Introduction

Canine diabetes mellitus (CDM) affects between 0.0005% and 1.5% of the population (Catchpole et al. 2005), and the number of diagnosed cases has progressively increased in recent decades (Guptill et al. 2003). Although the pathogenesis of CDM is similar to type I diabetes mellitus in humans, associated with immunomediated destruction of beta cells and the presence of anti-islet autoantibodies (Hoenig 2002), a high proportion of dogs that develop CDM show similar characteristics to those observed in human gestational diabetes mellitus (GDM) (Rand et al. 2004). Various studies have shown that about 70% of CDM diagnosed cases are in female dogs (Catchpole et al. 2005, Guptill et al. 2003, Mattheeuws et al. 1984). In a population of diabetic females, about 70% of the animals develop diabetes during the diestrus phase (Pöppl & González 2005). Ryan and Enns (1998) showed a decreased of tyrosine kinase activity (TK) of the insulin receptor (IR) in a culture of rat adipocytes in response to progesterone. Eigenmann et al. (1983), like other investigators (Rijnberk et al. 2003, Selman et al. 1994), associated insulin resistance during diestrus in dogs with the increase in the release of growth hormone (GH) by the mammary gland. However, the development of CDM during the diestrus phase appears to be multifactorial (Scaramal et al. 1997). Only two articles correlating diabetes and pyometra in female dogs were found in literature (Klinkenberk et al. 2006, Krook et al. 1960). Pöppl & González (2005) have described simultaneous diabetes and pyometra diagnosis in a bitch, as well as Pöppl et al., (2005) has described a hyperosmolar non ketotic diabetes mellitus in a bitch with pyometra, condition associated to a severe insulin resistance. Not much is known about the insulin-receptor (IR) binding and transduction of the insulin signal in dogs (Feldman & Nelson, 2004). Therefore, the objective of this study was to evaluate the TK activity of the insulin receptor, as well as insulin binding affinity and capacity in muscle tissue of female dogs during the estrus cycle and in bitches with pyometra.

Materials and Methods

Thirty-two female dogs were evaluated in the Veterinary Clinics Hospital of the Federal University of Rio Grande do Sul (HCV/UFRGS) in the Breeding Control of Dogs and Cats university extension service, during the period from 23 January 2006 to 18 January 2007. In parallel, thirteen bitches with pyometra diagnosis where also evaluated. This study was approved by the Research Ethics Committee of the University (CEP/UFRGS) and by the Research Commission of the Institute of Basic Health Sciences (COMPESQ/ICBS). Permission for the animals to participate in the study was obtained from their owners, who signed a form indicating their consent. For tissue collection, patients received 3 mg/kg of meperidine (Dolosal, Cristália, São Paulo, Brazil) intramuscularly, and 22 mg/kg of sodium ampicillin (Ampicilina Veterinária, Univet, São Paulo, Brazil). Anesthesia was then induced with 5 mg/kg of propofol (Provine 1%, Claris, São Paulo, Brazil), followed by orotracheal intubation and maintenance of the anesthetic plane with 2% isoflurane (Forane, Abbot, Rio de Janeiro, Brazil) in oxygen (White Martins, Triunfo, Brazil). After ovariohysterectomy, samples of 1 g of rectoabdominal muscle were collected, immediately frozen in liquid nitrogen, and then stored at 80°C until the membranes were prepared. Immediately postoperatively, the patients received 40,000 U/kg of benzathine penicillin (Pentabiótico Veterinário, Fort Dodge, Campinas, Brazil) and 2 mg/kg of ketoprofen (Ketofen, Merial, Campinas, Brazil), both intramuscularly. Female dogs with pyometra received different postoperative protocols according to each case. Muscle membranes were prepared as previously described (Orcy et al. 2005) with certain modifications. Muscle tissues were homogenized in Ultra-Turrax® at 4°C in a buffer containing 100 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM TRIS, 1 mM EDTA, and 250 mM sucrose, pH 7.4. The eluate was then centrifuged for 5 min at 3,000 x g, at 4°C. The supernatant was collected and centrifuged for 20 min at 30,000 x g, and the pellet was resuspended in 250-350 μL of buffer. The protein concentration of the samples was measured according to Bradford (Bradford 1976), using bovine serum albumin as a control. To evaluate tyrosine kinase activity, plasma membranes (40 μg protein) were pre-incubated with 100 nM of bovine serum albumin (BSA) for 30 min at 25°C, and then incubated with [γ³²P] ATP (0.5 μCi--Amersham Biosciences, Little Chalfont, United Kingdom--3,000 Ci/mmol) together with unlabeled ATP (5 μM) in 25 mM HEPES buffer, pH 7.4, containing 10 mM MgCl2, 10 mM MnCl2 and 1 mM sodium orthovanadate, for 10 min at 25°C. Subsequently, 1 mM poly (Glu 4: Tyr 1, Sigma, Steinheim, Germany) was added and incubated for 1 h at 25°C. The reaction was stopped by application of the sample onto phosphocellulose paper (Gibco BRL, Gaithersburg, USA). The filter was rinsed with 10% TCA for 10 min, and three more times with 75 mM H3PO4 for 10 min each. The incorporated γ³²P was measured with scintillation liquid [toluene--Triton® X-100 (2:1), PPO (0.4%), and POPOP (0.01%)] in the LKB counter. Binding experiments were performed according to Kucharski et al. (1997). 400 μg of membrane proteins were used per tube with Mammalian Krebs-Ringer buffer (MKR) pH 7.4 together with 1% BSA and in presence of crescent concentrations of human regular insulin (Eli Lilly--0,1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml, 250 μg/ml e 500 μg/ml) plus 20,000 cpm from human ¹²⁵I-insulin (Amersham Biosciences--2,000 Ci/mmol) or only with radioactive insulin (total binding). After 2 hours incubating at 25°C, tubes contents were filtered in a glassed microfiber filter (Whatman GF/B 2,4 cm). Each filter was rinsed five times with 1 ml of MKR 0,1% BSA. After dried, the formation of human ¹²⁵I-insulin/receptor complexes in filters were measured in a LKB counter. Insulin competition curves were analyzed with the computer program Kell for Windows version 6 (Biosoft), according to Munson & Rodbard (1980) for generation of Scatchard plots, dissociation constants, as well as, total binding capacity. The results of the experiments were expressed as the mean (±) standard deviation. A one-way ANOVA was applied, followed by Tukey's test to compare the results obtained in anestrus, estrus and diestrus groups. Data from pyometra group were compared against diestrus group by Student's t test, once it is a condition typical from this phase. The computer program used for these analyses was Sigma Stat 2.0 for Windows.

