DIABETES AND ESTROUS CYCLE INTERACTION IN DOGS
NATURAL ESTROUS CYCLE IN NORMAL AND DIABETIC BITCHES IN RELATION
TO GLUCOSE AND INSULIN TESTS
JORGE D. SCARAMAL, AURORA
RENAULD*, NELIDA V. GOMEZ**, DELIA GARRIDO, MARIA M. WANKE, ADRIAN G.
Departamento de Medicina,
Facultad de Ciencias Veterinarias; Departamento de Fisiología,
Facultad de Medicina; Cátedra de Matemática, Facultad de Farmacia y
Bioquímica, Universidad de Buenos Aires
* Member of Research Career of CONICET (Consejo Nacional de
Investigaciones Científicas y Técnicas)
** Fellow of CONICET
Key words: diabetes, estrous cycle, metabolism in bitches
influence of spontaneous «sex seasons» on blood sugar (BS) and se-
rum insulin levels was studied in bitches with natural diabetes
mellitus (DM) and normal controls, in the basal condition and during
glucose and insulin tests, was studied. DM increased basal BS, reduced
glucose tolerance, distribution space (DS) and clearance from blood,
and induced resistance to insulin hypoglycemic action. In normals,
occurrence of «seasons», inconsistently modified basal BS, increased
glucose tolerance and DS; during estrogenic phase (EP), these
variables were above those during luteal phase (LP). In diabetics at
LP, BS found in fasting condition and during glucose test were higher
than in diabetic bitches at EP (respective values at anestrous (A) in
between) and glucose DS was smaller. Rate of glucose clearance from
blood remained unaffected by «seasons» in both dog groups. Basal
serum IRI was not modified by DM or «seasons». In normals, serum IRI
response to glucose load was nonsignificant during A and increased
during the «seasons»; either insulin DS or the rate of insulin
clearance from blood stream remained unchanged under the
circumstances, the increase being mediated by insulin secretion.
During EP, the increase was particularly intense and mean
insulinogenic index (MII) rose. During LP, MII returned to A value,
whereby diabetic states might be manifest. Serum IRI profiles during
insulin test were not modified by «seasons» in normal bitches; such
response in diabetic bitches was intense during A, then decreased (EP)
or was later abolished (LP). Either in normal or diabetic bitches, the
sensitivity to exogenous insulin hypoglycemic action remained
unchanged in spite of «seasons». In diabetic bitches at A, serum IRI
after glucose challenge peaked higher than in respective normal
controls (insulin clearance and insulin DS were similar): they
exhibited relative insulin shortage and resistance to insulin
hypolgycemic action partly compensated by promoted insulin secretion.
Along with «season», abolished serum IRI response to glucose load in
diabetics was observed. During EP, extrapancreatic factors regulating
serum IRI concentration and MII did not change in respect to A,
whereby abolishment appears mediated by depressed insulin secretion.
During LP, insulin antagonism in conjunction with 1) absolute insulin
deficiency and 2) intense decrease in MII appears as a powerful factor
exposing diabetic bitches to a severe or fatal derangement in diabetic
estral espontáneo en perras normales y diabéticas en relación con
pruebas de glucosa e insulina. Se estudió la influencia de los ciclos
sexuales espontáneos sobre la glucemia (G) y niveles de insulina
sérica inmunoactiva (IRI) en perras con diabetes mellitus (DM)
espontánea y en controles normales, en la condición basal y durante
las pruebas de glucosa e insulina. DM aumentó la G basal, redujo la
tolerancia, espacio de distribución (ED) y el índice de aclaramiento
de la glucosa, induciendo resistencia a la acción hipogluce-miante de
la insulina. En normales, las «estaciones sexuales» modificación
inconsistentemente la G basal, aumentaron la tolerancia a la glucosa y
su ED; durante la fase estrogénica (FE), estas variables estuvieron
por encima que las de la fase luteal (Fl). En diabéticas en FL, la
glucemia basal o durante la prueba de glucosa estuvieron por encima de
las halladas en diabéticas en FE, con los valores de anestro (A)
entre ellas y el ED de la glucosa fue menor. El índice de
aclaramiento de glucosa de la sangre no fue afectado por el ciclo en
ambos grupos de perros. La IRI sérica basal no se modificó por DM o
ciclos. En normales, la respuesta de IRI sérica a la sobrecarga de
glucosa fue no significativa durante el A y aumentó durante los
ciclos; tanto el ED de insulina como el aclaramiento de insulina de la
corriente sanguínea quedaron no afectadas en esas circunstancias; el
aumento es mediado por la secreción de insulina. Durante la FE, el
aumento fue particularmente intenso y el índice insulinogénico medio
(IIM) aumentó. Durante la fase luteal, el IIM retornó al valor de A,
pudiéndose presentar crisis diabéticas. Los perfiles de IRI sérica
durante la prueba de insulina no se modificaron por los ciclos en
perras normales; dicha respuesta en perras diabéticas fue intensa
durante A, luego decreció (FE) y fue más tarde abolida (FL). Tanto
en perras normales como diabéticas, la sensibilidad a la acción
hipolucemiante de la insulina exógena no cambió a pesar de los
ciclos. En perras diabéticas en A, el pico de IRI sérica después de
la sobrecarga de glucosa fue más alto que en controles normales
respectivos (aclaramiento y ED de insulina no variaron): ellas
mostraban una privación parcial de insulina y resistencia a la
acción hipoglucemiante de esta hormona parcialmente compensada por
aumentada secreción de insulina. A lo largo del ciclo, se observó
abolición de la respuesta de IRI sérica a la glucosa exógena.
