Effect of contraceptive steroids on arginine-stimulated glucagon and insulin secretion in women: I-lipid physiology

Effect of Contraceptive Steroids on Arginine-Stimulated Glucagon and Insulin Secretion in Women: I-Lipid Physiology Paul Beck, R. Philip Eaton,

D. M. Arnett,

To evaluate the effect of contraceptive steroids on endogenous glucagon and insulin secretion,&-arginine was infused intravenously in normal young women before and during selective steroid treatment. The effect of the combination of an estrogen derivative (mestranol), plus norethindrone (NorinylR, Syntax) was compared to the effect of ethinyl estradiol alone and to norethindrone alone. All three steroid schedules resulted in suppression of aminogenic insulin secretion. However, glucagon secretion was reduced only with ethinyl estradiol alone or the combination of mestranol plus norethin-

and R. N. Alsever

drone. In accordance with previous reports, treatment with an ethinyl estradiol derivative alone or in combination with norethindrone resulted in a tendency for elevated serum lipid concentration, while norethindrone alone resulted in a significant reduction in serum lipid concentration. These observations suggest an inverse relationship between aminogenic glucagon secretion and serum lipid concentration as influenced by contraceptive steroids. It is suggested that the metabolic effects of these steroids may be mediated in part by the associated alterations in pancreatic hormone secretory capacity.

0

RAL CONTRACEPTIVE ADMINISTRATION results in a variety of metabolic side effects.’ Perhaps the most frequent change is an increase in plasma triglyceride (TG) concentration often with an associated rise in cholesterol and very low-density lipoproteins (VLDL).lm6 Because of the epidemiological association between hyperlipemia and premature atherosclerotic cardiovascular disease,7 the widespread consumption of these contraceptive steroids for prolonged periods of time may have major potential public health significance. While the average rise in serum TG is reported to be 40 mg/ 100 ml after 1 mo in normal women, in certain susceptible individuals the oral agents result in striking hyperlipemia. **’Moreover, with prolonged use, a continued rise in TG concentration occurs in normal women as reported by Kekki and Nikkila over 6 yr of observation.6 The mechanisms mediating this gonadal steroid induced lipemia are not established, but the estrogenic component has been implicated from studies in several laboratories.6*8-10 In contrast, the progestogen components (nortestosterone derivatives) are generally considered to exert either neutral” or even lipid lowering effects. ‘IX’)Recent studies in this laboratory14 have led to the sug-

From the Department of Medicine, University of New Mexico School of Medicine, Albuquerque, and the University of Colorado School ofMedicine,Denver. Receivedfor publication May 5.1975. Supported in part by grants (HD-02455. FR-00051, and HE-12085) from the U.S. Public Health Service and by grant RR-51 from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health, and by a grant from the KROC Foundation. Presented in part at the Western Section of the AFCR. February, 1974. Reprint requests should be addressed to R. Philip Eaton, M.D.. University of New Mexico School of Medicine, Department of Medicine. Albuquerque, New Mex. 87131. o 1975 by Grune & Stratton, Inc. Metabolism, Vol. 24, No. 9 (September), 1975

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gestion that estrogen containing contraceptive steroids may elevate serum lipids by compromising pancreatic glucagon secretion, a hormone with recognized lipid-reducing potential in man. I5 Such an event would not only create a deficiency of this hypolipemic hormone which may result in hyperlipemia,44 but also a relative excess of insulin with the potential for insulin-augmented hepatic TG production.‘6q’7 The present investigation describes experiments designed to explore the influence of commonly prescribed gonadal steroids upon the glucagon and insulin secretory capacity of normal young women after arginine stimulation. By examining the component steroids as well as the “combination steroid,” we have attempted to delineate the specific role of the estrogenic component upon pancreatic hormonal secretory potential. MATERIALS

