Porcine leptin alters insulin inhibition of lipolysis in porcine adipocytes in vitro

The present study determined whether porcine leptin can alter the lipolytic rate in porcine adipocytes produced in vitro. The stromal-vascular cell fraction of neonatal subcutaneous adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. These stromal-vascular cells were seeded on 25-cm2 tissue culture flasks and proliferated to confluency in 10% fetal bovine serum in DMEM/F12 (50:50). Cultures were differentiated using 2% pig serum + 10 mM isobutyl methylxanthine + 1 microM dexamethasone for 48 h. This medium was replaced with 5% pig serum + 1 microM insulin to promote lipid filling of adipocytes for 7 d. Adipocyte-containing cultures were incubated overnight in serum-free medium and then used for experiments. Acute experiments assessed lipolysis in cultures exposed to porcine leptin (0 to 1,000 ng/mL medium) for 2 h. Chronic experiments used cultures incubated with 100 ng porcine leptin/mL of medium for 72 h prior to lipolysis measurements. Direct effects of leptin were examined by incubating cultures in DMEM/F12, 25 mM HEPES, 3% bovine serum albumin, 20 mU of adenosine deaminase/mL of medium in the presence of 0 to 1,000 ng of porcine leptin/mL of medium. Indirect effects of leptin were examined using the same incubation medium but also supplemented with 1 microM isoproterenol +/- 10 nM insulin in the presence of 0 to 1,000 ng of porcine leptin/mL of medium. Media glycerol concentration was measured at the end of 2-h incubations. Acute leptin exposure induced up to a 76% increase in lipolysis (P < 0.05) but had no effect on insulin's inhibition of lipolysis. Chronic exposure to leptin produced up to a 56% increase in lipolysis (P < 0.05) and reduced insulin's inhibition ofisoproterenol-stimulated lipolysis by up to 31% (P < 0.05). These data demonstrate leptin functions to promote the partitioning of energy away from lipid accretion within porcine adipose tissue by promoting lipolysis directly and indirectly by reducing insulin-mediated inhibition of lipolysis.     

Porcine leptin inhibits lipogenesis in porcine adipocytes

The present study examined whether recombinant porcine leptin alters lipid synthesis in porcine adipocytes. The stromal-vascular cell fraction of neonatal pig subcutaneous adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. These cells were seeded on 25-cm2 tissue culture flasks and proliferated to confluency in 10% (vol/vol) fetal bovine serum in Dulbecco's modified Eagle medium/F12 (DMEM/F12, 50:50). Cultures were differentiated using 2.5% pig serum (vol/vol), 10 nM insulin, 100 nM hydrocortisone. After 7 d of lipid filling, cultures were washed free of this medium, incubated overnight in DMEM/F12 containing 2% pig serum (vol/vol), and then used for experiments. Acute experiments assessed U-(14)C-glucose or 1-(14)C-palmitate metabolism in cultures exposed to porcine leptin (0 to 1,000 ng/mL medium) for 4 h. Chronic experiments used cultures incubated with 0 to 1,000 ng porcine leptin/mL medium for 44 h before measurements of U-(14)C-glucose and 1-(14)C-palmitate oxidation and incorporation into lipid. Another experiment examined whether chronic leptin treatment alters insulin responsiveness by including insulin (10 nM) with incubations containing leptin. Leptin had no acute effects on glucose oxidation or conversion to lipid (P > 0.05). Acute leptin treatment decreased palmitate incorporation into lipids up to 45% (P < 0.05). Chronic leptin exposure decreased glucose oxidation (21%), total lipid synthesis (18%), and fatty acid synthesis (23%) at 100 ng/mL medium (P < 0.05). Insulin increased rates of glucose oxidation, total lipid, and fatty acid synthesis (P < 0.05); however, chronic exposure to 10 ng leptin/mL medium decreased the effectiveness of 10 nM insulin to affect these measures of glucose metabolism by approximately 18 to 46% (P < 0.05). Higher concentrations of leptin inhibited all effects of insulin on glucose metabolism (P < 0.05). Chronic exposure to leptin increased palmitate oxidation by 36% (P < 0.05). Chronic leptin exposure decreased palmitate incorporation into total lipids by 40% at 100 ng/mL medium (P < 0.05). Lipoprotein lipase activity was not affected (P > 0.05) by leptin. These data indicate that leptin functions to promote partitioning of energy away from lipid accretion within porcine adipose tissue by inhibiting glucose oxidation and lipogenesis indirectly, by decreasing insulin-mediated stimulation of lipogenesis, and by stimulating fatty acid oxidation while inhibiting fatty acid esterification.     

