.4cta pharmacol. et toxicol. 1976,38, 10-16.
From the Division of Hepatology, Medical Department A and the Department of Psychiatry, Rigshospitalet, DK-2100 Copenhagen 0, Denmark
The Influence of Lithium on Carbohydrate and Lipid Metabolism in the Perfused Rat Liver BY P. B. Vendsborg and H. Vilstrup (Received April 29, 1975; Accepted May 26, 1975)
Abstract: Isolated rat liver was perfused with a once-through technique. Glycogen synthesis was not affected by the presence of lithium (1 or 5 mM) in the perfusion medium. The rate of glycogen breakdown both without and with glucagon stimulation was not affected by lithium (1 mM). The uptake of free fatty acids was stimulated by lithium (5 mM) in the perfusion medium and a trend towards an increase in deposition of triglyceride in the same order of magnitude was seen.
Key-words: Lithium - liver
- liver
glycogen - free fatty acids
- triglycerides.
Several investigators have shown that lithium influences the metabolism of glucose. The uptake of glucose in tissues is increased both in vitro (BHATTACHARYA 1959, 1961 & 1964; CLAUSEN 1968a & b), and in intact man (VAN DER VELDE & GORDON 1969; VENDSBORG & RAFAELSEN 1973). An increased amount of glycogen in muscle and brain has been found after lithium administration (PLENGEet al. 1970). Glycogen in liver has been reported to be decreased after a single lithium dose (PLENGEet al. 1970), but increased (KRULIK& Zvo~sIck1970) or unchanged (OLESEN& THOMSEN 1974) after longer periods of lithium administration. Our previous findings of increased glucose disposal in man (VENDSBORG & RAFAELSEN 1973) pointed to the liver as one of the sites of extra glucose uptake. The mechanism by which lithium influences fat metabolism has not been investigated in detail although obesity is the most serious side effect to et al., unpublished lithium treatment (KERRYet al. 1970), (VENDSBORG results). In animals given lithium for longer periods increased weight gain et al. 1973). has been found (PLENGE The purpose of the present investigation was to estimate the influence of lithium on the metabolism of glucose and free fatty acids in the liver. No
LITHIUM AND HEPATIC METABOLISM
11
influence of lithium on glucose uptake could be demonstrated in the isolated liver but the uptake of free fatty acids (FFA) was increased.
Materials and Methods Female Wistar rats weighing about 200 g were used. The fasted rats were fasted for 20 hours. Isolation of the liver for perfusion was performed by the technique of HEMS et al. (1966), except that the perfusion was started immediately after cannulation of the portal vein. The once-through technique was used for the perfusion. The medium consisted of Krebs-Henseleit-buffer, 3 per cent (w/v) bovine serum albumin powder fraction V, and about 7 mmoVl of haemoglobin as bovine red cells washed three times with saline. Lactate was not added to the medium but was present possibly due to the bovine erythrocytes in a final concentration of about 1 mM. Lithium was added as a 150 mM lithium chloride solution. In control experiments the same amount of 150 mM sodium chloride solution was added. Free fatty acids (FFA) were added to the medium as a complex of palmitic acid and albumin, prepared according to the method of VAN HARKEN et al. (1969). The medium was oxygenated with 95 per cent atmospheric air and 5 per cent carbon dioxide. The pH was adjusted to 7.4. The perfusion was performed in a thermostated box of plexiglass at 37'. The flow was 8-10 ml/min. and the pressure in the portal vein was 10-15 cm of water. Samples for measurements were taken from the reservoirs before and after the liver. The substrate determinations were performed according to the following methods: Lactate (HOHORST 1962), glucose by the hexokinase method (BERGMEYER1970) and FFA by the method of FALHOLT et al. (1973). The oxygen consumption was calculated from measurements of oxygen saturation (haemoreflector). Furthermore tissue samples for glycogen determination were taken with the freeze clamp technique, and for determination of triglyceride a small sample (20-40 mg) was quickly frozen in a deep freeze. The tissue concentration of glycogen was determined as glucose after hydrolysis (PLENGEet al. 1970), and the tissue concentration of triglyceride by the method of CARLSON(1963) after sonification in the extraction solution. The rate of metabolism was estimated from the difference between the concentration of metabolites in affluent and effluent medium and perfusion flow. The t-test was used to test statistical significance.
