Increase in human skeletal muscle lactate produced by fenfluramine

617 Nature Vol. 262 August 12 1976 • Krebs, H. A., and Henseleit, K., Z. physio/. Chem., 210, 33-66 (1932). Morgan, H. E., Henderson, M. J., Regen, D. M., and Park, C.R.,./. biol. Chem., 236, 253-261 (1961). 11 Moir, A. J. G., Wilkinson, J.M., and Perry, S. V., FEBS Lett., 42, 253-256 (1974). 12 Huang, T. S., Bylund, D. B., Stull, J. T., and Krebs, E. G., FEBS Lett., 42, 249-252 (1974). 13 Syska, H., Wilkinson, J. M., Grand, R. J. A., and Perry, S. V., Biochem . ./., 153, 375-387 (1976). 1 4 Rubio, R., Bailey, C., and Villar-Palasi, C., J. Cyclic Nucleotide Res., I, 143-150 (1975). 15 Perry, S. V., and Cole, H. A., Biochem. J., 141, 733-743 (1974). 10 Increase in human skeletal muscle lactate produced by fenfluramine OuR recent hyipothesis' tha,t "anorectic agents" such as fenfluramine and maZJindol owe at le,ast part of their antiobesity prnpenties to a peripheral actfon on glucose metabolism by causing an increase in glucose uptake into skeletal muscle and a subsequent "wasting of calories" has been criticised' on the ,grounds ,that there is no information on the f1ate of the glucose so taken up, nor convincing evidence for an increase in metabolic rate with these drugs. We have now studied ,the effect of fenfluramine on lactate production in isolated human skeletal muscle, our previous work having shown no increase in glycogen levels' nor in carbon dioxide producrtion'. An ,increase ,in lactate production would indicate metaJbo1ism of the glucose entering the cell under ,the influence of fenfluramine. Human skeletal muscle was obtained at surgery for total hip joint replacement. The preparation and incubation of the tissue {in Krebs-bica11bonate buffer with added bovine serum a,lbumin 2 mg ml- 1 and glucose 16.67 x 10-, M) was carried out using the method of ~irby, Leighton and Turner'. Muscle strips, wet weight 80~140 mg, we,re prepared from each sample and incubated in 2 ml of media containing (l) 100 µU m1· 1 ,insulin, (2) 100 µU ml- 1 insulin plus 100 ng ml- 1 fenfluramine (prev,1ously shown to cause a maximal increase in glucose uptake'), (3) 100 µU ml- 1 insulin ,plus 100 ngml- 1 ,amphetamine (amphetamine has no significant effect on glucose urptak,e into isolated muscle'). For each muscle sample ,two stnips were prepared and used for control laotate determinations; they were not incubated. Flasks were flushed with a mixture of 95 % oxygen and 5 % carbon dioxide for 10 s before ,incubation at 37 °c for 90 min. The lactate content of the muscle and the residual media were then determined. The method for measurement of muscle ,and residual media laotate was based on that for blood ,lactate using NAD/LDH enzymes7 and the Boehringer Mannheim Biochemica Test Combinaition for lactate. Musde strips Table 1 Lactate formed by isolated human skeletal muscle incubated with insulin, fenfluramine or amphetamine Insulin* 13.85 14.11 9.79 9.12 10.97 8.78 7.68 8.58 7.48 8.79 Means±s.e.m. t Significance Fenfluramine plus insulin : change compared with insulin alone* Amphetamine plus insulin: change compared with insulin alone* +1.04 -2.09 +2.33 +0.23 +0.10 +1.67 +1.07 +1.53 +2.53 +1.07 +0.95±0.42 2.26 P = 0.05 -0.10 +1.82 +0.84 -3.00 -0.53 +2.07 -1.67 -1.57 -0.08 -0.70 -0.29±0.50 0.58 NS All values are means of two incubations. *mmol lactate per kg wet weight skeletal muscle per 90 min; 100 µU ml - 1 insulin, I 00 ng ml - 1 fenfluramine and I 00 ng ml - 1 amphetamine. were removed from t,he media and homogenised for 5 min a,t 25,000 r.p.m. with ,a hlade homogeniser in 2 ml of 0.6 M perchloric a:cid. The homogenate was centrifuged for 10 min a,t 3,000 r.p.m. and 0.2 ml of the supernatant used for the lactate determination. One millilitre of the residual medium was added ,to 1 ml of 0.6 M perchloric acid and centrifuged as before, 0.2 ml of the supernatant being used for the subsequent ,analysi,s, ,with ,the Biochemka Test Combination. A lacta,te standard (0.5 mM) was included as a blank. Preliminary work had demonstrated a linear relations?iP between a:bsorbance at 340 nm and Iact,a,te concentration over the ,range O~I.O mM. Good ,reproducibiJiity was obtained with the method, the mean of six muscle lactate determinations in strips from ,the same sample being 6.91 ±0.21 mmol per kg wet weight of muscle. T'he results (Table 1) demonstrate that fenfluramine produced an increase in muscle lactate concentrations. Amphetamine, which has no effect on glucose uptake, has no significant effect on lactate concentrntion. Further studies are needed to determine in more detail ,the metabolic fate of Vhe glucose taken up under the influence of fenfluramine, but these results are further evidence for a peripheral metabolic mechanism in its anti-obesity activity. M. J. K. is a redpient of ,the Williams fellowship for scientific and medical researioh, awarded by London University. MARILYN J. KIRBY P. TURNER Department of Clinical Pharmacology, St Bartholomew's Hospital Medical College, London EClA 7BE, UK Received May 19; accepted June 8, 1976. Kirby, M. J., and Turner, P., Lancet, i, 566-567 (1976). Garrow, J. S., Lancet, i, 691-692 (1976). . Kirby, M. J., and Turner, P., in Ret:ent Advances in Obesity Research:1 (edit. by Howard, A.), 378-380 (Newman Publishing Ltd, London, 1975). 4 Kirby, M. J., thesis, London Univ. (1975). 5 Kirby, M. J., Leighton, M., and Turner, P., Br.J. c/in. Pharmac., 3, 299-304(1976). • Kirby, M. J .. Br. J. din. Pharmac., 1, 511-512 (1974). . 7 Hohorst, H.J., in Bergmeyer, H. U., Methods of Enzymatic Analyszs, first edn., 622 (Verlag Chemie, Weinheim, 1962). I 2 3 Ribosome slowed by mutation to streptomycin resistance AL THOUGH the basic mechanisms of protein synthesis are now rather well understood, particularly in Escherichia coli, it is less clear how the high accuracy of this process is achieved. (The error rate in protein synthesis in E. coli seems to be about one in IO' amino acid misincorporations (Edelman and Gallant, unpublished results, and our own unpublished data)). A major site determining accuracy in protein synthesis is the ribosome, and in addition to ,the ribosome-mediated effects on accuracy (in vivo and in vitro) of various metal ions, aminoglycoside antibiotics and organic solvents, there are ribosomal mutations which increase or decrease accuracy'. Certain mutations of the 30S ribosomal protein S12 conferring streptomycin resistance (the strA locus) enhance accuracy'-', whereas certain mutations in the S4 protein (the ram locus) are known to decrease accuracy'. The strA mutations are restrictive (reducing) in their effect on both missense and nonsense suppression in vivo and on miscoding in vitro', the ram mutation, on the other hand, has the opposite effect in both cases•. It is possible that it is the kinetics of polypeptide synthesis at the rtbosome that determines accuracy and that the kinetics in turn are affected by the strA and ram mutations. Because the same transfer RNA (tRNA) discrimination kinetics that may determine ac (...truncated)