Loyola University Chicago Loyola eCommons Master's Theses Theses and Dissertations 1965 Effect of Some Oral Hypoglycemic Agents on Hepatic Protein Synthesis Lawrence Dechatelet Loyola University Chicago Follow this and additional works at: https://ecommons.luc.edu/luc_theses Part of the Biochemistry, Biophysics, and Structural Biology Commons Recommended Citation Dechatelet, Lawrence, "Effect of Some Oral Hypoglycemic Agents on Hepatic Protein Synthesis" (1965). Master's Theses. 1976. https://ecommons.luc.edu/luc_theses/1976 This Thesis is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Master's Theses by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. Copyright © 1965 Lawrence Dechatelet EJ'FEC'l' OF SOME ORAL HYPOGLYCEMIC ABUTS ON HEPATIC PROTEIN SYNTHESIS by LAWRENCB R. DECHA'l'ELBT A Thesis Submitted to the 'aculty of the Graduate School of Loyola University in Partial Fulfillment of the Requirements for the Degree of Master of Science June 1965 LIFE Lawrence Robert DeChatelet was born in Chicago, Illinois on December 24, 1940. In June 1959, he graduated from Saint Ignatius High School, Chicago, Illinois and then attended Loyola University, Chicago, Illinois, from where he received the Degree of Bachelor of Science in Chemistry in June, 1963. In September 1963, the author began his advanced studies in the Graduate School of Loyola University, Department of Bio­ chemistry. From September 1963, to September 1964, he held the position of Research Assistant in the Department of Biochemistry at the Stritch School of medicine of Loyola University, Chicago, Illinois. The position was supported financially by a grant from the Chicago Heart Association. From September 1964, to the pres­ ent he was a Cooperative Graduate Fellow of the National Science Foundation, Washington, D.C. On December 26, 1964, the author married the former Miss Mary Patricia Dolan of Chicago, Illinois. ACKNOWLEDGEMENT The author wishes to thank his advisor, Dr. H.J. Me Donald, not only for his time and effort in developing this thesis, but perhaps more importantly, for his role in developing the author's scientific maturity. Thanks are due likewise to the entire staff of the de­ partment of Biochemistry and Biophysics as well as to my fellow graduate students for their many helpful comments and suggestions Finally, an inexpressible debt is due to my wife Pat, who willingly put up with the many inconveniences that are the 10 of a graduate student's wife. Without her encouragement, this thesis might well have never been written. TABLE OF CONTENTS CHAPTER -PAGE I DIABETES AND THE ACTION OF INSULIN. • • • • • • • 1 II THE CHEMISTRY OF INSULIN AND THE ORAL HYPOGLYCEMIC AGENTS • • • • • • • • • • • • • • • 16 III MATERIALS AND METHODS • • • • • • • • • • • • • • 35 General Experimental Procedure • • • • • • • 35 Chemicals ••• • • • • • • • • • • • • • • • 37 Composition of Solutions • • • • • • • • • • 40 Preparation of Liver Homogenates • • • • • • 42 Isolation of Beta-Lipoprotein. • • • • • • • 43 Cardiac Puncture • • • • • • • • • • • • • • 46 Protein Isolation. • • • • • • • • • • • • • 4'7 Protein Determination. • • • • • • • • • 48 14 C 02 Collection and Measurement • • • • • • 52 IV EXPERIMENTAL RESULTS • • • • • • • • • • • • • • 56 Experiment 1: Change in pH During the Course of the Incubation • • • • •• 56 .K.xperiment 2: The Incorporation of Leucine -C14 into Beta-Lipoprotein: Effect of Differing Isotope. 