Hostname: page-component-7c8c6479df-7qhmt Total loading time: 0 Render date: 2024-03-29T13:25:12.127Z Has data issue: false hasContentIssue false

Influence of Ramadan-type fasting on enzymes of carbohydrate metabolism and brush border membrane in small intestine and liver of rat used as a model

Published online by Cambridge University Press:  15 March 2007

Rights & Permissions [Opens in a new window]

Abstract

During Ramadan, Muslims the world over abstain from food and water from dawn to sunset for a month. We hypothesised that this unique model of prolonged intermittent fasting would result in specific intestinal and liver metabolic adaptations and hence alter metabolic activities. The effect of Ramadan-type fasting was studied on enzymes of carbohydrate metabolism and the brush border membrane of intestine and liver from rat used as a model. Rats were fasted (12 h) and then refed (12 h) daily for 30 d, as practised by Muslims during Ramadan. Ramadan-type fasting caused a significant decline in serum glucose, cholesterol and lactate dehydrogenase activity, whereas inorganic phosphate increased but blood urea N was not changed. Fasting resulted in increased activities of intestinal lactate (+34 %), isocitrate (+63 %), succinate (+83 %) and malate (+106 %) dehydrogenases, fructose 1,6-bisphosphatase (+17 %) and glucose-6-phosphatase (+22 %). Liver lactate dehydrogenase, malate dehydrogenase, glucose-6-phosphatase and fructose 1,6-bisphosphatase activities were also enhanced. However, the activities of glucose-6-phosphate dehydrogenase and malic enzyme fell significantly in the intestine but increased in liver. Although the activities of alkaline phosphatase, γ-glutamyl transpeptidase and sucrase decreased in mucosal homogenates and brush border membrane, those of liver alkaline phosphatase, γ-glutamyl transpeptidase and leucine aminopeptidase significantly increased. These changes were due to a respective decrease and increase of the maximal velocities of the enzyme reactions. Ramadan-type fasting caused similar effects whether the rats fasted with a daytime or night-time feeding schedule. The present results show a tremendous adaptation capacity of both liver and intestinal metabolic activities with Ramadan-type fasting in rats used as a model for Ramadan fasting in people.

Type
Research Article
Copyright
Copyright © The Authors 2006

The digestion and absorption of food components are major functions of the intestinal mucosa. These functions are dramatically altered by dietary status, including fasting, restricted energy intake and other dietary manipulations (Budhoski et al. Reference Budhoski, Challis and Newsholme1982; Mayhew, Reference Mayhew1987; Dou et al. Reference Dou, Gregersen, Zhao, Zhuang and Gregersen2001; Martins et al. Reference Martins, Hipolito-Reis and Azevedo2001). Short-term fasting for a few days causes a significant decrease in glucose degradation with a concomitant increase in its production in the intestine and other tissues (Shen & Mistry, Reference Shen and Mistry1979; Farooq et al. Reference Farooq, Yusufi and Mahmood2004), whereas refeeding fasted rats resulted in a reversal of these effects (Buts et al. Reference Buts, Vijverman, Barudi, De Keyser, Maldague and Dive1990).

Ramadan fasting is a unique model of fasting that is quite different from widely studied total fasting or starvation (Malhotra et al. Reference Malhotra, Scott, Scott, Gee and Wharton1989; Nomani et al. Reference Nomani, Hallack, Nomani and Siddiqui1989; Cheah et al. Reference Cheah, Ch'ng, Husain and Duncan1990). During the Islamic month of Ramadan, which lasts for 29 or 30 d each year, millions of Muslims all over the world observe total abstention from food and water from dawn to sunset. Food and water is, however, permitted ad libitum between sunset and dawn (Husain et al. Reference Husain, Duncan, Cheah and Ch'ng1987; Toda & Morimoto, Reference Toda and Morimoto2000). Hence, Ramadan fasting is in fact repeated cycles of fasting followed by refeeding every day and night for about 30 d.

Ramadan fasting in man results in increased serum lipids, uric acid, urea (Gumaa et al. Reference Gumaa, Mustafa, Mahmoud and Gader1978; Fedail et al. Reference Fedail, Murphy, Salih, Bolton and Harvey1982; Hallack & Nomani, Reference Hallack and Nomani1988; Nomani et al. Reference Nomani, Hallack, Nomani and Siddiqui1989), NEFA and 3-OH butyrate, and a decrease in blood glucose, lactate and pyruvate (Malhotra et al. Reference Malhotra, Scott, Scott, Gee and Wharton1989; Nomani et al. Reference Nomani, Hallack, Nomani and Siddiqui1989), indicating alterations in metabolic activities. The changes in urine volume, osmolarity, total solutes and ions (Na+, K+) and urea produce no adverse health effects on renal function (Cheah et al. Reference Cheah, Ch'ng, Husain and Duncan1990; Leiper et al. Reference Leiper, Molla and Molla2003). Basal metabolism slows down (Husain et al. Reference Husain, Duncan, Cheah and Ch'ng1987), whereas body fat is utilised efficiently during Ramadan fasting (El Ati et al. Reference El Ati, Beji and Danguir1995). HDL cholesterol increases whereas LDL cholesterol decreases with Ramadan fasting (Adlouni et al. Reference Adlouni, Ghalim, Benslimane, Lecerf and Saile1997; Benli Aksungar et al. Reference Benli Aksungar, Eren, Ure, Teskin and Ates2005). Owing to different dietary habits and physical activities, both a gain (Frost & Pirani, Reference Frost and Pirani1987) and a loss (El Ati et al. Reference El Ati, Beji and Danguir1995), and sometimes no change (Husain et al. Reference Husain, Duncan, Cheah and Ch'ng1987), in body weight have been reported after Ramadan fasting.

