Serum insulin, glucose and non esterified fatty acids after administration of follicle-stimulating and luteinizing hormones in bitches

A. Renauld¹, N. V. Gomez², J. D. Scaramal², D. Garrido³, M. M Wanke²

¹ Departamento de Fisiología, Facultad de Medicina,  
² Departamento de Medicina, Facultad de Ciencias Veterinarias,
³ Cátedra de Matemática, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires

Postal address: Dr. Aurora Renauld, Departamento de Fisiología,  Facultad de Medicina, Paraguay 2155, 1121 Buenos Aires, Argentina.

Abstract
This paper reports the effect of the simultaneous administration of follicle-stimulating (FSH) and luteinizing hormones (LH) on serum glucose, insulin and nonesterified fatty acid responses after glucose or insulin challenge. The animals were originally at anestrous. FSH (dose 2.5 U/kg body wt.) and LH (0.27 U/kg body wt.) were sc injected on days 1, 4, 8 and 11. Vaginal smears were obtained daily. Six untreated controls at anestrous and six treated bitches reaching proestrous were used. Glucose tolerance tests were done with a dose of 1 g of glucose per kg of body weight. Bovine insulin was administered at the dose of 0.25 U/kg body wt. During these tests, neither serum glucose and nonesterified fatty acids nor glucose distribution space and glucose clearance were affected by the treatment. The serum insulin response to hyperglycemia was greatly increased. The distribution space and clearance rate of this hormone were not affected by FSH + LH treatment. We conclude that, in the bitch, FSH + LH  treatment, at doses that trigger «sex seasons», increases the serum insulin response to glucose load and produces a moderate resistance to the hypoglycemic, lipogenic and antilipolytic insulin actions. These phenomena are evident during hyperglycemia.

Key words: FSH. LH; Glucose; Nonesterified fatty acids; Insulin.

Resumen
Modificaciones de la glucemia, insulina y ácidos grasos no esterificados durante la sobrecarga de glucosa o insulina en perras tratadas con hormona folículo-estimulante y luteinizante. Este trabajo describe el efecto de la administración simultánea de FSH y LH sobre los niveles de glucemia e insulina y ácidos grasos no esterificados séricos luego de una sobrecarga de glucosa o insulina. Los animales se encontraban originalmente en anestro, controlado por extendidos vaginales diarios. FSH (2.5 U/kg peso corp./día y LH (0.27 U/kg peso corp./día) se inyectaron por vía subcutánea en los días 1, 4, 8 y 11 del tratamiento. Cada grupo experimental estaba formado por seis perros en anestro y seis en proestro. Las sobrecargas de glucosa (1g/kg peso corp.) fueron  administradas por vía endovenosa rápida. Las concentraciones de glucosa en sangre o ácidos grasos no esterificados séricos durante los tests de sobrecarga, los espacios de distribución de glucosa en sangre e insulina sérica o sus clearances plasmáticos no se vieron afectados por el tratamiento. Concluimos que la secreción de insulina como respuesta a una sobrecarga de glucosa  aumenta significativamente en perras en anestro tratadas con FSH + LH. Al mismo tiempo se observa una moderada resistencia a la insulina, en los efectos hipoglucemiantes, lipogénicos y antilipolíticos de esta hormona durante esta prueba.

Palabras claves: FSH. LH; Insulina; Glucosa; Ácidos grasos no esterificados.

Houssay and Biasotti¹ were the first to show the important role played by the hypophysis in the development of diabetes mellitus. Revision of the scientific literature reveals that the influence of FSH and LH on glucose homeostasis is still an undefined issue. Some studies in the bitch have demonstrated that there is a close relationship of the estrous cycle  with the onset of diabetes mellitus and the insulin requirements during overt diabetes^2,3. Ovariectomy greatly stimulates the insulin response to hyperglycemia in normal dogs⁴. This phenomenon does not appear to be a primary action of estrogen deficiency, though it is reverted by 17-beta-estradiol therapy probably by feed-back inhibition of gonadotrophin secretion⁴.
On the other hand, the spontaneous activation of the hypothalamus-pituitary-ovarian axis in normal bitches at anestrous produces complete estrous cycles. In this state, the tolerance to glucose of these animals is not affected but hyperglycemia promotes an intense secretion of insulin⁵, entirely different from those when the entire axis is in operation⁶. This phenomenon is particularly evident during the estrogenic phase.
Prolactin has been reported to have no effect on metabolic variables during glucose or insulin tolerance tests⁷.
The simultaneous administration of FSH and LH triggers estrous cycles in canines^{8, 9}. This paper reports the effects of these hormones on serum insulin, non esterified fatty acids and blood sugar during glucose and insulin tolerance tests.

