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Abstract
OBJECTIVE
To develop a technique for isolation and culture of canine insulinoma cells and assess expression of cellular hexokinases (glucokinase and hexokinase I) and expression and secretion of insulin from these cells in vitro.
SAMPLE
Pancreatic insulinomas and normal pancreatic tissue from 4 and 3 dogs, respectively.
PROCEDURES
Tissues were collected by surgical excision or at necropsy. Insulinoma cells from 2 dogs were cultured for up to 10 weeks with standard techniques; insulin synthesis in vitro was confirmed by immunohistochemical analysis of freshly prepared slides of cultured cells, and insulin secretion was assessed by measurement of insulin concentrations in culture medium with an ultrasensitive mouse insulin ELISA. Expression of cellular hexokinases in insulinomas and adjacent normal (nontumor) pancreatic tissue from the same dog (n = 3) was examined by quantitative reverse transcriptase PCR assay.
RESULTS
Insulinoma cells survived for up to 10 weeks but did not proliferate in culture. Insulin was detected in isolated cells and secreted into culture medium for up to 10 weeks. Both cellular hexokinases were expressed; glucokinase appeared to be overexpressed in insulinomas, compared with normal pancreatic tissue from the same dogs.
CONCLUSIONS AND CLINICAL RELEVANCE
Canine insulinomas expressed hexokinases responsible for glucose responsiveness. Insulinoma cells were successfully maintained in short-term culture; cultured cells remained functional for 10 weeks as evidenced by cellular insulin content and had detectable secretion of insulin into the culture medium for ≥ 5 weeks. Apparent glucokinase overexpression by insulinomas suggested a possible mechanism underlying excessive insulin release by these tumors.
Abstract
OBJECTIVE
To evaluate the performance of 2 assays for measurement of serum fructosamine (SF) and glycated hemoglobin (HbA1c) values in dogs and to compare the usefulness of the 2 glycated proteins for assessment of glycemic control in dogs with diabetes mellitus (DM).
SAMPLE
Blood samples from 40 healthy dogs, 13 diabetic dogs, and 23 anemic normoglycemic nondiabetic dogs and results of 200 assessments of glycemic control in 46 diabetic dogs.
PROCEDURES
Colorimetric and immunoturbidimetric methods were used for measurement of SF and HbA1c values, respectively. Linearity and precision were determined. The usefulness of SF and HbA1c values for assessment of glycemic control was evaluated with a clinical scoring method used as the reference standard. Cutoff values obtained from receiver operating characteristic curves were used to identify the percentage of dogs correctly categorized by means of SF and HbA1c values.
RESULTS
Mean intra-assay and interassay coefficients of variation were 3.8% and 2.5%, respectively, for the SF assay, and 1.2% and 1.8%, respectively, for the HbA1c assay. Excellent linearity (R 2 > 0.99) was obtained for both assays. Values for SF and HbA1c were inversely correlated (r = −0.40 and −0.33, respectively) with clinical score and correctly indicated glycemic control in 99 of 200 (50%) and 88 of 200 (44%) assessments, respectively.
CONCLUSIONS AND CLINICAL RELEVANCE
The SF and HbA1c assays were precise, had good linearity, and appeared to be suitable for routine use in veterinary medicine. However, they performed poorly for classifying glycemic control in diabetic dogs.
Abstract
OBJECTIVE
To assess effects of basal-bolus insulin treatment (BBIT) with lispro and neutral protamine Hagedorn (NPH) insulins, compared with NPH insulin alone, on serum fructosamine concentration (SFC) and postprandial blood glucose concentration (BGC) in dogs with clinically well-controlled diabetes mellitus and postprandial hyperglycemia fed a high insoluble fiber–content diet.
ANIMALS
6 client-owned dogs with diabetes mellitus.
PROCEDURES
Blood samples were collected for BGC and SFC measurement in hospitalized dogs just before feeding and routine SC NPH insulin administration (time 0); samples were collected for BGC measurement every 30 minutes for 2 hours, then every 2 hours for up to 10 additional hours. Postprandial hyperglycemia was identified when BGC 30 minutes after insulin administration exceeded BGC at time 0 or the 1-hour time point. For BBIT, owners were instructed to continue NPH insulin administration at the usual dosage at home (q 12 h, with feeding) and to administer lispro insulin (0.1 U/Kg, SC) separately at the time of NPH injections. Two weeks later, SFC and BGC measurements were repeated; results at the start and end of the study were compared statistically.
