The effecT of fasTing on The glycogen meTabolism in heaT-acclimaTed raTs �IljANA

We investigated the influence of successive fasting for �4�� 48�� 7��� and 96 h on some key enzymes and substrates of liver�� kidney�� and muscle in control and heat-acclimated (3� days at 35 ± 1°C� rats� Short-term fasting (for �4 and 48 h� resulted in decrease of liver glycogen content�� blood glucose level�� and concentration of glucose-6-phosphate�� as well as increase of glucose-6-phosphatase activity�� regardless of the previous temperature of acclimation� During a period of prolonged fasting (for 7� and 96 h��� there was a rebound of liver glycogen content only in animals kept at room temperature� Fasting induced increase of renal glycogen content in animals kept at room temperature and increase of renal glucose-6-phosphatase activity in both experimental groups� As for muscle metabolism�� endogenous nutrition resulted in decrease of muscle glycogen content in heat-acclimated animals� Activity of muscle glycogen phosphorylase (a+b� was decreased in the control and increased in heat-acclimated animals� The obtained results indicate that the examined carbohydrate-related parameters show time-dependent changes during 4 days of fasting� Twenty-fourand 48-h fasting intensifies glycogenolytic processes�� while 7�and 96-h fasting intensifies gluconeogenic processes�� doing so to a lesser extent in heat-acclimated animals� The changes caused by the fasting were modified by acclimation to moderate heat�� primarily in the liver and to a lesser extent in the kidney and muscle�


INTRODUCTION
Fasting is a specific metabolic state of the organism accompanied by a number of hormonal and enzymatic disturbances which cause changes in the direction of metabolic processes (� u y s e et al��� ������ � u y s e et al��� ������ et al��� ������ The effects of fasting�� i�e��� of endogenous nutrition�� are mostly manifested in changes of carbohydrate metabolism� In humans�� total endogenous production of glucose is known to decrease during prolonged fasting (O w e n �� 1969�� This decrease is essentially due to decrease of hepatic gluconeogenesis�� which is partially balanced by increase of gluconeogenesis in the kidney� Glucose production by the human kidney in the postapsorptive state accounts for about 5% of total endogenous glucose production (O w e n e t a l � �� 1969��� whereas during fasting for 6� h the net renal glucose output was reported to be ��% of total glucose production (E c k b e r g e t a l � �� 1999�� As for muscle carbohydrate metabolism�� 18 to 36% contributions of glucose production through the Cory cycle were reported during 1�-and 4�-h fasting�� respectively (K a t z and Ta y e k �� 1998�� Carbohydrate metabolism is also affected by acclimation of the organism to moderate environmental heat� Acclimation to moderate heat alters metabolic pathways in the direction of rebound of hepatic glycogen stores (M i t e v�� 1983; M i t e v and � u z a l k o v�� 1985; M i t e v et al��� 1991; D i n e v s k a �� 199�; D i n e v s k a -K j o v k a r o v s k a �� 1998��� increased gluconeogenesis (D i n e v s k a -K j o v k a r o v s k a �� 1998��� and decrease in the rate of metabolism (K a t s u m a t a et K a t s u m a t a et al��� 199�; H o r o w i t z 1994�� The effecT of fasTing on The glycogen meTabolism in heaT-acclimaTed raTs Since both fasting and acclimation to heat have specific effects on the effect of fasting on carbohydrate metabolism in animals acclimated to moderate heat�� there is a need for further research on the common effect of both factors� Such research would contribute to clarification of the metabolic changes caused by high environmental temperature during endogenous nutrition� To our knowledge�� there are no published data on the effect of fasting on carbohydrate metabolism in animals acclimated to moderate heat� Our goal was to estimate the effect of long-term heat acclimation on changes of some glycogen-metabolizing enzymes and substrates in the liver�� kidney�� and muscle during four days of endogenous nutrition�� with special emphasis on distinguishing changes caused by short-term (�4-and 48-h� as opposed to long-term (7�-and 96-h� fasting�

