonald /R ebello/Keyser
in the liver TG was significantly increased in the fructose-I and the sucrose-1 groups compared to the values 28 days after returning to a control diet, at which time the values were similar to control. The value o f the fructose-I group was significantly greater than that o f either the glucose-I or sucrose-I groups. Serum Cholesterol (table III) No significant change was found in the serum-cholesterol concentration within any group, nor was any difference found between the groups. Triglyceride (table III) Concentration. No significant difference was found between the I and D groups or within groups. Specific activity. The TG-specific activity was significantly lower after re turning to a normal diet in the glucose and fructose groups, and, although lower
« 001 >
I
0
Fig. 3. Mean amount o f radio-activity (with SE) in TG in 100 ml serum .------- = Mean o f three controls. Abbreviations as in figure 2.
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dpm
289
Consequences of Dietary Carbohydrates in Rats
in the sucrose group, this difference was not significant. 28 days after returning to the control diet, the TG-specific activity was significantly less in the glucose compared to the sucrose and fructose groups. Am ount incorporated, dpm/total TG/100 ml serum (fig. 3). There was a tendency for more l4C-carbohydrate to be incorporated into serum TG at the end o f the carbohydrate-consuming period than after the return to the control diet, but this only achieved statistical significance in the fructose group. The fructose-1 group incorporated significantly more carbohydrate into the serum TG than the glucose-I or sucrose-I groups. Adipose Tissue (fig. 4) The specific activity o f the adipose tissue was considerably greater at the end of the period on high carbohydrate diets than 4 weeks later, after the return to the control diet. Though the specific activity was greater with glucose than with sucrose and fructose, these differences were not significant.
dpm/mg
60
Fig. 4. Mean specific activity (with SE) o f lipid from peri-renal adipose tissue.------- = Mean o f three controls. Abbreviations as in figure 2.
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D
Macdonald ¡Rebello /Keyser
290
Fatty A cid Profile Liver TG (table IV) While on the high carbohydrate diet, the proportions o f 16:0, 16:1 and 18:1 acids were significantly greater, compared to those found in the control animals, whereas with 18:2 fatty acid the reverse was found. The proportions o f 16:0 and 18:1 fatty acid had not altered significantly 4 weeks later, after re-
Table IV. Mean percentage o f fatty acids in liver T G in rats killed while on a high carbo hydrate diet (immediate) and 4 weeks after returning to a control diet (delayed) Diet
14:0 X
16:0 SE
18:0
16:1
18:1
18:2
X
SE
X
SE
X
SE
X
SE
X
SE
0.16 0.34 0.10 0.30
33.5 36.4 42.0 39.9
0.57 0.53 3.17 1.92
6.6 10.0 13.2 12.2
0.28 0.38 0.42 0.54
2.5 2.5 2.6 2.2
0.08 0.40 0.16 0.22
27.9 38.5 37.4 41.9
0.66 2.48 1.23 2.52
27.7 11.3 2.0 1.6
0.92 0.84 0.32 0.24
0.15 0.07 0.45
37.6 38.8 34.6
0.90 0.74 2.70
9.2 7.6 8.8
0.96 0.49 0.59
2.3 3.9 3.6
0.34 0.89 0.43
31.7 34.3 40.6
1.27 1.22 2.50
17.6 13.9 10.2
1.84 0.77 1.99
Immediate animals Control Glucose Sucrose fructose
1.9 1.3 2.8 2.2
Delayed animals Glucose Sucrose Fructose
1.6 1.5 2.2
Table V. Mean percentage o f fatty acids in adipose tissue from rats killed while oni a high carbohydrate diet (immediate) and 4 weeks after returning to a control diet (delayed) Diet
14:0 X
16:1
16:0
18:0
18:1
18:2
X
SE
X
SE
X
SE
X
SE
X
SE
0.54 0.30 0.54 0.90
35.7 39.3 33.6 34.0
1.32 2.68 0.40 3.76
12.6 12.0 13.9 17.9
0.54 1.03 0.61 2.28
2.9 6.0 4.1 2.7
0.20 1.41 0.38 0.46
24.2 34.7 38.3 33.0
1.15 2.27 0.42 5.32
18.4 5.2 6.4 5.5
0.96 0.70 0.54 0.84
0.16 0.85 0.15 0.49
38.8 37.2 32.9 37.2
1.12 1.57 0.56 3.73
11.2 11.3 11.2 12.0
0.31 0.60 0.23 1.38
3.4 4.1 4.2 3.6
0.27 0.29 0.41 0.35
24.9 29.6 33.4 29.2
0.65 1.81 0.86 2.94
17.3 12.9 15.7 14.1
0.69 0.50 0.45 1.68
SE
Immediate animals Control Glucose Sucrose Fructose
6.2 2.8 3.7 6.9
Control Glucose Sucrose Fructose
4.4 4.9 2.6 3.9
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Delayed animals
Consequences o f Dietary Carbohydrates in Rats
291