As millions of Muslims (young and old) have for centuries abstained from food and water in the daytime during the Islamic month of Ramadan, it seems important to examine the influence of this fasting schedule on human health, especially with respect to nutrition and energy metabolism. Although studying human biology is ideal, such studies are neither feasible nor ethical. Thus, the vast majority of current biomedical research is conducted using laboratory animals such as rats. In the present study, Ramadan-type fasting (RTF) was mimicked experimentally in rats used as a model for Ramadan fasting in man. Rats were fasted (12 h) and then refed (12 h) daily for 30 d, as practised by Muslims during the month of Ramadan. We hypothesised that RTF would result in specific intestinal and liver adaptations and alter metabolic activities.

To address this hypothesis, the influence of RTF on enzymes of carbohydrate metabolism and brush border membrane (BBM) in rat intestine and liver was determined. The activities of enzymes involved in glucose oxidation (e.g. lactate dehydrogenase (LDH), isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH)) and its production (e.g. fructose 1,6-bisphosphatase, glucose-6-phosphatase) markedly increased in mucosal and liver homogenates in RTF compared with control rats. However, the enzymes of the BBM involved in the terminal digestion and/or absorption of nutrients decreased in intestine but increased in liver.

Materials and methods

Chemicals

Sucrose, p-nitrophenyl phosphate, sodium succinate, NADH and NADP+ were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals used were of analytical grade and were purchased from either Sigma Chemical Co. or Sisco Research Laboratory (Mumbai, India).

Experimental design

Unlike people, rats are nocturnal feeders, and it may be considered unphysiological to fast them in the daytime. It has, however, been reported that the rhythmic pattern of certain intestinal enzymes disappears in rats when they are fasted (for up to 5 d) on a daytime or night-time feeding schedule, and instead increases or changes either in anticipation of or in the presence of food (Saito et al. Reference Saito, Murakami, Nishida, Fujisawa and Suda1976). It has also been reported that a monosodium-glutamate-induced increase in alkaline phosphatase activity was not a consequence of actual day/night intake variations but due to a more general effect of monosodium glutamate characterised by neurohormonal and metabolic disturbances (Martinkova et al. Reference Martinkova, Lenhardt and Mozes2000).

Considering the importance of Ramadan fasting, the effect of RTF was determined initially in rats that were fasted for 12 h followed by 12 h refeeding with either a daytime or a night-time feeding schedule for 30 d. The rats were killed at the end of last fast in the morning (day-fasters) or in the evening (night-fasters) after a stabilisation period of 10–12 h. It was noted that rats rushed to eat and drink immediately when food or water was given to them at the end of fasting period and then ate intermittently during the refeeding (12 h) time.

The results summarised in Table 1 shows that day/night RTF resulted in similar alterations in serum glucose, cholesterol, blood urea N, inorganic phosphate and LDH activity. As there was no significant difference between the respective controls, the values were pooled to make one control value. Various tissue enzymes also showed a similar pattern irrespective of day/night fasting–refeeding variations (see Results). It appeared that rats, irrespective of whether they were fasted by day or by night, showed adaptations similar to those observed earlier (Saito et al. Reference Saito, Murakami, Nishida, Fujisawa and Suda1976). Therefore a comprehensive effect of RTF was determined, as described later, by a daytime fasting (12 h) followed by a night-time refeeding (12 h) schedule; the results are compared with the nocturnal fasting schedule where appropriate.

Table 1 Effect of daytime and night-time Ramadan-type fasting on serum parameters(Mean values with their standard errors and percentage change from control values (%) for 12 rats per group)

* Mean values were significantly different from control at P < 0·05 or higher degree of significance by group t test and ANOVA.

Young adult Wistar rats weighing 135–155 g, fed with a standard pellet diet (Amrut Laboratories, Pune, India) and water ad libitum, were conditioned for 1 week before the start of the experiment. All animals were kept under conditions that prevented them from experiencing unnecessary pain and discomfort according to the guidelines approved by Ethical Committee. The rats were separated into two groups. One group was put on RTF (12 h fasting/12 h refeeding) for 30 d. The other group received their diet and water ad libitum both day and night and were used as a control. After 30 d, the rats were killed under light ether anaesthesia. The liver and entire small intestine, from the ligament of Trietz to the end of ileum, was removed. The intestines were washed by flushing them with ice-cold buffered saline (1 mmol/l Tris-HCl, 9 g/l NaCl, pH 7·4). The weights of the animals were recorded at the beginning and end of the experiment.

Preparation of homogenate

The washed intestines were slit in the middle, and the entire mucosa was gently scraped with a glass slide and weighed. A 15 g/l homogenate of this mucosa was prepared in ice-cold 100 mmol/l Tris-HCl, pH 7·4, using a Potter-Elvehejem homogeniser (Remi Motors, Mumbai, India) by passing five pulses. The homogenate was centrifuged at 2000 g at 4 °C for 10 min to remove cell debris, and the supernatant thus obtained was used for assaying enzymes of carbohydrate metabolism. Liver homogenates were similarly prepared and analysed simultaneously. Aliquots of these homogenates were saved and kept at − 20 °C until analysis.