Durante la FE, factores extra-pancreáticos que regulan el nivel de
IRI sérica y el IIM no cambiaron respecto de A, por lo que la
abolición parece mediada por depresión en la secreción de insulina.
Durante la FL, el antagonismo insulínico junto con 1) deficiencia
absoluta de insulina, y 2) intensa disminución en el IIM parecen
factores poderosos que exponen a las perras diabéticas a un severo o
fatal empeoramiento de su enfermedad diabética.
Postal address: Dr. Aurora Renauld, Departamento de
Fisiología, Paraguay 2155, 1121 Buenos Aires, Argentina
Received: 12-IV-1995 Accepted: 5-IX-1996
The spontaneous activation of hypothalamus-pituitary-ovarian axis
has been reported to induce glucose intolerance and increased serum
immu-noreactive insulin (IRI) responses to glucose challenge in
ovariectomized rats1 and normal bitches2.
The canine species shows spontaneous diabetes mellitus (DM), which is
seldomly pancreatic in origin but frequently secondary to other marked
endocrine changes. For instance, in bitch, there is a great body of
evidence supporting the influence of the activation of gonadal axis on
onset or deterioration of diabetic states3-8, which may be observed
during the estrogenic phase of estrous cycle (EP)8, but becomes highly
conspicuous or fatal in aged intact bitches during the course of their
luteal phases (LP)3-8. Although these observations have been well
documented, the pathogenesis of the disorder remains unknown.
According to Eigenmann et al3-5, 7, the high protracted
hyperpro-gesteronemia characterizing the long-lasting canine
metadiestrous might be responsible for the elevated serum growth
hormone (GH) levels observed in diabetic and in some normal bitches at
LP; as time elapses, in certain predis-ponsed animals, this disorder
would result in Langerhans islet histological lesions, exhaustion or
deterioration of pancreatic insulin stores and settlement of
ketose-prone, insulin-dependent diabetic states, similar to those
exhibited by normal dogs under GH treatment for several days9.
According to results obtained in our laboratory2,10-13, we decided to
check Eigenmann’s suggestion from an ampler standpoint. This
appeared pertinent because the involvement of menstrual cycle in
outset or deterioration of a diabetic state in women still remains
unclear. Therefore, we studied the influence of spontaneous estrous
cycle (also referred to as «sex season») upon some pancreatic and
extrapancreatic mechanisms regulating blood sugar (BS) and serum IRI
levels in bitches with naturally occurring DM and in normal controls,
fasting and during the course of glucose and insulin tests. Thus, BS
and serum IRI were measured and then we calculated either glucose or
insulin distribution spaces in body tissues and clearance rates from
blood stream and also respective mean insulinogenic index. The results
obtained are discussed in relation to the patho-genesis of outset or
deterioration of a diabe- tic state on the estrous cycle in the canine
Material and methods
Animals. Thirty female dogs, of unknown ages, weighing 8-28 kg,
were used in the experiments. Fifteen of them were mixed-breed mongrel
normal controls which were disinfested, kept in individual kennels,
and fed on dog chow pellets and «ad libitum» water, for a 3
month-period before the tests performance. The remaining 15 bitches
were privately owned, ambulatory patients attending to Dr. Ernesto
Cánepa Small Animal Hospital, Veterinary Sciences Faculty, Buenos
Aires University, for naturally occurring DM care; most of them had
never received insulin therapy; only 2 were injected N.P.H. insulin
for a few days till the 5th day preceding the tests performance: they
then received only chrystalline insulin for 1 day, which was later
interrupted until the tests were over.
Every bitch was checked for estrous cycle through exfoliative vaginal
cytology13. They were randomly used for these experiments as they
reached anestrous (A), e (eosinophillic index = 20-100%) and LP (early
metadies-trous). Continuous A for 3 months was required by us to
include a bitch in the A group. The bitches that were to be tested on
the following day fasted overnight (18-22 h fast); meanwhile, they had
free access to water.