turning to a control diet. The proportions of 16:1 and 18:2 fatty acids showed significantly greater deviation from the control in the sucrose-I and fructose-1 groups than in glucose-I group. Adipose Tissue (table V) At the end o f the period on the high carbohydrate diet, the levels o f 14:0 and 18:2 fatty acids were significantly lower in all the carbohydrate groups, compared to the control group, whereas with 18:1 fatty acid the reverse was found. At the end of 4 weeks on a control diet, the 18:2 fatty acid in the adipose tissue in all three carbohydrate groups had returned to levels not mark edly different from the control group. The changes in the fatty acid profile, found in the carbohydrate groups, all showed a tendency, after 4 weeks on a control diet, to return to the levels found in those rats which had consumed the control diet throughout.
The measurements made in these experiments do not support the view that an abnormal intake o f dietary carbohydrate by a young rat would result in permanent metabolic changes throughout the remainder of the animal’s life. The measurements made were of some of the end products o f dietary carbohydrate metabolism, and it could be reasoned that if the dramatic changes seen in the animals while on a high carbohydrate diet were no longer present after 4 weeks on a normal diet, then the changes in metabolic activity, which presumably were present during the period o f high carbohydrate intake, did not persist. In a similar, but not comparable study, Moser and Berdanier (9) placed weanling rats on high starch or sucrose diets and then switched the type o f carbohydrate given. They found differences in liver lipid content and enzyme activity between the carbohydrates, but the animals did not at any time return to a control diet. Unlike the experiments described here, their rats were killed after a 16-hour fast and were o f a strain known to be carbohydrate-sensitive. Also the plan o f their experiments was not such as to be able to determine whether the young animals on high carbohydrate diets had values different from control, and if they were different, whether the animals’ metabolism had the capability of returning to values found in control diets. Many o f the findings in the liver and serum relating to TG have been reported frequently in rats which have been on high carbohydrate diets. The increase in level o f TG is greater with dietary fructose than with glucose, the results from the high sucrose diet being in between. Also the radio-activity, following 14C carbohydrate, in the liver and serum TG is greater in those animals
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Discussion
Macdonald /R ebello/Keyser
292
which were on a high fructose diet than in those on a high glucose diet, with again sucrose in between. However, 4 weeks after the return to a control diet all measurements were very similar to those in the control group, with the excep tion o f the specific activity o f serum TG following 14C fructose. The changes in the fatty acid profile o f the liver and adipose tissue were relatively rapid while on the high carbohydrate diets with much slower return to control levels, when the control diet was substituted for the high carbohydrate diet. It is possible that the youthfulness of the animals was responsible for the rapid change in fatty acid profile o f both liver and adipose T G , because when the rats at the end o f their period on a high carbohydrate diet were given the control diet for 4 weeks, the changes in the fatty acids had not all returned to control levels. The drop in proportion o f 18:2 fatty acid in liver and adipose tissue TG was not expected, as each rat was given approximately 50 mg linoleic acid/day. Also it seemed that the drop in the proportion o f this fatty acid in liver was greater and the recovery less rapid in those rats with fructose as the dietary carbohydrate, when compared to the glucose fed animals. This would suggest that there is some link between the type o f dietary carbohydrate and the metab olism o f the ‘essential’ fatty acid, linoleic acid. Needs for linoleic acid are greatest when an animal is young, and it is possible that an increased intake of carbohydrate and especially fructose, could make greater demands on linoleic acid metabolism. Thus, the experiments described here indicate that in weanling rats no dra matic changes in lipid metabolism could be found after periods o f living on high carbohydrate containing diets, followed by 4 weeks on a standard laboratory diet. Whether a longer period on the experimental diet, or a more precise search for metabolic ‘hangovers’ would have revealed permanent differences could be the basis for further studies.
