Effect of contraceptive steroids on arginine-stimulated glucagon and insulin secretion in women. II. Carbohydrate and lipid physiology in insulin-dependent diabetics

Effect of Contraceptive Steroids on Argininestimulated Glucagon and Insulin Secretion in Women. II. Carbohydrate and Lipid Physiology in Insulin-dependent Diabetics Paul

Beck,

David

M.

Arnett,

Robert

The effect of contmceptive steroids on aminogenic glucagon secretion was studied in six insulin-dependent diabetic women. After 2 wk treatment with combined mestmnol (80 pg) plus norethindrone( 1 mg) daily, the mean pk plasma glucagon response to arginine infusion was suppressed to one-fourth of control levels. This was associated with a small but significant decrease in mean basal plasma cholesterol concentmtions. There were no changes in basal plasma triglyc-

N. Alsever,

and

R. Philip

Eaton

eride, free fatty acid, glucose, insulin, or a-amino nitrogen concentrations or in daily insulin requirements during met tmnol plus norethindrone treatment. These results confirm previous reports of no consistent changes in the insulin requirements of insulin-dependent diabetic women using contmceptive steroids and suggest that these women may not experience dmmatic changes in their lipid metabolism during contmceptive thempy.

A

LTHOUGH THE EFFECTS of contraceptive steroids on carbohydrate and lipid metabolism in normal women are well described,’ their effects on the metabolic processes of diabetic women are less clear. Szabo et al. reported that subclinical diabetic women exhibited deterioration of glucose tolerance when treated with combined mestranol plus norethindrone;2 Gold et a1.3 reported that glucose tolerance deteriorated (together with a decrease in the insulin response to glucose) in a group of maturity-onset diabetics during mestranol plus ethynodiol diacetate treatment. By contrast, neither Steindel et al.4 or Mendner5 observed a significant change in daily insulin requirements of insulin-dependent diabetic women treated with birth control pills containing a synthetic estrogen in combination with nortestosterone or 17 cu-acetoxyprogesterone derivatives. With regard to lipid metabolism, Hassing Nielsen6 has reported that there was a significant increase in plasma triglyceride concentrations during treatment with ethinyl estradiol plus norgestrel in women predisposed to diabetes (gestational diabetes, history of excessive-weight baby, family history of diabetes). However, no comparable studies of lipid metabolism in

From the Departments of Medicine, University of Colorado School of Medicine, Denver, Colo. and the University of New Mexico School of Medicine, Albuquerque, N.M. Receivedfor publication March 18.1975. Supported in part by USPHS Grams HDJ32455. FR-ooO51. and HE-12085, by Grant RR-51 from the General Clinical Research Cenler Program of the Division of Research Resources, National Institutes of Health, and by a gram from the Kroc Foundation. Presenred in part at the Western Section of the American Federation for Clinical Research, February. 1974. Reprint requests should be addressed to Dr. Paul Beck, University of Colorado School of Medicine. 4200 East Ninth Avenue, Denver, Colo. 80220. o 1976 by Grune Cc Stratton. Inc.

Metabolism,

Vol. 25, No. 1 (January),

1976

23

24

BECK ET At.