AND METHODS

Subjects. Studies were performed upon 18 healthy females with a mean age of 22.6 yr (range 21-25) who had no family history of diabetes mellitus, no evidence of diabetes as determined by cortisone glucose tolerance tests,18 and who were all within 5% of ideal body weight (as determined by Metropolitan Life Insurance Tables, 1959). None of the subjects had previously taken oral contraceptives and dietary habits were not altered in this study. No evidence of preexisting lipemia was detected, with no subject demonstrating chylomicron or prebeta migrating lipoprotein on fasting serum lipoprotein electrophoresis. Fasting serum triglyceride (105 + 13 mg/lOO ml) and cholesterol (I 15 =t IO mg/lOO ml) concentration were within the normal range of our laboratory. Procedures. The subjects were randomly assigned by chance to one of three daily treatment schedules: (1) combined estrogen and norethindrone treatment (Norinyl 1 f 80R, Syntex, 0.08 mg mestranol* and 1.0 mg norethindrone), (2) ethinyl estradiol (0.10 mg), or (3) norethindrone (10.0 mg). Each subject underwent a control arginine tolerance test in the untreated state between the 6th and 16th day of the normal menstrual cycle; and a “treatment” arginine tolerance test after 14 days of the above steroid treatment schedule during the subsequent menstrual cycle. In this way, each woman served as her own normal control, and statistical comparisons were made using the Student’s “t” test for paired samples. The data thus represent paired statistical comparison of six women in each treatment group using their own matched control data only. All studies were performed in the Clinical Research Center between 7 and 9 a.m. after a lo-12 hr overnight fast. Each arginine infusion was carried out as follows: an indwelling venous catheter for frequent blood sampling was placed in the forearm and kept patent with small amounts of normal saline. Two baseline blood samples were obtained 10 min apart. J-arginine (5% R-GeneR, Cutter Laboratories) was then infused at a rate of 1.O grams/min (total &-arginine 30 grams) through a large bore needle into the opposite arm. Blood samples were obtained at 10 min intervals for I hr beginning at the onset of the arginine infusion, and they were analyzed for glucose, free fatty acids (FFA), triglyceride, cholesterol, glucagon, insulin, and a-amino nitrogen. All plasma assays were performed in duplicate, and basal values represent the mean of both preinfusion control samples. Anulyses. Plasma glucose was measured in samples preserved in NaF by Technicon Autoanalyzer using the ferricyanide method.19 Samples for triglycerides, FFA, cholesterol, a-amino nitrogen, glucagon, and insulin were drawn into heparinized tubes and placed immediately on ice. Upon completion of the study, all samples were centrifuged at 2000 RPM for 10 min at 4°C and aliquots pipetted for appropriate determinations. TrasylolR was added to the aliquots for glucagon assay, and all samples were frozen at -20°C until the chemical analyses were performed. Plasma glucagon was measured by double antibody radioimmunoassay” using Unger’s K-30 antiserum specific for pancreatic glucagon. Insulin levels were measured by the method of Morgan and

*Mestranol

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derivative

of ethinyl estradiol.

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Lazarow.” Methods for the determination of plasma FFA, triglycerideqz3 and cholesterol” have been previously described, and a-amino nitrogen was measured colorimetrically.25 RESULTS

Arginine Tolerance Test

Thirty-six paired arginine infusions were performed in the 18 normal women in both the control and post “contraceptive-steroid” state. The steroid administration was well tolerated with no change in dietary habits, and no change in body weight. Plasma glucagon. Mean basal glucagon concentration was 50 =t 20 pg/ml (mean f SEM) in the control studies, and was unchanged by either “combination steroid” administration (32 f 10 pg/ml, t = 0.84, p = N.S.), ethinyl estradiol administration (41 f 10 pg/ml, t = 1.4, p = N.S.), or norethindrone administration (100 f 50 pg/ml, f = 1.8, p = N.S.). In response to+arginine infusion, glucagon concentration rose promptly, attaining a maximum level at the completion of the infusion, and returning to basal values over the subsequent 30 min (Fig. 1). The mean peak rise in plasma glucagon response during “combination steroid” administration (202 f 51 pg/ml) was only one-third of the mean peak rise in glucagon concentration obtained during the paired control infusion (753 f 140 pg/ml, 2 = 3.49, p < 0.005). Administration of ethinyl-estradiol alone also resulted in a reduced plasma glucagon response, with the peak rise in plasma glucagon concentration of 447 f 138 pg/ml representing a 50% reduction compared to the paired control maximum rise of 985 f 148 pg/ml, t = 3.82,~ < 0.005) (Fig. 1). In contrast, administration of norethindrone was not associated with a reduction in aminogenic glucagon secretion. In fact, there was a tendency for a more rapid rise in hormone concentration during the first 10 min of the arginine

Fig. 1. Effect of gonadal steroid administration on the plasma glucagon nsponse to intravenous 4 -arginine in 18 normal women studied in groups of six (left panel = “combination steroid;” middle panel = ethinyl estradiol; right panel = norethindrone). The rise in plasma a-amino nitrogen in response to aginine infusion is plotted below. All values mpmsent the mean f SEM, with the control studios shown with cross-hatching.