Results Table 1 shows glycogen synthesis with 30 mM glucose in the perfusion medium. There was an average glycogen synthesis of about 0.3 pmoVmin. per gram of wet liver weight. Both with 1 and 5 mM of lithium chloride, control experiments were run because of the addition of different amounts of 150 mM sodium chloride to the controls. Neither 1 nor 5 mM lithium in the perfusion medium changed the rate of glycogen synthesis. The release of glucose from perfused fed rat livers to a medium without glucose was about the same in two consecutive 60 minutes' periods and unaffected by lithium (table 2). A small uptake of lactate took place, decreasing with time, but was not changed by the presence of lithium.
P. B. VENDSBORG AND H. VILSTRUP
12
Table 1. Glycogen synthesis in perfused livers from fasted rats without and with lithium in the medium. Results are expressed as means f S.E.M. with number of experiments in brackets. The rats were starved for 20-24 hours. The livers were perfused for 70 minutes. Liver lobes for glycogen estimation were taken at 10 and 70 minutes. Perfusion medium Glucose Lithium
Glycogen synthesis pmol glucose/min./g liver wet weight
f 0.06 (11) If: 0.06 (9)
30 mM 30 mM
0 1mM
0.29 0.27
30 mM 30 mM
0 5 mM
0.29 k 0.08 (11) 0.29 k 0.08 (11)
Table 3 shows the release of glucose before and after stimulation with two different doses of glucagon. The high dose which was calculated to give maximum glycogen break-down resulted in a 4 fold increase in glucose output. The smaller submaximal dose gave a 3 fold increase. Lithium did not affect the glucagon response. The uptake of free fatty acids in perfused livers from fasted rats is seen in table 4. In the perfusions with lithium in the medium the uptake was about 20 higher than in the controls (P < 0.05). The amount of triglycerides in the liver before and after perfusion with FFA containing medium is also seen in table 4. In the livers perfused with lithium there was about 20 per
Table 2. Release of glucose and uptake of lactate by perfused livers from fed rats without and with lithium in the medium. Results are expressed as means +. S.E.M. with number of experiments in brackets. Each perfusion lasted for 120 minutes. The medium was changed at 60 minutes from lithium free to lithium containing or vice versa. No glucose was present in the affluent medium.
Time (min.)
Lithium
Glucose release pmol/min./g liver wet weight
Lactate uptake pmol/min./g liver wet weight
0-60
1mM 0
1.38 k 0.10 (3) 1.06 f 0.32 (3)
0.37 k 0.05 (2) 0.21 k 0.07 (2)
0 1mM
1.41 1.45
60-120
0-60 60-120
f 0.17 (3) f 0.43 (3)
0.45 0.21
f 0.01 (2) f 0.02 (2)
LITHIUM AND HEPATIC METABOLISM
13
Table 3 . Release of glucose after stimulation with glucagon from perfused livers of fed rats without and with lithium in the medium. Results are expressed as means k S.E.M. with number of experiments in brackets. The livers were perfused for 40 minutes. After 20 minutes perfusion glucagon was added to the perfusion medium. Glucose was estimated in the perfusion medium used from 0 to 20 and from 20 to 40 minutes. No glucose was present in the affluent medium. Glucagon
Lithium
Nil Nil 3 * 10-"JM 3 . 10-1OM 10-QM 10-9~
0 1 mM 0
Glucose release pmol/min./g liver wet weight 1.31 ? O.lO(5) 1.29 k 0.06 ( 5 ) 4.05 C 0.69 (5 ) 3.92 f 0.65 (5) 4.83 ? 0.49 (4) 5.50 C 0.28 (4)
1 mM 0 1 mM
cent more triglyceride than in the controls, but the difference did not reach statistical significance. No measurable triglyceride was present in the perfusion medium either before or after it had passed through the liver. Discussion