58 Experiment 3: The Incorporation of Leucine _C14 into Beta-Lipoprotein: Effect of Tolbutamide Addition and of Omission of Premix • • 60 Experiment 4: Distribution of Radioactivity in Various Protein Fractions. 62 CHAPTER -PAGE IV Experiment 5: A Comparison of the Effect of Tolbutamide and Phenethyl­ biguanide on the Incorporation of Leucine into Various Protein Fractions. • • • • • • • • • • 66 Experiment 6: Effect of Tolbutamide and Phen­ ethylbiguanide on c140 Produc- 2 tion from Leucine-l-C14• • •• 70 Experiment 7: The Effect of Three Hypo- glycemic Agents on Leucine Anabolism and Catabolism • • • '{3 Experiment 8: The Etfect of the Concentra- tion of Phenethylbiguan1de on 140 C 2 Evolution. • • • • • •• 75 Experiments 9 and 10: The Effect of the Concentrati en of Phenethylb1guanide on c140 2 Evolution and Protein Incorpo- ration • • • • • • • • • • •• 78 Experiment 11: The Effect of the Concentra­ tion of Tolbutamide on C140 2 Evolution and Protein Incorpo- ration • • • • • • • • • • •• 78 Experiment 12: The Effect of Glucose on Inhi­ bition by Hypoglycemic Agents of Leucine Anabolism and Catabolism • • • • • • • • •• 87 SUMMARY AND CONCLUSIONS. • • • • • • • • • • • • • go APPENDIX ••• • • • • • • • • • • • • • • . • • 92 BIBLIOGRAPHY • • • • • • • • • • • • • • • • • • • 96 LIST OF TABLES TABLE -PAGE I STUDIES OF THE INFLUENCE OF INSULIN ON PROTEIN METABOLISM • • • • • • • • • • • • • • • • • •• 11 II THE ARYLSULrONYLUREAS. • • • • • • • • • • • • • 22 III GUAHIDlNE AND SOME DERIVATIVES • • • • • • • • • 23 IV COMPOSITION OF PREMIX. • • • • • • • • • • • • • 41 V STANDARD CURVE DATA FOR PROTEIN DETERMINATION.. 50 VI SELP-ABSORPTIOH CORRECTIONS FOR BaC03. • • • •• 55 VII THE pH OF THE INCUBATION MIXTURES. • • • • • • • VIII INCORPORATION OF LEUCINE INTO BETA-LIPOPROTEIH: EFFECT OF SPECIFIC ACTIVITY OF ISOTOPE • • • •• 59 IX INCORPORATION OF LEUCINE INTO BETA-LIPOPROTEIN: EfFECT OF TOLBUTAMIDE ADDITION AND OF OMISSION OF PREMIX. • • • • • • • • • . • • • • • • • • • 61 X DISTRIBUTION OF RADIOACTIVITY. • • • • • • • •• 65 XI DISTRIBUTION OF RADIOACTIVITY: INCUBATION WITH TOLBUTAMIDE AND PHENETHYLBIGUANIDE 6'{ XII SUMMARY or EXPERIMENTS 4 AND 5 • • • • • • • • • 68 XIII CO2 RELEASE AND PROTEIN INCORPORATION FROM EXPERI~ 6 • • • • • • • • • • • • • • • • • • 71 XIV EFFECT 01' THREE AGENTS ON ANABOLISM AND CATABOLISM OF LEUCINE. • • • • • • • • • • • • • '{4 xv EFFECT OF CONCENTRATION OF PHENE'l'HYLBIGUANIDE ON 14 C O2 EVOLUTION (EXPERIMENT 8) • • • • • • • •• '(6 TABLI!: -PAGE XVJC ltFFECT OF CONCENTRATJCON OF PHENETHYLBIGUANJCDE ON C140 It''O~U'l'JCON (EXPERJCMEN'l' 9). • • • • • •• 79 2 XVJCJC EFFECT OF CONCltNTRATION OF PHENETHYLBJCGUANJCDE ON JCNCO~PORATJCON OF LBUCINE-I-C14 JCNTO PROTEJCN OF JlJl~ ~JC"lt~ • • • • • • • • • • • • • • • • • • 80 XVIJCJC EFFECT or CONCENTRATJCON or TOLBUTAMIDE ON c1402 EVO~UT ION. • • • • • • • • • •• • • • • •• 81 XJCX ltPFECT OF CONCENTRATJCON OF TOLBUTAMIDE ON JCNCOR­ PORATION 0' LltUCJCNE-I-c14 JCNTO PROTEIN OF RAT LIVER. • • • • • • • • • • . • • • • • • • •• 82 :xx ErnCT OF GLUCOSE AND ORAL HYPOGLYCEMIC AGENTS 14 ON C 02 RELEASE • • • • • • • • • • • • • • •• 88 XXI EFFECT OF GLUCOSE AND ORAL HYPOGLYCEMIC AGENTS ON JCNCORPORATION OF LKUCIHE-c14 INTO PROTEIN .