Tests. Every dog was tested for glucose (IVGTT) and insulin (ITT)
tolerances on consecutive days; glucose tests were performed first.
Glucose (dose 1 g/kg body wt.) was dissolved in distillated water
(conc. 20 g/dl). Glucagon free chrystalline ox insulin, potency 27.5
IU/mg protein, was dissolved (conc. 1 mg/ml) in 0.005N HCl, pH 2.4,
and was 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. Every
dog received quick-, alternatively one of these solutions into a
peripheral (external saphenous, median or radial) vein. Blood samples
were taken from these veins (except that used for injection) basally,
5, 15, 25, 45, and 60 min after glucose challenge (IVGTT) and at
15,20, 25, 30, 35, and 40 min from insulin load (ITT).
Assays. All blood samples were assayed for BS in a Technicon
Autoanalyzer14; some NaF was added to a small portion of every sample
to prevent coagulation, and then it was diluted (5 per cent) in
distillated water for the assay. The remaining portion of the samples
was allowed to clot for 2 h at room temperature, it was then
centrifuged at 2500 r.p.m. for 5 min at the same temperature, its
serum was separated and stored at -25°C. Serum IRI was later measured
by using a commercial kit (C.N.E.A., Argentina); pork insulin standard
and guinea pig anti-(pork insulin) serum were used in the assay, for
there is a reasonable cross-reaction rate between dog and pork
insulins15. In a basal serum sample from every bitch, beta-estradiol16
and progesterone17 levels were measured.
Statistical evaluation. All missing values were estimated first18. As
the influence of sex stages (Phase=P) in normal and diabetic bitches
(Group=G) on Bs and serum IRI levels, fasting and at several time
intervals from glucose and insulin load (Time = T) was to be studied,
in analysis of variance (ANOVA) of three factor experiments (factors:
G,P.T) with repeated measures on one factor (T), was applied19; the
original data had been submitted to log transformation for homogeneity
of variance; mean comparisons by « a posteriori» one-tailed Dunnett
test19 or Tuckey test18 were carried out; whenever not indicated, the
latter was used.
Equations for the log-linear regressions representing both glucose20
and insulin21 disappearance from blood stream in every dog during
glucose and insulin test were respectively calculated. The equations’
general form was Y = loge V = logea-kt, where Y = variable response, V
= variable concentration, logea = constant (Y axis intercept), k =
constant, and t = time elapsed from glucose and insulin loads,
respectively; the significance of regression line deviations from
linearity was tested22. Either glucose or insulin, half-life time in
blood stream (t1/2 = 0.69/k min) and distribution space (DS) in body
tissues per dog were calculated; aware of Y0 value by extrapolating
the mean regression line for t = 0, glucose or insulin total dose
injected and dog body weight, the respective distribution space was
The insulinogenic index (II) at every time during glucose test per dog
from the following formula was calculated.
II = Serum IRI (µU/ml)/Blood sugar (mg/dl)
MII was then estimated by calculating the respective area according to
To study the influence of sex stages (P) in normal and diabetic
bitches (G) on both glucose and insulin, t1/2 and DS, and also on MII,
an ANOVA of two factor (G,P) experiments with 5 observations per cell
was applied19; t1/2 values first underwent inverse transformation, and
DS and MII data were submitted to log transformation for variance
homogeneity; mean comparisons were then performed (Tuckey test)18.
In this study, a mean difference was considered significant as equal
or as P > 0.05.
1. Influence of diabetes mellitus and estrous cycle on several
variables in bitches
DM did not modify the mean basal serum concentrations of 17 beta -
estradiol and progesterone in bitches at the EP and LP of their
estrous cycles, respectively (Table 1, Fig. 1). The diabetic condition
significantly increased either the mean basal BS level or glucose t1/2
and diminished glucose distribution space in body tissues (Tabla 1
Fig. 3). DM failed to affect the mean fasting serum IRI concentration,
insulin t1/2 as well as the mean of insulinogenic index.
The occurrence of estrous cycle modified these variables in the
diabetic dogs and in the normal controls, except for the basal serum
IRI level. Thus, in the latter, the cycles decreased the mean basal BS
level and increased their glucose distribution spaces in body tissues.
In the diabetic dogs, the fasting BS concentration decreased during EP
in respect to A but increased during the LP, and just a small decrease
in glucose distribution space as estrous cycle progressed from EP to
LP was observed. In the normal controls, estrous cycle failed to
modify glucose t1/2 whereas in the diabetic dogs during LP this
variable intensely rose as compared with A (Table 1, Fig. 3).