In a previous investigation (VENDSBORG & RAFAELSEN 1973) we have found that lithium caused an increased glucose tolerance in man (20-100 & per cent) with a very low concomitant phosphate uptake. FORSHAM THORN(1949) have shown that the phosphate uptake following hepatic
Table 4. Uptake of free fatty acids and triglyceride synthesis in perfused livers from fasted rats without and with lithium in the medium. Results are expressed as means k S.E.M. with number of experiments in brackets. Rats were fasted for 20-24 hours. Glucose concentration was 10 mM and FFA concentration 1.4 mM. Liver samples for triglyceride estimation were taken at 0 and 120 minutes. Lithium
F F A uptake pmol/min./g liver wet weight
Triglyceride synthesis pg/min./g liver wet weight
5 mM 0
1.22 k 0.063 (18) 1.02 f 0.058 (18)
139 C 9 (3) 119 f 6 (3)
14
P. B. VENDSBORG AND H.VILSTRUP
glucose disposal is smaller than following muscular glucose disposal. The small extra uptake of phosphate which followed the increase in glucose uptake induced by lithium therefore pointed to the liver as the site of the phenomenon. We were neither able to induce glycogen synthesis with physiological glucose concentrations nor with 2-3 times normal fasting level. When the glucose concentration was raised to 30 mM glycogen synthesis took place as reported by other investigators (WHITTON & HEMS1971). It was not possible to stimulate the synthesis either with 1 or with 5 mM lithium chloride. This is in contrast with in vitro findings in muscle tissue (CLAUSEN 1968a & b) and in rat muscle, adipose tissue and brain in vivo (BHATTACHARYA 1964; PLENGE et al. 1907). Two main mechanisms have been proposed for the increased glucose uptake and glycogen synthesis caused by lithium in some tissues: Increase in membrane permeability (CLAUSEN 1972; KOHN & CLAUSEN 1972) and et al. 1970). It is not stimulation of glycogen synthesizing enzymes (WALAAS known which mechanism has the greater importance. As the liver is almost freely permeable to glucose an effect in that organ must be due to changes in enzyme activities. Since no change in glycogen synthesis was found in liver by us this might indicate that the effect seen in other tissues is mainly due to the membrane effect although differences in enzyme sensitivity to lithium are also possible. Instead of increasing glycogen synthesis lithium might inhibit glycogen breakdown resulting in greater glycogen concentration in tissue. A possible mechanism is through the adenyl-cyclase system. Inhibition of adenyl-cyclase by lithium is found in several tissues, e. g. thyroid membranes, brain, renal cortex (WOLFFet al. 1970; FORN & VALDECASAS 1971; MARCUS & AURBACH 1971) and possibly also in liver tissue (OLESENet al. 1974). An inhibition of liver adenyl-cyclase would give less cyclic AMP and less degradation of glycogen. The possible inhibition of glycogen breakdown through the adenyl-cyclase system was tested under basal conditions and after glucagon stimulation in experiments reported in table 2. Two doses giving maximal and submaximal stimulation (EXTON& PARK1971) were used to make sure that the chance of an inhibition was not masked by an unphysiological stimulus. Addition of lithium to the medium did not decrease the rate of glucose liberation from the liver. This is in accordance with in vivo findings of OLESEN & THOMSEN (1974). We found an increased rate of uptake of free fatty acids and a trend towards an increase in triglyceride deposition. The rate of uptake without lithium in the medium was about the same as reported by others (VAN HARKEN et al. 1969). The findings are in accordance with findings in lithium
LITHIUM AND HEPATIC METABOLISM
15