insulin-dependent diabetic women using birth control pills are as yet recorded in the literature. Recently, Beck et al.’ have observed that glucagon secretion in response to IV arginine was blunted in normal women treated with mestranol plus norethindrone. A comparable suppression of aminogenic glucagon secretion was observed in normal women during treatment with ethinyl estradiol alone, but not during norethindrone alone. Plasma triglyceride and cholesterol concentrations increased during treatment with the estrogen-containing preparations, either alone or in combination with norethindrone, but not with norethindrone alone. These findings suggest an inverse relationship between plasma lipid concentrationsand aminogenic glucagon secretion as influenced by contraceptive steroids. However, contraceptive steroid-induced changes in insulin secretion must be considered in evaluating their effect on lipid physiology, since Olefsky, Farquhar, and Reaven* have shown a clear-cut relation between basal serum insulin concentrations and hepatic production of very-low-density lipoprotein triglycerides and circulating triglycerides concentration. Arginine-stimulated insulin secretion was blunted by each of the three contraceptive steroid treatment regimens examined by Beck et al.’ Nevertheless, when the lipid-elevating effect of insulin and the apparent lipid-lowering effect of glucagon were examined in terms of their bihormonal balance (i.e., insulin:glucagon (I/G) molar ratio as suggested by Unger9), a significant linear relationship was observed between the change in I/G ratio and the change in fasting plasma cholesterol during contraceptive steroid treatment.’ A similar association between changes in I/G molar ratio and changes in basal triglyceride concentrations has been observed in normal subjects treated with clofibrate, a hypolipemic drug.‘O This raises the question whether contraceptive steroids would also alter circulating lipid levels in subjects with impaired insulin reserve and relatively constant endogenous insulin production, viz., insulin-dependent diabetic subjects. The purpose of the present study is (1) to examine the effect of combined mestranol plus norethindrone treatment on plasma glucose, triglyceride, and cholesterol concentrations and daily insulin requirements in insulin-dependent diabetic women, (2) to determine whether these changes are related solely to alterations in the plasma glucagon response to arginine, and (3) to ascertain whether the lack of endogenous insulin might modify the changes in basal lipid concentrations produced in normal women by these steroids. MATERIALS

AND

METHODS

Studies were performed in six otherwise healthy juvenile-onset, ketosis-prone, insulin-dependent diabetic women with a mean age of 21.3 (range 17-26). All of the subjects were within 10% of the ideal body weight (as determined by Metropolitan Life Insurance Tables, 1959). None of the subjects had hypertension or renal disease, and all had been well controlled as determined by plasma glucose and urinary glucose and ketone measurements during the 6 mo before study. During the course of the study, none of the subjects demonstrated changes in dietary habits or body weight. Each subject underwent a control arginine tolerance test in the untreated state between the sixth and eleventh day of the normal menstrual cycle. A second arginine tolerance test was performed on the fourteenth day of daily treatment with mestranol (80 pg) plus norethindrone (I mg) (Norinyl 1 + SO’). In this way, each woman served as her own control, and statistical comparisons were made using Student’s t-test for paired samples” and Spearman’s rank order correlation coefficient.‘* All studies were performed in the Clinical Research Center between 7 and 9 a.m.

CONTRACEPTIVE

25

STEROIDS

after a IO-12-hr overnight fast. The usual daily insulin dosage was withheld until the completion of the arginine tolerance test. Daily insulin requirements ranged from 35-70 units prior to the study and were kept constant in each subject during the interval between arginine tolerance tests. Subsequently, while still on oral contraceptives, the daily insulin dosage was adjusted on the basis of fractional urine glucose and ketone analyses to account for any changes in glucose homeostasis. Insulin doses were changed only if the patient demonstrated symptoms suggesting hypoglycemia, glycosuria in all four daily urinalyses, or a consistent increase in the fasting morning urine glucose concentration. Each arginine tolerance test was performed as follows. An indwelling venous catheter for frequent blood sampling was placed in the forearm and kept patent with small amounts of saline. Two baseline samples were obtained IO min apart. I-Arginine (5% R-Gene,’ Cutter Laboratories) was then infused at a rate of 1.2 g/min (total I-arginine = 30 g) through a large bore needle into the opposite arm. Blood samples were obtained at IO-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, and o-amino nitrogen. Plasma glucose was measured in samples preserved by NaF using the ferricyanide method.13 Samples for triglycerides, FFA, cholesterol, a-amino nitrogen, and glucagon were drawn into heparinized tubes and placed immediately on ice. On completion of the study, all samples were centrifuged at 2,OLXlRPM for IO min at 4’C, and aliquots were pipetted for appropriate determinations. Trasylol” was added to the aliquots for glucagon assay, and all samples were frozen at -20°C until assayed. Plasma glucagon was measured by double antibody radioimmunoassay’ using Unger’s K-30 antiserum which is specific for pancreatic glucagon. Methods for the determination of plasma FFA,” triglyceridesI cholesterol,” and a-amino nitrogen’* have been previously described. RESULTS