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Fig. 2. Effect of gonadal steroid administration on the plasma insulin and glucagon response to intravenous J,-arginine in 18 normal women studied in #roups of six (see Iwad to Fig. 1.).

infusion (Fig. 1). The peak rise in glucagon concentration during the control infusion (749 f 190 pg/ml) was indistinguishable from that rise seen during the norethindrone study (741 f 152 pg/ml, t = 0.40,~ = N.S.). Plasma insulin. Mean basal insulin concentration was 16 f 5 rcU/ml in the control studies, and was unchanged by either “combination steroid” administration (20 f 6 pU/ml, t = 0.42, p = N.S.), ethinyl estradiol administration (12 f 4 pU/ml, t = 0.28, p = N.S.), or norethindrone administration (10 f 4 pU/ml, t = 0.17, p = N.S.). As shown in Fig. 2, upon infusion ofaarginine, mean plasma insulin concentration began to rise by 5 min, continuing still higher at 10 min, and attaining a maximum at the termination of the 30-min infusion. Mean maximum rise in concentration was 107 f 20 pU/ml in the control studies, and was slightly reduced by all three contraceptive steroid schedules (Fig. 2). Following “combination steroid” administration, the mean peak rise was 51 + 5 rU/ml (t = 4.78, p < 0.001); following ethinyl estradiol the mean peak rise was 99 f 22 pU/ml (t = 1.20, p = N.S.); and following norethindrone the mean peak was 59 f 15 pU/ml (t = 3.91,~ < 0.01). Mean basal glucose concentration was 83 + 6 mg/ 100 ml Plasma glucose. in the control studies, and was unchanged by either “combination steroid” administration (76 f 4 mg/ 100 ml, t = 1.40, p = N.S.), ethinyl estradiol administration (84 + 5 mg/lOO ml, t = 0.45, p = N.S.), or norethindrone administration (87 & 2 mg/lOO ml, t = 0.32, p = N.S.) (Fig. 2). In response to &arginine infusion, the maximum level of 111 + 11 mg/ 100 ml was attained at 20 min of infusion, and began to fall prior to completion of the infusion. By 60 min, plasma glucose levels were well below basal levels, averaging 66 f 5 mg/lOO ml. As plotted in Fig. 2, this glucose response was not significantly altered by any of the contraceptive steroid schedules.

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Plasma alpha-amino nitrogen (Fig. I). Both basal and infusion levels of aamino nitrogen were indistinguishable in the control and post contraceptive steroid studies. Basal concentrations averaged 2.2 f 0.3 pU/ml and attained a maximum level of 8.4 f 0.6 &j/ml in all arginine tolerance tests. The peak concentration uniformly occurred between 20 and 30 min, and was reduced to levels of 4.6 & 0.9 pU/ml by 60 minutes. Plasma free fatty acids (FFA). Mean basal FFA concentration was 770 f 75 pU/L in the control studies and was not significantly changed by either “combination steroid” administration (692 + 80 rU/L, t = 1.50, p = N.S.), or ethinyl estradiol administration (932 f 90 pU/L, t = 1.25, p = N.S.). However, in the six women receiving norethindrone, a small but significant reduction was observed(714 f 55 rU/L, t = 3.75,~ < 0.005). Plasma lipids (Fig. 3). The mean basal concentrations of plasma triglyceride (TG) and cholesterol demonstrated a consistent rise in concentration in all women treated with the “combination steroid,” or with ethinyl estradiol alone. While the rise was consistent, the absolute change was small and thus of questionable physiological significance in these normal women. Mean basal TG concentration was 105 f 13 mg/lOO ml in the 18 subjects in the control study, and increased +8 mg/ 100 ml (t = 2.64, p < 0.05) following “combination steroid” administration, and + 14 mgf 100 ml (t = 0.91, p = N.S.) following ethinyl estradiol administration (note, data represent paired statistical comparison of the six women in each treatment group using their own matched control data only). Similarly, mean basal cholesterol concentration was 115 f 10 mg/ 100 ml in the control studies, and increased + 13 mg/ 100 ml (t = 2.72,