MATERIAlS AND METHODS
Experimental animals.Experiments were performed on 11� female Wistar laboratory rats�� weighing 17�-�1� g� The animals were kept under conditions of a 1�-h light regime (light from 6:�� to 18:�� h�� Experimental conditions.The effects of �4-�� 48-�� 7�-�� and 96-h fasting were estimated in control rats (kept at room temperature of �� ± �°C� and heat-acclimated animals� Heat acclimation for 3� days was performed in a special heated chamber at a temperature of 35±1°C and relative air humidity of 3�-4�%� The fasting animals received water ad libitum during the whole experimental period and were sacrificed after fasting for �4�� 48�� 7��� and 96 h� Comparisons were drawn between fed animals from room temperature and ones heat-acclimated for 3� days�� as well as between the respective groups at different tempera-tures� Isolation and preparation of material for analysis� The experimental animals were anesthetized with ether� After laparatomy�� blood was taken from the v� cava posterior� Pieces of the liver�� right kidney�� and right m� gastrocnemius were isolated and immediately frozen in liquid nitrogen� The frozen tissues were kept at -8�°C until the analyses� Sacrifice of the ani-mals was performed always in the period between 8:�� and 9:�� h� Homogenization of the material for analysis was performed with Cole-Parmer 471� ultrasonic homogenizator for a period of 1�-15 s� The whole procedure was performed at a temperature of � -4°C (on ice�� Analytical methods.The activity of glucose-6-phosphatase in the liver and kidney (H e r s �� 1959� and that of muscle glycogen phosphorylase (a + b� (M o r g a n and P a r m e g g i a n i �� 1964� were determined in tissue homogenates� Enzyme activity was determined indirectly through the quantity of inorganic phosphate produced (F i s k e and S u b b a r o w�� 19�5�� Protein content was determined by the l o w r y method (1951�� The concentration of glucose-6-phosphate was indirectly calculated through changes in the concentration of NADPH (l a n g and M i c h a l �� 1974�� Glycogen content was determined by the anthrone method (S e i f t e r et al��� 1951��� the blood glucose level by the enzyme-colorimetric method with a GOD-PAP instrument (Chronolab�� Statistics.Results are presented as means ± SD� To examine statistical differences between each group and the control�� we used one way ANOvA analysis with the Newman-Keuls post-hoc test� linear regression and correlation analyses between the parameters were performed and only significant coefficients are presented� In all tests�� differences with a probability level of p<���5 were considered significant� RESUlTS �lood glucose level� The obtained results (Fig� 1� showed that fasting for �4 to 96 h causes a continuous and significant decrease of the blood glucose level in animalskept at room temperature (from -13�7 to -31�1%�� p<���5��� whereas in heat-acclimated animals significant changes occur only during prolonged fasting for 7� and 96 h (from -1��5% to -17�7%�� p<���5�� As for the effects of high environmental temper-ature�� we found that only the control-fed and �4-h fasted heat-acclimated animals have a significantly lower glucose level compared to the corresponding groups from room temperature (C:C' �� p<���5; �4 : �4' �� p<���5�� Liver Glycogen content� Our results showed that 4-day fasting leads to a significant decrease of hepatic glycogen content (Fig� �� regardless of previous thermal acclimation (from -83�4% to -97�4%�� p<���5 for animals from room temperature and from -95�7% to -96�7%�� p<���5 for heat acclimated animals�� In comparison with short-term fasting�� during prolonged fasting (for 7� and 96 h� there was a significant increase of liver glycogen content in the animals from room temperature (�4:7��� +38���7%�� p<����5 and �4:96�� +155���%�� p<����5��� even though significantly less marked than in the control-fed ani-mals� It is important to note that in heat-acclimated rats subjected to prolonged fasting there were no significant changes of liver glycogen content in com-parison with the short-term fasted animals (�4':7�' and �4':96' �� n�s�� With respect to heat acclimation�� we found higher hepatic glycogen content in fed heat-acclimated animals compared to those from room temperature (C:C' �� p<���5�� Short-term fasted (for �4 and 48 h� heat-acclimated animals have a significantly higher hepatic glycogen content (+56�8 to +11���%�� p <���5��� while ones fasted for 7� and 96 h have significantly lower glycogen content compared to animals kept at room temperature (from -5��1% to -7���%�� p <���5�� Glucose-6-phosphatase activity.It can be seen from the obtained results (Fig� 3� that fasting caused increase in the activity of glucose-6-phosphatase in animals from both thermal environments� As for animals from room temperature�� there was a less intensive increase during the first �4 and 48 hours (from +43�4 to +58��%�� p<���5� and a more intensive one during prolonged fasting (from +1�9�5 to
Table 3. Statistical analysis of differences between experimental groups with respect to liver glucose-6-phosphatase activity.Table 4. Statistical analysis of differences between experimental groups with respect to glucose-6-phosphate concentration.