treated rats reported by KRULIKel al. (1971). Since the overall effect of lithium treatment is obesity in both rats (PLENGEet al. 1973) and man (KERRYet al. 1970; MELLERUP et al. 1972; VENDSBORG et al. unpublished results) lithium must influence weight regulation, but the mechanism is still unknown. Acknowledgements This investigation was supported in part by grants from ’Fonden ti1 k g e videnskabens Fremme’, ’Statens Laegevidenskabelige Forskningsrbd’ and NOVO’s Fond. Thanks are due to Glenna Skouboe for skillful technical assistance. REFERENCES Bhattacharya, G.: Influence of ions on the uptake of glucose and on the effect of insulin on it by rat diaphragm. Nature 1959, 183, 324-325. Bhattacharya, G.: Effects of metal ions on the utilization of glucose and on the influence of insulin on it by the isolated rat diaphragm. Biochem. J. 1961, 79, 369377. Bhattacharya, G.: Influence of Li+ on glucose metabolism in rats and rabbits. Biochim. Biophys. Acta 1964, 93, 644-646. Bergmeyer, H. V.: Methoden der enzyrnatischen Analyse. Verlag Chemie, Weinheiml Bergstr. 1970, 1162-1190. Carlson, L. A.: Determination of serum triglycerides. J. Atheroscler. Res. 1963, 3, 334-336. Clausen, T.: The relationship between the transport of glucose and cations across cell membranes in isolated tissues. IIL Effect of Na+ and hyperosmolarity on glucose metabolism and insulin responsiveness in isolated rat hemidiaphragm. Biochim. Biophys. Acta 1968a, 150, 56-65. Clausen, T.: The relationship between the transport of glucose and cations across cell membranes in isolated tissues. IV. The ’insulin-like’ effect of Li+. Biochim. Biophys. Acta 1968b, 150, 66-72. Clausen, T.: Cations, glucose metabolism, and insulin action. A review. Aarhus 1972. Exton, J. H. & C. R. Park. In: Cyclic AMP. Eds.: G. A. Robinson, R. W. Butcher & E. W. Sutherland. Academic Press 1971, pp. 237. Falholt, K., B. Lund & W. Falholt: An easy colorimetric micromethod for routine determination of free fatty acids in plasma. Clin. Chim. Acta 1973, 46, 105-111. Forn, J. & F. G. Valdecasas: Effects of lithium on brain adenyl cyclase activity. Biochem. Pharmacol. 1971, 20, 2773-2779. Forsham, P. H. & G. W. Thorn: Changes in inorganic serum phosphorus during the intravenous glucose tolerance test as an adjunct to the diagnosis of early diabetes mellitus. Proc. Amer. Diab. Ass. 1949, 9, 99-122. Hems, R., B. D. Ross, M. N. Berry & H. A. Krebs: Gluconeogenesis in the perfused rat liver. Biochem. J. 1966,101, 284-292. Hohorst, H. J.: In: Methoden der enzymatischen Analyse. Ed.: H. V. Bergmeyer. WeinheidBergstr., Verlag Chemie, 1962, pp. 215. Kerry, R. J., L. I. Liebling & G. Owen: Weight changes in lithium responders. Acta psychiat. scand. 1970, 46, 238-243.
16
P. B. VENDSBORG AND H. VILSTRUP
Kohn, P. G. & T. Clausen: The relationship between the transport of glucose and cations across cell membranes in isolated tissues. VII. The effects of extracellular Na+ and K+ on the transport of 3-0-methylglucose and glucose in rat soleus muscle. Biochim. Biophys. Acta 1972, 255,798-814. Krulik, R. & P. Zvolsk$ The effect of lithium on the metabolism of experimental animals. Activ. nerv. sup. 1970, 12,279-283. Krulik, R., L. Janko & M. Cerny: Metabolism of lipids in lithium administered rats. Acta Univ. Car. Med. 1971, 17,533-540. Marcus, R. & G. D. Aurbach: Adenyl cyclase from renal cortex. Biochim. Biophys. Acta 1971,242, 410-421. Mellerup, E. T., H. Gr@nlundThomsen, N. Bj@rum& 0. J. Rafaelsen: Lithium, weight gain, and serum insulin in manic-depressive patients. Acta psychiat. scand. 1972, 48, 332-336. Olesen, 0. V.,J. Jensen & K. Thomsen: Effect of lithium on glucagonstimulated cyclic AMP excretion in rats. Acta pharmacol. et toxicol. 