Insulin Requirements Paired arginine tolerance tests were performed in the six insulin-dependent diabetic women in both the control and the contraceptive steroid treatment state. The birth control pills were well’tolerated, and there was no change in appetite, daily food intake, or body weight. As shown in Table 1, there was no consistent change in the amount of insulin used daily by individual subjects, and the amount of change was small. As shown in Table 2, mean basal plasma glucose concentration during the control tests was 203 mg/dl and increased to 226 mg/dl during contraceptive treatment, but this difference was not statistically significant. It should be noted that the plasma glucose concentrations rose continuously during the 30 min after completion of the arginine infusions (Table 2), whereas plasma glucose concentrations in normal women return to baseline levels following arginine administration.’ Table

1.

Change Womon

in Daily

Insulin

During

Contmcoptive

Doses in Insulin-Dependent Steroid

Diabetic

Treatment

Dailv Total Insulin Dose (Units) Subject

Before

During

LB FC MC JL JM

NPH 35 U NPH 30 U Reg. 10 U NPH25UReg.15.U Lente30UReg.15U NPH 70 U

NPH 35 U NPH 30 U Reg. 10 U

DW

Lente 45 u

Lente 45 u

NPH 25 U Reg. 20 U hte 35 U Reg. 15 U NPH 70 U

26

BECK ET AL.

Table

2.

Mean Fasting, 30- and 60-min Concentrations Arginine Infusion in Diabetic Women Before Contmceptive Fasting

Glucose

(mg/dl)

Mean A Variance P

A

from

IV

During 60 min

239

269

226

+ 36 f 25.4 < 0.01

+ 66 f 25.1
-

60 min

30 min

Fasting

-

fosting

During

Tmatment

203 -

= change

Steroid

Before 30 min

of Glucose and During

254

274

+ 20 f 9.7 < 0.001

+ 48 k19.2
value.

Plasma Lipids and FFA (Table 3)

Mean basal plasma triglyceride concentration was 217 mg/dl in the control studies and was not significantly different during mestranol plus norethindrone treatment. Mean fasting plasma cholesterol concentration decreased from 240 mg/dl during the control tests to 215 mg/dl during contraceptive treatment. Mestranol plus norethindrone treatment produced a decrease in the mean basal plasma FFA concentrations which was not statistically significant. Nevertheless, there was a significant correlation between the degree of decrease in basal FFA concentrations and the decrease in basal cholesterol concentration (r, = 0.943, p < OS), but not with the changes in basal triglycerides. Plasma (~-Amino Nitrogen

Both basal and infusion levels of o-amino nitrogen during the arginine infusions were indistinguishable in the control and contraceptive steroid treatment studies. The peak concentrations uniformly occurred at 20 or 30 min and returned to baseline levels by 60 min. . Plasma Glucagon (Table 4)

Mean basal glucagon concentration was 49 pg/ml in the control studies and was virtually identical during mestranol plus norethindrone treatment. In response to I-arginine infusion, plasma glucagon concentration rose promptly to loble

3.

Before

Fasting and

During

Plasma

Concentmtions

Treatment

with

of Cholesterol, Mestmnol

in Diabetic

JL LB JM FC MC DW Mean Mean A Variance P

Triglycerides, Nomthindrone

and (M

FFA

+ N)

Women

Triglycerides Subject

plus

Cholesterol

Bafore

During

Bafore

330 140 190 232

260 200 158 178

252 293 247 247

312 100

285 121

262 131

217 -

200 - 17 * 49.9

239 -

-

NS

FFA During

Before

During

235 275 188

766 605 1103

576 835 709

203 264 131

1188 817 773

773 605 787

216 - 23 f 24.3 <.05

875 -

714 -

161

f

247 NS

CONTRACEPTIVE

Table

STEROIDS

27

Mean Fasting and 3D-min Plasma Glucagon Concentrations (pg/ml) 4. During IV Arginine Infusions in Diabetic Women More and During Treatment with Mestmnol and Norethindrone (M + N) Fasting