References 1 2 3
Ahrens, E .H .; Hirsch, S .; Oettle, K .; Farquhar, J.W ., and Stein, Y .: Carbohydrateinduced and fat-induced lipemia. Trans. Ass. Am . Physns 74: 134 146 (1961). Briggs, G.M. and Williams, M .A .: A new mineral mixture for experimental rats diet and evaluation o f other mixtures. Fed. Proc. Am . Socs exp. Biol. 22: 261 (1963). Carlson, L .A .: Determination o f serum triglycerides. J . Atheroscler. Res. 3: 334 - 336 (1963).
5 6
Eggstein, M. und Kreutz, F .H .: Eine neue Bestimmung der Neutralfette im Blutserum und Gewebe. Klin. Wschr. 44: 262 267 (1966). Folch, ] .; Lees, M ., and Sloan-Stanley, G .H .: A simple method for the isolation and purification o f total lipids from animal tissues. J . biol. Chem. 226: 497 509 (1957). Jeanrenaud, B.: Lipid components o f adipose tissue: in Adipose tissue. Handbook o f physiology, vol. 5, pp. 169
176(1965).
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Consequences of Dietary Carbohydrates in Rats 7
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Kaufrnann, N .A .; Poznanski, R .; Blondlieim, S .A ., and Stein, Y.: Changes in scrum
Dr. I. Macdonald, Department o f Physiology, G u y ’s Hospital Medical School, London S E I 9RT (England)
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lipid levels o f hyperlipidemic patients following the feeding o f starch, sucrose and glucose. Am . J . clin. Nutr. 18: 261-169 (1966). 8 Levine, J .: Morgenstern, S.. and Vlastelica, D .: A direct Liebermann-Burchard method for serum cholesterol. Automn anal. Chem. /: 25 -2 8 (1967). 9 Moser, P.B. and Berdanier, C .D .: Effect o f early sucrose feeding on the metabolic patterns o f mature rats. J . Nutr. 104: 687 -694 (1974). 10 Schlierf, G. and Wood, P.: Quantitative determination o f plasma free fatty acids and triglycerides by thin layer chromatography. J . Lipid Res. 6: 317-319 (1965).
An Investigation in Rats, into the Metabolic Consequences of Early Ingestion of Dietary Carbohydrates I. Macdonald, T. Rebella and Anne Keyser' Department o f Physiology, G u y ’s Hospital Medical School, London
Key Words. Triglyceride • Cholesterol • Liver-fatty acids - Serum-fatty acids • Adiposefatty acids • Glucose - Sucrose • Fructose • Rats
Abstract. Weanling rats were given diets containing a high proportion o f either glucose, sucrose or fructose until they reached 200 g weight, when they were either transferred to a control (chow) diet for 4 weeks or killed. The results were compared with rats receiving a control diet throughout. The triglyceride (TG ) and cholesterol levels in liver and in serum had returned to values similar to the control animals 4 weeks after being given the control diet. The fatty acid profile o f liver TG and adipose lipid tended to take longer to return to normal.
In some people normal intakes o f carbohydrate may aggravate disorders of lipid metabolism, and moreover, at least in the short term, intakes o f sucrose appear to emphasise pre-existing abnormalities o f the serum lipids (7). The aim o f the present experiments was to determine whether or not dif ferent dietary carbohydrates, taken by young animals, would cause metabolic changes which persisted or first appeared after sexual maturity. This question is clinically relevant since some children take large amounts o f sucrose (glucose and fructose) in confectionery and beverages.
1 Wc arc grateful to Beccham Products Ltd. for a research grant which enabled these studies to be carried out. We should also like to thank Dr. A. MacRae for his help.
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Received: March 17, 1975 ; accepted: May 12, 1975.