• 89 EFPICACY OF WASHINGS: LJCPOPROTEJCN. • • • • • • • • • • • • • • • • • • 92 XXJCJCJC EPFJCCACY OF WASHINGS: PROTEIN. • • • • • • • • • • • • • • • • • • • • 93 XXJCV EFFECT OP CONCENTRATJCON OF PHENETHYLBJCGUANIDE ON 14 C O2 RELEASE (EXPERIMENT 10). • • • • • • • •• 94 xxv EFFECT OF CONCENTRATJCON OF PHENETHYLBJCOUANJCDE ON JCNCORPORATJCON OF LEUCJCNE-I-c14 JClft'O PROTEIN OF RAT LIVER (EXPERIMBNT 10). • • • • • • • • • •• 95 LIST OF FIGURES FIGURE I THE STRUCTURE OF INSULIN. • • • • • • • • • • 1'7 II THE METABOLISM OF TOLBUTAMIDE • . • • • • • • 2 rr III STANDARD CURVE FOR PROTEIN ANALYSIS • • • • • • • 51 IV SELP-ABSORPTION CORRECTION CURVE FOR BaC0 . • . 54 3 V SEPARATION OF PROTEIN FRACTIONS • • • • • • • • • 64 VI THE CATABOLISM OF LEUCINE • . • • • • • • • • • • 69 VII INHIBITION OF C140 PRODUCTION FROM LEUCINE-1-C14 2 BY PHENETHYLBIGUANIDE • • • • • • • • • • • • • • 77 VIII COMPARISON OF INHIBITION OF C140 RELEASE FROM 2 LEUCINE-1-C14 BY TOLBUTAMIDE AND PHENETHYL- BIGUANIDE • • • • • • • • • • • • • • • • • • • • 83 IX COMPARISON OF INHIBITION OF PROTEIN SYNTHESIS BY TOLBUTAMIDE AND PHENETHYLBIGUANIDE. • • • • • •• 84 ABSTRACT The action of the hypoglycemi.c compounds, tolbutamide and phenethylbiguanlde, on protein biosynthesis was investigated by the use of rat liver homogenates, the necessary cofactors and 1euc1ne-c14. The results show that the incorporation of leuclne­ C14 into he~atic protein is inhibited by both compounds, though in a strikingly different manner. The inhibition by tolbutamide is linear with respect to concentration whereas the inhibition by phenethylbiguanide is not evident until a certain concentration is reached. At this pOint, the amount of incorporation decreases markedly until it levels off at a very low value. It has been postulated that there 1s some substance present which combines with phenethylblguanide and renders it inactive toward inhibition of protein synthesis. Glucose was investigated in tnis respect and found to have no effect on either tolbutamide or phenethyl­ biguanide induced inhibition. The catabolism of leucine was measured by the amount of C140 liberated in the course of the incubation. Both tolbutamide 2 and phenethylbiguanide were found to inhibit CO production. 2 Again, inhibition by tolbutamld.,3 was linear with respect to con­ centration. Phenethylbiguanld:a caused a pronounced initial inhi­ bition which tended to level off near the same concentration where the protein inhibition first occurs. This suggests that one of the reasons that low concentrations of PESO does not inhibit pro­ tein incorporation is due to the great decrease in leucine catab­ olism. Since less leucine is broKen down (catabolized), there is ecessarily more present to be incorporated