Porcine preadipocyte proliferation and differentiation: a role for leptin?

The present study was designed to determine whether porcine leptin can alter the proliferation and differentiation of the porcine preadipocyte. The stromal vascular cell fraction of neonatal pig s.c. adipose tissue was isolated by collagenase digestion, filtration, and subsequent centrifugation. For differentiation studies, cells were seeded on six-well tissue culture plates and proliferated to confluency in 10% (vol/vol) fetal bovine serum (FBS) in Dulbecco's modified Eagle medium/F12 (DMEM/F12; 50:50). Cultures were differentiated using 2.5% pig serum (vol/vol) and recombinant porcine leptin at concentrations of 0 to 1,000 ng/mL alone or in combination with porcine insulin (100 nM), dexamethasone (1 microM), or IGF-1 (250 ng/mL). After 7 d of lipid filling, cultures were harvested for analysis of sn-glycerol 3 phosphate dehydrogenase (GPDH) and lipoprotein lipase (LPL). The GPDH and LPL activities are measures of preadipocyte differentiation. Data were corrected for protein content of the cultures. For proliferation experiments, 24 h after seeding cells with 10% FBS in DMEM/F12 in 25-cm2 tissue culture flasks, cells were switched to 5% FBS and supplemented with 0 to 1,000 ng of porcine leptin or 1,000 ng of murine leptin. Cell proliferation was measured by 3H-thymidine incorporation in preconfluent cultures over 24 h on d 4 of culture. At confluency, cells were switched to a medium to promote differentiation and lipid filling (2.5% pig serum, 100 nM insulin, 1 microM dexamethasone) for 7 d. Cells were harvested from the flasks and adipocytes were separated from stromal cells by Percoll gradient centrifugation. In a series of experiments, leptin alone or in combination with insulin, dexamethasone, or IGF-I did not affect differentiation as measured by the activity of GPDH and LPL. Leptin at any concentration did not inhibit differentiation induced by insulin, dexamethasone, or IGF-I; however, leptin at 1,000 ng/mL stimulated a 30% increase in preadipocyte proliferation (P = 0.007; n = 6) and a 27% increase in stromal cell proliferation (P < 0.001; n = 6). These results indicate that, at most, porcine leptin may contribute to the recruitment of new adipocytes within the adipose tissue.     

Effects of an intravenous injection of NPY on leptin and NPY-Y1 receptor mRNA expression in ovine adipose tissue

Neuropeptide Y (NPY) is highly expressed in hypothalami of undernourished and genetically obese animals, and is a potent regulator of food intake and reproduction. Leptin, a protein expressed by adipocytes, has been reported to reduce hypothalamic NPY expression. We recently detected (by ribonuclease protection assay [RPA]) expression of the NPY receptor subtype Y1 (but not Y2) mRNA in adipose tissue. Based on these observations we hypothesized that NPY-Y1 receptors in adipose may represent a peripheral mechanism by which NPY can regulate leptin expression in a direct and rapid manner. To test this hypothesis, adipose samples were biopsied from the tailhead region of 48 ± 3 kg wether lambs immediately before and 30 min after a single intravenous injection of 50 μg porcine NPY (“treated” animals, n = 5), or vehicle (“control” animals, n = 4). Injection of NPY resulted in an increase in expression (P = 0.013; as measured by RPA) of both leptin and NPY-Y1 mRNA. In treated animals, negative correlations were found between response in leptin expression and body weight (r = −0.82, P = 0.092), and between leptin response and initial leptin mRNA levels (r = −0.81, P = 0.097). These data provide evidence of a peripheral mechanism by which NPY may regulate adipocyte expression of both leptin and NPY-Y1 receptor mRNA.     