1974, 35, 403-411. Olesen, 0. V. & K. Thomsen: Effect of prolonged lithium ingestion on glucagon and parathyroid hormone responses in rats. Acta pharmacol. et toxicol. 1974, 34, 225-231. Plenge, P., E. T. Mellerup & 0. J. Rafaelsen: Lithium action on glycogen synthesis in rat brain, liver, and diaphragm. J . psychiat. Res. 1970, 8, 29-36. Plenge, P. K., E. T. Mellerup & 0. J. Rafaelsen: Weight gain in lithium-treated rats. Int. Pharmacopsychiat. 1973, 8, 234238. van der Velde, C. D. & M. W. Gordon: Manic-depressive illness, diabetes mellitus, and lithium carbonate. Arch. gem Psychiat. 1969, 21,478-485. van Harken, D. R., C. W. Dixon & M. Heimberg: Hepatic lipid metabolism in experimental diabetes. J . Biol. Chem. 1969, 244,2278-2285. Vendsborg, P. B. & 0. J. Rafaelsen: Lithium in man: Effect on glucose tolerance and serum electrolytes. Acta psychiat. scand. 1973, 49,601410. Whitton, P. D. & D. A. Hems: Glycogen synthesis in the perfused liver of starved rats. Biochem. J . 1971, 125,76. Walaas, E., 0. Walaas & R. S . Horn: In: The mechanism of insulin action. Symposium, Oslo, 1970, pp. 93-98. Wolff, J., S . C. Berens & A. B. Jones: Inhibition of thyrotropinstimulated adenyl cyclase acticity of beef thyroid membranes by low concentration of lithium ion. Biochem. Biophys. Res. Comrizun. 1970, 39, 77-82.
From the Division of Hepatology, Medical Department A and the Department of Psychiatry, Rigshospitalet, DK-2100 Copenhagen 0, Denmark
The Influence of Lithium on Carbohydrate and Lipid Metabolism in the Perfused Rat Liver BY P. B. Vendsborg and H. Vilstrup (Received April 29, 1975; Accepted May 26, 1975)
Abstract: Isolated rat liver was perfused with a once-through technique. Glycogen synthesis was not affected by the presence of lithium (1 or 5 mM) in the perfusion medium. The rate of glycogen breakdown both without and with glucagon stimulation was not affected by lithium (1 mM). The uptake of free fatty acids was stimulated by lithium (5 mM) in the perfusion medium and a trend towards an increase in deposition of triglyceride in the same order of magnitude was seen.
Key-words: Lithium - liver
- liver
glycogen - free fatty acids
- triglycerides.
Several investigators have shown that lithium influences the metabolism of glucose. The uptake of glucose in tissues is increased both in vitro (BHATTACHARYA 1959, 1961 & 1964; CLAUSEN 1968a & b), and in intact man (VAN DER VELDE & GORDON 1969; VENDSBORG & RAFAELSEN 1973). An increased amount of glycogen in muscle and brain has been found after lithium administration (PLENGEet al. 1970). Glycogen in liver has been reported to be decreased after a single lithium dose (PLENGEet al. 1970), but increased (KRULIK& Zvo~sIck1970) or unchanged (OLESEN& THOMSEN 1974) after longer periods of lithium administration. Our previous findings of increased glucose disposal in man (VENDSBORG & RAFAELSEN 1973) pointed to the liver as one of the sites of extra glucose uptake. The mechanism by which lithium influences fat metabolism has not been investigated in detail although obesity is the most serious side effect to et al., unpublished lithium treatment (KERRYet al. 1970), (VENDSBORG results). In animals given lithium for longer periods increased weight gain et al. 1973). has been found (PLENGE The purpose of the present investigation was to estimate the influence of lithium on the metabolism of glucose and free fatty acids in the liver. No
LITHIUM AND HEPATIC METABOLISM
11
influence of lithium on glucose uptake could be demonstrated in the isolated liver but the uptake of free fatty acids (FFA) was increased.