The occurrence of estrous cycle modified the mean fasting serum IRI
level in neither dog group except for the diabetic bitches at EP, in
which a moderate increase in relation to respective A was observed. As
for insulin distribution space in body tissues and insulin t1/2 in
blood stream of normal bitches, they were very small and remained
unaffected by estrous cycle. Either were these variables in the
diabetic bitches at A and EP, whereas during the LP, such variables
were both quite increased. In the normal group, the insulinogenic
index intensely peaked during the EP whereas, in the diabetic dogs, it
significantly decreased only during the LP as compared with the
respective controls at A (Table 1, Fig. 3).
2. Blood sugar
A. Glucose test. DM influenced the mean BS concentration during the
test (P < 0.01); this level was increased. The mean BS level varied
with time elapsed after glucose challenge (P < 0.01); hyperglycemia
was observed. The mean BS profiles in normal and diabetic bitches
differ (P < 0.01); in the normal controls, hyperglycemia between 5
and 45 min from glucose load was observed (P < 0.01) and base line
was again reached at 60 min; in the diabetic group, hyperglycemia
throughout this test was found (P < 0.01) (Dunnett test) (Fig. 1,
The sex stages influenced the mean BS levels in normal and diabetic
bitches, either combined (P < 0.01) or separately considered (P
< 0.01). Thus, in the normal ones at every sex stage, the mean BS
concentrations during glucose test were different (P <0.01); a
relative maximum at A -and a minimum during EP- was observed. In the
diabetic group, only EP and LP affected these levels differently (P
< 0.01); a relative maximum during the course of LP -and a minimum
during EP- was found. On the other hand, the effects of DM on the mean
BS profiles during this test in bitches at every sex stage were
significant (P < 0.01); at every sex stage, DM raised them. (Fig.
1, Table 2).
B. Insulin test. DM influenced the mean BS levels during this test (P
< 0.01); which were increased. The mean BS concentration varied
with time after glucose challenge (P < 0.01); hypo-glycemia was
observed. The mean BS curves in normal and diabetic bitches differed
(P < 0.05); in the normal group, hypoglycemia at every time
throughout this test was found (P < 0.05) whereas in the diabetic
bitches the mean BS levels reached the respective base line from 25
min till the test was over (P < 0.05) (Dunnett test) (Fig. 2, Table
2). The effect of sex stages on the mean BS level of every group
during this test was nonsignificant and therefore they were not
compared in particular.
3. Serum immunoreactive insulin
A. Glucose test. DM did affect the mean serum IRI concentration
during the course of this test (P < 0.05). The mean serum IRI level
varied with the time after glucose challenge (P < 0.01), and the
shape of serum IRI curves obtained in normal and diabetic bitches
differed (P < 0.05). (Fig. 3, Table 3).
The effect of sex stages on the mean serum IRI concentrations during
this test was signifi- cant (P < 0.01). In normal and diabetic
bitches at every sex stage, the shapes of the mean serum IRI profiles
during the test differed (P < 0.05). Thus, in the normal bitches a)
at A, the mean serum IRI concentration did not change significantly (P
> 0.05), b) at EP, this concentration intensely rose above the
respective base line at 5, 15, 25 and 45 min from glucose load (P <
0.05): base line was again reached at 60 min, and c) at LP, such
concentration was moderately above the respective basal level only at
45 min from the load (P < 0.01). In contrast, in the diabetic
bitches a) at A, the mean serum IRI concentration was moderately above
the respective base line at 5 and 15 min from glucose load (P <
0.01), whereas b) at EP and LP, there was no significant change in
this variable (P > 0.05) (Dunnett test). (Fig. 3, Table 3).
B. Insulin test. III. DM influenced the mean serum IRI level during
this test (P < 0.01). This level changed with the time after
insulin load (P < 0.01); the shape of serum IRI profile in normal
and diabetic bitches did not differ (P > 0.05). (Fig. 4, Table 3).
The sex stages influenced either the mean serum IRI concentration
found during insulin test (P < 0.05) or the shape of the respective
serum IRI profile (P < 0.01). In normal and diabetic bitches, this
shape was differently affected by sex stages (P < 0.01). Thus, in
normal bitches at every sex stage, these profiles coincided, and they
were above base line throughout the test (P < 0.01, Dunnett test).
In contrast, in the diabetic group at every sex stage, the profiles
differed (P < 0.01); thus, a) at A, the mean serum IRI
concentration at every time throughout the test was far above the
respective basal value (P < 0.05), b) during EP, the concentrations
between 15 and 35 min were moderately above this value (P < 0.01),
which was again reached at 40 min, and c) during LP, the concentration
overpassed significantly the basal level at no time during the test (P
> 0.05). (Fig. 4, Table 3).