RESULTS
Median SFC was significantly higher at the start (400 μmol/L) than at the end (390 μmol/L) of the study. Median 1-hour (313 mg/dL) and 1.5-hour (239 mg/dL) BGC measurements at the start of the study were significantly higher than those at the end of the study (117 and 94 mg/dL, respectively).
CONCLUSIONS AND CLINICAL RELEVANCE
In this sample of dogs with well-controlled diabetes mellitus, addition of lispro insulin to an existing treatment regimen of NPH insulin and dietary management significantly decreased postprandial BGCs. Further study of BBIT for dogs with diabetes mellitus is warranted.
Abstract
OBJECTIVE
To determine effects of hydrocortisone administration on serum leptin and adiponectin concentrations, abdominal fat distribution, and mRNA expression of leptin and adiponectin in abdominal adipose tissue of dogs.
ANIMALS
12 healthy dogs.
PROCEDURES
Dogs received hydrocortisone (8.5 mg/kg; n = 6) or a placebo (6) orally every 12 hours for 90 days. Serum leptin and adiponectin concentrations were measured with a canine-specific ELISA on the day before (day 0; baseline) and during (days 1, 3, 7, 30, 60, and 90) administration. On days 0, 30, 60, and 90, abdominal fat mass was quantified with CT, and mRNA expression of leptin and adiponectin in abdominal fat was analyzed by use of a PCR assay.
RESULTS
Hydrocortisone administration resulted in an increase in visceral fat mass on days 60 and 90, compared with the mass at baseline. Visceral fat mass at the level of L3 increased during hydrocortisone administration. Serum leptin concentration began to increase on day 1 and was significantly higher than the baseline concentration on days 30 and 60. Serum adiponectin concentration on days 30, 60, and 90 was significantly lower than the baseline concentration. Leptin and adiponectin mRNA expression in abdominal fat was greater on day 30, compared with expression at baseline, but lower on days 60 and 90, compared with expression on day 30. Serum leptin concentration and visceral fat mass were correlated.
CONCLUSIONS AND CLINICAL RELEVANCE
Hydrocortisone administration affected abdominal fat distribution and serum leptin and adiponectin concentrations through dysregulation of leptin and adiponectin expression.
Abstract
OBJECTIVE
To investigate the effect of high doses of orally administered levothyroxine sodium (LT4) on serum concentrations of triiodothyronine (T3) and thyroxine (T4) in euthyroid horses.
ANIMALS
12 healthy adult horses.
PROCEDURES
10 horses initially received water (vehicle) or 240 mg (5X treatment) or 480 mg (10× treatment) of LT4, and blood samples were collected at baseline (0 hours) and 0.5, 1, 2, 4, 6, 8, 10, 12, 18, 24, 48, 72, 96, and 120 hours after treatment to measure serum T3 and T4 concentrations. Three horses then received 480 mg of LT4 for 14 days, and T4 concentration was measured on days 0, 14, 21, 28, and 35. Changes in T3 and T4 concentrations were compared over time and among treatments.
RESULTS
One-time administration of LT4 resulted in variable but significant increases in both T3 and T4 concentrations for up to 120 hours; however, T3 and T4 concentrations rarely exceeded reference intervals with either treatment. Prolonged administration of 480 mg of LT4 resulted in a 15-fold increase in T4 concentration after 14 days, but concentration returned to day 0 values within 21 days after LT4 administration was discontinued.
CONCLUSIONS AND CLINICAL RELEVANCE
In euthyroid horses, administration of a high dose of LT4 resulted in mild increases in thyroid hormone concentrations; however, prolonged administration of high doses of LT4 resulted in markedly increased thyroid hormone concentrations that returned to pretreatment values within 3 weeks after discontinuation of LT4 administration. These results indicated complex kinetics of LT4 and suggested a possible saturation of T4 excretion in euthyroid horses.
