affected subjects are usually more than 5 years of age , and there is a predisposition in females . in dogs , insulin resistance ( ir ) is suspected when hyperglycemia is still detected despite administration of insulin doses greater than 1.0 to 1.5 iu / kg .
definitions of resistance to exogenously administered insulin vary , but all are based on the dose insulin administered and the resultant blood glucose concentrations .
ir is caused by an increase in circulating counter regulatory hormones ( glucagon , glucocorticoids , catecholamines and growth hormone ) .
these stress - related diabetogenic hormones increase as a result of concurrent diseases , endocrine disorders or exogenous administration .
progesterone ( p4 ) also induces ir by stimulating growth hormone ( gh ) production in mammary glands .
increases in circulating p4 concentration are associated with exogenous administration and physiologic conditions such as diestrus and pregnancy . in pregnant and non - pregnant bitches , increases in gh are related to increases in circulating p4 and appears to be secondary to synthesis of gh in mammary glands .
it has been clearly demonstrated that p4 can stimulate mammary gh hypersecretion during the non - pregnant luteal phase .
elevated gh plasma concentrations are characterized by the absence of a pulse pattern and insensitivity to stimulation and inhibition tests , with the exception of inhibition by the progesterone antagonist ru486 . in hypophysectomized dogs , no absence or decrease in plasma gh concentrations
selman et al .   demonstrated that mammary glands were effectively the source of gh concentrations . in human and felines , increased gh concentrations have been observed after progestin treatment .
aglepristone , which is the first p4 receptor blocker licensed for veterinary use , has been successfully used for pregnancy interruption , pyometra medical therapy and treatment of feline fibroadenomatous mammary hyperplasia .
aglepristone binds to uterine p4 receptors with affinity three times greater than p4 itself in bitches and nine times greater in queens .
aglepristone does not modify plasma concentrations of p4 , but indirectly induces uterine contractions and cervix dilatation after multiple injections .
.   demonstrated that aglepristone in dogs reduces the length of p4 secretion by cl and accelerates the luteolytic process , and that this effect persists for about 6 to 8 days .
several side effects have been observed after administration of aglepristone , such as anorexia , restlessness , depression , vomiting , diarrhea decrease in body temperature and local inflammatory reaction after injection .
the present study was conducted to investigate the use of aglepristone for the treatment of insulin - resistant diabetes mellitus during the luteal phase .
all bitches included in the study were diabetic subjects under insulin therapy that were referred to our veterinary teaching hospital over a period of two years for insulin - resistant diabetes mellitus that developed during mid - luteal phase of the estrous cycle ( 25th~30th day of diestrus ) .
the luteal phase was established by vaginal cytology examined upon hematoxylin - eosin staining and blood p4 concentrations .
criteria for selection of patients were : glycemia persistently > 200 mg / dl over a 12 h period , despite treatment with more than 1.5 iu / kg bid of insulin ; clinical and laboratory findings ( p4 > 2 ng / ml ) indicative of diestrus .
diet , training , environmental factors and the insulin protocol administration were similar for all subjects .
exclusion criteria were the presence of other disorders or treatments that can cause insulin resistance .
eight intact bitches of different breeds and ages ( 9 to 15 years ; mean  standard deviation [ sd ] : 10.9  1.46 years ) met the inclusion criteria , as treatment group ( group t ; n = 8) and were therefore classified as having a p4 induced insulin - resistant diabetes mellitus ( table 1 ) .
the control group ( group c ; n = 6 ) was composed by six diabetic bitches of mixed breeds and ages ( 8 to 12 ; mean  sd : 10  1.41 ) under insulin therapy in the same phase of the estrous cycle ( 25th~30th day of diestrus , table 2 ) .
the study was carried out in accordance with the italian legislation on animal care ( dl 116/92 ) .
blood samples ( 4 ml ) for the determination of p4 concentration ( a commercial available radioimmunoassay - ria for canine ) and gh ( commercially available ria for canine and porcine gh ; linco research , usa ) were collected before and after treatment with aglepristone by cephalic venipuncture and immediately transfer to ice - chilled edta - coated tubes for gh determination , as well as to tubes without anticoagulant for p4 determination .
all samples were centrifuged at 4 for 10 min , after which plasma was stored at -25 until assayed .
blood samples for determination of the pulsatile plasma profile of gh were collected at 15 min intervals between 8:00 am and 14:00 pm before aglepristone administration and five days after the last aglepristone treatment .
serial blood glucose curves were performed on day 0 , 5 , 12 and 20 after the beginning of treatment .
all subjects were treated with porcine insulin zinc suspension ( caninsulin ; intervet , italy ) as reported in table 1 .
bitches in group t were treated with 10 mg / kg subcutaneously ( sc ) aglepristone ( alizin ; virbac , italy ) on day 1 , 2 , 9 and 17 in diestrus bitches .
the control group received porcine insulin zinc suspension 1 iu / kg ( caninsulin ; intervet ) as well as saline solution ( 0.3 ml / kg , sc ) on days 1 , 2 , 9 , 17 .