Materials and Methods

Animals. Twenty mongrel, disinfested, female dogs, of unknown ages, weighing 7.3-16.0 kg, were used in these experiments. They were housed in metabolic cages, fed on  dog chow pellets and «ad libitum»  water, for 3 months. During this period they were at anestrous. It was expected that the high progesterone levels of the early stages of the preceding metadiestrous would prevent the occurrence of new estrous cycles¹⁰.
Six out of the fourteen treated bitches (43%) responded to FSH + LH treatment producing proestrous. Later, for a few days, these bitches showed atypical vaginal smears, and then  returned to anestrous. This reaction was taken as evidence for the adequacy of the dose of gonadotrophins.
Two groups were then formed, receiving (on days 1, 4, 8 and 11 of the experiments) either 1 ml saline (six control bitches) or a mixture of FSH and LH, in 1 ml saline (14 treated bitches). The dose of FSH was 2.5 U/kg body wt. and that of LH 0.27 U/kg body wt. FSH was from Serono Laboratory, Argentina, highly purified of human origin. LH was from UIAMDD (Batch 13, biopotency: 15 N.I.H. U/mg protein; negligible contamination with pituitary GH, TSH and PRL). Only the six bitches reaching proestrous just after treatment completion and the six saline treated controls were submitted to the tests described below.  Anestrous and occurrence of estrous cycles were checked by vagina smears according to Schutte⁹.
The bitches  were to be tested on the following day fasted overnight for 18-22 h. They had free access to tap water.
Tests. Every dog was submitted to glucose tolerance tests and insulin tolerance tests on consecutive days, in that order.
Glucose tolerance tests. Glucose solution (200 g/L) was administered i.v. at  the dose of 1 g/kg body weight. Glucose was administered as a bolus injection through a peripheral vein (external saphena, median or radial). Blood samples were drawn from a peripheral vein not used for injection, before and at 5, 15, 25, 45, 60 and 90 minutes after glucose load.
Insulin tolerance Tests.  Insulin, (from ox origin, crystalline, potency: 27.5 IU/mg protein, glucagon-free) was dissolved in 0.005 N HCl, pH 2.4, at a concentration of 1 mg/mL, and further diluted with saline so that the total dose per animal (0.25 IU/kg body wt.) was contained in a final volume of 5.0 ml. Blood samples was drawn from a peripheral vein (not used for injection) before and at 15, 20, 25, 30 and 35 minutes after insulin challenge.
Blood was allowed to clot for two hours at room temperature and centrifuged for 5 minutes at 3000 g. Serum was stored at -25 °C. Glucose was measured in a Technicon Autoanalyzer¹¹. Serum insulin was measured with a commer-cial kit (C.N.E.A.) and according of the cross-reaction between dog, ox and pork insulins^{12, 13}. Nonesterified fatty acids were measured according to Itaya and Ui method¹².
Equations for the regressions between the natural logarithms of glucose¹⁴ and insulin¹⁵ concentrations as a function of time were calculated. The general form of these equations was Y = a x e^-kt, where Y = concentration of the variable at time t, a = concentration at time zero, k = time coeficient. Glucose and insulin half-life times in blood stream were calculated according to t_1/2 = ln 2 / k. Their distribution spaces were calculated as: total amount of glucose or insulin divided by a.
Statistical analysis. An analysis of variance (ANOVA) of two factors per variable -dog, group (G), time (T) -with repeated measures on one factor (T) was performed¹⁶.
As a significant G-effect on a variable was found, the mean data per group at every time were compared with the  respective basal value, and between groups, following one-tailed, Dunnett test¹⁶. The same test was used to assess the significance of G X T. The significance of deviations from the mean regression line per group was tested¹⁶. The G  effects on the respective t_1/2 or distribution spaces were assessed by ANOVA of one factor (G). A difference was considered not significant (NS) as P > 0.05.

Results

Glucose Load

As shown in Fig.1, the levels of blood glucose in control and LH + FSH treated bitches did not differ significantly before or after glucose load (G x T, P > 0.05 ). The results of both bitches groups were pooled. Hyperglycemia was then observed at 5, 15 and 25 minutes; the basal values were again reached at 45 minutes.

The rate of glucose disappearance from blood in both groups, estimated between 5 and 45 minutes, decayed following the function stated in Material and Methods¹⁴. The t_1/2  of glucose did not significantly differ between groups (Controls: 22.03 ± 1.73 minutes, Treated bitches: 30.91 ± 4.85; F_1;10 = 2.97, mean ± S.E.M.).
Glucose distribution space (mean ± S.E.M.)was not significantly different between groups (Controls dogs: 36.92 ± 4.52 % body wt., Treated bitches: 70.70 ± 22.82;  F_1,10 = 2.11, P > 0.05).
As expected, glucose administration increased insulin serum levels (T, P < 0.01). Gonadotrophins treatment did not affect the basal serum insulin (P > 0.05) but it did increase the mean serum insulin (G, P < 0.01) and the shape of serum insulin profile (G x T, P < 0.05) during the test.
Since G x T interaction is significant, the effect of time alone was assessed. Gonadotrophins treatment further increased insulin levels at 5 (P < 0.01), 15 (P < 0.01), 25 (P < 0.01) and 45 (P < 0.05) minutes.
As expected, glucose administration reduced the serum concentration of nonesterified fatty acids. (T, P < 0.01). Gonadotrophins treatment affected neither the basal concentration of this variable (P > 0.05), their mean concentrations during the test (G,  P > 0.05) nor the shape of the curve.