Abstract
OBJECTIVE
To investigate luteinizing hormone (LH) receptor expression in canine nonneoplastic and neoplastic lymph nodes, circulating nonneoplastic lymphocytes, and T-cell lymphoma (TCL) cell lines.
SAMPLE
Formalin-fixed, paraffin-embedded lymph nodes (5 neoplastic and 3 nonneoplastic) from 6 dogs, circulating lymphocytes from venous blood specimens obtained from 12 healthy dogs, and 3 TCL cell lines derived from 3 dogs with primary lymphoma.
PROCEDURES
Lymph node specimens were immunohistochemically stained for determination of LH receptor expression. Circulating nonneoplastic lymphocytes and TCL cell lines were evaluated for LH receptor expression by use of flow cytometry; circulating lymphocytes were also immunophenotyped. The mean percentage of cells positive for LH receptors was determined for each type of specimen. For the healthy dogs, percentages of circulating B and T lymphocytes that expressed LH receptors were assessed on the basis of sex and reproductive status.
RESULTS
The mean percentage of LH receptor-positive cells in canine neoplastic and nonneoplastic lymph nodes was 12.4% and 4.1%, respectively. For the healthy dogs, the mean percentage of circulating LH receptor-positive T lymphocytes was significantly higher in gonadectomized dogs (16.6%) than in sexually intact dogs (10.5%); the percentages of circulating LH receptor-positive B lymphocytes did not significantly differ by reproductive status. Among the 3 canine TCL cell lines, LH receptor expression ranged from 10% to 45%.
CONCLUSIONS AND CLINICAL RELEVANCE
In this study, LH receptor expression by canine neoplastic and nonneoplastic lymphocytes was detected. Research into the effects of downregulation of LH receptor activation in dogs with lymphoma is warranted.
Abstract
OBJECTIVE To assess insulin, glucagon, and somatostatin expression within pancreatic islets of horses with and without insulin resistance.
ANIMALS 10 insulin-resistant horses and 13 insulin-sensitive horses.
PROCEDURES For each horse, food was withheld for at least 10 hours before a blood sample was collected for determination of serum insulin concentration. Horses with a serum insulin concentration < 20 μU/mL were assigned to the insulin-sensitive group, whereas horses with a serum insulin concentration > 20 μU/mL underwent a frequently sampled IV glucose tolerance test to determine sensitivity to insulin by minimal model analysis. Horses with a sensitivity to insulin < 1.0 × 10−4 L•min−1•mU−1 were assigned to the insulin-resistant group. All horses were euthanized with a barbiturate overdose, and pancreatic specimens were harvested and immunohistochemically stained for determination of insulin, glucagon, and somatostatin expression in pancreatic islets. Islet hormone expression was compared between insulin-resistant and insulin-sensitive horses.
RESULTS Cells expressing insulin, glucagon, and somatostatin made up approximately 62%, 12%, and 7%, respectively, of pancreatic islet cells in insulin-resistant horses and 64%, 18%, and 9%, respectively, of pancreatic islet cells in insulin-sensitive horses. Expression of insulin and somatostatin did not differ between insulin-resistant and insulin-sensitive horses, but the median percentage of glucagon-expressing cells in the islets of insulin-resistant horses was significantly less than that in insulin-sensitive horses.
CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that, in insulin-resistant horses, insulin secretion was not increased but glucagon production might be downregulated as a compensatory response to hyperinsulinemia.
Abstract
OBJECTIVE To assess the discriminatory value for corticosteroid-induced alkaline phosphatase (CiALP) activity and other variables that can be measured routinely on a CBC and biochemical analysis for the diagnosis of hypoadrenocorticism in dogs.
SAMPLE Medical records of 57 dogs with confirmed hypoadrenocorticism and 57 control dogs in which hypoadrenocorticism was suspected but ruled out.
PROCEDURES A retrospective case-control study was conducted. Dogs were included if a CBC and complete biochemical analysis had been performed. Dogs with iatrogenic hypoadrenocorticism and dogs treated previously with glucocorticoids were excluded. Cortisol concentration for dogs with hypoadrenocorticism was ≤ 2 μg/dL both before and after ACTH administration. Cortisol concentration for control dogs was > 4 μg/dL before or after ACTH administration.