Preparation of brush border membrane

BBM was prepared at 4 °C using differential precipitation by CaCl2 (Kessler et al. Reference Kessler, Acuto, Storelli, Murer, Muller and Semenza1978). Mucosa scraped from 4–5 washed intestines was used for each BBM preparation. This was homogenized in 50 mmol/l mannitol, 2 mmol/l Tris-HCl buffer, pH 7·5, in a glass homogeniser (Wheaton, IL, USA) with five complete strokes. The homogenate was then subjected to high-speed Ultra-Turrex Kunkel (Janke & Kunkel GmbH & Co. KG, Staufen, Germany) homogenation for three strokes of 15 s each with an interval of 15 s between each stroke. Solid CaCl2 was added to the homogenate to a final concentration of 10 mmol/l, and the mixture stirred for 20 min on ice. The homogenate was centrifuged at 2000 g in a J2-21 Beckman centrifuge (J2 MI; Beckman lnstruments, Palo Alto, CA, USA), and the supernatant was then recentrifuged at 352 000 g for 30 min. The pellet was resuspended in 50 mmol/l sodium maleate buffer, pH 6·8, with four passes by a loose-fitting Dounce homogeniser (Wheaton) in a 15 ml corex tube and centrifuged at 352 000 g for 20 min. The outer white fluffy pellet of BBM was resuspended in a small volume of sodium maleate buffer.

The membrane preparations were purified several magnitudes as the specific activities of the BBM enzymes were increased 7–10-fold compared with the homogenate. Aliquots of homogenates (after high-speed homogenisation) and BBM thus prepared were saved and stored at − 20 °C until further analysis for the BBM enzymes sucrase, alkaline phosphatase and γ-glutamyl transpeptidase (GGTase).

Enzyme assays

The activities of marker enzymes in the homogenate and BBM fraction were determined by standard methods. The activity of alkaline phosphatase was measured by the method of Kempson et al. (Reference Kempson, Kim, Northrup, Knox and Dousa1979) using p-nitrophenyl phosphate as a substrate, whereas sucrase was assayed by the method of Bernfeld (Reference Bernfeld, Colowick and Kaplan1955). GGTase was measured by the method of Glossmann & Neville (Reference Glossmann and Neville1972) and leucine aminopeptidase (LAP) by the method of Goldmann et al. (Reference Goldmann, Schlesinger and Segal1976). The Michaelis Menton constant (K m) and maximal velocity of the enzyme reaction (V max) were determined by assaying these enzymes at various substrate concentrations (0·6–5·0 mmol/l for alkaline phosphatase, 5–160 mmol/l for sucrase, 0·1–0·4 mmol/l for LAP) and analysing the data by Lineweaver–Burk plot. Protein concentrations in BBM preparations and homogenates were determined by the method of Lowry et al. (Reference Lowry, Rosebrough, Farr and Randall1951) as modified by Yusufi et al. (Reference Yusufi, Low, Turner and Dousa1983).

The activities of LDH, MDH, glucose-6-phosphate dehydrogenase (G6PHD), malic enzyme and ICDH, involved in the oxidation of NADH or reduction of NADP+, were determined by measuring extinction changes at 340 nm in a spectrophotometer (Cintra 5; GBC Scientific Equipment Pty, Victoria, Australia) using 3·0 ml assay mixture in a 1 cm cuvette at room temperature (28–30 °C). The net reaction rate was measured by the difference between the extinction values obtained prior to the addition of substrate and the values for the actual enzymic reaction following addition of the substrate. Appropriate blanks, in which the substrate was added after stopping the reaction, were run simultaneously.

All enzyme activities were measured under conditions in which enzyme reaction rates were linear with respect to incubation time and protein concentration using the method mentioned against each enzyme: LDH, E.C. 1·1·1·27 (Kornberg, Reference Kornberg, Colowick and Kaplan1955); MDH, E.C. 1·1·1·37 (Meyer et al. Reference Meyer, Kornberg, Grisolia and Ochoa1948); G6PDH, E.C. 1·1·1·49 (Shonk & Boxer, Reference Shonk and Boxer1964); SDH, E.C. 1·3·99·1 (Szczepanska-Konkel et al. Reference Szczepanska-Konkel, Yusufi and Dousa1987); ICDH, E.C.1·1·1·42 (Ochoa, Reference Ochoa, Colowick and Kaplan1955a); malic enzyme, E.C. 1·1·1·40 (Ochoa, Reference Ochoa, Colowick and Kaplan1955b). Glucose-6-phosphatase E.C. 3·1·3·9 and fructose-1,6-bisphosphatase E.C 3·1·3·11 were assayed by the method of Shull et al. (Reference Shull, Ashmore and Mayer1956). The inorganic phosphate liberated was measured by the method of Tausky & Shorr (Reference Tausky and Shorr1953).

Analysis of serum parameters

The serum samples were deproteinated with 3 % trichloroacetic acid in a ratio of 1:3 v/v. The samples were centrifuged at 2000 g (Remi centrifuge, India) for 10 min. The protein-free supernatant was used to estimate inorganic phosphate by the method of Tausky & Shorr (Reference Tausky and Shorr1953). Total serum cholesterol was estimated directly in serum samples by the method of Zlatkis et al. (Reference Zlatkis, Zak and Boyle1953). Urea was measured by the method of Fingerhut et al. (Reference Fingerhut, Ferzola, Marsh and Miller1966); glucose was estimated by o-toluidine method using kit from Span diagnostics (Surat, India).

Definition of unit

One unit of enzyme activity is the amount of enzyme required for the formation of 1 μmol product/h under specified experimental conditions. Specific activity is enzyme units/mg protein.

Statistical analysis

Results are expressed as means with their standard errors for at least three separate experiments. There were two groups of rats in each experiment: control and RTF group. Each sample of BBM and homogenate was prepared by pooling tissues from 4–5 rats. The data are representative of 12–15 rats per group per experiment. Where appropriate, statistical evaluation was conducted by group t test and ANOVA.

Results

The effect of RTF with 12 h daytime fasting and 12 h night-time refeeding or vice versa for 30 d was studied in detail by assessing some serum parameters as well as the activities of certain enzymes from liver and small intestinal mucosa of rats that were involved in terminal digestion, absorption and carbohydrate metabolism.