Before During Mean At Variance

f

‘A = change t A = change

from from

Mean A*

49

832

+ 783

48 -1

315 - 517

+ 267 -

223.7

f

NS

P

30 Min

567.0 < .05

-

Variance f f

645.9 273.1 -

P < .02 < .05 -

fasting value. pretreatment value.

a peak value at the end of the infusion and returned to baseline levels within the subsequent 30 min. The mean peak plasma glucagon concentration during mestranol plus norethindrone administration (314 pg/ml) was only one-fourth of the mean peak glucagon level observed during the paired control infusion (832 pg/ml). The magnitude of the rise in circulating glucagon concentrations during the arginine infusion did not correlate significantly with the degree of change in circulating FFA, triglycerides, or cholesterol in the individual subjects, either in the basal state or during arginine infusion. Similarly, during mestranol plus norethindrone treatment, there was no consistent relationship between the degree of decline in arginine-stimulated plasma glucagon production and the degree of decline in basal triglyceride, cholesterol, or FFA concentrations. DISCUSSION

These studies demonstrate that arginine-stimulated glucagon secretion is significantly reduced in insulin-dependent diabetic women during treatment with mestranol plus norethindrone. The degree of suppression of aminogenic glucagon secretion in these diabetic women is comparable to that previously observed in normal women with the same combination of contraceptive steroids and with ethinyl estradiol alone.’ The mechanism responsible for contraceptive steroid suppression of glucagon secretion is unknown. Somatostatin has been reported to lower circulating glucagon acutely, and this may be associated with a profound decrease in blood glucose concentrations both in insulin-deficient diabetic animalsI and in insulin-dependent diabetic patients. 2o It seems unlikely that estrogens or contraceptive steroids stimulate somatostatin secretion, since combined ethinyl estradiol plus norethindrone derivative treatment has been reported to increase basal circulating serum growth hormone,2’ while somatostatin lowers it?* Unfortunately, we did not measure growth hormone or somatostatin levels in this study. The reduction in aminogenic glucagon secretion during mestranol plus norethindrone treatment does not appear to be mediated by reduced insulin secretion; the plasma glucose concentrations during both control and treatment arginine infusions were virtually identical, and they continued to rise even after completion of the infusion (Table 2). The latter finding implies that these subjects were not secreting any quantity of biologically active insulin. It is worth noting that basal plasma glucagon concentrations were within

28

BECK ET Al.

normal limits7 in our insulin-dependent diabetic subjects, particularly since it has been previously reported that diabetics have inappropriately high circulating glucagon levels in relation to their hyperglycemiaP Gerich et a1.23have also observed normal fasting glucagon levels in non-ill insulin-dependent young men who were maintained in a euglycemic state by continuous insulin infusions. These findings suggest that hyperglucagonemia develops in diabetic subjects only when they are “out of control,” i.e., when their physiologic needs require the catabolism of FFA and amino acids as an alternate or substitute for glucose as a metabolic fuel. Of additional interest is the fact that neither daily insulin requirements nor fasting blood glucose concentrations changed significantly in these insulindependent women while on mestranol plus norethindrone. This confirms the previous work of Steindel et al.4 and MendnerS and is consistent with the relatively small changes in glucose tolerance of normal women treated with EE + N derivatives.’ On the other hand, Gershberg et a1.24observed some amelioration of hyperglycemia in maturity-onset diabetics treated with mestranol alone, and Gerich et al.2o have reported the lowering of blood glucose concentrations in insulin-dependent subjects whose circulating glucagon levels were reduced, by somatostatin administration after ingestion of breakfast. In addition, Matute and KalkhoffZS have reported that estradiol inhibits gluconeogenesis and alanine conversion to glucose in rats. These effects may be due to estrogen blunting of endogenous glucagon secretion. In the present study, the failure to observe a decrease in fasting glucose and insulin requirements may be due to increased secretion of contrainsulin hormones, such as the secretion of growth hormone, cortisol, or epinephrine,26s27 either as a result of the contraceptive steroids or as a consequence of long-term insulin therapy. The present study shows for the first time that short-term combined mestranol plus norethindrone treatment does not produce an increase in basal circulating triglyceride or cholesterol concentrations in insulin-dependent diabetic women. These findings were unexpected since (1) the same treatment produced an increase in basal circulating triglyceride concentrations in nondiabetic, nonhyperlipidemic women7 and (2) the synthetic estrogens in contraceptive steroids have been clearly correlated with increased hepatic production and increased serum concentrations of triglycerides and lipoproteins in normal women.28*29 Moreover, since the basal lipid concentrations of the diabetic subjects during the control tests were already twice normal, one might expect a greater increase in fasting plasma triglyceride concentrations after exposure to estrogens. In the studies of Gershberg, Hulse, and Javier,30 subjects with high basal lipid levels developed marked hyperlipidemia after exposure to estrogens. The reason for the failure to see a further increase in basal triglyceride concentrations in our diabetic subjects during mestranol plus norethindrone treatment is not clear from the present studies. One possible explanation is that the norethindrone component of their birth control pills stimulated plasma lipoprotein clearance sufficiently to overcome any hyperlipidemic effect of the estrogen component. Norethindrone has been shown to increase postheparin lipoproteinlipase activity (PHLA) in hyperlipemic subjects.3’ In addition, Glueck et al.32 have reported a marked increase in PHLA and triglyceride iipase activity plus improved efficiency of VLDL triglyceride removal in subjects treated