Fig. 7.
Renal glucose-6-phosphatase activity during fasting in control and heat-acclimated rats.Legend as in Fig. 1.

DISCUSSION
The liver and fasting.The obtained results point to significant changes in all examined parameters in the liver�� which vary depending on previous thermal acclimation and the duration of fasting� The observed changes occurred in two phases -during short-term fasting (for �4 to 48 h� and during long- Table 7. Statistical analysis of differences between experimental groups with respect to muscle glycogen content.term (prolonged� fasting (for 7� to 96 h�� It can be seen that intensive mobilization of liver glycogen content occurs in the first 48 h of fasting� This decrease of glycogen reserves in the liver is almost the same in animals from both thermal environments (about 95%�� Fig� ��� However�� it is very important to note that at this time (�4 and 48 h� heat-acclimated fasted animals have a higher glycogen content compared to the corresponding groups from room temperature (�4:�4'�� +58��6% and 48:48'�� +11�%�� p<����5��� This might be a result of acclimation changes in animals before the period of fasting�� i�e��� higher glycogen content was recorded in heat-acclimated fed rats in comparison with the fed rats kept at the room temperature� Decrease of liver glycogen content in the first 48 h of fasting is accompanied by increase of glucose-6-phosphatase activity in animals of both experimental groups (Fig� 3��� but this increase is more pronounced in heat-acclimated ones� According to some previous results obtained on fasted rats from room temperature (M i n a s s i a n and M i t h i e u x �� 1994; M i t h i e u x �� 1996; M i n a s s i a n et al��� 1999��� glucose-6-phosphatase activity increases in the first 48 h of endogenous nutrition�� which is a result of induction of the mRNA responsible for synthesis of glucose-6-phosphatase (D h a h b i et al��� ���1�� In the same experimental period�� we recorded decrease in the concentration of hepatic glucose-6-phos-phate�� regardless of previous thermal acclimation (Fig� 4�� Our results show that changes in the concentration of glucose-6-phosphate are more intensive in heat-acclimated animals�� which might be a consequence of the higher glucose-6-phosphatase activity observed in these animals� This�� together with decreased glycogen content in the liver�� points to increased glycogenolysis in the first 48 h of the fasting period� As for glucose levels in the blood�� during shortterm fasting we found significant decrease only in rats kept at room temperature�� non-significant changes being recorded in heat-acclimated ones� This might be a consequence of the fact that heat acclimation by itself probably causes decrease of the blood glucose level in fed animals (C:C' �� -����1%�� p<����5�� In contrast to short-term fasting�� during prolonged fasting (for 7� and 96 h� we observed a rebound of hepatic glycogen�� but only in the animals kept at room temperature (Fig� ���� which probably is a result of increased intensity of gluconeogenesis (j a h o o r et al��� 199�; l a n d a u et al��� 1996; C h a n d r a m a u l i et al�� 1997�� According to va n d e We r v e and j e a n r e n a u d (1987��� increased re-synthesis of glycogen in the liver does not involve activation of glycogen synthesis or inactivation of glycogen phosphorylation�� but is regulated through the concentration of glucose-6-phosphate as a substrate precursor for synthesis of glycogen� However�� contrary to the findings of M i n a s s i a n and Mithieux�� (1994��� instead of increased concentration of glucose-6-phosphate and decreased activity of glucose-6-phosphatase after 3-4 days of fasting�� we found decrease of the substrate and increase of the enzyme in the same experimental period� We assume that the process of gluconeogenesis is accelerated and glucose-6-phosphate is intensively consumed by the increased glucose-6-phosphatase activity� Moreover�� it might be that not only gluconeogenesis to glucose�� but also resynthesis of glycogen from gluconeogenic substrates and glucose-6-phosphate is intensified�� while glycogenolysis is decreased� It is important to note that there is no rebound of hepatic glycogen in heat-acclimated rats during a period of prolonged fasting (Fig� ��� According to C h a y o t h et al� (198���� �4-h fasting caused sixfold