Effect of Ramadan-type fasting on serum parameters in daytime and night-time fasting conditions

As shown in Table 1, an effect of RTF was observed on various serum parameters during a daytime compared with night-time fasting schedule. Serum glucose, cholesterol and LDH activity significantly lowered, whereas inorganic phosphate increased under both the daytime and night-time fasting schedule. Blood urea N was not changed. The activity of serum alkaline phosphatase was, however, significantly increased with daytime fasting but only slightly increased with night-time fasting.

Effect of Ramadan-type fasting on body and mucosal weight of rats

The young adult rats used in the study showed a significant increase in body weight in both the control (+68 %) and the fasted (+55 %) rats compared with the weight recorded at the start of the experiment (145·97 (se 7·75) g). The gain in body weight at the end of 30 d fasting period was slightly but not significantly lower in the RTF than the control rats. The mucosal weight was also lowered (–22 %; Table 2).

Table 2 Effect of Ramadan-type fasting on body and mucosal weight of rats(Mean values with their standard errors for three different experiments with four rats per group and change from control values (%))

The initial mean body weight was 145·95 (se 7·75)g for n 24 rats.

Effect of Ramadan-type fasting on brush border membrane enzymes in mucosal homogenates and isolated brush border membrane

RTF resulted in significant decrease in the activities of alkaline phosphatase, GGTase and sucrase in mucosal homogenates and in the isolated BBM preparations (Table 3). The enzyme activities similarly increased (7–10-fold) in the membrane preparations compared with respective values for the homogenate in both the control and RTF rats, indicating that the quality of membranes prepared by the procedure was similar for control and RTF rats. The specific activities of alkaline phosphatase, GGTase and sucrase all fell significantly (by approximately 25 %) in the homogenates. However, alkaline phosphatase activity decreased to greater extent (–38 %) than the activities of GGTase (–25 %) and sucrase (–20 %) in BBM preparations. In a preliminary experiment, it was observed that the activities of both alkaline phosphatase (–25 %) and sucrase (–20 %) declined similarly in daytime-fasted as well in night-time-fasted rats (data not shown). The kinetic parameters (K mV max) of alkaline phosphatase and sucrase were also determined by assaying the enzymes in BBM preparations at different substrate concentrations. The results summarised in Table 4 show that the decrease in the activity of both alkaline phosphatase and sucrase caused by RTF was due mainly to a decrease in the V max of the enzyme rather than to changed values of the Michaelis Menton constant (K m).

Table 3 Effect of Ramadan-type fasting on the activities of alkaline phosphatase, γ-glutamyl transpeptidase and sucrase in the mucosal homogenates and brush border membrane preparations(Mean values with their standard errors for specific activities (μmol/mg protein per h) for three different experiments with their respective change from control values (%))

* Means were significantly different from control at P < 0·05 or higher degree of significance by group t test and ANOVA.

Table 4 Effect of Ramadan fasting on the kinetic parameters of alkaline phosphatase and sucrase(Means values with their standard errors for three different experiments with their respective change from control values (%))

K m (Michaelis Menton constant) and V max (maximal velocity of enzyme reaction) were determined in brush border membrane preparations.

* Means were significantly different from control at P < 0·05 or higher degree of significance by group t test and ANOVA.

Effect of Ramadan-type fasting on enzymes of carbohydrate metabolism in rat intestine

The specific activities of various enzymes involved in carbohydrate metabolism were determined in mucosal homogenates of control and RTF rats (daytime-fasted) after 30 d fasting. The activity of LDH, a representative of anaerobic glycolysis, markedly increased (+34 %) with RTF (LDH activity being similarly enhanced by night-time fasting). However, the activities of ICDH, SDH and MDH, enzymes of glucose oxidation, profoundly increased after the 0 d RTF period. The activity of ICDH increased significantly (+63 %), whereas the activities of SDH (+83 %) and MDH (+106 %) increased to much greater extent compared with control values (Table 5).

Table 5 Effect of Ramadan type fasting on the specific activities of lactate dehydrogenase (LDH), isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) in homogenates of intestinal mucosa(Mean values with their standard errors for specific activities (μmol/mg protein per h) for three different experiments with their respective change from control values (%))

* Means were significantly different from control at P < 0·05 or higher degree of significance by group t test and ANOVA.

The effect of RTF on the activities of gluconeogenic enzymes fructose 1,6-bisphosphatase and glucose-6-phosphatase was also determined. The activities of these enzymes were also enhanced during RTF compared with values in the control rats, although the increase was smaller compared with that seen with the enzymes involved in glycolysis (LDH) and the tricarboxylic acid cycle (ICDH, SDH, MDH). The activities of G6PDH (hexose monophosphate shunt) and malic enzyme, which play important role in reducing anabolic pathways by supplying NADPH, were also studied. In contrast to the enzymes of glucose oxidation and production, the activities of both G6PDH (–33 %) and malic enzyme (–36 %) significantly declined in rat mucosa after RTF (Table 6).

Table 6 Effect of Ramadan-type fasting on the specific activities of fructose 1,6-bisphosphatase, glucose-6-phosphatase (G6Pase), glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme in homogenates of intestinal mucosa(Mean values with their standard errors for specific activities (μmol/mg protein per h) for three different experiments with their respective change from control values (%))

* Means were significantly different from control at P < 0·05 or higher degree of significance by group t test and ANOVA.

