Influence of phenformin on fat and lactate metabolism and insulin production in starved normal subjects

Metabolism Clinical and Experimental MARCH 1972

VOL. XXI, NO. 3

Influence of Phenformin on Fat and Lactate Metabolism and Insulin Production in Starved Normal Subjects By Jens Lyngsoe, Vibeke Bitsch, In starved but not in fed, normal subjects, phenformin increases the level of plasma FFA and P-hydroxybutyrate and augments the plasma triglyceride increase that occurs during starvation. These findings suggest that phenformin increases the release and degradation of fatty acids in starved subjects. Arterial blood lactate levels were increased by phenformin in fed sub-

and Jens Trap-Jensen

jects. In starved persons phenformin inhibited the fall in blood lactate values induced by i.v. glucose administration. Neither in fed nor in starved, normal subjects, did phenformin decrease the insulin response to i.v. glucose administration. A higher insulin response was found in the phenformintreated, starved subjects than in the controls.

I

N FED, NORMAL SUBJECTS, phenformin increases glucose turnover and Cori cycle activityl‘s without having any hypoglycemic effect.4-6 In starved normal subjects we found a hypoglycemic effect of phenformin with a concomittant fall in urinary nitrogen excretion after 40 hr of starvation, suggesting that phenformin decreases gluconeogenesis from proteins in starved subjects.(j This theory finds support in the results of animal studies that have shown that phenformin induces a decrease in liver gluconeogenesis from alanine. In light of our previous findings, it seemed of interest to investigate whether phenformin had other metabolic effects in starved subjects. In the present investigation, we present evidence that phenformin also induces changes in the metabolism of fat and lactate and in the production of insulin in starved, normal individuals. From the Departments

of internal

Medicine

C, Clinical

Chemistry,

and Clinical

Physiol-

ogy, Bispebjerg Hospital, Copenhagen, Denmark, Received for publication September 3, 1971. Supporfed by a grunt from the King Christian X Foundation. Jens Lyngsoe, M.D.: Head, Department of Infernul Medicine C, Bispebjerg Hospital, Copenhagen, Denmark. Vibeke Bitsch, MS.: Chemist, Department of Clinical Chemisfry, Bispebjerg Hospital, Copenhagen, Denmark. Jens Trap-Jensen, M.D.: Research Fellow, Departments of Internal Medicine C and Clinical Physiology, Bispebjerg Hospital, Copenhagen, Denmark.

Metabolism, Vol. 21, No. 3 (March), 1972

179

180

LYNGSOE,BITSCH, AND TRAP-JENSEN

Fig. 1. Arterial plasma free fatty acids before and after i.v. administration of glucose to phenformin-treated subjects (n = 5) controls (n = 5). Mean values 1- SEM. Where no p value is shown, no statistically differences significant were found between values in the two groups (p >O.l). MATERIALS

AND METHODS

Thirteen healthy, nonobese male volunteers (ages 19-31) with normal i.v. glucose tolerance and without a family history of diabetes mellitus, acted as experimental subjects. The informed consent of each person was obtained. Seven subjects were given phenformin (50 mg) in timed disintegration capsules orally every 12 hr. On the third day of medication the subjects were hospitalized in late afternoon. The following morning, after an overnight fast, an i.v. glucose tolerance test was performed by giving 25 g of glucose i.v. at a constant rate during 2 min. The phenformin treatment continued during the following 3 days of complete starvation whereafter the i.v. glucose tolerance test was repeated in the morning. The six remaining subjects acted as a control group. They were interviewed 3 days prior to hospitalization in late afternoon. Thereafter they underwent the same experimental procedure: starvation for 3 days and a glucose tolerance test before and after the period of starvation. The control subjects had no medication before nor during hospitalization. Smoking was not allowed during the period of starvation. In conjunction with the i.v. glucose tolerance tests, arterial blood was sampled from a catheter introduced percutaneously into the brachial artery. The following parameters were measured: blood lactate,8 plasma FFAg plasma P-hydroxybutyrate,lo plasma glycerol,11 plasma triglycerides,12 and immunoreactive plasma insulin.13 Venous blood was collected every 6 hr during the starvation period to determine plasma ,8-hydroxybutyrate.

Fig. 2. Arterial plasma glycerol before and after i.v. administration of gluto phenformincose treated subjects (n = 7) and controls (n 6). Mean values * SEM. No significant statistically differences were found between values in the two groups (p >O.l).