elevation in hepatic gluconeogenesis in the control�� whereas only fourfold enhancement of this pathway was found in heat-acclimated animals�� which suggests that a decrease of hepatic gluconeogenesis in heat acclimation occurs in fasted animals� Also�� lowered levels of branched-chain amino acids and arginine were found in heat-acclimated hamsters�� which implies that this lower level of gluconeogenic substrates causes decrease in the intensity of the gluconeogenesis (C h a y o t h et al��� 1984�� In the same experimental period�� heat-acclimated rats showed a tendency toward normalization (decrease� of glucose-6-phosphatase activity that resulted in normalization (increase� of glucose-6-phosphate� We found that heat-acclimated animals We found that heat-acclimated animals (fasted for 7�-and 96 h� have a lower activity of glucose-6-phosphatase and higher concentration of glucose-6-phosphate compared to the same animals from room temperature� This indicates a high negative correlation between the enzyme and the substrate regardless of thermal acclimation (Fig� 5�� From the above data�� it can be assumed that endogenous glucose production during short-term fasting is due to intensive liver glycogenolysis�� whereas during prolonged fasting the process of gluconeogenesis is favored� The intensity of glycogenolysis and gluconeogenesis depends on previous thermal acclimation� The kidney and fasting.As one of the gluconeogenic organs�� the kidney plays a significant part in the maintenance of glucose homeostasis in fasting con-ditions� It is well known that in the fed condition kidney gluconeogenesis provides about 5-1�% of total glucose (O w e n et al��� 1969� and about ��-�5% during 6�-h fasting (E k b e r g et al��� 1999�� We investigated the biochemical pathways involved in the kidney carbohydrate metabolism of fasted heat-acclimated rats through glycogen content (Fig� 6� and the activity of glucose-6-phosphatase (Fig� 7�� During 4 days of fasting�� glycogen content shows a tendency to increase in animals of both experimental groups (but with significantly lower values in heat-acclimated ones�� Thus�� as in the liver�� the increase of kidney glycogen in heatacclimated rats is less marked than in animals kept at room temperature� With respect to renal glucose-6-phosphatase�� we observed a continuous increase in activity of the enzyme throughout the whole period of fasting (Fig� 7� in both thermal groups�� but lower enzyme activity was recorded in the heat-acclimated ones� There are also time-dependent differences in the contributions of hepatic and renal gluconeogenesis during fasting� To be specific�� hepatic glucose-6phosphatase is dominant in the first 48 h of the fasting�� the renal enzyme only during 7�-and 96-h fasting (M i n a s s i a n and M i t h i e u x �� 1994�� It was previously reported that contributions of these Changes in the examined parameters caused by exposure to high environmental temperature are of special interest� In animals previously acclimated to moderate heat�� we found decrease of muscle glycogen content (Fig� 8� and increase in the activity of glycogen phosphorylase a+b (Fig� 9�� Compared to the animals exposed to room temperature�� heatacclimated animals have lower glycogen content in muscle tissue and somewhat higher activity of muscle glycogen phosphorylase� Increased activity of glycogen phosphorylase in these animals probably results in additional decrease of glycogen content in the muscles� On the basis of the obtained results�� we can conclude that in the condition of endogenous nutrition�� carbohydrate-metabolizing enzymes and substrates in the liver�� kidney�� and muscle are highly influenced by temperature as one of the most important ecological factors of the environment� role of glucose-6-phosphatase in the repletion of liver glycogen during refeeding in fasted rats� Biochim.Biophys.Добијени резултати указују да испитивани параметри угљених хидрата показују временом условљене промене током четири дана гладовања� Гладовање у трајању од �4 и 48 часова интензификује гликогенолитичке процесе�� док гладовање током 7� и 96 часова интензификује глуконеогене процесе�� не у мањој мери код топло аклимованих пацова� Промене узроковане гладовањем мењају се током аклимације на умерену топлоту�� првенствено у јетри�� а у мањем обиму и у бубрезима и мишићима�

Fig. 6 .
Fig. 6.Renal glycogen content during fasting in control and heat-acclimated rats.Legend as in Fig. 1.

Fig. 8 .
Fig. 8. Muscle glycogen content during fasting in control and heat-acclimated rats.Legend as in Fig. 1.
two organs during prolonged fasting are almost the same (O w e n et al��� 1969�� According to C l u t t e r and C r y e r (1994��� increased renal gluconeogenesis during endogenous nutrition is a mechanism for combating the production of ammonia and metabolic acidosis characteristic of fasting� To be specific�� it is known that production of ammonia is associated with intensive gluconeogenesis from glutamine (P o g s o n et (P o g s o n et et al��� 1976�� Metabolic acidosis causes induction of ��� 1976�� Metabolic acidosis causes induction of � Metabolic acidosis causes induction of the PEPCK gene and increases the activity of this enzyme (K a i s e r and C u r t h o y s �� 1991�� (K a i s e r and C u r t h o y s �� 1991�� � The above-mentioned findings emphasise the importance of kidney gluconeogenesis in the condition of prolonged fasting� The obtained results -lower glycogen content and lower activity of glucose-6-phosphatase in heat-acclimated fasted rats -indicate reduced intensity of kidney gluconeogenesis in these animals� Muscle tissue and fasting.Since muscle has a glycogenolytic enzyme profile�� we studied changes caused by fasting and heat acclimation through its glycogen content and the activity of total glycogen phosphorylase (a+b� � It can be seen that fasting causes decrease of muscle glycogen content and glycogen phosphorylase activity (Figs� 8 and 9�� According to S a k a d a et al� (1987��� muscle glycogen participates in the maintenance of glycemia in fasting rats (through the Cory cycle��� but only during prolonged fasting�� after consumption of hepatic glycogen reserves� It is important to note that in humans�� the major part of glucose production after overnight fasting derives from lactate and alanine�� the rest consisting of the glutamine flux emerging from muscle proteins (S a k a d a et al��� 1987� Other investigators found a progressive decrease of muscle glycogen in fasting rats (H i r o s h e et al��� 1986; O i et al��� 1997��� the indicated decrease being accompanied by an increase in the activity of glycogen phosphorylase (H i r o s h e et al��� 1986��

Table 1 .
Statistical analysis of differences between experimental groups with respect to blood glucose level.Fig. 2. Liver glycogen content during fasting in control and heat-acclimated rats.Legend as in Fig. 1.

Table 2 .
Statistical analysis of differences between experimental groups with respect to liver glycogen content.

Table 8 .
Statistical analysis of differences between experimental groups with respect to muscle glycogen phosphorylase (a+b) activity.