284
Macdonald/Rebello/Key ser Table I. Composition o f the experimental diets Parts by weight Glucose, sucrose or fructose Calcium caseinate (//-Methionine Dried yeast Salt mixture (see reference 2) Methyl cellulose Sunflower seed oil
70 21 0.5 4 3.5 3 1
12,000 III vitamin A . 1,200 IU vitamin D, 30 mg vitamin C/kg food
Methods Three groups o f 10 weanling male rats o f the Wistar strain were given a diet containing either glucose, sucrose or fructose as the main carbohydrate. The composition o f the diets is given in table 1. The animals were weighed weekly, and the food (presented ad libitum) taken from the feeding pot was estimated by weighing the pots each day. When each animal reached 200 g in body weight it was either killed (immediate group = I) or placed on a control (chow) diet for 4 weeks before being killed (delayed group = D). Three groups o f 6 animals were given a control (chow) diet throughout and the amount o f food eaten by this group was not measured. In the morning, 7 h prior to death, the food was removed from the cage and 5 h later, under light ether anaesthesia, each animal was given, by stomach tube, 5 uCi o f uniformally labelled 14C glucose, sucrose or fructose (with 0.5 g 'cold’ carbohydrate in 2 ml water). The carbohydrate was the same as that- which formed the major part o f the diet. Each o f the three radioactive carbohydrates was given to a group o f three rats which had been on a control (chow) diet. At post-mortem the liver was removed, blotted and weighed, and serum was obtained, as was a small piece o f peri-renal fat.
cation o f the Eolch method. The portion o f peri-renal fat was mixed with chloroform and. after filtration, the solvent was evaporated. Triglyceride and cholesterol estimation. Determination o f the concentration o f triglyc eride (TG) in the liver and in the serum was carried out using a modified semi-automated method (4). The radio-activity in the TG fraction was measured after separation by thin layer chromatography (10) from a known amount o f total lipid. With adipose tissue, a known weight of lipid was counted without prior separation, it being assumed that 90 99 % of the lipid extracted was TG (6). An automated method (8) was used for the cholesterol estimation. Fatty acid composition. Liver T G separated by thin-layer chromatography, was saponi fied with alcoholic KO H and the fatty acids were extracted and methylated. Analysis was
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A nalytical Methods Liver, serum and adipose tissue. After homogenising the liver the lipid was extracted by the method o f Folch et al. (5). The serum lipid was extracted using the Carlson (3) modifi
Consequences o f Dietary Carbohydrates in Rats
285
carried out by gas-liquid chromatography (G LC ) using a flame ionisation detector. The fatty acid profile o f the adipose tissue was also determined by G L C . Radio-activity. After the TG band on the thin-layer chromatograph plate (identified by iodine staining) had been scraped o ff, it was mixed with approximately 0.5 g o f ‘Cab-O-Sil’ (Koch-Light) and 6 ml o f scintillant. Counting was carried out in a Nuclear Enterprises 8102 with 93 % efficiency. Statistical comparisons were made using Student’s t test.
Results F ood Intake and Weight Gain It took 28 days for the first animals to reach 200 g body weight, and only the data up to this time are considered for the estimation of food intake and weight gain (The longest time to reach 200 g was 61 days). The mean food consumption (excluding controls) was greatest for those with glucose as the dietary carbohydrate and least for those eating sucrose (fig. 1). The mean intake of 13.2 g/day/animal (SE ± 0.91) on the glucose diet was significantly greater than the mean sucrose intake (9.5 g/day/animal, SE ± 0.61; p = 0.005-0.001). Despite this there was no significant difference between the groups in mean body weight at 28 days (fig. 1). No group had diarrhoea and it is not possible to state the reason for the similar weight gain despite the difference in food intake.
T riglyceride Total amount (table II). The amount of liver TG was significantly greater in the fructose group, compared with each o f the other three groups at the end o f the period on a high carbohydrate diet. In the group o f animals on the fructose diet there was a significant fall in total TG 4 weeks after returning to the control diet. Specific activity, dpm/mg TG (table II). The fructose-I group had a signifi cantly greater TG-specific activity than either the glucose-I or sucrose-I groups. There was a reduction in the specific activity after the animals had returned to a control diet, a reduction which was significant in the sucrose and fructose groups (p < 0.025). Am ount incorporated, dpm/total TG (fig. 2). The amount o f radio-activity
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Liver Wet Weight and Cholesterol (table II) The wet weight was significantly greater in the animals which were returned to a control diet for 4 weeks before being killed. There was a tendency for the total* amount o f cholesterol in the liver to be greater in the D group. The amount o f cholesterol in the fructose-I group was significantly greater than in the compa rable control group.