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p < 0.05) following “combination steroid” administration and +25 mg/ 100 ml (t = 3.48, p < 0.001) following ethinyl estradiol administration (see Fig. 3). The response to norethindrone administration differed both qualitatively and quantitatively from that seen with “combination steroid” or ethinyl estradiol administration. Mean basal TG was reduced 26 mg/lOO ml (t = 3.68, p < 0.001) and mean basal cholesterol was reduced 25 mg/lOO ml (t = 3.48, p < O.OOl), as has previously reported by other investigators.‘C’3 Since plasma FFA represent one substrate for TG synthesis, an evaluation of this potential determinant of TG concentration was performed by regression analysis. As expected from the essentially unaltered basal FFA levels (see previous paragraph), the alterations in basal serum TG concentration were not proportional to the simultaneously determined serum FFA levels. For each gonadal steroid schedule regression analysis of paired FFA: TG concentration in the control and treated state failed to meet statistical significance; viz., “combination steroid” administration correlation coefficient = 0.126 (p = N.S.); norethindrone administration corr. coef. = 0.47 (p = N.S.); ethinyl estradiol administration corr. coef. = 0.08 (p = N.S.); corr. coef. of all studies together = 0.274 (p = N.S.). Similarly, regression analysis of the change in FFA versus the change in TG concentration failed to reveal a statistically significant relationship. DISCUSSION

These studies demonstrate that arginine-stimulated glucagon secretion is significantly reduced following initiation of “combination steroid” administration. A similar effect is demonstrated following ethinyl estradiol administration, but not with norethindrone, suggesting that the estrogenic component of the “combination steroid” may be responsible for this phenomenon. The consequences of this reduced glucagon secretion are not defined by our studies. However, the synchronous elevation in basal serum lipids and reduction in arginine-stimulated glucagon secretion in the estrogen studies, and the reduction in basal serum lipids with failure of suppression of arginine-stimulated glucagon secretion in the norethindrone studies, directs attention to a possible relationship between these events. While current understanding of the quantitative role of endogenous glucagon in lipid regulation is not clear, the hypolipemic action of the hormone through reduction in hepatic lipoprotein production is a well defined pharmacological property. Glucagon exposure is reported to inhibit apolipoprotein formation in the liver15 and depress hepatic triglyceride synthesis.26 In perfused liver systems, the hormone has been shown to reduce the conversion of FFA to TG by stimulating the synthesis of ketone bodies, thus modulating the availability of FFA substrate for lipid synthesis, independent of plasma FFA concentration.” Direct intravenous injection of glucagon in man results in a prompt elevation in the concentration of ketones and a reduction in serum TG consistent with the perfused liver data.ZB*29 Recognizing this lipid-lowering potential of glucagon, it has been suggested that a reduction in glucagon secretion and/or activity might be expected to pattern is recognized result in a tendency for hyperlipemia. 44Such an hormonal