Relationship between leptin content, metabolic hormones and fat deposition in three beef cattle breeds

The aim of the study was to determine if cattle breeds differing in their carcass characteristics also differ in the profiles of their leptin and metabolic hormones. Three breeds, Belgian Blue (BB) (n = 12), Limousin (L) (n = 12) and Aberdeen Angus (AA) (n = 12) with varying ability to deposit fat and protein were compared. Blood, muscle and subcutaneous (SC) adipose tissue were sampled. Animal performance, carcass and meat characteristics were determined as well as plasma leptin concentration, leptin gene expression in SC adipose tissue, leptin-receptor gene expression in SC adipose tissue and plasma concentration of insulin, tri-iodothyronin (T3), thyroxin (T4) and cortisol. The BB bulls showed the lowest values of leptin gene expression (P < 0.05). Values of plasma leptin concentration and of leptin-receptor gene expression tended to be lower in BB than in the other breeds. For a similar amount of adipose tissue (after normalisation), BB bulls showed a higher ratio of plasma leptin (P < 0.05), whereas normalised leptin gene and leptin-receptor gene expressions did not significantly differ between breeds. Belgian Blue bulls also differed in their metabolic hormone profile, tending to show lower values of insulin, T3 and T4 than the two other breeds. Cortisol levels were significantly lower (P < 0.05) in BB than in L and AA animals.     

Macroscopic and Histological Morphology of the Equine Digital Cushion and Its Association with Subcutaneous Fat Thickness

This study evaluated the influence of the Body Condition Score (BCS) and subcutaneous fat thickness on the tissue composition of the digital cushion in horses. Sixty mixed breeds of Criollo horses (21 males and 39 females) were sent for slaughter. All animals were submitted to BCS analysis, through visual antemortem evaluation, and then ultrasound evaluation to estimate the subcutaneous fat thickness. Macroscopic analyses of the thoracic and a pelvic limb (weight, volume, and density of the hooves and digital cushions) were performed. In addition, measurements of the area of internal structures to the hoof and histological analyzes were carried out to measure the areas of adipose, fibroelastic, and collagen fibers of the digital cushion. There were no macroscopic differences in the digital cushion between thoracic and pelvic limbs or between genders (P > .05). Likewise, the histological characteristics between the limbs and the genders were similar (P > .05). There was no correlation between the weight, volume, and density of the digital cushion with the BCS (P > .05). A positive correlation was observed with the area of adipose tissue (P = .038, R² = 0.28) and a negative correlation with area of fibroelastic tissue (P = .005, R² = −0.37) and collagen fibers (P = .003, R² = −0.39). In conclusion, the adipose tissue, fibroelastic tissue, and collagen fibers of equine digital cushion alter their areas in the functions of the various subcutaneous fat patterns in horses.     