PHENFORMIN

Fig. 3. Arterial plasma P-hydroxybutyrate before and after i.v. administration of glucose to phenformin-treated subjects (n =7) and controls (N = 6). Mean values * SEM. Before starvation, no statistically significant differences were found between the two groups. After starvation, between differences values in the two groups were significant during entire observation period (p <0.005). RESULTS Before

Starvation

The mean value for fasting arterial blood lactate after 3 days of phenformin medication and normal diet was significantly higher than that found in the control group (Fig. 5). Mean plasma triglyceride values were lower in the phenformin-treated group, but the difference was not statistically significant (Table 1).The simultaneously measured fasting mean values for arterial plasma FFA, glycerol, /3-hydroxybutyrate (Figs. I-S), and insulin (Fig. 6) did not differ significantly in the two groups. During the first i.v. glucose test performed after an overnight fast, both groups showed decreasing plasma levels of FFA, glycerol, and P-hydroxybutyrate and increasing values of plasma insulin and blood lactate. During Starvation

During the 72-hr starvation period, the venous plasma P-hydroxybutyrate level increased gradually in all 13 subjects. After 44 hr and more the mean

1 Fig. 4. Venous plasma in P-hydroxybutyrate phenformin-treated subjects (n =7) and controls (n = 6) during starvation. Mean values f SEM. Where no p value is shown, no statistically significant difference was found between the two groups. (p >O.l).

182

LYNGSOE, Table 1. Arterial

Plasma Triglycerides and Control

(mmoleiliter)

BITSCH, AND TRAP-JENSEN in Phenformin-treated

Subjects

Control Group

Phenformin-treated Group

Before starvation (mean +- SEM)

0.96 * 0.17

0.68 * 0.11

p > 0.100 t = 1.400

After starvation (mean t SEM)

1.16 -t 0.17

1.35 + 0.12

Increase during starvation (mean * SEM)

0.20 C 0.08

0.67 * 0.13

p> f= p< f=

0.1 0.827 0.02 2.918

values had risen to a significantly higher level in the phenformin-treated group (Fig. 4). At the end of the starvation period, the mean arterial plasma FFA and P-hydroxybutyrate levels were significantly higher in the phenformin-treated subjects as compared to the levels in the controls (Figs. 1 and 3). Arterial plasma glycerol was somewhat higher in the phenformin-treated group, but the differences were not statistically significant (Fig. 2). Both groups showed increased triglyceride values after starvation. The net increase was significantly greater in the phenformin-treated group, although the absolute values after starvation were not significantly different statistically (Table I). The arterial blood lactate levels showed considerable individual variation within the phenformin-treated group, but the mean value was not significantly different from the control group (Fig. 5). The insulin response to starvation showed no significant difference between the two groups (Fig. 6). During the i.v. glucose test performed at the end of the 72-hr fast, there were no statistically significant differences between the two groups in the decrease of plasma FFA, glycerol, and ,O-hydroxybutyrate (Figs. l-3). A significant fall in bIood lactate was found in control subjects (p
The present study showed that in starved normal subjects phenformin increased plasma ,8-hydroxybutyrate and FFA. The increase in plasma triglycerides that normally occurs during starvation was greater in the phenformintreated subjects. In contrast, phenformin had no significant effect on plasma levels of FFA and P-hydroxybutyrate in fed subjects. No phenformin effect could be shown on the levels of plasma glycerol either in fed or starved subjects. Searle et al.14 have reported that phenformin causes a decrease in plasma FFA and triglycerides in fed normal subjects. This was not quite so in the present study although the triglyceride level after 3 days of phenformin medication and normal diet was somewhat lower than in the controls. The interindividual deviations were rather large and as the subjects did not act as their own controls the differences between the two groups were not statistically

PHENFORMIN

Fig. 5. Arterial blood lactate before and after i.v. administration of gluphenformincose to treated subjects (n = 7) and controls (n -6). Mean values * SEM. Where no p value is shown, no statistically significant differences were found between values in the two groups. (p >O.l). Starved control subjects showed statistically significant fall in blood lactate (t6.858, p O.l).

183

1 ., 50

’ O”

050

significant. No action of the drug on the FFA levels could be detected in the fed state. During starvation the phenformin-induced changes in FFA and P-hydroxybutyrate levels suggest that the drug increases lipolysis and degradation of FFA. Phenformin did not alter the glycerol level in the starved subjects, but since in such subjects glycerol acts as an important substrate for gluconeogenesis, the unaltered glycerol level does not contradict the above hypothesis. In vitro studies on rat adipose tissue have shown that biguanides in concentrations considerably higher than the normal human therapeutic level inhibit lipolysis’5-17 while lower and more pharmacological concentrations are without effect.15*‘s These findings do not exclude that phenformin in vivo may stimulate lipolysis but suggest that it does not directly effect the fat cell. A recent

Fig. 6. Arterial plasma insulin before and after i.v. administration of glucose to phenformintreated subjects (n = 7) and controls (n =6). Mean values * SEM. Where no p value is shown, no statistically significant difference (p >O.l) was found between values in the two groups.