Effect of Ramadan-type fasting on enzymes of carbohydrate metabolism in rat liver homogenates

The effect of RTF in both a daytime and night-time fasting schedule are shown in Table 7. The specific activities of LDH and MDH, enzymes involved in glucose degradation, profoundly increased in both fasting schedules. The activities of gluconeogenic enzymes glucose-6-phosphatase and fructose 1,6-bisphosphatase also markedly enhanced after RTF in the liver homogenates irrespective of day/night variations in the feeding schedule. The effect was more prominent in the liver than in intestinal enzymes. In contrast to intestine, where the activities of G6PDH and malic enzyme declined, the activities of these enzymes profoundly increased to a similar extent in both fasting conditions. Compared with intestinal enzymes, the activities of alkaline phosphatase, GGTase and LAP were significantly increased in the liver homogenates (Table 8) from both daytime-fasted and night-time-fasted RTF rats. Kinetic analysis showed that the increase in LAP activity (+71 %) was mainly due to an alteration in the V max of LAP activity (RTF: 3·33 (se 0·08) v. control: 1·96 (se 0·02), whereas K m was unchanged (data not shown).

Table 7 Effect of daytime and night-time Ramadan-type fasting on the specific activities of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), fructose 1,6-bisphosphatase (FBPase), glucose-6-phosphatase (G6Pase), gluose-6-phosphate dehydrogenase (G6PDH) and malic enzyme in liver homogenate(Mean values with their standard errors for specific activities (μmol/mg protein per h) for three different experiments with their respective change from control values (%))

* Means were significantly different from control at P < 0·05 or higher degree of significance by group t test and ANOVA.

Table 8 Effect of daytime and night-time Ramadan-type fasting on the specific activities of alkaline phosphatase, γ-glutamyl transpeptidase and leucine aminopeptidase in liver homogenate(Mean values with their standard errors for specific activities (μmol/mg protein per h) for three different experiments with their respective change from control values (%))

* Means were significantly different from control at P < 0·05 or higher degree of significance by group t test and ANOVA.

Discussion

The main purpose of the present study was to determine the influence of RTF on certain enzymes involved in carbohydrate metabolism and terminal digestion in the intestine and liver of rats used as a model of human Ramadan fasting. The present results in part support our hypothesis that RTF results in specific intestinal and liver metabolic adaptations. Indeed, the activities of enzymes belonging to various metabolic pathways, for example glycolysis, the tricarboxylic acid cycle and gluconeogenesis, profoundly increased in both the intestine and liver with RTF.

In general, a 30 d 12 h fasting/12 h feeding schedule was associated with a steady gain in body weight in both control (+68 %) and fasted (+55 %) young adult rats compared with their starting weights (Table 2). At the end of 30 d period, however, body weight was slightly ( − 8 %) but insignificantly lower in RTF compared with control rats, as has previously been observed in some studies on people (Hallack & Nomani, Reference Hallack and Nomani1988; Adlouni et al. Reference Adlouni, Ghalim, Benslimane, Lecerf and Saile1997, 1998; Maislos et al. Reference Maislos, Abou-Rabiah, Iordash and Shany1998; Yucel et al. Reference Yucel, Degirmenci, Acar, Albayrak and Haktanir2004). The loss of body weight in human subjects has been attributed to either dehydration or loss of body fat during the course of fasting (Roky et al. Reference Roky, Houti, Moussamih, Qotbi and Aadil2004). The lowering of serum glucose, cholesterol and LDH activity in rats is in partial agreement with the reported decrease in blood glucose, lactate and pyruvate in people after Ramadan fasting (Fedail et al. Reference Fedail, Murphy, Salih, Bolton and Harvey1982; Hallack & Nomani, Reference Hallack and Nomani1988; Malhotra et al. Reference Malhotra, Scott, Scott, Gee and Wharton1989).

The results of the present study demonstrate that the enzymes involved in glucose degradation and production were significantly enhanced in both the mucosa and the liver (Tables 57). Anaerobic glycolysis, compared with oxidative metabolism, has been shown to be the major source of energy in the rat intestine (Hubscher & Sherrat, Reference Hubscher and Sherrat1962). The marked increase in ICDH, SDH and MDH together with LDH activity in the present study, however, suggests that oxidative metabolism in addition to glycolysis becomes involved in increased energy production during prolonged intermittent RTF. Increased fatty acid oxidation (El Ati et al. Reference El Ati, Beji and Danguir1995) may result in enhanced activities of enzymes in the tricarboxylic acid cycle. The activities of G6PDH and malic enzyme, which act to produce NADPH, were differentially altered by RTF. Although the activities of these enzymes significantly declined in the intestine, they were profoundly increased in the liver. Increased NADPH thus produced at least in the liver may have supported many reducing anabolic reactions needed during prolonged intermittent fasting.

The underlying mechanism by which RTF may cause alterations in liver and intestinal metabolic activities seems the composite effects of fasting and refeeding. Fasting is known to produce extensive morphological as well as biochemical changes in the small intestine and liver (Dou et al. Reference Dou, Gregersen, Zhao, Zhuang and Gregersen2001; Martins et al. Reference Martins, Hipolito-Reis and Azevedo2001); these include decreased mucosal weight, a reduction in the number and length of microvilli and hence a lowering of the total surface area of the villi and microvilli (Geyra et al. Reference Geyra, Uni and Sklan2001). Refeeding, in contrast, causes a reversal of these effects (Butzner & Gall, Reference Butzner and Gall1990).