differences among groups were assessed by anova , and a p < 0.05 was considered to indicate statistical significance .
all bitches included in the study were diabetic subjects under insulin therapy that were referred to our veterinary teaching hospital over a period of two years for insulin - resistant diabetes mellitus that developed during mid - luteal phase of the estrous cycle ( 25th~30th day of diestrus ) .
the luteal phase was established by vaginal cytology examined upon hematoxylin - eosin staining and blood p4 concentrations .
criteria for selection of patients were : glycemia persistently > 200 mg / dl over a 12 h period , despite treatment with more than 1.5 iu / kg bid of insulin ; clinical and laboratory findings ( p4 > 2 ng / ml ) indicative of diestrus .
diet , training , environmental factors and the insulin protocol administration were similar for all subjects .
exclusion criteria were the presence of other disorders or treatments that can cause insulin resistance .
eight intact bitches of different breeds and ages ( 9 to 15 years ; mean  standard deviation [ sd ] : 10.9  1.46 years ) met the inclusion criteria , as treatment group ( group t ; n = 8) and were therefore classified as having a p4 induced insulin - resistant diabetes mellitus ( table 1 ) .
the control group ( group c ; n = 6 ) was composed by six diabetic bitches of mixed breeds and ages ( 8 to 12 ; mean  sd : 10  1.41 ) under insulin therapy in the same phase of the estrous cycle ( 25th~30th day of diestrus , table 2 ) .
the study was carried out in accordance with the italian legislation on animal care ( dl 116/92 ) .
blood samples ( 4 ml ) for the determination of p4 concentration ( a commercial available radioimmunoassay - ria for canine ) and gh ( commercially available ria for canine and porcine gh ; linco research , usa ) were collected before and after treatment with aglepristone by cephalic venipuncture and immediately transfer to ice - chilled edta - coated tubes for gh determination , as well as to tubes without anticoagulant for p4 determination .
all samples were centrifuged at 4 for 10 min , after which plasma was stored at -25 until assayed .
blood samples for determination of the pulsatile plasma profile of gh were collected at 15 min intervals between 8:00 am and 14:00 pm before aglepristone administration and five days after the last aglepristone treatment .
serial blood glucose curves were performed on day 0 , 5 , 12 and 20 after the beginning of treatment .
all subjects were treated with porcine insulin zinc suspension ( caninsulin ; intervet , italy ) as reported in table 1 .
bitches in group t were treated with 10 mg / kg subcutaneously ( sc ) aglepristone ( alizin ; virbac , italy ) on day 1 , 2 , 9 and 17 in diestrus bitches .
the control group received porcine insulin zinc suspension 1 iu / kg ( caninsulin ; intervet ) as well as saline solution ( 0.3 ml / kg , sc ) on days 1 , 2 , 9 , 17 .
differences among groups were assessed by anova , and a p < 0.05 was considered to indicate statistical significance .
prior to aglepristone administration , the mean serum p4 concentrations averaged 9.25  3.15 and 10.50  2.06 ng / ml in group t and group c , respectively ( p > 0.05 ) ( tables 1 and 2 ) , while the mean plasma gh concentrations were 2.37  0.17 g / l in group t and 1.80  0.11 g / l in group c ( p < 0.05 ) . following treatment , the plasma gh concentration in group t was significantly lower 1.7  0.6 g / l ( tables 3 and 4 ) ; however , in group group c it was 1.68  0.07
g / l , which was not a significant difference ( table 4 ) . at the end of treatment ,
the mean p4 serum concentrations were 8.37  2.24 and 9.33  1.79 ng / ml in group t and group c , respectively ( tables 5 and 6 ) .
the final p4 values of group t and c did not differ significantly . in ir subjects ( group t ) , no significant variations in glycemia mean values were observed between day 0 and day 5 ( p < 0.05 ) . at day 12 and 20 ,
the mean concentration of blood glucose was significantly lower ( p < 0.05 ) than on day 0 ( table 5 ) . in the control group ( table 6 ) , between the animals ,
blood glucose means concentrations were not significant ( p > 0.05 ) . on day 20
, diabetes mellitus was well controlled in all the bitches ( group t ; table 5 ) .
this results in group t allowed us to keep insulin dosage less than 1 iu / kg during the following 6 weeks , while before the aglepristone treatment the mean insulin dosage was 1.98  0.68 iu / kg ( tables 1 and 5 ) .
glucose is the main source of energy for all body tissue , except cardiac and skeletal muscle .
blood glucose concentration is a reflection of gastrointestinal absorption , glycogenolysis , gluconeogenesis and glucose consumption by tissues .
glucose production and its metabolism occur as a result of interaction of hormones , cytokines and intracellular transport .
several glucose transporters have been identified , such as glut-4 in skeletal muscle , cardiac and adipose cells , as well as other glucose transporters ( glut-1 ; glut-2 ) independent from insulin action in the brain , liver , kidney , placenta , sperm , adipocytes and erythrocytes . increasing blood glucose concentrations
stimulate insulin secretion , while low blood glucose concentrations suppress insulin secretion and stimulate production of various hormones ( glucagon , epinephrine , norepinephrine , growth hormone , cortisol ) .