I-LIPID PHYSIOLOGY

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in animal models of carbohydrate-induced hyperlipemia,% and cobalt-induced hyperlipemia,” where plasma lipid characteristics are indistinguishable from human contraceptive-induced lipemia. The reduction in aminogenic glucagon secretion following estrogen administration with an associated elevation in serum lipids is similar to the glucagon suppression reported following ingestion of high carbohydrate containing diets3’ which regularly induce human lipemia,3’ and to the reduced glucagon secretion in human obesity3* where a similar small elevation in plasma TG is commonly recognized. 33Thus, a role can be visualized for reduced glucagon secretory potential as a factor contributing to the genesis and/or maintenance of contraceptive induced lipemia. However, direct evidence for such a regulatory series of events is not available in man. As recently reviewed by Olefsky, Farquhar, and Reaven,16 progressive elevation in serum insulin concentration can be linearly correlated with progressively increased hepatic production of very low-density lipoprotein TG and with basal serum TG concentration. To simultaneously consider both the lipidelevating contributions of insulin and the lipid-lowering actions of glucagon, it may be instructive to examine the net change in the bihormonal balance generated by these three gonadal steroid regimens. If this relationship is expressed in terms of the insulin:glucagon molar ratio as suggested by Unger,35 this I/G ratio at the peak of arginine stimulation is 3.4 f 0.5 M/M in the control studies. Following either estrogen alone (4.4 M/M) or estrogen + norethindrone (7.6 M/M) the I/G ratio indicates net insulin excess. In contrast, following norethindrone administration, the I/G ratio is reduced (1.8 M/M), indicating net insulin reduction. The relationship between the net change in I/G ratio to the net change in plasma TG or plasma cholesterol is graphically depicted in Fig. 4. Linear regression analysis confirms a significant correlation between the change in I/G ratio and the change in plasma cholesterol (r = 0.828; p < 0.001) demonstrating a progressive rise in plasma lipid in association with the rise in plasma I/G ratio. A similar association is seen upon examination of the rise in plasma TG, but the variability of change in triglyceride concentration in the ethinyl-estradiol treated population does not demonstrate statistical significance in this relationship (I = 0.31; p < 0.05).This confirmation of an inverse relationship between the net insulin:glucagon balance and serum lipids is similar to that seen with the hypolipemic drug, clofibrate, which also reduces basal TG concentration in normal subjects by 25 + 9 mg/ 100 ml in association with a net reduction in I/G ratio (2.1 M/M).36 These combined changes in aminogenic glucagon and insulin secretion induced by specific gonadal steroids offer a bihormonal mechanism for the reported changes in lipid metabolism associated with ingestion of these agents. It may be important to note that previous investigations of the effects of gonadal steroids upon insulin secretion have not observed the reduction in concentration of this hormone noted in our studies.’ In fact, the administration of steroid” preparations have been reported to increase some “combination glucose-stimulated insulin secretion .& This difference may relate to the utilization by most investigators of glucose as a stimulus for insulin secretion, which can be expected to directly suppress pancreatic glucagon secretion. Our choice of arginine as an aminogenic hormone stimulus is based upon its ability to

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Fig. 4. Relationship between the gonadal steroid induced change in the Insulin: Glucagon molar ratio, (during ATT) and the basal plasma TO, (lower panel) or basal plasma cholesterol (upper panel). The data demonstmte the simultaneous reduction in I/G and plasma lipids following norethindrone, and elevation in I/G and plasma lipids following ethinyl estmdiol (see text).

simultaneously stimulate both insulin and glucagon, since both hormones are critical to lipid metabolism. These differences in pancreatic hormonal response to differing stimuli, illustrate the necessity for caution in extrapolating from pharmacologic tests to the physiologic responses to normal patterns of dietary behavior. The possibility remains that contraceptive steroids may also alter the plasma clearance rate of lipids. The progestogen, norethindrone, is an effective lipid

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lowering agent in both normal and hyperlipemic subjects.‘2*‘3 The decrements in TG induced by norethindrone are often associated with a simultaneous increase in post heparin lipolytic activity (PHLA) and in triglyceride lipase activity in the plasma. l2 More extensive studies have been reported concerning the structurally related carbon- 17 alkylated derivative of norestestosterone, oxandrolone, which has similar lipid lowering effects but which has no progesradioactive glycerol turnover studies, tational hormonal activity. 37~38Utilizing Glueck et a1.38 reported no consistent change in VLDL TG turnover, and attributed the lipid lowering effects to the marked increase in PHLA and triglyceride lipase activity and improved efficiency of VLDL TG removal, Our studies now extend these lipid lowering events by reporting an associated reduction of aminogenic insulin secretion resulting in relative glucagon excess. Caren and Carbo have reported that glucagon is capable of augmenting lipid removal from the plasma by means of increased binding to cellular elements of the blood.30 Thus, a glucagon potentiation of some modes of peripheral lipid clearance might contribute to the lipid lowering action of norethindrone in man. While no studies of the effect of glucagon on PHLA in vivo in man have been published, preliminary in vitro investigation is reported to demonstrate the opposite effect; i.e., inhibition of adipose tissue lipoprotein lipase.45 The effect of the estrogenic component of oral contraceptives on plasma lipid clearance is less well studied. PHLA activity is reported to be depressed by estrogen,N*4’ both when administered alone, and in combination with a progestogen.5,42 However, the simultaneous demonstration of unchanged plasma TG removal in spite of reduced PHLA has led to the suggestion that the estrogen component of oral contraceptives may stimulate endogenous TG synthesis and/or release into the plasma compartment. 42*43 This suggestion is strengthened by the observation that “combination steroid” administration results in a twofold increase in plasma TG turnover6 contrasting with the normal turnover reported with progestational-norestestosterone agents alone.43 In summary, our studies demonstrate that synthetic estrogens blunt aminogenie glucagon secretion, and suggest that this event contributes to ‘increasing the molar ratio of insulin versus glucagon presented to the liver. This altered bihormonal state is suggested as potentially contributing to the lipemia characteristic of estrogen administration, as previously suggested as an hormonal mechanism for human lipemia induced by carbohydrate ingestion and/or 44It is apparent that the 2 wk of exposure to conventional genetic predisposition. contraceptive steroids in these normal women did not result in a clinically significant change in serum triglyceride or cholesterol concentration in spite of the reduction in pancreatic glucagon secretion. As discussed previously, the lipidlowering actions of the nortestosterone component may have effectively neutralized any lipemic sequellae of the estrogen-induced suppression of glucagon. However, our studies only examined a brief exposure to the gonadal steroids, suggesting that the prolonged exposure utilized by conventional contraceptive therapy might result in a significant hormone induced lipemia as a result of the changes in glucagon secretion. Perhaps more likely is the possibility that significant lipemia may evolve in women treated with estrogen containing medications in whom a preexisting defect in plasma clearing lipases or excessive basal insulin production with limited glucagon secretory capacity may be present.8*9