CONTRACEPTIVE

STEROIDS

29

with oxandrolone, a 17-alkylated derivative of testosterone. Thus, the failure to detect an increase in basal triglyceride levels in the diabetics during mestranol plus norethindrone treatment does not preclude their ability to manifest an increased lipemic response to estrogen which could be neutralized by norethindrone. Alternately, it is possible that the liver of diabetic subjects may respond differently than normal to a lipogenic stimulus such as blunted glucagon secretion. Admittedly, it is not known whether increased hepatic production of VLDL lipoproteins contributes significantly to the hypertriglyceridemia observed in insulin-requiring diabetic patients. 33 Nevertheless, in normal subjects, Olefsky, Farquhar, and Reaven* have demonstrated that progressive elevation in serum insulin concentration can be linearly correlated with a progressive increase in hepatic production of VLDL lipoprotein production and with increased basal serum triglyceride concentrations. In the present study, the diabetic patients were constantly exposed to pharmacologic amounts of insulin because of exogenous insulin therapy. Although plasma insulin concentrations were not measured in the present study, Kuzuya et al.M have demonstrated that the plasma insulin concentrations of insulin-dependent diabetic patients greatly exceed the plasma insulin concentrations of subjects who have never received insulin, even after correction for anti-insulin antibodies. Thus, the organs and tissues of insulin-treated diabetic patients are probably exposed to high concentrations of insulin. Although glucagon has been shown to inhibit apolipoprotein formation in the liver,” to depress hepatic triglyceride synthesis,36 and to reduce the conversion of FFA to triglycerides by stimulating the synthesis of ketone bodies,37 insulin has been demonstrated to reverse these glucagon effects.37 Consequently, it is possible that the liver of insulin-dependent diabetic subjects is “hyperinsulinized” which could lead to a constant overproduction of triglycerides and to the hypersecretion of VLDL lipoproteins. If the “hyperinsulinization” of the liver were maximally effective, it is quite likely that decreased delivery of glucagon to the liver (as a consequence of estrogen treatment) would have no effect on hepatic triglyceride production. Nevertheless, the possibility remains that some poorly-controlled, hyperglucagonemic diabetic subjects might benefit from a reduction of endogenous glucagon secretion, particularly if this led to reduction in hepatic production of ketones from FFA oxidation. Schade and Eaton3* have demonstrated an acute increase in plasma concentrations of acetoacetate and &hydroxybutyrate in insulin-deficient diabetics following glucagon administration. By contrast, Gerich and co-workersz3 have reported suppression of plasma &hydroxybutyrate FFA and glycerol concentrations, as well as glucagon, during somatostatin infusion in human juvenile-type diabetic patients who were withdrawn from their exogenous insulin therapy. Since these patients did not develop ketoacidosis, it appears that glucagon is a major participant in the development of diabetic ketoacidosis. Thus, in the ketosis-prone, so-called “brittle” diabetic, chronic suppression of endogenous glucagon secretion might be clinically beneficial. However, until somatostatin becomes widely available clinically, chronic estrogen administration might prove useful in reducing the frequency of ketoacidosis episodes in nonpregnant brittle diabetics. In summary, the present study shows that short-term administration of