Pharmacokinetics and physiologic effects of alprazolam after a single oral dose in healthy mares

The objective of this study was to evaluate the pharmacokinetic properties and physiologic effects of a single oral dose of alprazolam in horses. Seven adult female horses received an oral administration of alprazolam at a dosage of 0.04 mg/kg body weight. Blood samples were collected at various time points and assayed for alprazolam and its metabolite, α‐hydroxyalprazolam, using liquid chromatography/mass spectrometry. Pharmacokinetic disposition of alprazolam was analyzed by a one‐compartmental approach. Mean plasma pharmacokinetic parameters (±SD) following single‐dose administration of alprazolam were as follows: C_max 14.76 ± 3.72 ng/mL and area under the curve (AUC_0–∞) 358.77 ± 76.26 ng·h/mL. Median (range) T_max was 3 h (1–12 h). Alpha‐hydroxyalprazolam concentrations were detected in each horse, although concentrations were low (C_max 1.36 ± 0.28 ng/mL). Repeat physical examinations and assessment of the degree of sedation and ataxia were performed every 12 h to evaluate for adverse effects. Oral alprazolam tablets were absorbed in adult horses and no clinically relevant adverse events were observed. Further evaluation of repeated dosing and safety of administration of alprazolam to horses is warranted.     

Effects of short-term feed deprivation and melatonin implants on circadian patterns of leptin in the horse

Leptin is a protein hormone produced by adipose tissue that influences hypothalamic mechanisms regulating appetite and energy balance. In species tested thus far, including horses, concentrations of leptin increase as animal fat mass increases. The variables and mechanisms that influence the secretion of leptin are not well known, nor is it known in equine species how the secretion of leptin is influenced by acute alterations in energy balance, circadian patterns, and/or reproductive competence. Our objectives were to determine in horses: 1) whether plasma concentrations of leptin are secreted in a circadian and/or a pulsatile pattern; 2) whether a 48-h period of feed restriction would alter plasma concentrations of leptin, growth hormone, or insulin; and 3) whether ovariectomy and/or a melatonin implant would affect leptin. In Exp. 1, mares exposed to ambient photoperiod of visible light (11 h, 33 min to 11 h, 38 min), received treatments consisting of a 48-h feed restriction (RES) or 48 h of alfalfa hay fed ad libitum (FED). Mares were maintained in a dry lot before sampling and were tethered to a rail during sampling. Analyses revealed that leptin was not secreted in a pulsatile manner, and that mean leptin concentrations were greater (P < 0.001) in FED vs. RES mares (17.20 +/- 0.41 vs. 7.29 +/- 0.41 ng/mL). Plasma growth hormone was pulsatile, and mean concentrations were greater in RES than FED mares (2.15 +/- 0.31 vs. 1.08 +/- 0.31 ng/mL; P = 0.05). Circadian patterns of leptin secretion were observed, but only in FED mares (15.39 +/- 0.58 ng/mL for morning vs. 19.00 +/- 0.58 ng/mL for evening; P < 0.001). In Exp. 2, mares that were ovariectomized or intact received either a s.c. melatonin implant or a sham implant. Thereafter, blood was sampled at weekly intervals at 1000 and 1700. Concentrations of leptin in samples collected at 1700 were greater (P < 0.001) than in those collected at 1000 (28.24 +/- 1.7 vs. 22.07 +/- 1.7 ng/mL). Neither ovariectomy nor chronic treatment with melatonin affected plasma concentrations of leptin or the circadian pattern of secretion. These data provide evidence that plasma leptin concentrations in the equine are sensitive to acute changes in nutritional status and vary in a circadian pattern that is sensitive to fasting but not to melatonin treatment or ovariectomy.     

Pharmacokinetics and effects on thromboxane B2 production following intravenous administration of flunixin meglumine to exercised thoroughbred horses

