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 diabetes2,3. 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 canines8, 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 insulins12, 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 t1/2 = 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 t1/2 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 t1/2 of glucose did
not significantly differ between groups (Controls: 22.03 ± 1.73 minutes,
Treated bitches: 30.91 ± 4.85; F1;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; F1,10 =
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 (F2,20 =
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; F1,9 =
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 (F1,9 =
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 concentrations10, 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 vitro24-26.
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