BECK ET Al.

30

mestranol plus norethindrone suppresses arginine-stimulated rises in plasma glucagon concentrations without any increase in circulating lipids or in daily insulin requirements. Nevertheless, in view of the facilitatory role of glucagon in gluconeogenesis and ketogenesis, it is possible that estrogen therapy may have a useful role in the management of ketosis-prone juvenile-type diabetic patients. REFERENCES 1. Beck P: Contraceptive steroids: Moditications of carbohydrate and lipid metabolism. Metabolism 22:841-855, 1973 2. Szabo AJ, Cole HS, Grimaldi RD: Glucose tolerance in gestational diabetic women during and after treatment with a combinationtype oral contraceptive. N Engl J Med 282:646650,197O 3. Gold EM, Carvajal J, Rudnick PA, Gerszi KE: Insulin-production in overt (maturityonset) diabetes: Absence of hyperinsulinemia despite hyperglycemia induced by contraceptive steroids, in Salhanick HA, Kipnis DM, Vande Wiele PL (eds): Metabolic Effects of Gonadal Hormones and Contraceptive Steroids. New York, Plenum, 1969, pp 144156 4. Steindel E, Mohnike A, Langreck G: Erfahrungen bei der ambulaten Behandlung von Diabetikerren mit dem Ovulationshemmer Ovosiston. Z. Aerztl Fortbild (Jena) 65: 159, 1971 5. Mendner K: Zur Frage der Beeinflussung des KohlenhydratstolTwechsels bei Diabetikerinnen durch Ovulationshemmer. Med Welt 22:828, 1971 6. Hassing Nielsen F: Serum triglyceride in women with and without a predisposition to diabetes during short-term administration of a combined oral contraceptive. Acta Obstet Gynecol Stand 53:13-19.1974 7. Beck P, Eaton RP, Arnett DM, Alsever RN: Effect of contraceptive steroids on arginine-stimulated glucagon and insulin secretion in women. I. Lipid physiology. Metabolism 24:1055-1056, 1975 8. Olefsky JM, Farquhar JW, Reaven GM: Reappraisal of the role of insulin in hypertriglyceridemia. Am J Med 57:551-560, 1974 9. Unger RH: Glucagon and the insulin: Glucagon ratio in diabetes and other catabolic illnesses. Diabetes 20:834-838, 197 1 IO. Eaton RP, Schade DS: Effect of clofibrate on arginine-stimulated glucagon and insulin secretion. Metabolism 23:445-454, 1974 11. Worcester J: The statistical method. N Engl J Med 27427-36, 1966 12. Siegel S: Non-Parametric Statistics. New York, McGraw-Hill, 1956, pp 203-212