Furthermore, the activities of certain enzymes involved in carbohydrate metabolism decrease after fasting (Anderson & Zakim, Reference Anderson and Zakim1970; Budhoski et al. Reference Budhoski, Challis and Newsholme1982; Farooq et al. Reference Farooq, Yusufi and Mahmood2004) and are restored upon refeeding (Stifel et al. Reference Stifel, Rosenweig, Zakim and Herman1968, Reference Stifel, Herman, Rosensweig and Zakim1969; Shakespeare et al. Reference Shakespeare, Srivastava and Hubscher1969; Kotler et al. Reference Kotler, Kral and Bjorntorp1982). In contrast, gluconeogenesis is known to increase with fasting and decrease upon refeeding. A rapid degradation of various proteins and enzymes has also been reported by fasting, whereas refeeding results in increased protein/enzyme synthesis (Holt & Yeh, Reference Holt and Yeh1992; Boza et al. Reference Boza, Moennoz, Vuichoud, Jarret, Gaudard-de-Weck, Fritsche, Donnet, Schiffrin, Perruisseau and Ballevre1999). The profound increase in the activities of the enzymes of glycolysis, of the tricarboxylic acid cycle and to some extent of gluconeogenesis by RTF might be due to an enhanced synthesis of these enzymes resulting from repeated 12 h fasting/12 h refeeding for 30 d. The respective increase or decrease in the activities of alkaline phosphatase, GGTase, LAP and/or sucrase in liver and intestine caused by RTF was found to be due to alterations in the V max rather than K m values. These observations also indicate adaptive but specific alterations in protein/enzyme synthesis. The other regulatory mechanisms might be activated by repeated cycles of a 30 d fasting/refeeding schedule. Elevated serum thyroid hormone levels, as observed in human subjects during Ramadan fasting, might be one of such factors responsible for enhanced metabolic activity (Fedail et al. Reference Fedail, Murphy, Salih, Bolton and Harvey1982).

It has been reported that short-term fasting followed by refeeding gave rise to a disappearance of circadian activity and that the alterations observed were actually produced in anticipation of food, rather than in its presence, by specific adaptative mechanisms (Saito et al. Reference Saito, Murakami, Nishida, Fujisawa and Suda1976) similar to the learning reflexes put forward long ago by Pavlov. It seems reasonable to suggest that rats can be used as a model of human Ramadan fasting because of the similar alterations observed in some blood parameters in rats and reported in man, and also because of similar metabolic changes observed in the daytime and night-time fasting schedules.

We therefore conclude that RTF in rats results in specific adaptive changes in the metabolic activities of both the intestine and the liver. The increased activities of enzymes involved in the degradation as well as the production of glucose suggest that RTF enhances nutrition and energy metabolism. The results provide useful information of significant clinical importance on adaptations to unusual eating habits with restricted energy intake.

Acknowledgements

The Council of Scientific and Industrial Research, New Delhi, India, is acknowledged for its award of a Research Associate fellowship to N. F. Financial support to the department from the University Grant Commission and the Department of Science and Technology, and a research grant (SO/SO/B-93/89) from the Department of Science and Technology to A. N. K. Y. is also gratefully acknowledged.