286
Macdonald¡Rebello ¡K ey ser
Fig. 1. Mean body weight (g) o f the rats during the first 28 days on the diets, together with the mean food intake (and SE). Values in parentheses indicate the number o f animals.
d p m / t o t a l TG
X 1,000 120
J
100
80
60
40
T
20
T
_x
—
G
s 1
....... F
± 3 G
S
F
0
Fig. 2. Mean amount (with SE) o f 14C incorporated in liver T G . G = Glucose; S = sucrose; F = fructose; 1= immediate group; D = delayed g ro u p .--------= Mean o f three controls.
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0
Consequences o f Dietary Carbohydrates in Rats
287
Table II. Mean values for the amount and specific activity o f T O , amount o f choles terol and liver weight in animals killed while on the high carbohydrate diet (immediate) and 4 weeks after returning to a chow diet (delayed) Diet
T G , mg
X
SE
39 30 45 87*
6.4 5.3 4.5
54 45 44 43
TG-specific activity, dpm/mg
Choles terol, mg
Liver weight, g
X
SE
X
SE
X
SE
1.2
169.6 62.5 147.9
13* 16 15* 18*
1 .1
632 455* 1,263*
9.9 7.7 8.5 9.4
0.39 0.47 0.59 o.so
7.1 8.3 5.6 3.9
20
1.4
270 246 270
115.3 46.0 106.2
17
1.6
22
1.6
23
1.7
11.9 10.5 11.1 12.4
0.65 0.71 0.43 0.71
Immediate animals Control Glucose Sucrose Fructose
1.4
1.0 1.0
Delayed animals Control Glucose Sucrose Fructose
* p < 0.025 when compared with delayed group.
Table III. Mean values o f the serum T G and cholesterol concentrations, together with the specific activity o f the T G , in rats killed while on a high carbohydrate diet (im mediate) and 4 weeks after returning to a control diet (delayed) 1 --------------------------------
Diet
TG-specific activity, dpm/mg
T G . mg
Cholesterol, mg/100 ml
X
SE
X
SE
X
SE
91 64 96 117
8.2 12.1 9.7 27.3
456* 565 2.033*
77.6 110.1 642.2
58 63 63 79
2.9 7.6 3.9 8.9
116 97 95 141
15.5 18.0 12.1 23.2
238 368 572
9.0 17.8 143.6
67 65 58 60
2.6 6.4 4.3 3.3
Immediate animals Control Glucose Sucrose Fructose
Control Glucose Sucrose Fructose
* p < 0.025 when compared with delayed group.
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Delayed animals
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in the liver TG was significantly increased in the fructose-I and the sucrose-1 groups compared to the values 28 days after returning to a control diet, at which time the values were similar to control. The value o f the fructose-I group was significantly greater than that o f either the glucose-I or sucrose-I groups. Serum Cholesterol (table III) No significant change was found in the serum-cholesterol concentration within any group, nor was any difference found between the groups. Triglyceride (table III) Concentration. No significant difference was found between the I and D groups or within groups. Specific activity. The TG-specific activity was significantly lower after re turning to a normal diet in the glucose and fructose groups, and, although lower
« 001 >
I
0
Fig. 3. Mean amount o f radio-activity (with SE) in TG in 100 ml serum .------- = Mean o f three controls. Abbreviations as in figure 2.
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dpm
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Consequences of Dietary Carbohydrates in Rats
in the sucrose group, this difference was not significant. 28 days after returning to the control diet, the TG-specific activity was significantly less in the glucose compared to the sucrose and fructose groups. Am ount incorporated, dpm/total TG/100 ml serum (fig. 3). There was a tendency for more l4C-carbohydrate to be incorporated into serum TG at the end o f the carbohydrate-consuming period than after the return to the control diet, but this only achieved statistical significance in the fructose group. The fructose-1 group incorporated significantly more carbohydrate into the serum TG than the glucose-I or sucrose-I groups. Adipose Tissue (fig. 4) The specific activity o f the adipose tissue was considerably greater at the end of the period on high carbohydrate diets than 4 weeks later, after the return to the control diet. Though the specific activity was greater with glucose than with sucrose and fructose, these differences were not significant.
dpm/mg
60
Fig. 4. Mean specific activity (with SE) o f lipid from peri-renal adipose tissue.------- = Mean o f three controls. Abbreviations as in figure 2.