Flunixin meglumine is commonly used in horses for the treatment of musculoskeletal injuries. The current ARCI threshold recommendation is 20 ng/mL when administered at least 24 h prior to race time. In light of samples exceeding the regulatory threshold at 24 h postadministration, the primary goal of the study reported here was to update the pharmacokinetics of flunixin following intravenous administration, utilizing a highly sensitive liquid chromatography–mass spectrometry (LC‐MS). An additional objective was to characterize the effects of flunixin on COX‐1 and COX‐2 inhibition when drug concentrations reached the recommended regulatory threshold. Sixteen exercised adult horses received a single intravenous dose of 1.1 mg/kg. Blood samples were collected up to 72 h postadministration and analyzed using LC‐MS. Blood samples were collected from 8 horses for determination of TxB₂ and PGE₂ concentrations prior to and up to 96 h postflunixin administration. Mean systemic clearance, steady‐state volume of distribution and terminal elimination half‐life was 0.767 ± 0.098 mL/min/kg, 0.137 ± 0.12 L/kg, and 4.8 ± 1.59 h, respectively. Four of the 16 horses had serum concentrations in excess of the current ARCI recommended regulatory threshold at 24 h postadministration. TxB₂ suppression was significant for up to 24 h postadministration.     

Adiponectin and leptin are related to fat mass in horses

Plasma concentrations of adiponectin and leptin were measured in 23 mature Standardbred mares (age: 10 ± 3 years) and 12 weanling fillies (10 Quarter Horse/Belgian crossbreds and two Quarter Horses; aged: 4 ± 3 months) to test the hypothesis that adipocytokines are proportional to adiposity in horses. Rump fat thickness was measured using B-mode ultrasound and percent body fat (% fat) calculated using a published formula for the estimation of fatness in horses. Plasma adiponectin and leptin were determined using radioimmunoassay. In the absence of purified equine adiponectin or leptin, results were expressed as human equivalents (HE) of immunoreactive (ir) adipocytokines.Plasma ir-leptin HE concentration was positively correlated (r = 0.543; P < 0.001) with percent body fat and (r = 0.556; P < 0.001) to fat mass in all horses. The plasma ir-leptin HE concentration was lower (P = 0.03) in weanlings (1.90 ± 0.34 ng/mL HE) than in mature mares (3.47 ± 0.50 ng/mL HE). The ratio of ir-adiponectin HE to ir-leptin HE was negatively correlated (r = −0.621; P < 0.001) to percent fat and (r = −631; P < 0.001) to fat mass in all horses. The ratio of ir-adiponectin HE to ir-leptin HE was higher (P < 0.001) in weanlings (3.04 ± 0.51) than mature mares (1.03 ± 0.13). These data suggest that leptin is proportional while adiponectin is inversely proportional to adiposity in horses.     

Effects of Intravenous Detomidine on Intraocular Pressure Readings Obtained by Applanation Tonometry in Clinically Normal Horses

This study was conducted to determine effects of intravenous detomidine on intraocular pressure (IOP) readings obtained by applanation tonometry in clinically normal horses. Twenty horses were randomly divided into two groups of 10 each (treatment and control). All horses in the treatment group received intravenous detomidine alone (20 μg/kg). The horses in the control group received only intravenous saline (0.2 mL/100 kg). The IOP values were measured before the treatment (T₀) and then at 5 (T₅), 20 (T₂₀), 60 (T₆₀), and 120 (T₁₂₀) minutes after drug administration in both groups. A significant decrease in IOP values was observed in both right and left eyes of the horses in the treatment group at T₅, T₂₀, and T₆₀ in comparison with the baseline values (P < .001). The observed decrease was only statistically significant in the right eyes of the treatment group horses at T₁₂₀ (P = .044). Mean IOP was not significantly altered at any time point during the treatment period compared with the baseline evaluations in both eyes of the horses in the control group. This study demonstrates that the use of intravenous detomidine lowers IOP quickly.     