References

Adlouni, A, Ghalim, N, Benslimane, A, Lecerf, JM & Saile, R (1997) Fasting during Ramadan induces a marked increase in high-density lipoprotein cholesterol and decrease in low-density lipoprotein cholesterol. Ann Nutr Metab 41, 242249CrossRefGoogle ScholarPubMed
Anderson, JW & Zakim, D (1970) The influence of alloxan-diabetes and fasting on glycolytic and gluconeogenic enzyme activities of rat intestinal mucosa and liver. Biochim Biophys Acta 201, 236241CrossRefGoogle ScholarPubMed
Benli Aksungar, F, Eren, A, Ure, S, Teskin, O & Ates, G (2005) Effects of intermittent fasting on serum lipid levels, coagulation status and plasma homocysteine levels. Ann Nutr Metab 49, 7782CrossRefGoogle Scholar
Bernfeld, P (1955) α and β Amylases. In Methods in Enzymology vol. 1, pp. 149151 [Colowick, SP and Kaplan, NO, editors]. New York: Academic PressCrossRefGoogle Scholar
Boza, JJ, Moennoz, D, Vuichoud, J, Jarret, AR, Gaudard-de-Weck, D, Fritsche, R, Donnet, A, Schiffrin, EJ, Perruisseau, G & Ballevre, O (1999) Food deprivation and refeeding influence growth, nutrient retention and functional recovery of rats. J Nutr 129, 13401346CrossRefGoogle ScholarPubMed
Budhoski, L, Challis, RA & Newsholme, EA (1982) Effects of starvation on the maximal activities of some glycolytic and citric acid cycle enzymes and glutaminase in mucosa of the small intestine of the rat. Biochem J 206, 169172CrossRefGoogle Scholar
Buts, JP, Vijverman, V, Barudi, C, De Keyser, N, Maldague, P & Dive, C (1990) Refeeding after starvation in the rat: comparative effects of lipids, proteins and carbohydrates on jejunal and ileal mucosal adaptation. Eur J Clin Invest 20, 441452CrossRefGoogle ScholarPubMed
Butzner, JD & Gall, DG (1990) Impact of refeeding on intestinal development and function in infant rabbits subjected to protein-energy malnutrition. Pediatr Res 27, 245251CrossRefGoogle ScholarPubMed
Cheah, SH, Ch'ng, SL, Husain, R & Duncan, MT (1990) Effects of fasting during Ramadan on urinary excretion in Malaysian Muslims. Br J Nutr 63, 329337CrossRefGoogle ScholarPubMed
Dou, Y, Gregersen, S, Zhao, J, Zhuang, F & Gregersen, H (2001) Effect of re-feeding after starvation on biomechanical properties in rat small intestine. Med Eng Phys 23, 557566CrossRefGoogle ScholarPubMed
El Ati, J, Beji, C & Danguir, J (1995) Increased fat oxidation during Ramadan fasting in healthy women: an adaptative mechanism for body weight maintenance. Am J Clin Nutr 62, 302307CrossRefGoogle ScholarPubMed
Farooq, N, Yusufi, ANK & Mahmood, R (2004) Effect of fasting on enzymes of carbohydrate metabolism and brush border membrane in rat intestine. Nutr Res 24, 407416CrossRefGoogle Scholar
Fedail, SS, Murphy, D, Salih, SY, Bolton, CH & Harvey, RF (1982) Changes in certain blood constituents during Ramadan. Am J Clin Nutr 36, 350353CrossRefGoogle ScholarPubMed
Fingerhut, B, Ferzola, R, Marsh, WH & Miller, AB Jr (1966) Automated methods for blood glucose and urea with adaptation or simultaneous determination. Clin Chem 12, 570576CrossRefGoogle ScholarPubMed
Frost, G & Pirani, S (1987) Meal frequency and nutritional intake during Ramadan: a pilot study. Hum Nutr Appl Nutr 41, 4750Google ScholarPubMed
Geyra, A, Uni, Z & Sklan, D (2001) The effect of fasting at different ages on growth and tissue dynamics in the small intestine of the young chick. Br J Nutr 86, 5356CrossRefGoogle ScholarPubMed
Glossmann, H & Neville, DM (1972) γ Glutamyl transferase in kidney brush border membranes. FEBS Lett 19, 340344CrossRefGoogle ScholarPubMed
Goldmann, DR, Schlesinger, H & Segal, S (1976) Isolation and characterization of the brush border fraction from newborn rat renal proximal tubule cells. Biochim Biophys Acta 419, 251260CrossRefGoogle ScholarPubMed
Gumaa, KA, Mustafa, KY, Mahmoud, NA & Gader, AMA (1978) The effects of fasting in Ramadan 1. Serum uric acid and lipid concentrations. Br J Nutr 40, 573581CrossRefGoogle ScholarPubMed
Hallack, MH & Nomani, MZ (1988) Body weight loss and changes in blood lipid levels in normal men on hypocaloric diets during Ramadan fasting. Am J Clin Nutr 48, 11971210CrossRefGoogle Scholar
Holt, PR & Yeh, KY (1992) Effects of starvation and refeeding on jejunal disaccharidase activity. Dig Dis Sci 37, 827832CrossRefGoogle ScholarPubMed
Hubscher, G & Sherrat, HAS (1962) Oxidation and glycolysis in subcellular fractions from small intestinal mucosa. Biochem J 84, 24Google Scholar
Husain, R, Duncan, MT, Cheah, SH & Ch'ng, SL (1987) Effects of fasting in Ramadan on tropical Asiatic Moslems. Br J Nutr 58, 4148CrossRefGoogle ScholarPubMed
Kempson, SA, Kim, JK, Northrup, TE, Knox, FG & Dousa, TP (1979) Alkaline phosphatase in adaptation to low dietary phosphate intake. Am J Physiol 237, E465E473Google ScholarPubMed
Kessler, M, Acuto, O, Storelli, C, Murer, H, Muller, M & Semenza, G (1978) A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems. Biochim Biophys Acta 506, 136154CrossRefGoogle ScholarPubMed
Kornberg, A (1955) Lactic dehydrogenase of muscle. In Methods in Enzymology vol. 1, pp. 441443 [Colowick, SP and Kaplan, NO, editors]. New York: Academic PressCrossRefGoogle Scholar
Kotler, DP, Kral, JG & Bjorntorp, P (1982) Refeeding after a fast in rats: effects on small intestinal enzymes. Am J Clin Nutr 36, 457462CrossRefGoogle ScholarPubMed
Leiper, JB, Molla, AM & Molla, AM (2003) Effects on health of fluid restriction during fasting in Ramadan. Eur J Clin Nutr 57, S30S38CrossRefGoogle ScholarPubMed
Lowry, OH, Rosebrough, NJ, Farr, AL & Randall, RJ (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193, 265275CrossRefGoogle ScholarPubMed
Maislos, M, Abou-Rabiah, Y, Iordash, S & Shany, S (1998) Gorging and plasma HDL-cholesterol – the Ramadan model. Eur J Clin Nutr 52, 127130CrossRefGoogle ScholarPubMed
Malhotra, A, Scott, PH, Scott, J, Gee, H & Wharton, BA (1989) Metabolic changes in Asian Muslim pregnant mothers observing the Ramadan fast in Britain. Br J Nutr 61, 663672CrossRefGoogle ScholarPubMed
Martinkova, A, Lenhardt, L & Mozes, S (2000) Effect of neonatal MSG treatment on day-night alkaline phosphatase activity in rat duodenum. Physiol Res 49, 339345Google ScholarPubMed
Martins, MJ, Hipolito-Reis, C & Azevedo, I (2001) Effect of fasting on rat duodenal and jejunal microvilli. Clin Nutr 20, 325331CrossRefGoogle ScholarPubMed
Mayhew, TM (1987) Quantitative ultrastructural study on the responses of microvilli along the small bowel to fasting. J Anat 154, 237243Google Scholar
Meyer, AH, Kornberg, A, Grisolia, S & Ochoa, S (1948) Enzymic mechanism of oxidation reactions between malate or isocitrate and pyruvate. J Biol Chem 174, 961977Google Scholar
Nomani, MZA, Hallack, MH, Nomani, S & Siddiqui, IP (1989) Changes in blood urea and glucose and their association with energy containing nutrients in men on hypocaloric diets during Ramadan fasting. Am J Clin Nutr 49, 11411145CrossRefGoogle ScholarPubMed
Ochoa, S (1955a) Isocitrate dehydrogenase system (TPN) from pig heart. In Methods in Enzymology vol. 1, pp. 699704 [Colowick, SP and Kaplan, NO, editors]. New York: Academic PressCrossRefGoogle Scholar
Ochoa, S (1955b) Malic enzyme from pigeon liver and wheat germ. In Methods in Enzymology vol. 1, pp. 739747 [Colowick, SP and Kaplan, NO, editors]. New York: Academic PressCrossRefGoogle Scholar
Roky, R, Houti, I, Moussamih, S, Qotbi, S & Aadil, N (2004) Physiological and chronobiological changes during Ramadan intermittent fasting. Ann Nutr Metab 48, 296303CrossRefGoogle ScholarPubMed
Saito, M, Murakami, E, Nishida, Y, Fujisawa, Y & Suda, M (1976) Circadian rhythms of digestive enzymes in the small intestine of rat. II. Effects of fasting and refeeding. J Biochem (Tokyo) 80, 563568zzCrossRefGoogle ScholarPubMed
Shakespeare, P, Srivastava, LM & Hubscher, G (1969) Glucose metabolism in the mucosa of the small intestine: the effect of glucose on hexokinase activity. Biochem J 111, 6367CrossRefGoogle ScholarPubMed
Shen, CS & Mistry, SP (1979) Effects of fasting and refeeding on hepatic and renal gluconeogenic enzymes in chicken. Poult Sci 58, 890895CrossRefGoogle ScholarPubMed
Shonk, CC & Boxer, GE (1964) Enzyme patterns in human tissues. I. Methods for determination of glycolytic enzymes. Cancer Res 24, 709721Google ScholarPubMed
Shull, KH, Ashmore, J & Mayer, J (1956) Hexokinase, glucose-6-phosphatase and phosphorylase levels in hereditarily obese, hyperglycemic mice. Arch Biochem Biophysics 62, 210216CrossRefGoogle ScholarPubMed
Stifel, FB, Herman, RH, Rosensweig, NS & Zakim, D (1969) Dietary regulation of glycolytic enzymes. III. Adaptive changes in rat jejunal pyruvate kinase, phosphofructokinase, fructosediphosphatase and glycerol-3-phosphate dehydrogenase. Biochim Biophysics Acta 184, 2934CrossRefGoogle Scholar
Stifel, FB, Rosenweig, NS, Zakim, D & Herman, RH (1968) Dietary regulation of glycolytic enzymes. I. Adaptive changes in rat jejunum. Biochim Biophysics Acta 170, 221227CrossRefGoogle ScholarPubMed
Szczepanska-Konkel, M, Yusufi, ANK & Dousa, TP (1987) Interaction of [14C] phosphonoformic acid with renal cortical brush border membrane. J Biol Chem 262, 80008010CrossRefGoogle Scholar
Tausky, HH & Shorr, EA (1953) A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem 202, 675685CrossRefGoogle Scholar
Toda, M & Morimoto, K (2000) Effects of Ramadan fast on health of Muslims. Nippon Eiseigaku Zasshi 54, 592596CrossRefGoogle ScholarPubMed
Yucel, A, Degirmenci, B, Acar, M, Albayrak, R & Haktanir, A (2004) The effect of fasting month of Ramadan on the abdominal fat distribution: assessment by computed tomography. Tohoku J Exp Med 204, 179187CrossRefGoogle ScholarPubMed
Yusufi, ANK, Low, MG, Turner, ST & Dousa, TP (1983) Selective removal of alkaline phosphatase from renal brush border membrane and sodium dependent brush border membrane transport. J Biol Chem 258, 56955701CrossRefGoogle ScholarPubMed
Zlatkis, A, Zak, B & Boyle, AJ (1953) A new method for the direct determination of serum cholesterol. J Lab Clin Med 41, 486492Google ScholarPubMed
Figure 0