184

LYNGSOE,

BITSCH, AND TRAP-JENSEN

study performed on perfused rat liver showed a stimulating effect of phenformin on /3-hydroxybutyrate production in this preparation,lg but the phenformin concentration used was also considerably higher than the pharmacological level in humans. It remains an open question whether phenformin has a comparable pharmacological effect in man. No study concerning the effects of phenformin on fat metabolism in starved normal subjects has yet been published. In diabetics phenformin increases the level of FFA and ketone bodies in plasma, 14120while different effects have been found on plasma tripllycerides.12s20v21 Searle et al.14 found an increased rate of FFA oxidation and FFA incorporation into triglycerides in diabetics, while in normal subjects the effect was just the opposite. These findings as well as the present study may indicate that the effects of phenformin on the metabolism of fatty acids are secondary in nature. Searle et al.14 suggest that the different findings in normal subjects and diabetics are related to the physiological presence and action of insulin. Effects of phenformin on fat metabolism were in the present study found only in starved subjects. In this condition the insulin level was practically the same in the phenformin-treated subjects and in the controls. It seems most probable, therefore, that it is the metabolic state of the tissues and not the level of circulating insulin that determines the response of fat metabolism to phenformin. In fed, normal subjects we found that phenformin increased the lactate level in arterial blood. This finding differs from previous studies in which venous blood was used and which showed no significant changes in lactate level after phenformin.4j22‘24 It must be emphasized that peripheral venous blood can hardly be used as a measure of the average blood lactate concentration. It seems reasonable, therefore, to ascribe the difference between our results and those of others to the different blood sampling technique. After glucose administration we found a normal increase in blood lactate in phenformin-treated subjects, a finding compatible with previous studies on this drug showing increased glucose turnover and lactate production from glucose in both normal and obese subjects.2s24s25 After starvation the arterial lactate level was elevated in both groups. The glucose load was followed by decreasing lactate levels in the control subjects, while the lactate level in the phenformin-treated group remained practically constant. Previous studies have shown that the conversion of glucose to lactate is inhibited during starvation,24 which is in good agreement with our finding that blood lactate values did not increase in response to glucose after starvation. The falling blood lactate levels in the control subjects are not easily explained, but the decrement could be due to an increased lactate metabolism by means of a glucose effect on the Krebs cycle. In the starved phenformin treated subjects, no fall in blood lactate was seen after glucose. This might reflect an inhibition of lactate degradation but it could also be caused bv an increased degradation of glucose to lactate, because Kreisberg et al.25 have shown that phenformin has this effect in fed subjects. The insulin level and the insulin response to iv. administered glucose was practically identical in control and phenformin-treated subjects, but the starved phenformin-treated group had a slightly higher insulin response than the con-

185

PHENFORMIN

trols. This finding shows that phenformin does not decrease the insulin sensitivity of the starved organism and suggests that the effect of phenformin in starved subjects is different from that in obese subjects in whom phenformin increases insulin sensitivity.26 REFERENCES 1. Searle, G. L., Schilling, 5, Porte, D., Barbaccia, J., De Grazia, J., and Cavalieri, R. R.: Body glucose kinetics in non-diabetic human subjects after phenethylbiguanide. Diabetes 15:173, 1966. 2. -, Gulli, R., and Cavalieri, R. R.: Effect of phenformin in non-diabetic humans. Estimation of glucose turnover rate and Cori cycle activity. Metabolism 18:148, 1969. 3. Kreisberg, R. A.: Kinetics of glucose utilization in obesity: The Effect of Phenformin. Diabetes 17:481, 1968. 4. Fajans, S. S., Moorhouse, J. A., Doorenbos, H., Louis, L. H., and Conn, J. W.: Metabolic effects of Phenethylbiguanide in normal subjects and in diabetic patients. Diabetes 9:194, 1960. 5. Madison, L. L., and Unger, R. H.: Effect of Phenformin on peripheral glucose utilization in human diabetic and nondiabetic subjects. Diabetes 9:202, 1960. 6. Lyngsoe, J., and Trap-Jensen, J.: Phenformin induced hypoglycemia in normal subjects. Brit. Med. J. 2:224, 1969. 7. Meyer, F., Ipaktchi, M., and Clausen, H. : Specific inhibition of gluconeogenesis by Biguanides. Nature (London) 213:203, 1967. 8. Horn, D. D., and Bruns, F. H.: Quantitative Bestimmung von L. (+)-MilchsZure mit MilchsHuredehydrogenase. (Quantitative estimation of 1 (+)-lactic acid with lactic acid dehydrogenase). Biochem. Biophys. Acta 21~378, 1956. 9. Laurell, S., and Tibbling, C.: Colorimetric micro-determination of free fatty acids in plasma. Clin. Chim. Acta 16 57, 1967. 10. Bitsch, V.: A modification of the Gibbard and Watkins method for determination of D-P-hydroxybutyrate. Unpublished. 11. Laurell, S., and Tibbling, G.: An enzymatic fluorometric micromethod for the determination of glycerol. Clin. Chim. Acta 13:317, 1966. 12. -: A method for routine determination of plasma triglycerides. Stand. J. Clin. Lab. Invest. 18:668, 1966. 13. Orskov, H. : Wick-chromatography for