Effects of short-term infusion with propionate on the mRNA expression of a putative G-protein coupled receptor 41 (GPR41) in adipose tissue of goats

Short chain fatty acids (SCFA) represent the main source for energy supply in ruminants. Propionate up-regulates leptin synthesis through the G protein-coupled receptor 41 (GPR41) in mice but the importance of the GPR41 in ruminants is not yet clarified. Here we characterise the short-term effects of intravenously infused propionate on a putative GPR41 mRNA in goat adipose tissue. Castrated male goats (Capra hircus) received propionate infusion or NaCl solution with equivalent sodium content for 260 min. A putative GPR41 mRNA was quantified in subcutaneous and perirenal adipose tissue by real-time RT-PCR. The mRNA concentration of the putative GPR41 mRNA increased (p = 0.029) in subcutaneous but not in perirenal adipose tissue (p = 0.756) of propionate-infused animals versus the NaCl group. We hypothesise that the differential response of the putative GPR41 mRNA in subcutaneous versus perirenal adipose tissue towards short-term propionate infusion could be involved in a differential nutrient sensing of SCFA in the two adipose depots of goats.     

Transition period-related changes in the abundance of the mRNAs of adiponectin and its receptors,of visfatin,and of fatty acid binding receptors in adipose tissue of high-yielding dairy cows

Adipose tissue expresses adipokines, which are involved in regulation of energy expenditure, lipid metabolism, and insulin sensitivity. To adapt for the transition from pregnancy to lactation, particularly in high-yielding dairy cows, adipokines, their receptors, and particular G-protein coupled receptors (GPRs) are of potential importance. Signaling by GPR 41 stimulates leptin release via activation by short-chain fatty acids; GPR 43/109A inhibits lipolysis, and GPR 109A thereby mediates the lipid-lowering effects of nicotinic acid and β–hydroxybutyrate. The aim of this study was to compare the mRNA expression of adiponectin and visfatin, adiponectin receptors 1 and 2 (AdipoR1/2), leptin receptor (obRb), insulin receptor as of the aforementioned GPRs during the transition period in high-yielding dairy cows. Biopsies from subcutaneous fat and blood samples were obtained from 10 dairy cows 1 week before and 3 weeks after calving. For AdipoR1 and AdipoR2 mRNA abundance as well as for leptin concentrations in plasma, a reduction (P ≤ .05) was observed postpartum; for visfatin and putative GPR 109A mRNA abundance in adipose tissue, there was a trend (P < .1) for analogous changes. In contrast, the mRNA content of obRb and GPR 41 in adipose tissue was higher (P ≤ .05) in samples from early lactation than in those from late gestation. Our results indicate decreasing adiponectin sensitivity in adipose tissue after calving, which might be involved in the reduced insulin sensitivity of adipose tissue during early lactation. In addition, visfatin, GPR 41, and GPR 109A may further modulate insulin sensitivity.     

N-carbamoylglutamate improves lipid metabolism,inflammation, and apoptosis responses in visceral adipocytes of Japanese seabass (Lateolabrax japonicus), in vivo and in vitro

This study applied in vivo and in vitro methods to investigate the effect of dietary N-carbamoylglutamate (NCG) on lipid metabolism, inflammation and apoptosis related-gene expression in visceral adipose tissue and isolated adipocytes of Japanese seabass (Lateolabrax japonicus). A basal diet and a test diet supplemented with 720 mg/kg NCG were fed to the fish for 10 weeks. During the growth trial, no mortality and no significant differences in growth performance were observed in fish between the 2 groups (P > 0.05). Plasma Arg content and mRNA level of argininosuccinate synthetase (ASS) in adipose tissue were significantly increased, which indicated that NCG inclusion promoted endogenous Arg synthesis. Thereafter, the potential effects of NCG treatment on lipid metabolism-related genes expression were studied through in vivo and in vitro methods. In the present study, we successfully established a primary adipocytes culture system and isolated pre-adipocytes in vitro of Japanese seabass for the first time. Both the results in vivo and in vitro showed that NCG treatment decreased the mRNA levels of genes related to adipogenesis (fatty acid synthase, FASN), cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase, HMGCR) and fat deposition (lipoprotein lipase [LPL] and leptin), which revealed the underlying mechanism of NCG on reducing fat deposition. The results of this study demonstrated that NCG inclusion reduced the expression of inflammatory and apoptosis cytokines markedly in vivo and in vitro. In conclusion, NCG did exert beneficial effects on ameliorating adipogenesis, inflammation and apoptosis via promoting Arg endogenous synthesis in Japanese seabass.     