13. Grady HJ, Lamar MA: Glucose determination by automatic chemical analysis. Clin Chem 5:542-550, 1959 14. Eaton RP: Glucagon secretion and activity in the cobalt chloride treated rat. Am J Physiol225:67-72, 1973 15. Duncombe WG: The calorimetric microdetermination of long-chain fatty acids. Biochem J 88:7-10, 1963 16. Carlson LA: Determination of serum triglycerides. J Atheroscler Res 3:334-336, 1963 17. Leffler HH: Estimation of cholesterol in serum. Am J Clin Pathol31:310-313, 1963 18. Frame EG, Russel JA, Wilhelmi AE: The calorimetric estimation of aminonitrogen in blood. J Biol Chem 149:255-270, 1943 19. Dobbs R, Sakurai H, Unger RH: The essential role of glucagon in diabetic hyperglycemia and the effect of glucagon blockade in diabetes mellitus. Clin Res 22:648A, 1974 20. Gerich JE, Lorenzi M, Schneider V, Karam JH, Rivier J, Guillemin R, Forsham PH: Effects of somatostatin on plasma glucose and glucagon levels in human diabetes mellitus. N Engl J Med 291:544547, 1974 21. Spellacy WN, Carlson KL, Schade SL: Human growth hormone levels in normal subjects receiving an oral contraceptive. JAMA 202:45 l-454, 1967 22. Parker DC, Rossman LG, Siler T, Rivier J, Yen S, Guillemin R: Inhibition of physiologic hGH release in early sleep by somatostatin. Clin Res 22: 1 I8A, 1974 Lorenzi M, Bier DM, 23. Gerich JE, Schneider V, Tslakian E, Karam JH, Forsham PH: Prevention of human diabetic ketoacidosis by somatostatin: Role of glucagon. N Engl J Med 292:985-989, 1975 24. Gershberg H, Javier Z, Hulse M, Cohane J: Improvement of glucose tolerance with estrogen treatment in maturity-onset diabetics. Diabetes 16525.1967 25. Matute ML, Kalkhoff RK: Sex steroid influence on hepatic gluconeogenesis and glycogen formation. Endocrinology 92:762-768, 1973 26. Kipnis DM, Stein MF: Insulin antag-

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onism: Fundamental considerations. Ciba Found Colloquia Endocrinol 15:156-191, 1964 27. Daughaday WH, Kipnis DM: The growth-promoting and anti-insulin actions of somatotropin. Ret Prog Horm Res 22:49-99, 1966 28. Kekki M, Nikkila EA: Plasma Triglyceride turnover during use of oral contraceptives. Metabolism 20~878-889, 1971 29. Kissebah AH, Harrigan P, Wynn V: Mechanism of hypertriglyceridemia associated with contraceptive steroids. Horm Metab Res 5:184190, 1970 30. Gershberg H, Hulse M, Javier Z: Hypertriglyceridemia during treatment with estrogen and oral contraceptives. Obstet Gynecol 31: 186189.1968 31. Glueck CJ, Levy RI, Frederickson DS: Norethindrone acetate, post-heparin lipolytic activity, and plasma triglycerides in familial types I, III, IV, and V hyperlipoproteinemia. Ann Intern Med 75:345-352, 1971 32. Glueck CJ, Ford S Jr, Steiner P, Fallat R: Triglyceride removal efficiency and lipoprotein lipases: Effects of oxandrolone. Metabolism 22:807-814, 1973

31

33. Have1 RJ: Hypertriglyceridemia, in Stollerman GH (ed): Adv Int Med. vol. 15. Chicago, Year Book Medical, 1969, pp 117-154 34. Kuzuya H, Lewis SB, Murray WK, Coustan DR. Daane TA, Wallin JD, Rubenstein AH: Circadian variation of serum glucose, C-peptide immunoreactivity and free insulin in normal and insulin-treated pregnant subjects. Program of the 57th Annual Meeting of the Endocrine Society, 1975, p. 87 35. Amatuzio DW, Grande F, Wada S: Effect of glucagon on the serum lipids in essential hyperlipemia and in hypercholesterolemia. Metabolism 11:1240-1249, 1962 36. Penhos JC, Wu CH, Daunas J, Reitman M, Levine R: The effect of glucagon on the metabolism of lipids and on urea formation by the perfused rat liver. Diabetes 15:74&748, 1966 37. Heimberg M, Weinstein I, Kohout M: The effects of glucagon, dibutyiyl cyclic adenosine 3.5 monophosphate, and concentration of free fatty acid on hepatic lipid metabolism. J BiolChem 244~5131-5139, 1969 38. Schade DS, Eaton RP: Modulation of fatty acid metabolism by glucagon in man. II. Effects in insulin-deficient diabetics. Diabetes 24:510-515, 1975