Results

In patients in anestrus (7328 ± 1191 cpm), TK activity was 42% and 46% higher than those observed in patients in estrus (4180 ± 513 cpm) or diestrus (3924 ± 582 cpm), respectively (p < 0.05). However, in pyometra group (4803 ± 1613 cpm) no significant difference (p > 0.05) was observed against diestrus group. Binding data indicates the presence of two insulin sites, being one (1) high affinity / low capacity site, whereas the second (2) it is a low affinity / high capacity site. Dissociation constant (Kd2), as well as total binding capacity (Bmax2) for low affinity / high capacity sites do not differ among the different groups (p > 0,05). Kd1 for anestrus group was 6.54 ± 2.77 fM/mg of protein, whereas Kd1 for groups estrus (28.54 ± 6.94 fM/mg of protein) and diestrus (15.56 ± 3.88 fM/mg of protein) were significantly higher (p < 0.001). Bmax1 from group estrus (0.83 ± 0.42 fM/mg of protein) and group diestrus (1.24 ± 0.24 fM/mg of protein) were also higher (p < 0.001) than values observed in anestrus group (0.35 ± 0.061 fM/mg of protein). Bitches with pyometra have show a marked increase (p < 0.01) in Kd1 (41.04 ± 16.95 fM/mg of protein) against diestrus group, however there was no significant increase (p > 0,05) in binding capacity (2.09 ± 1.35 fM/mg of protein) when compared with diestrus group.

Discussion and Conclusions

The first step in insulin signaling after the binding of the hormone to the alpha subunit of insulin receptor (IR) is the induction of a complex cascade of tyrosine phosphorylation (the 1158, 1160 and 1163 residues being the regulatory domains) in the beta subunit. When phosphorylated at these specific sites, the beta subunit initiates a series of phosphorylations in intracellular substrates (Saltiel & Kanh 2001). The phosphorylation of a synthetic substrate such as the oligopeptide Poly (Glu 4: Tyr 1) provides a good way to evaluate the TK activity of the IR and the capacity for phosphorylation of intracellular substrates by the insulin receptor, in both mammals and invertebrates (Kucharski et al. 1999, Orcy et al. 2005). The lower phosphorylation of the insulin receptor and the lower translocation of GLUT 4 to plasmatic membrane have previously been demonstrated in a culture of rat adipose cells treated with progesterone (Ryan & Enns 1998). In dogs, it has been shown that chronic administration of estradiol and progesterone causes insulin resistance in muscle tissue (Batista et al. 2008). These findings corroborate our results of lower TK activity observed in estrus and diestrus, and suggest that the changes in the postreceptor steps are involved in the occurrence of insulin resistance in estrus and diestrus. The lower TK activity observed in female dogs with pyometra may be a somatic effect of the hormonal environment plus inflammatory and septic state typical of this condition. Das (2003) cited lower insulin receptor phosphorylation, lower IRS-1 phosphorylation, as well as IRS-1 phosphorylation in serine residues due to higher concentrations of IL-1 in sepsis. The Kd value means the insulin concentration needed to bind half of the IRs. The higher the Kd value, higher the insulin resistance, once higher concentrations of the hormone are needed to stimulate the same effect. This results of Kd from high affinity / low capacity insulin binding sites clearly demonstrates an insulin resistance status in female dog's muscle tissue during estrus, diestrus and pyometra. However, bitches in estrus and diestrus are able to compensate this status by increasing the total insulin binding capacity (higher Bmax1 values). The same was not observed during pyometra. Jonhston et al., (1991) have described the same population of insulin receptors (high affinity / lower capacity and low affinity / higher capacity sites) in dog's muscle, heart and liver and found also modulation only on high affinity sites comparing newborn and adult dogs. In conclusion, this study reports, for the first time, evidence of lower activation of post receptor steps in insulin signaling in dogs. Indeed, a modulation of the insulin receptor affinity and binding capacity was clear during estrus cycle and in a septic state such as pyometra. A lack in this increase on insulin binding capacity during estrus and diestrus, associated with other risk factors may be involved in diabetes mellitus onset in bitches. In addition, a new field of investigation is opened on the molecular mechanisms involved in the development of diabetes in dogs.

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