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D
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Fatty A cid Profile Liver TG (table IV) While on the high carbohydrate diet, the proportions o f 16:0, 16:1 and 18:1 acids were significantly greater, compared to those found in the control animals, whereas with 18:2 fatty acid the reverse was found. The proportions o f 16:0 and 18:1 fatty acid had not altered significantly 4 weeks later, after re-
Table IV. Mean percentage o f fatty acids in liver T G in rats killed while on a high carbo hydrate diet (immediate) and 4 weeks after returning to a control diet (delayed) Diet
14:0 X
16:0 SE
18:0
16:1
18:1
18:2
X
SE
X
SE
X
SE
X
SE
X
SE
0.16 0.34 0.10 0.30
33.5 36.4 42.0 39.9
0.57 0.53 3.17 1.92
6.6 10.0 13.2 12.2
0.28 0.38 0.42 0.54
2.5 2.5 2.6 2.2
0.08 0.40 0.16 0.22
27.9 38.5 37.4 41.9
0.66 2.48 1.23 2.52
27.7 11.3 2.0 1.6
0.92 0.84 0.32 0.24
0.15 0.07 0.45
37.6 38.8 34.6
0.90 0.74 2.70
9.2 7.6 8.8
0.96 0.49 0.59
2.3 3.9 3.6
0.34 0.89 0.43
31.7 34.3 40.6
1.27 1.22 2.50
17.6 13.9 10.2
1.84 0.77 1.99
Immediate animals Control Glucose Sucrose fructose
1.9 1.3 2.8 2.2
Delayed animals Glucose Sucrose Fructose
1.6 1.5 2.2
Table V. Mean percentage o f fatty acids in adipose tissue from rats killed while oni a high carbohydrate diet (immediate) and 4 weeks after returning to a control diet (delayed) Diet
14:0 X
16:1
16:0
18:0
18:1
18:2
X
SE
X
SE
X
SE
X
SE
X
SE
0.54 0.30 0.54 0.90
35.7 39.3 33.6 34.0
1.32 2.68 0.40 3.76
12.6 12.0 13.9 17.9
0.54 1.03 0.61 2.28
2.9 6.0 4.1 2.7
0.20 1.41 0.38 0.46
24.2 34.7 38.3 33.0
1.15 2.27 0.42 5.32
18.4 5.2 6.4 5.5
0.96 0.70 0.54 0.84
0.16 0.85 0.15 0.49
38.8 37.2 32.9 37.2
1.12 1.57 0.56 3.73
11.2 11.3 11.2 12.0
0.31 0.60 0.23 1.38
3.4 4.1 4.2 3.6
0.27 0.29 0.41 0.35
24.9 29.6 33.4 29.2
0.65 1.81 0.86 2.94
17.3 12.9 15.7 14.1
0.69 0.50 0.45 1.68
SE
Immediate animals Control Glucose Sucrose Fructose
6.2 2.8 3.7 6.9
Control Glucose Sucrose Fructose
4.4 4.9 2.6 3.9
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Delayed animals
Consequences o f Dietary Carbohydrates in Rats
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turning to a control diet. The proportions of 16:1 and 18:2 fatty acids showed significantly greater deviation from the control in the sucrose-I and fructose-1 groups than in glucose-I group. Adipose Tissue (table V) At the end o f the period on the high carbohydrate diet, the levels o f 14:0 and 18:2 fatty acids were significantly lower in all the carbohydrate groups, compared to the control group, whereas with 18:1 fatty acid the reverse was found. At the end of 4 weeks on a control diet, the 18:2 fatty acid in the adipose tissue in all three carbohydrate groups had returned to levels not mark edly different from the control group. The changes in the fatty acid profile, found in the carbohydrate groups, all showed a tendency, after 4 weeks on a control diet, to return to the levels found in those rats which had consumed the control diet throughout.