there is growing evidence suggesting that gh modulates insulin sensitivity via multiple mechanisms due to the influence of crosstalk between gh / insulin - like growth factor-1 ( igf-1 ) and insulin signaling , including reduced tyrosine kinase ( tk ) activity .
however , insulin resistance alone appears likely to cause diabetes , once only few bitches in diestrus develop canine diabetes mellitus , which is undoubtedly a multifactorial disease , because there is growing evidence suggesting that gh modulates insulin sensitivity by multiple mechanisms , due to the influence of crosstalk between gh / insulin - like growth factor1 ( igf-1 ) .
pregnancy is associated with ir in humans and dogs , which occurs in response to suppression of the intracellular transport of glucose and its increasing concentration in blood .
p4 , estradiol , growth hormone , placental lactogen and placental cytokines all play important roles in causing insulin resistance .
there are several causes of resistance to exogenous insulin that do not lead to ir , such as improper handling and administration of insulin .
somogyi effect , which occurs when pronounced hyperglycemia develops in response to severe insulin - induced hypoglycemia , may also cause misdiagnosis of insulin resistance . in diabetic patients , sudden severe insulin - induced hypoglycemia results in development of protective mechanisms involving secretion of catecholamines , glucocorticoids , glucagon , and gh , which lead to pronounced hyperglycemia .
actually , in subjects showing somogyi effect , the activity of insulin is high . in our cases ,
gestational diabetes mellitus is a clinical condition characterized by a variable degree of glucose intolerance that can develop during pregnancy , parturition and diestrus . during pregnancy
it is likely that gestational diabetes mellitus exerts a negative impact on fetuses and can compromise pregnancy and newborn viability .
a high risk of adverse fetal events , including abortion , small unthrifty pups and overly large pups ( macrosomia ) , has been described in diabetics bitches .
vascular effects of diabetes may reduce placental blood supply , contributing to abortion or poorly - grown pups .
pregnant bitches show greater insulin resistance than non - pregnant diestrous bitches . during the luteal phase basal gh secretion and p4 concentrations
the canine mammary gland expresses genes encoding gh , and its expression is strongly stimulated by p4 .
the long exposure to high circulating levels of p4 during the luteal phase may even result in excess gh with acromegaly and/or diabetes mellitus in bitches .
previous studies identified foci of hyperplastic ductular epithelium of the mammary gland as the site of origin of gh excess induced by progestins .
these observations are consistent with the central role of progestins in gh gene expression in canine mammary gland and allow for a target endocrine therapy with progesterone receptor blockers with progestin - induced mammary - derived gh hypersecretion .
some authors have also found that administration of antiprogestin resulted in decreased plasma gh concentrations and normalization of plasma igf - i concentrations in bitches with progestin - induced acromegaly .
the insulin - antagonist action of gh ( progestin - induced hypersecretion of gh ) may result in hyperglycemia and diabetes mellitus .
additionally , treatment with aglepristone ( ru 46534 ) was useful to control the hyperglycemia levels in diestrus bitches , which was clearly demonstrated by the p4 levels ( > 2 ng / ml ) at the end of the aglepristone treatment .
our findings are in agreement with those of watson et al . , who found that administration of the mifepristone resulted in a decreased gh plasma concentration in bitches with progestin - induced acromegaly .
.   showed lower progesterone levels after aglepristone administration , which was likely related to acceleration of the luteolytic process .
the gradual decline of progesterone observed in treated dogs suggested that antiprostagen triggers an anticipated , physiological - like luteolytic process . in our opinion
, the results of this prospective study suggest that aglepristone represents an effective therapeutic choice .
nevertheless , the best and definitive treatment for insulin resistance due to progesterone is gonadectomy as soon as possible , while the use of aglepristone should be reserved only for cases in which surgery is not possible or authorized by the owners .
ovariohysterectomy promotes a quick drop in p4 levels , which is of great interest to managing diabetic bitches . in conclusion
, the results of the present study demonstrated that aglepristone treatment significantly decreased blood glucose concentration in bitches with progestin - induced insulin - resistance .
 insulin resistance ( ir ) in dogs is suspected when hyperglycemia is present despite administration of insulin doses greater than 1.0 to 1.5 ui / kg . 
 ir is caused by increases in counter regulatory hormones concentrations ( glucagon , glucocorticoids , catecholamines and growth hormone ) . 
 this study was conducted to investigate the use of aglepristone ( ru 46534 ) , a p4 receptor antagonist , for the treatment of ir diabetes mellitus in bitches during the luteal phase . 
 all animals were treated with porcine insulin zinc suspension ( caninsulin ) and aglepristone ( alizin ) 10 mg / kg subcutaneously at day 1 , 2 , 9 and 17 from diagnosis . at day 5 , no significant variation in glycemia was shown . at day 12 and 20 , serum glucose concentrations were significant lower ( p < 0.05 ) . from day 12 
 the insulin dose was reduced to 0.8 iu bid . 
 insulin was reduced in the following weeks and glycemia was controlled . 