It is well known that the intense hypoglycemia evoked by insulin
load during insulin test in the normal animals is primarily
counteracted through a dissipation of the injected insulin; some quick
enhancement in the blood concentration of insulin antagonists usually
occurs as well23-27. It is likely that the same did occur in the
bitches studied herein; we have demonstrated that at least insulin,
epinephrine and cortisol participate in this concern in the canine
species28. On the other hand, glucose challenge during glucose test
provoked hyperglycemia and hyperinsulinemia in many groups of bitches
studied herein, and other investigators have demonstrated that a
depression in GH and in glucagon secretion occurs6. Later,
hyperinsulinemia exerts its anabolic action as observed in several
species of mammals including dogs29, and a compensation of the
excessively brisk fall of the Bs level by various insulin antagonistic
hormones is usually developed9, 24, 30, GH appearing excluded in this
respect20. Although glucose is a major pancreas stimulus for insulin
secretion in canines2, 10, 11, 13, 30-32, the normal bitches at A
exhibited nonsignificant serum IRI responses to hyperglycemia, thereby
confirming our previous reports2, 10, 12, on account of a marked sex
dimorphism in this variable behavior during glucose test11.
As expected, the diabetic bitches showed in general altered regulation
in BS levels certainly related to their relative or absolute insulin
deficiency. During A, this deficiency was only relative; apparently,
the «in vivo» insulin secretion in these animals was more intense
than in the respective normal controls; their major extrapancreatic
factors normally regulating serum IRI levels, viz. insulin
distribution space in body tissues and insulin clearance rate from
blood stream, were within their respective normal ranges. On the other
hand, the diabetic state induced in the bitches at A a resistance to
insulin hypoglycemic action. It is impossible for the diabetic bitches
to have developed insulin antibodies as a consequence of insulin
therapy because most of them had never been submitted to such therapy
or had eventually been given the hormone just for a few days so as to
insure survival during the severe acute diabetic crises characterizing
the «seasons», mainly LP. Such resistance appears rather related to
a deranged insulin-antiinsulin balance in their body tissues. Although
the intrinsec nature of the insulin antagonist(s) provoking the
disorder remains unknown, its occurrence is in keeping with
observations made in humans. Thus, type I or II diabetic subjects
exhibit basal hyperglucagonemia and lack of glucagon suppressibility
by hyper-glycemia24; hyperglucagonemia potentiates epine-phrine
hyperglycemic action in both insulin-dependent humans and dogs23, 24;
type I diabetic patients show also basal GH overproduction and poor
blood GH suppressibility by hyperglycemia33, but in type II this
suppressibility is adequate9. Apparently, at least glucagon,
epinephrine and the permissive action of cortisol can be blamed for
the quick insulin resistance observed in the diabetic bitches at A; GH
share in this respect appears remote6. In agreement with results shown
here, the literature reports that the insulin resistance exhibited by
diabetic human subjects appears lower as studied during insulin test.
Thus, in recent-onset type I DM, glucagon secretory response to
hypoglycemia decreases being only in part transiently compensated by
epinephrine, GH and cortisol23, 33; in type II diabetic humans this
response is normal.
The increased serum IRI response to glucose challenge observed in the
normal bitches «in season» (either phase) was in keeping with our
previous findings2, and both glucose, tolerance and distribution space
in body tissues varied accordingly. This increase did not result from
a favorable stimulation by the BS level nor was it mediated by any
change in the extrapancreatic factors normally regulating serum IRI
concentration (see above); it seems more conceivably related to
enhanced «in vivo» insulin secretion. In contrast, serum IRI
response to stimulation by glucose load was abolished in the diabetic
bitches «in season». The mechanism whereby this absolute insulin
shortage occurs remains obscure, but it is quite clear that the
abolishment starts during EP, as expected from clinical studies8. The
shortage is definitely not related to a deficient pancreas stimulation
for insulin secretion by glucose because in these animals the BS peak
was certainly above (LP) or just hardly below (EP) that observed in
the diabetic controls at A. During EP, one might expect that an
intensely depressed «in vivo» insulin secretion provoked the
shortage, because the major extrapancreatic factors regulating serum
IRI (see above) remained unchanged despite that this phase was
triggered. As for LP, inferring the state of insulin secretion from
“in vivo” insulin response to glucose challenge, it is
unfortunately unattainable because of the huge share of the
tremendously broadened insulin distribution space in body tissues —an
extrapancreatic factor— in the development of the insulin shortage.