RESULTS Area under the receiver operating characteristic (ROC) curve for CiALP activity was low (0.646; 95% confidence interval, 0.494 to 0.798). Area under the ROC curve for a model that combined the CiALP activity, Na-to-K ratio, eosinophil count, activity of creatine kinase, and concentrations of SUN and albumin was high (0.994; 95% confidence interval, 0.982 to 1.000). Results for this model could be used to correctly classify all dogs, except for 1 dog with hypoadrenocorticism and no electrolyte abnormalities.
CONCLUSIONS AND CLINICAL RELEVANCE CiALP activity alone cannot be used as a reliable diagnostic test for hypoadrenocorticism in dogs. Combined results for CiALP activity, Na-to-K ratio, eosinophil count, creatine kinase activity, and concentrations of SUN and albumin provided an excellent means to discriminate between hypoadrenocorticism and diseases that mimic hypoadrenocorticism.
Abstract
OBJECTIVE To assess effects of major abdominal surgery on serum cortisol and aldosterone and plasma canine ACTH (cACTH) concentrations.
ANIMALS 39 healthy dogs undergoing laparotomy during veterinary student surgical laboratories.
PROCEDURES Blood samples were obtained before and at completion of surgery. Serum cortisol and aldosterone and plasma cACTH concentrations were measured by use of validated radioimmunoassays. Changes in concentrations (postoperative concentration minus preoperative concentration) were calculated. Data were analyzed by use of the Wilcoxon signed rank test, Pearson correlation analysis, and Mann-Whitney rank sum test.
RESULTS Cortisol, aldosterone, and cACTH concentrations increased significantly from before to after surgery. Although cortisol and aldosterone concentrations increased in almost all dogs, cACTH concentrations decreased in 6 of 32 (19%) dogs. All dogs had preoperative cortisol concentrations within the reference range, but 24 of 39 (62%) dogs had postoperative concentrations above the reference range. A correlation between the change in cACTH concentration and the change in cortisol concentration was not detected.
CONCLUSIONS AND CLINICAL RELEVANCE Laparotomy caused a significant increase in serum cortisol and aldosterone concentrations. In most dogs, but not all dogs, plasma cACTH concentrations increased. Lack of correlation between the change in cACTH concentration and the change in cortisol concentration suggested that increased postoperative cortisol concentrations may have been attributable to ACTH-independent mechanisms, an early ACTH increase that caused a sustained cortisol release, or decreased cortisol clearance. Further studies are indicated to evaluate the effects of various anesthetic protocols and minimally invasive surgical techniques on the stress response.
Abstract
OBJECTIVE To compare tear cortisol concentrations between horses and ponies with pituitary pars intermedia dysfunction (PPID) and healthy nonaged (≤ 15 years old) and aged (≥ 20 years old) horses and to determine whether serum and tear cortisol concentrations were correlated.
ANIMALS 11 horses and ponies with PPID and 20 healthy control horses and ponies (11 nonaged and 9 aged).
PROCEDURES Paired tear and serum samples were obtained from PPID and control animals. All animals were free of active ocular disease. Tear and serum cortisol concentrations were measured with an ELISA and chemiluminescent assay, respectively. Groups were compared with Kruskal-Wallis and Mann-Whitney U tests, and Spearman correlation analysis was used to examine relationships between tear and serum cortisol concentrations within groups.
RESULTS Median tear cortisol concentration was significantly higher in PPID animals than in aged control animals, despite comparable serum cortisol concentrations in PPID and aged control animals. Median tear-to-serum cortisol concentration ratios were also significantly higher in PPID animals than in aged control animals. Serum and tear cortisol concentrations were not significantly correlated in PPID or control animals.
CONCLUSIONS AND CLINICAL RELEVANCE Some horses and ponies with PPID had increased tear cortisol concentrations, compared with concentrations in healthy aged animals. Localized cortisol production in the tear film or altered cortisol binding dynamics could have contributed to this increase. Further studies are warranted to evaluate these mechanisms and to determine whether increased tear cortisol concentrations are associated with delays in corneal wound healing in horses and ponies with and without PPID.