Materials and Methods Female Wistar rats weighing about 200 g were used. The fasted rats were fasted for 20 hours. Isolation of the liver for perfusion was performed by the technique of HEMS et al. (1966), except that the perfusion was started immediately after cannulation of the portal vein. The once-through technique was used for the perfusion. The medium consisted of Krebs-Henseleit-buffer, 3 per cent (w/v) bovine serum albumin powder fraction V, and about 7 mmoVl of haemoglobin as bovine red cells washed three times with saline. Lactate was not added to the medium but was present possibly due to the bovine erythrocytes in a final concentration of about 1 mM. Lithium was added as a 150 mM lithium chloride solution. In control experiments the same amount of 150 mM sodium chloride solution was added. Free fatty acids (FFA) were added to the medium as a complex of palmitic acid and albumin, prepared according to the method of VAN HARKEN et al. (1969). The medium was oxygenated with 95 per cent atmospheric air and 5 per cent carbon dioxide. The pH was adjusted to 7.4. The perfusion was performed in a thermostated box of plexiglass at 37'. The flow was 8-10 ml/min. and the pressure in the portal vein was 10-15 cm of water. Samples for measurements were taken from the reservoirs before and after the liver. The substrate determinations were performed according to the following methods: Lactate (HOHORST 1962), glucose by the hexokinase method (BERGMEYER1970) and FFA by the method of FALHOLT et al. (1973). The oxygen consumption was calculated from measurements of oxygen saturation (haemoreflector). Furthermore tissue samples for glycogen determination were taken with the freeze clamp technique, and for determination of triglyceride a small sample (20-40 mg) was quickly frozen in a deep freeze. The tissue concentration of glycogen was determined as glucose after hydrolysis (PLENGEet al. 1970), and the tissue concentration of triglyceride by the method of CARLSON(1963) after sonification in the extraction solution. The rate of metabolism was estimated from the difference between the concentration of metabolites in affluent and effluent medium and perfusion flow. The t-test was used to test statistical significance.
Results Table 1 shows glycogen synthesis with 30 mM glucose in the perfusion medium. There was an average glycogen synthesis of about 0.3 pmoVmin. per gram of wet liver weight. Both with 1 and 5 mM of lithium chloride, control experiments were run because of the addition of different amounts of 150 mM sodium chloride to the controls. Neither 1 nor 5 mM lithium in the perfusion medium changed the rate of glycogen synthesis. The release of glucose from perfused fed rat livers to a medium without glucose was about the same in two consecutive 60 minutes' periods and unaffected by lithium (table 2). A small uptake of lactate took place, decreasing with time, but was not changed by the presence of lithium.
P. B. VENDSBORG AND H. VILSTRUP
12
Table 1. Glycogen synthesis in perfused livers from fasted rats without and with lithium in the medium. Results are expressed as means f S.E.M. with number of experiments in brackets. The rats were starved for 20-24 hours. The livers were perfused for 70 minutes. Liver lobes for glycogen estimation were taken at 10 and 70 minutes. Perfusion medium Glucose Lithium
Glycogen synthesis pmol glucose/min./g liver wet weight
f 0.06 (11) If: 0.06 (9)
30 mM 30 mM
0 1mM
0.29 0.27
30 mM 30 mM
0 5 mM
0.29 k 0.08 (11) 0.29 k 0.08 (11)
Table 3 shows the release of glucose before and after stimulation with two different doses of glucagon. The high dose which was calculated to give maximum glycogen break-down resulted in a 4 fold increase in glucose output. The smaller submaximal dose gave a 3 fold increase. Lithium did not affect the glucagon response. The uptake of free fatty acids in perfused livers from fasted rats is seen in table 4. In the perfusions with lithium in the medium the uptake was about 20 higher than in the controls (P < 0.05). The amount of triglycerides in the liver before and after perfusion with FFA containing medium is also seen in table 4. In the livers perfused with lithium there was about 20 per
Table 2. Release of glucose and uptake of lactate by perfused livers from fed rats without and with lithium in the medium. Results are expressed as means +. S.E.M. with number of experiments in brackets. Each perfusion lasted for 120 minutes. The medium was changed at 60 minutes from lithium free to lithium containing or vice versa. No glucose was present in the affluent medium.
Time (min.)