Insulin Load

Insulin  administration  produced   the   expected   hypoglycemic response (T, P < 0.01) in both dogs groups, without significant differences between them (G, P > 0.05). Gonadotrophins treatment did not  affect the basal insulin levels nor the concentration of this hormone during the tests (P > 0.05).
During this test, the rate of disappearance of  serum insulin followed the function stated in Material and Methods, with good fitting between 15 and 30 minutes after insulin injection. The F value for respective deviations from the regression line was very low (F_2,20 = 0.29, P > 0.05). Half life time of insulin in blood stream did not differ between groups (Controls = 8.94 ± 1.19 minutes, Treated = 30.67 ± 12.76 minutes; F_1,9 = 3.51, P > 0.05).
The distribution space of insulin was not affected by treatment (Controls = 31.04 ± 7.85% body wt. (mean + S.E.M.); Treated = 111.26 ± 64.75 (F_1,9 = 2.08, P > 0.05).
The insulin load increased significantly serum nonesterified fatty acids levels (T, P < 0.05), without difference between groups.

Discussion

The bitches treated with FSH + LH share some metabolic features characteristic of obese women with polycystic ovaries. These patients show increased serum levels of LH together with normal or low serum FSH concentration¹⁷ and exhibit variable degrees of peripheral resistance to insulin.
The serum concentrations of FSH and LH were not measured in this study. Literature reports indicate for the bitch at anestrous, low or negligible serum FSH, LH, progesterone and estrogens concentrations^{10, 18-20}. This hormonal state provokes ovary quiescence and sex rest.
FSH²⁰ and LH¹⁹ administration are known to promote the secretion of estrogens and progesterone in the bitch. These hormones characterize early spontaneous proestrous in these animals, which could be reasonably extended to our bitches at proestrous triggered by FSH + LH treatment. These two groups of bitches exhibit at proestrous similar pictures of the vaginal epithelium, but these pituitary hormones fail to evoke further estrous and metadiestrous,  then leading to atypical, highly misleading vagina smear pictures.
The regulatory role played by the estrogens on the variables studied here has been analyzed elsewhere²¹. The data indicate that estrogenic mediation in the effect of FSH + LH on serum insulin during the glucose tolerance tests is deemed unlikely.
That FSH + LH are directly responsible for  the strong stimulation of insulin secretion is only a hypothesis. However, Bailey and Matty²² have observed raised serum insulin responses to glucose in ovariectomized rats trea-ted with Pergonal.
Althouth the pituitary gonadotrophins actions on the metabolism of ovarian and testicular cells have been extensively studied during the last past years, the role played by FSH and LH in the regulation of lipid and carbohydrate metabolism in vivo is largely unknown. Our experiments show clearly the lack of action of FSH + LH on glucose homeostasis of intact bitches during glucose and insulin load. These findings agree with observations made in ketoacidotic human patients²³ and also in ovariectomized rats²². Gonadotrophins did not exert a direct lipolitic action on isolated fat cells of rats and rabbits as incubated in vitro^24-26.
It can be concluded that the results reported here show that FSH + LH treatment increases the serum insulin response to hyperglycemia in the bitch at anestrous. However, the treatment fails to affect two major extrapan-creatic factors regulating serum IRI: the distribution space and the insulin rate of clearance from the blood stream. These observations suggest that the treatment with FSH + LH would stimulate the in vivo secretion of insulin without affecting the blood sugar and serum nonesterified fatty acid levels. These findings are interpreted as evidence for the development of a moderate resistance to the hypoglycemic, lipogenic and antilipogenic actions of insulin in these animals.

Acknowledgements: The authors feel indebted to Alicia Agüero V.M. for helpful advice on dog estrous cycle artificial induction by FSH and LH. The skilled technical assistance of Miss M. N. Jofré, Mr. H. Cabrera and Miss Marisa M. Moriondo is fully acknowledged. LH was donated by the National Institute of Arthitis, Metabolism and Digestive Diseases (National Pituitary Agency, Maryland University Medical School, Baltimore, Md.,U.S.A.) through the kindness of Salvatore Raiti M.D. Glucagon free, ox insulin was provided by Mr. H. Schneider, from Química Hoechst Argentina. The present study was financed with funds from The National Council of Scientific and Technical Research (CONICET), Res. Grant N° PIA 004-1042/87, Argentina.

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Recibido: 9 de Agosto de 2002
Aceptado: 24 de Octubre de 2002