The measurements made in these experiments do not support the view that an abnormal intake o f dietary carbohydrate by a young rat would result in permanent metabolic changes throughout the remainder of the animal’s life. The measurements made were of some of the end products o f dietary carbohydrate metabolism, and it could be reasoned that if the dramatic changes seen in the animals while on a high carbohydrate diet were no longer present after 4 weeks on a normal diet, then the changes in metabolic activity, which presumably were present during the period o f high carbohydrate intake, did not persist. In a similar, but not comparable study, Moser and Berdanier (9) placed weanling rats on high starch or sucrose diets and then switched the type o f carbohydrate given. They found differences in liver lipid content and enzyme activity between the carbohydrates, but the animals did not at any time return to a control diet. Unlike the experiments described here, their rats were killed after a 16-hour fast and were o f a strain known to be carbohydrate-sensitive. Also the plan o f their experiments was not such as to be able to determine whether the young animals on high carbohydrate diets had values different from control, and if they were different, whether the animals’ metabolism had the capability of returning to values found in control diets. Many o f the findings in the liver and serum relating to TG have been reported frequently in rats which have been on high carbohydrate diets. The increase in level o f TG is greater with dietary fructose than with glucose, the results from the high sucrose diet being in between. Also the radio-activity, following 14C carbohydrate, in the liver and serum TG is greater in those animals
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which were on a high fructose diet than in those on a high glucose diet, with again sucrose in between. However, 4 weeks after the return to a control diet all measurements were very similar to those in the control group, with the excep tion o f the specific activity o f serum TG following 14C fructose. The changes in the fatty acid profile o f the liver and adipose tissue were relatively rapid while on the high carbohydrate diets with much slower return to control levels, when the control diet was substituted for the high carbohydrate diet. It is possible that the youthfulness of the animals was responsible for the rapid change in fatty acid profile o f both liver and adipose T G , because when the rats at the end o f their period on a high carbohydrate diet were given the control diet for 4 weeks, the changes in the fatty acids had not all returned to control levels. The drop in proportion o f 18:2 fatty acid in liver and adipose tissue TG was not expected, as each rat was given approximately 50 mg linoleic acid/day. Also it seemed that the drop in the proportion o f this fatty acid in liver was greater and the recovery less rapid in those rats with fructose as the dietary carbohydrate, when compared to the glucose fed animals. This would suggest that there is some link between the type o f dietary carbohydrate and the metab olism o f the ‘essential’ fatty acid, linoleic acid. Needs for linoleic acid are greatest when an animal is young, and it is possible that an increased intake of carbohydrate and especially fructose, could make greater demands on linoleic acid metabolism. Thus, the experiments described here indicate that in weanling rats no dra matic changes in lipid metabolism could be found after periods o f living on high carbohydrate containing diets, followed by 4 weeks on a standard laboratory diet. Whether a longer period on the experimental diet, or a more precise search for metabolic ‘hangovers’ would have revealed permanent differences could be the basis for further studies.
References 1 2 3
Ahrens, E .H .; Hirsch, S .; Oettle, K .; Farquhar, J.W ., and Stein, Y .: Carbohydrateinduced and fat-induced lipemia. Trans. Ass. Am . Physns 74: 134 146 (1961). Briggs, G.M. and Williams, M .A .: A new mineral mixture for experimental rats diet and evaluation o f other mixtures. Fed. Proc. Am . Socs exp. Biol. 22: 261 (1963). Carlson, L .A .: Determination o f serum triglycerides. J . Atheroscler. Res. 3: 334 - 336 (1963).
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Eggstein, M. und Kreutz, F .H .: Eine neue Bestimmung der Neutralfette im Blutserum und Gewebe. Klin. Wschr. 44: 262 267 (1966). Folch, ] .; Lees, M ., and Sloan-Stanley, G .H .: A simple method for the isolation and purification o f total lipids from animal tissues. J . biol. Chem. 226: 497 509 (1957). Jeanrenaud, B.: Lipid components o f adipose tissue: in Adipose tissue. Handbook o f physiology, vol. 5, pp. 169
176(1965).
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4
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Kaufrnann, N .A .; Poznanski, R .; Blondlieim, S .A ., and Stein, Y.: Changes in scrum
Dr. I. Macdonald, Department o f Physiology, G u y ’s Hospital Medical School, London S E I 9RT (England)
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lipid levels o f hyperlipidemic patients following the feeding o f starch, sucrose and glucose. Am . J . clin. Nutr. 18: 261-169 (1966). 8 Levine, J .: Morgenstern, S.. and Vlastelica, D .: A direct Liebermann-Burchard method for serum cholesterol. Automn anal. Chem. /: 25 -2 8 (1967). 9 Moser, P.B. and Berdanier, C .D .: Effect o f early sucrose feeding on the metabolic patterns o f mature rats. J . Nutr. 104: 687 -694 (1974). 10 Schlierf, G. and Wood, P.: Quantitative determination o f plasma free fatty acids and triglycerides by thin layer chromatography. J . Lipid Res. 6: 317-319 (1965).