Lithium
Glucose release pmol/min./g liver wet weight
Lactate uptake pmol/min./g liver wet weight
0-60
1mM 0
1.38 k 0.10 (3) 1.06 f 0.32 (3)
0.37 k 0.05 (2) 0.21 k 0.07 (2)
0 1mM
1.41 1.45
60-120
0-60 60-120
f 0.17 (3) f 0.43 (3)
0.45 0.21
f 0.01 (2) f 0.02 (2)
LITHIUM AND HEPATIC METABOLISM
13
Table 3 . Release of glucose after stimulation with glucagon from perfused livers of fed rats without and with lithium in the medium. Results are expressed as means k S.E.M. with number of experiments in brackets. The livers were perfused for 40 minutes. After 20 minutes perfusion glucagon was added to the perfusion medium. Glucose was estimated in the perfusion medium used from 0 to 20 and from 20 to 40 minutes. No glucose was present in the affluent medium. Glucagon
Lithium
Nil Nil 3 * 10-"JM 3 . 10-1OM 10-QM 10-9~
0 1 mM 0
Glucose release pmol/min./g liver wet weight 1.31 ? O.lO(5) 1.29 k 0.06 ( 5 ) 4.05 C 0.69 (5 ) 3.92 f 0.65 (5) 4.83 ? 0.49 (4) 5.50 C 0.28 (4)
1 mM 0 1 mM
cent more triglyceride than in the controls, but the difference did not reach statistical significance. No measurable triglyceride was present in the perfusion medium either before or after it had passed through the liver. Discussion
In a previous investigation (VENDSBORG & RAFAELSEN 1973) we have found that lithium caused an increased glucose tolerance in man (20-100 & per cent) with a very low concomitant phosphate uptake. FORSHAM THORN(1949) have shown that the phosphate uptake following hepatic
Table 4. Uptake of free fatty acids and triglyceride synthesis in perfused livers from fasted rats without and with lithium in the medium. Results are expressed as means k S.E.M. with number of experiments in brackets. Rats were fasted for 20-24 hours. Glucose concentration was 10 mM and FFA concentration 1.4 mM. Liver samples for triglyceride estimation were taken at 0 and 120 minutes. Lithium
F F A uptake pmol/min./g liver wet weight
Triglyceride synthesis pg/min./g liver wet weight
5 mM 0
1.22 k 0.063 (18) 1.02 f 0.058 (18)
139 C 9 (3) 119 f 6 (3)
14
P. B. VENDSBORG AND H.VILSTRUP
glucose disposal is smaller than following muscular glucose disposal. The small extra uptake of phosphate which followed the increase in glucose uptake induced by lithium therefore pointed to the liver as the site of the phenomenon. We were neither able to induce glycogen synthesis with physiological glucose concentrations nor with 2-3 times normal fasting level. When the glucose concentration was raised to 30 mM glycogen synthesis took place as reported by other investigators (WHITTON & HEMS1971). It was not possible to stimulate the synthesis either with 1 or with 5 mM lithium chloride. This is in contrast with in vitro findings in muscle tissue (CLAUSEN 1968a & b) and in rat muscle, adipose tissue and brain in vivo (BHATTACHARYA 1964; PLENGE et al. 1907). Two main mechanisms have been proposed for the increased glucose uptake and glycogen synthesis caused by lithium in some tissues: Increase in membrane permeability (CLAUSEN 1972; KOHN & CLAUSEN 1972) and et al. 1970). It is not stimulation of glycogen synthesizing enzymes (WALAAS known which mechanism has the greater importance. As the liver is almost freely permeable to glucose an effect in that organ must be due to changes in enzyme activities. Since no change in glycogen synthesis was found in liver by us this might indicate that the effect seen in other tissues is mainly due to the membrane effect although differences in enzyme sensitivity to lithium are also possible. Instead of increasing glycogen synthesis lithium might inhibit glycogen breakdown resulting in greater glycogen concentration in tissue. A possible mechanism is through the adenyl-cyclase system. Inhibition of adenyl-cyclase by lithium is found in several tissues, e. g. thyroid membranes, brain, renal cortex (WOLFFet al. 1970; FORN & VALDECASAS 1971; MARCUS & AURBACH 1971) and possibly also in liver tissue (OLESENet al. 1974). An inhibition of liver adenyl-cyclase would give less cyclic AMP and less degradation of glycogen. The possible inhibition of glycogen breakdown through the adenyl-cyclase system was tested under basal conditions and after glucagon stimulation in experiments reported in table 2. Two doses giving maximal and submaximal stimulation (EXTON& PARK1971) were used to make sure that the chance of an inhibition was not masked by an unphysiological stimulus. Addition of lithium to the medium did not decrease the rate of glucose liberation from the liver. This is in accordance with in vivo findings of OLESEN & THOMSEN (1974). We found an increased rate of uptake of free fatty acids and a trend towards an increase in triglyceride deposition. The rate of uptake without lithium in the medium was about the same as reported by others (VAN HARKEN et al. 1969). The findings are in accordance with findings in lithium
LITHIUM AND HEPATIC METABOLISM
15
treated rats reported by KRULIKel al. (1971). Since the overall effect of lithium treatment is obesity in both rats (PLENGEet al. 1973) and man (KERRYet al. 1970; MELLERUP et al. 1972; VENDSBORG et al. unpublished results) lithium must influence weight regulation, but the mechanism is still unknown. Acknowledgements This investigation was supported in part by grants from ’Fonden ti1 k g e videnskabens Fremme’, ’Statens Laegevidenskabelige Forskningsrbd’ and NOVO’s Fond. Thanks are due to Glenna Skouboe for skillful technical assistance. REFERENCES Bhattacharya, G.: Influence of ions on the uptake of glucose and on the effect of insulin on it by rat diaphragm. Nature 1959, 183, 324-325. Bhattacharya, G.: Effects of metal ions on the utilization of glucose and on the influence of insulin on it by the isolated rat diaphragm. Biochem. J. 1961, 79, 369377. Bhattacharya, G.: Influence of Li+ on glucose metabolism in rats and rabbits. Biochim. Biophys. Acta 1964, 93, 644-646. Bergmeyer, H. V.: Methoden der enzyrnatischen Analyse. Verlag Chemie, Weinheiml Bergstr. 1970, 1162-1190. Carlson, L. A.: Determination of serum triglycerides. J. Atheroscler. Res. 1963, 3, 334-336. Clausen, T.: The relationship between the transport of glucose and cations across cell membranes in isolated tissues. IIL Effect of Na+ and hyperosmolarity on glucose metabolism and insulin responsiveness in isolated rat hemidiaphragm. Biochim. Biophys. Acta 1968a, 150, 56-65. Clausen, T.: The relationship between the transport of glucose and cations across cell membranes in isolated tissues. IV. The ’insulin-like’ effect of Li+. Biochim. Biophys. Acta 1968b, 150, 66-72. Clausen, T.: Cations, glucose metabolism, and insulin action. A review. Aarhus 1972. Exton, J. H. & C. R. Park. In: Cyclic AMP. Eds.: G. A. Robinson, R. W. Butcher & E. W. Sutherland. Academic Press 1971, pp. 237. Falholt, K., B. Lund & W. Falholt: An easy colorimetric micromethod for routine determination of free fatty acids in plasma. Clin. Chim. Acta 1973, 46, 105-111. Forn, J. & F. G. Valdecasas: Effects of lithium on brain adenyl cyclase activity. Biochem. Pharmacol. 1971, 20, 2773-2779. Forsham, P. H. & G. W. Thorn: Changes in inorganic serum phosphorus during the intravenous glucose tolerance test as an adjunct to the diagnosis of early diabetes mellitus. Proc. Amer. Diab. Ass. 1949, 9, 99-122. Hems, R., B. D. Ross, M. N. Berry & H. A. Krebs: Gluconeogenesis in the perfused rat liver. Biochem. J. 1966,101, 284-292. Hohorst, H. J.: In: Methoden der enzymatischen Analyse. Ed.: H. V. Bergmeyer. WeinheidBergstr., Verlag Chemie, 1962, pp. 215. Kerry, R. J., L. I. Liebling & G. Owen: Weight changes in lithium responders. Acta psychiat. scand. 1970, 46, 238-243.
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P. B. VENDSBORG AND H. VILSTRUP
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