The Use of a Metabolic Disorder Index as a Predictor for Metabolic Eliminations in Endurance Horses

The equine endurance race involves both aerobic and anaerobic metabolisms of the horse. The intense physical activity over an extended period often causes susceptible horses to develop metabolic signs or problems resulting in elimination from races. Thus, the objective of this study was to develop a method for prediction and validation of a metabolic disorder index (MDI) to be used before endurance races. Three hundred seventy-five Arabian (n = 152) and Arabian cross (n = 223) endurance horses aged from 6 to 15 years and weighing between 350 and 450 kg were selected for the study in Malaysia. Blood samples were collected at pre- and post-race periods. The significant (P < .05) findings in horses with metabolic disorder were packed cell volume (0.50 ± 0.06 LL−1), creatine kinase (1,275.89 ± 121.45 UL−1), interleukin-6 (2.01 ± 0.89 ng/mL), decreased glutathione reductase (26.57 ± 3.95 ng/mL), and chloride (94.98 ± 8.12 mmol/L). A new method called MDI was developed as a predictor for horses with the potential to develop metabolic disorders in endurance races. The MDI indicated a higher value greater than 5.5 for those eliminated and lower value below 5.5 for those that completed the race successfully, this proved to be accurate in the prediction of metabolic disorder in endurance horses. The MDI is an innovative and simple method used as a prediction method that will assist the equine endurance society to reduce the rate of elimination and to safeguard against serious medical problems during endurance races in the tropics.     

Pharmacokinetics and pharmacodynamics of tramadol in horses following oral administration

Tramadol is a synthetic opioid used in human medicine, and to a lesser extent in veterinary medicine, for the treatment of both acute and chronic pain. In humans, the analgesic effects are owing to the actions of both the parent compound and an active metabolite (M1). The goal of the current study was to extend current knowledge of the pharmacokinetics of tramadol and M1 following oral administration of three doses of tramadol to horses. A total of nine healthy adult horses received a single oral administration of 3, 6, and 9 mg/kg of tramadol via nasogastric tube. Blood samples were collected at time 0 and at various times up to 96 h after drug administration. Urine samples were collected until 120 h after administration. Plasma and urine samples were analyzed using liquid chromatography–mass spectrometry, and the resulting data analyzed using noncompartmental analysis. For the 3, 6, and 9 mg/kg dose groups, C_max, T_max, and the t_1/2λ were 43.1, 90.7, and 218 ng/mL, 0.750, 2.0, and 1.5 h and 2.14, 2.25, and 2.39 h, respectively. While tramadol and M1 plasma concentrations within the analgesic range for humans were attained in the 3 and 6 mg/kg dose group, these concentrations were at the lower end of the analgesic range and were only transiently maintained. Furthermore, until effective analgesic plasma concentrations have been established in horses, tramadol should be cautiously recommended for control of pain in horses. No significant undesirable behavioral or physiologic effects were noted at any of the doses administered.     