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TT, Blomstrand R, Peterson ML: The influence of dietary fat on serum lipid levels in man. Lancet 1:943, 1957 32. Schade DS, Eaton RP: The role of insulin and glucagon in obesity. Diabetes 23:657, 1974 33. Robertson RP, Gavareski DJ, Henerson JD, Porte FD, Bierman EL: Accelerated triglyceride secretion: A metabolic consequence of obesity. J Clin Invest 52: 1620, 1973 34. Eaton RP, Kipnis DM, Karl I, Eisenstein AB: Effects of glucose feeding in insulin and glucagon secretion and hepatic gluconeogenesis in the rat. Am J Physio1227: 1160, 1974 35. Unger RH: Glucagon and the insulin: glucagon ratio in diabetes and other catabolic illnesses. Diabetes 20~834, 1971 36. Eaton RP, Schade DS: Effect of clofibrate on arginine-stimulated glucagon and insulin secretion in man. Metabolism 23:445,1974 37. Glueck CJ: Effects of oxandrolone on plasma triglycerides and post-heparin lipolytic activity in patients with Types III, IV, and V familial hyperlipoproteinemia. Metabolism 20: 691, 1971 38. Glueck CJ, Ford S, Jr, Steiner P, Fallat R: Triglyceride removal efficiency and lipoprotein lipases: Effects of Oxandrolone. Metabolism 22:807, 1973 39. Caren R, Corbo L: Transfer of plasma lipid to platelets by action of glucagon. Metabolism 19:598, 1970

1065 40. Fabian E, Stork A, Kobilkova J, Sponarova J: The activity of the lipoprotein lipase and estrogens. Enzymol Biol Clin 8:45 1, 1967 41. Fabian E, Stork A, Kucerova L, Sponarova J: Plasma levels of free fatty acid, lipoprotein lipase and post-heparin esterase in pregnancy. Am J Obstet Gynecol 100904, 1968 42. Hazzard WR, Notter DR, Spiger MJ, Bierman EL: Oral conceptives and triglyceride transport: Acquired heparin resistance as the mechanism for impaired post-heparin lipolytic activity. J Clin Endocrinol Metab 35:425, 1972 43. Rossner S, Larsson-Cohn U, Carlson A, Boberg J: Effects of an oral contraceptive agent on plasma lipids, plasma lipoproteins, the intravenous fat tolerance and the post-heparin lipoprotein lipase activity. Acta Med Stand 190:301,1971 44. Eaton RP, Schade DS, Conway M: Decreased glucagon activity: A mechanism for both genetic and acquired endogenous hyperlipemia. Lancet I 1: 1545, 1974 45. Nestel P, Austin W: Relationship be tween adipose tissue lipoprotein lipase activity and compounds which affect intracellular lipolysis. Life Sci 8:157, 1969 46. Spellacy WN, Carlson KL, Birk SA: Carbohydrate metabolic studies after six cycles of combined type oral contraceptive tablets. Diabetes 16:590, 1967