However, either the basal GH overproduction, lack of serum GH
suppressibility by hyperglycemia or Langer-hans islet histological
lesions observed by well known investigators in diabetic bitches at
LP2-7 strongly suggest their pancreas to be exhausted for insulin
secretion. Unfortunately, we could not check these suggestions made by
Eigenmann et al6, 7, 9 because there is neither canine GH antibody nor
the respective kit for canine GH radioimmuno-assay ready for
All the disturbances in the regulation of BS and serum IRI levels
related to estrous cycle in normal and diabetic bitches are somehow
connected with the hormone changes in hypothalamus-pituitary-ovarian
axis which induce the «sex seasons». In normal bitches, these
changes are well known to be similar to those occurring in normal
women during menstrual cycle only more prolonged34-36, whereas in
diabetic dogs they have not been studied so far.
As demonstrated in Table 1, the concentrations of 17 beta-estradiol
and progesterone in diabetic bitches during the estrogenic and the
luteal phases of estrous cycles, respectively, are similar to those in
normal controls and therefore they do not seem to account for these
disturbances. The respective FSH, LH and GnRH levels could not be
measured (unavailable dog specific RIA kits), but since the
«seasons» in the bitches with DM used herein progressed normally as
proved by exfoliative vaginal cytology, these levels are expected to
have been within the normal range at the time of the experiments.
Nevertheless, it seems interesting to remark that, like in diabetic
women and rats, sex cycles occurrence in some diabetic bitches was
difficult (luteinization being most frequently impaired). On the other
hand, at present is is quite clear for us that the ovarian hormones
physiologically inducing either phase of the sex cycles should not be
blamed for any stimulatory action6, 25, 37-45 on serum IRI response to
hyperglycemia in the normal group35; prolactin mediation can be ruled
out as well46; in contrast, FSH and LH are major factors evoking this
stimulation during the EP10 and likely over the LP. As found by us,
neither do the ovarian hormones12 nor prolactin46 account for the
improved glucose tolerance observed in the normal dogs «in season»;
we observed also that combined FSH and LH nonsignificantly account for
it10, which might suggest that GnRH —whose role(s) in the regulation
of BS and serum IRI levels is still ignored— is responsible for
these variables displayed by the activation of the gonadal axis.
In the normal bitches, the progress of naturally triggered estrous
cycles from EP to LP is mildly diabetogenic. Thus, during the LP,
although glucose distribution space in body tissues and glucose
clearance rate from blood stream remained unchanged with respect to
EP, either glucose tolerance or serum IRI response to glucose
challenge moderately decreased, and the mean insulinogenic index then
approached its A value. These results strongly suggest that most
normal bitches at LP shall not become diabetic; in keeping with this
observation, clinical studies report that only a minor fraction of
intact bitches develop DM3, 4, 47, which is manifested during LP, i.e.
when 1) progesterone synthesis is maximal4, 6, and 2) basal
hypersomatotrophinemia and lack of GH suppressibility by hyperglycemia
occur6, 7, 48. The dog, like other carnivores, is highly sensitive to
GH diabetogenic action6. Although the diabetic symptoms and
hypersomatotrophinemia were described to reverse after
ovariohysterectomy3, 6, 47 in bitches at LP, the role played by
progesterone alone in the pathogenesis of DM under these circumstances
is still controversial. Apparently, there is no cause-effect
relationship of progeste-ronemia to GH levels; thus, some normal
bitches at LP showing signs of intense glucose intolerance (with or
without acromegaly) exhibit normal serum progesterone concentrations
but elevated serum GH levels6. GH production was then proposed to be
paradoxically controlled by normal blood progesterone levels6 through
an unclear mechanism, which develops only in older age6; in this
context, it was interestingly remarked that dog, in contrast to other
species, exhibits very high postestrous progesterone levels for about
two months whether pregnant or not; futhermore, in bitch, the
reproductive cycles do not cease in older age. Whether such life-long
exposure to high blood progesterone somehow favours the development of
DM remains unclear but is possible3-7. It has been suggested that
progesterone alone cannot account for the precipitation of the disease
in normal bitches, but it is apparent that in conjunction with a
genetically determined predisposition or another
progesterone-controlled diabetogenic factor it must be responsible for
the induction of the disease6. This key role played by progesterone
appears to deserve some comments because this possibility, based upon
the strongly diabetogenic action of synthetic proges-tagens in canine
females4, 5, does not fully match our results reported herein and
elsewhere12. Thus, although it is true that progesterone is
diabeto-genic when administered alone at physiological doses to normal
bitches at A, this effect is lost when the hormone is injected
according to the normal estrogen-progesterone sequence12. Furthermore,
the normal bitches at LP studied herein, in spite of their high
progesterone levels35, 36 showed better glucose tolerances as compared
with respective controls at A, expected to exhibit neglectable blood
progesterone34-36. Moreover, since apparently just diabetic bitches at
LP and some normal controls at undetailed sex state were studied for
serum GH levels by Eigenmann’s group3, 4, one might wonder whether
such hyper-somatotrophinemia could be only a characteristic of the
diabetic state itself, as recently observed in humans with type I
DM33. Therefore, our recent findings2, 10, 12, 41 suggest a more
complicated view of the pathogenesis of DM as triggered by estrous
cycle in bitch, not exclusively based upon the possibilities outlined
by Eigenmann et al.3-7.