Porcine leptin alters isolated adipocyte glucose and fatty acid metabolism

This study examined if leptin can acutely affect glucose or fatty acid metabolism in pig adipocytes and whether leptin's actions on lipogenesis are manifested through interaction with insulin or growth hormone. Subcutaneous adipose tissue was obtained from approximately 55 kg crossbred barrows at the USDA abattoir. Isolated adipocytes were prepared using a collagenase procedure. Experiments assessed U-14C-glucose or 1-14C-palmitate metabolism in isolated adipocytes exposed to: basal medium (control), 100 nM insulin, 100 ng/ml porcine growth hormone, 100 ng/ml recombinant porcine leptin, and combinations of these hormones. Treatments were performed in triplicate and the experiment was repeated with adipocytes isolated from five different animals. Cell aliquots (250 microl) were added to 1 ml of incubation medium, then incubated for 2h at 37 degrees C for measurement of glucose and palmitate oxidation or incorporation into lipid. Incubation of isolated adipocytes with insulin increased glucose oxidation rate by 18% (P<0.05), while neither growth hormone nor leptin affected glucose oxidation (P>0.5). Total lipid synthesis from glucose was increased by approximately 25% by 100 nM insulin or insulin+growth hormone (P<0.05). Insulin+leptin reduced the insulin response by 37% (P<0.05). The combination of all three hormones increased total lipid synthesis by 35%, relative to controls (P<0.05), a rate similar to insulin alone. Fatty acid synthesis was elevated by insulin (32%, P<0.05) or growth hormone (13%, P<0.05). Leptin had no effect on fatty acid synthesis (P>0.05). Leptin reduced the esterification rate by 10% (P<0.05). Growth hormone and insulin could overcome leptin's inhibition of palmitate esterification (P>0.05).     

Effects of Intravenous Detomidine on Schirmer Tear Test Results in Clinically Normal Horses

This study was conducted to determine the effects of intravenous detomidine on Schirmer tear test (STT) results in clinically normal horses. Eighteen adult horses were randomly divided into two groups of nine horses each. The treatment group was sedated with intravenous detomidine alone (20 μg/kg), and the control group received only intravenous saline (0.2 mL/100 kg). Schirmer tear test was performed just before intravenous administration of detomidine or saline in treatment and control groups, respectively. Schirmer tear tests were repeated 5, 20, 60, and 120 minutes later. Horses enrolled in this study consisted of nine males and nine females. Breeds were Arabian and Hanoverian, ranging from 3 to 6 years in age. In the treatment group, the pretreatment and subsequent posttreatment mean ± standard deviation values were 17.0 ± 6.9 (0 minutes), 11.8 ± 2.9 (5 minutes), 12.1 ± 2.0 (20 minutes), 12.1 ± 3.1 (60 minutes), and 15.0 ± 2.8 (120 minutes) mm wetting/min. In this group of horses, a significant reduction was observed in STT values at 5, 20, and 60 minutes after treatment with detomidine hydrochloride in comparison to the pretreatment values (analysis of variance with post hoc testing; P₅ = 0.004, P₂₀ = 0.007, P₆₀ = 0.006). There was no significant difference between baseline values and posttreatment values in the control saline group (P ≥ .08). We conclude that intravenous detomidine causes a significant reduction in STT values in clinically normal horses. In horses, practitioners should measure STT values before intravenous administration of detomidine to accurately assess the results.     

The pharmacokinetics of glycopyrrolate in Standardbred horses

The disposition of plasma glycopyrrolate (GLY) is characterized by a three‐compartment pharmacokinetic model after a 1‐mg bolus intravenous dose to Standardbred horses. The median (range) plasma clearance (Clp), volume of distribution of the central compartment (V₁), volume of distribution at steady‐state (Vss), and area under the plasma concentration–time curve (AUC_0‐inf) were 16.7 (13.6–21.7) mL/min/kg, 0.167 (0.103–0.215) L/kg, 3.69 (0.640–38.73) L/kg, and 2.58 (2.28–2.88) ng*h/mL, respectively. Renal clearance of GLY was characterized by a median (range) of 2.65 (1.92–3.59) mL/min/kg and represented approximately 11.3–24.7% of the total plasma clearance. As a result of these studies, we conclude that the majority of GLY is cleared through hepatic mechanisms because of the limited extent of renal clearance of GLY and absence of plasma esterase activity on GLY metabolism. Although the disposition of GLY after intravenous administration to Standardbred horses was similar to that in Thoroughbred horses, differences in some pharmacokinetic parameter estimates were evident. Such differences could be attributed to breed differences or study conditions. The research could provide valuable data to support regulatory guidelines for GLY in Standardbred horses.