Table 1 Effect of daytime and night-time Ramadan-type fasting on serum parameters(Mean values with their standard errors and percentage change from control values (%) for 12 rats per group)

Figure 1

Table 2 Effect of Ramadan-type fasting on body and mucosal weight of rats(Mean values with their standard errors for three different experiments with four rats per group and change from control values (%))

Figure 2

Table 3 Effect of Ramadan-type fasting on the activities of alkaline phosphatase, γ-glutamyl transpeptidase and sucrase in the mucosal homogenates and brush border membrane preparations(Mean values with their standard errors for specific activities (μmol/mg protein per h) for three different experiments with their respective change from control values (%))

Figure 3

Table 4 Effect of Ramadan fasting on the kinetic parameters of alkaline phosphatase and sucrase(Means values with their standard errors for three different experiments with their respective change from control values (%))

Figure 4

Table 5 Effect of Ramadan type fasting on the specific activities of lactate dehydrogenase (LDH), isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) in homogenates of intestinal mucosa(Mean values with their standard errors for specific activities (μmol/mg protein per h) for three different experiments with their respective change from control values (%))

Figure 5

Table 6 Effect of Ramadan-type fasting on the specific activities of fructose 1,6-bisphosphatase, glucose-6-phosphatase (G6Pase), glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme in homogenates of intestinal mucosa(Mean values with their standard errors for specific activities (μmol/mg protein per h) for three different experiments with their respective change from control values (%))

Figure 6

Table 7 Effect of daytime and night-time Ramadan-type fasting on the specific activities of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), fructose 1,6-bisphosphatase (FBPase), glucose-6-phosphatase (G6Pase), gluose-6-phosphate dehydrogenase (G6PDH) and malic enzyme in liver homogenate(Mean values with their standard errors for specific activities (μmol/mg protein per h) for three different experiments with their respective change from control values (%))

Figure 7

Table 8 Effect of daytime and night-time Ramadan-type fasting on the specific activities of alkaline phosphatase, γ-glutamyl transpeptidase and leucine aminopeptidase in liver homogenate(Mean values with their standard errors for specific activities (μmol/mg protein per h) for three different experiments with their respective change from control values (%))