the immunaossay of insulin. Stand. J. Clin. Lab. Invest. 20:297, 1967. 14. Searle, G. L., Felts, J. M., and Cavalieri, R. R.: Free fatty acids metabolism in non-diabetic and diabetic humans, effects of phenethylbiguanide. Diabetes 18:325, 1969. 15. Botterman, I’.: II Internationales Biguanid Symposium. Stuttgart, G. Thieme, 1968, p. 71. 16. Stone, D. B., and Brown, J. D.: In vitro effects of Phenformin hydrochloride: Observations using isolated fat cells. Ann. N.Y. Acad. Sci. 148~623, 1968. 17. Brown, J. D. Stone, D. B., and Steele, A. A.: Mechanism of action of antilipolytic agents: Comparison of the effects of insulin, Tolbutamide and Phenformin on lipolysis induced by Dibutyryl Cyclic AMP plus Theophylline. Metabolism 18:926, 1969. 18. Ditschuneit, H., Rott, W. H., and Faulhaber, J. D.: Effect von Biguaniden auf den Stoffwechsel isolierter Fettzellen. II. Internatoinales Biguanid Symposium. Stuttgart, G. Thieme, 1968, p, 62. 19. Connon, J. J., Kyner, J. L., and Toews, C. J.: The action of Phenformin on gluconeogenesis and Ketone body formation by the perfused rat liver. In Advance Abstracts, Sixth Annual Meeting of the European Association for the Study of Diabetes, Warsaw, 1970. 20. Kattermann, R., Appels, A., Hubrich, K., Proschek, H., Soling, H. D., and Creutzfeldt, W. : Untersuchungen iiber die Wirkung von DiIt, Tolbutamid und Buformin sowie deren Kombination auf Korpergewicht und verschiedene Stoffwechselgrossen bei Diabetikern. II. Freie Fettszuren, Ketonkorper, Triglyceride und Cholesterin im Blut. Diabetologia 4:221, 1968. 21. Schwartz, M. J., Mirsky, S., and Schaeffer, L. E.: The effect of Phenformin hydrochloride on serum cholesterol and triglyceride levels of the stable adult diabetic. Metabolism 15 :808,1966. 22. Giittler, F., Bonde Petersen, F., and Kjeldsen, K.: The influence of Phenformin on blood lactic acid in normal and diabetic

LYNGSOE,

subjects during exercise. Diabetes 12:420, 1963. 23. Appels, A., et al.: Untersuchungen iiber die Wirkung von Dlt, Tolbutamid und Buformin, sowie deren Kombination auf Korpergewicht und verschiedene Stoffwechselgrijssen bei Diabetikerern. I. KGrperKohlenhydratstoffwechsel und gewicht, immunologische reagierendes Insulin. Diabetologia 4:210,1968. 24. Kreisberg, R. A., Pennington, L. F.,

BITSCH, AND TRAP-JENSEN

and Boshell, B. R.: Lactate turnover and gluconeogenesis in normal and obese humans. Diabetes 19:53,1970. 25. -, -, and -: Lactate turnover and gluconeogenesis in obesity. Effect of Phenformin. Diabetes 19:64,1970. 26. Grodsky, G. M., Karam, J. H., Pavlatos, F. C., and Forsham, P. H.: Reduction by Phenformin of excessive insulin levels after glucose loading in obese and diabetic subjects. Metabolism 12:278,1963.