As far as is known today, we might suggest that, in normal bitches
«in season», both moderate insulin-resistance and pancreas
overstimu-lation for insulin secretion induced by high serum FSH and
LH from the very beginning of the «season»10, predispose them for a
diabetic onset in the long run, viz. as soon as 1) a pancreas failure
for compensation and/or 2) GH overproduction3-7 (if any) will occur.
It is apparent that these mechanisms most frequently occur in aged
animals (more exposure to diabetogenic hormones of gonadal axis, in
time and periodicity) and/or in bitches exhibiting a genetically
determined predisposition for insulin deficiency.
As for the diabetic bitches, during A either their glucose intolerance
or the resistance to insulin hypoglycemic action were at least partly
compensated by a moderate augmentation in the «in vivo» insulin
secretion, whereby the mean insulinogenic index was normal and they
showed a poor prospective tendency to exhibit severe ketose-prone
acidotic diabetic crises, such as found in clinical studies3-8.
Unfortunately, as «sex seasons» happened to be spontaneously
triggered in these animals, their serum IRI responses to glucose
stimulation were abolished and the outset of diabetic crises could
occur, albeit seldom during EP8, most surely during LP3-8. Our
observations explain these findings, partly at least. Thus, during EP,
despite the insulin secretion blockade, glucose tolerance and the
insulinogenic index remained nonsignificantly altered in respect to A:
it is likely that some adaptation in the insulin antagonism must have
occurred. In contrast, estrous cycle progress from EP to LP in the
diabetic bitches resulted in a marked deterioration in their disease.
Thus, in the diabetic group at LP, exhibiting a decreased glucose
clearance rate from blood stream already from respective A and also a
blocked serum IRI response to stimulation from EP, the deterioration
was at least in part related to 1) a reduction in glucose distribution
space in body tissues most probably depending upon their absolute
insulin deficiency, and 2) a tremendous enlargement in the insulin
distribution space in body fluids which, despite the modest reduction
in the insulin clearance rate from blood stream, continuously
contibutes to a decrease in serum IRI level which insulin secretion is
absolutely unable to overcome. The progressive pancreas histological
lesions, such as Langerhans islet vacuolization, B-cell hydropic
degeneration, and islet hypoplasia observed in diabetic bitches at
this phase48 appear to explain this unability. The combined actions of
all these diabetogenic factors provoke in these animals the worst
glucose tolerance found in this study, resulting in turn in a dramatic
decrease of the mean insulinogenic index with the subsequent
aggravation in their sickness. As far as is known it is apparent that,
in the diabetic bitches at LP, the synergy of high levels of various
insulin antagonists, such as combined FSH and LH10, 34-36, and GH3-7
and the absolute insulin shortage, is a powerful mechanism which,
without excluding other(s), induces the severe or fatal derangement in
their diabetic condition which is frequently observed in veterinary
In brief, the foregoing study leads us to conclude that the occurrence
of spontaneous estrous cycles in normal and diabetic bitches modifies
their glucose metabolism and their serum IRI responses to stimulation
which —mainly during LP— can result in outset or aggravation of
diabetic states thereby explaining the results of various clinical
observations made in canines, as reported in the literature.
Acknowledgements. The authors wish to express their
appreciation to Miss M.N. Jofré and Mr. H. Cabrera for skilled
technical assistance. The normal dogs were obtained from Pasteur
Institute, Buenos Aires, through the kindness of Dr. A.J. Carrugati.
The batch of insulin was given by Mr. H. Schneider, Química Hoechst.
This study was financed by CONICET and by Universidad de Buenos Aires.
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TABLE 1.— Variables studies in this paper. Mean values and
respective S.E.M. are given; in the last 5 variables, only mean values
Normal controls Diabetic dogs
Anestrous Estrogenic Luteal Anestrous Estrogenic Luteal
Phase Phase Phase Phase
Estradiol, pg/ml - 112±13 - - 161±65 -
Progesterone, ng/ml - - 2.1±0.7 - - 2.1±1.3
Fasting glycemia, 81±7 28±3t 45±9t 232±24## 191±60##t 355±45##t
Fasting serum insulin 14±5 15±5 7±3 2±1 23±3 8±2
Glucose distribution 32.8 87.04tt 58.5tt 20.1## 25.4## 16.4##
space, % body wt. *
Glucose t1/2, min 35.4 26.4 32.6 92,7## 65,0## 110.6##
Insulin distribution 15.2 22.4 36.5 38.4 20.1 743.0tt
space, % body wt. **
Insulin t1/2, min 12.2 10.3 12.7 14.4 7.3 39.7**
Insulinogenic index, 0.15 1.20t 0.48 0.10 0.12 0.01*
µU/ml x 100
## P < 0.01 Levels of significance of comparisons in respect to
respective normal controls.
t,tt P < 0.05, P < 0.01 Levels of significance of comparisons in
respect to respective anestrous.
*,** P < 0,05, P < 0.01 Levels of significance of comparisons in
respect to respective estrogenic phase.
TABLE 3.— ANOVA of serum immunoreactive insulin levels in normal
and diabetic bitches (Group), at anestrous and during natural estrous
cycle (both phases) (Phase), basally and during the course of
intravenous glucose and insulin tests (Time). Respective mean results
shown in Fig. 3 and 4. Missing data were estimated; log transformation
of original data; ANOVA of three factor experiment with repeated
measures on one factor: d.f.: degrees of freedom; MS mean square; F:
Glucose test Insulin test
Source of variation
d.f. MS F d.f. MS F
Group (G) 1 7.1469 7.38* 1 10.2231 8.36**
Phase (P) 2 5.5897 5.78** 2 5.3551 4.38*
G x P 2 0.5018 0.52 2 1.3041 1.07
Subj. within G 24 0.9678 22 1.2230
Time (T) 5 0.8809 6.45** 6 2.6709 62.64**
G x T 5 0.3327 2.43* 6 0.0353 0.83
P x T 10 0.2609 1.91 12 0.1465 3.44**
G x P x T 10 0.2649 1.94* 12 0.1009 2.37**
T x Subj. within G 120 0.1367 141 0.0426
* ** Levels of significance of F (P < 0.05 and P < 0.01
TABLE 2.— ANOVA of blood sugar levels in normal and diabetic
bitches (Group), at anestrous and during natural estrous cycle (both
phases) (Phase), basally and during the course of intravenous glucose
and insulin tests (Time). Respective mean results shown in Fig. 1 and
2. Missing data were estimated; log transformation of original data;
three factor ANOVA with repeated measures on one factor (conservative
test for insulin test only); d.f.: degrees of freedom; MS mean square;
F: Fisher value.
Glucose test Insulin test
Source of variation
d.f. MS F d.f. MS F
Group (G) 1 18.0753 167.49** 1 33.4065 151.93**
Phase (P) 2 1.6927 15.68** 2 0.6183 2.81
G x P 2 0.7132 6.61** 2 0.8033 3.65*
Subj. within G 24 0.1079 22 0.2199
Time (T) 5 0.8930 107.20** 6 0.1763 16.86**
G x T 5 0.1269 15.24** 6 0.0545 5.21*
P x T 10 0.0110 1.32 12 0.0246 2.35
G x P x T 10 0.0039 0.46 12 0.0128 1.22
T x Subj. within G 120 0.0083 136 0.0105
* ** Levels of significance of F (P < 0.05 and P < 0.01
Fig. 1.— Blood sugar levels in normal and diabetic bitches at
anestrous and during the estrogenic and luteal phases of estrous cycle
over intravenous glucose test. Glucose dose: 1 g/Kg body wt.; it was
injected at 0 time into a peripheral vein. Systemic venous blood
withdrawn in the basal condition and over the test. blood sugar assay
in Technicon Autoanalyzer. Means of 5 animals for group are shown.
Fig. 2.— Blood sugar levels in normal and diabetic bitches at
anestrous and during the estrogenic and luteal phases of estrous cycle
over intravenous insulin test. Insulin dose: 0.25 IU/Kg body wt; it
was injected at 0 time into a peripheral vein. Systemic venous blood
was withdrawn in the basal condition and during the test. Blood sugar
assay in Technicon Autoanalyzer. Means of 5 animals per group are
Fig. 3.— Serum immunoreactive insulin (IRI) levels in normal and
diabetic bitches at anestrous and during the estrogenic and luteal
phases of estrous cycle over intravenous glucose test. Glucose dose: 1
g/Kg body wt.; it was injected at 0 time into a peripheral vein.
Systemic venous blood was withdrawn in the fasting condition and
during the test. Serum insulin measured by radioimmu-noassay. Means of
5 animals per group are shown.
Fig. 4.— Serum immunoreactive insulin (IRI) levels in normal and
diabetic bitches at anestrous and during the estrogenic and luteal
phases of estrous cycle over intravenous insulin test. Insulin dose:
0.25 IU/Kg body wt.; injected at 0 time into a peripheral vein.
Systemic venous blood was withdrawn in the basal condition and during
the test. Serum IRI measured by radioimmu-noassay. Mean values of 5
animals per group are shown.