High-fat diet-induced insulin resistance does not increase plasma anandamide levels or potentiate anandamide insulinotropic effect in isolated canine islets.

BACKGROUND: Obesity has been associated with elevated plasma anandamide levels. In addition, anandamide has been shown to stimulate insulin secretion in vitro, suggesting that anandamide might be linked to hyperinsulinemia. OBJECTIVE: To determine whether high-fat diet-induced insulin resistance increases anandamide levels and potentiates the insulinotropic effect of anandamide in isolated pancreatic islets. DESIGN AND METHODS: Dogs were fed a high-fat diet (n = 9) for 22 weeks. Abdominal fat depot was quantified by MRI. Insulin sensitivity was assessed by the euglycemic-hyperinsulinemic clamp. Fasting plasma endocannabinoid levels were analyzed by liquid chromatography-mass spectrometry. All metabolic assessments were performed before and after fat diet regimen. At the end of the study, pancreatic islets were isolated prior to euthanasia to test the in vitro effect of anandamide on islet hormones. mRNA expression of cannabinoid receptors was determined in intact islets. The findings in vitro were compared with those from animals fed a control diet (n = 7). RESULTS: Prolonged fat feeding increased abdominal fat content by 81.3±21.6% (mean±S.E.M, P<0.01). In vivo insulin sensitivity decreased by 31.3±12.1% (P<0.05), concomitant with a decrease in plasma 2-arachidonoyl glycerol (from 39.1±5.2 to 15.7±2.0 nmol/L) but not anandamide, oleoyl ethanolamide, linoleoyl ethanolamide, or palmitoyl ethanolamide. In control-diet animals (body weight: 28.8±1.0 kg), islets incubated with anandamide had a higher basal and glucose-stimulated insulin secretion as compared with no treatment. Islets from fat-fed animals (34.5±1.3 kg; P<0.05 versus control) did not exhibit further potentiation of anandamide-induced insulin secretion as compared with control-diet animals. Glucagon but not somatostatin secretion in vitro was also increased in response to anandamide, but there was no difference between groups (P = 0.705). No differences in gene expression of CB1R or CB2R between groups were found. CONCLUSIONS: In canines, high-fat diet-induced insulin resistance does not alter plasma anandamide levels or further potentiate the insulinotropic effect of anandamide in vitro.

Modest hyperglycemia prevents interstitial dispersion of insulin in skeletal muscle.

UNLABELLED: Insulin injected directly into skeletal muscle diffuses rapidly through the interstitial space to cause glucose uptake, but this is blocked in insulin resistance. As glucotoxicity is associated with endothelial dysfunction, the observed hyperglycemia in diet-induced obese dogs may inhibit insulin access to muscle cells, and exacerbate insulin resistance. Here we asked whether interstitial insulin diffusion is reduced in modest hyperglycemia, similar to that induced by a high fat diet. METHODS: During normoglycemic (100 mg/dl) and moderately hyperglycemic (120 mg/dl) clamps in anesthetized canines, sequential doses of insulin were injected into the vastus medialis of one hindlimb; the contra-lateral limb served as a control. Plasma samples were collected and analyzed for insulin content. Lymph vessels of the hind leg were also catheterized, and lymph samples were analyzed as an indicator of interstitial insulin concentration. RESULTS: Insulin injection increased lymph insulin in normoglycemic animals, but not in hyperglycemic animals. Muscle glucose uptake was elevated in response to hyperglycemia, however the insulin-mediated glucose uptake in normoglycemic controls was not observed in hyperglycemia. Modest hyperglycemia prevented intra-muscularly injected insulin from diffusing through the interstitial space reduced insulin-mediated glucose uptake. CONCLUSION: Hyperglycemia prevents the appearance of injected insulin in the interstitial space, thus reducing insulin action on skeletal muscle cells.

Increase in visceral fat per se does not induce insulin resistance in the canine model.

OBJECTIVES: To determine whether a selective increase of visceral adipose tissue content will result in insulin resistance. METHODS: Sympathetic denervation of the omental fat was performed under general inhalant anesthesia by injecting 6-hydroxydopamine in the omental fat of lean mongrel dogs (n = 11). In the conscious animal, whole-body insulin sensitivity was assessed by the minimal model (SI ) and the euglycemic hyperinsulinemic clamp (SICLAMP ). Changes in abdominal fat were monitored by magnetic resonance. All assessments were determined before (Wk0) and 2 weeks (Wk2) after denervation. Data are medians (upper and lower interquartile). RESULTS: Denervation of omental fat resulted in increased percentage (and content) of visceral fat [Wk0: 10.2% (8.5-11.4); Wk2: 12.4% (10.4-13.6); P < 0.01]. Abdominal subcutaneous fat remained unchanged. However, no changes were found in SI [Wk0: 4.7 (mU/l)(-1) min(-1) (3.1-8.8); Wk2: 5.3 (mU/l)(-1) min(-1) (4.5-7.2); P = 0.59] or SICLAMP [Wk0: 42.0 × 10(-4) dl kg(-1) min(-1) (mU/l)(-1) (41.0-51.0); Wk2: 40.0 × 10(-4) dl kg(-1) min(-1) (mU/l) (-1) (34.0-52.0); P = 0.67]. CONCLUSIONS: Despite a selective increase in visceral adiposity in dogs, insulin sensitivity in vivo did not change, which argues against the concept that accumulation of visceral adipose tissue contributes to insulin resistance.

Essentiality of portal vein receptors in hypoglycemic counterregulation: direct proof via denervation in male canines.

A major issue of in the treatment of diabetes is the risk of hypoglycemia. Hypoglycemia is detected both centrally and peripherally in the porto-hepatic area. The portal locus for hypoglycemic detection was originally described using the "local irrigation of the liver" approach in a canine model. Further work using portal vein denervation (DEN) in a rodent model characterized portal hypoglycemic sensing in detail. However, recent controversy about the relevance of rodent findings to large animals and humans prompted us to investigate the effect of portal DEN on the hypoglycemic response in the canine, a species with multiple similarities to human glucose homeostasis. Hypoglycemic hyperinsulinemic clamps were performed in male canines, before (PRE) and after (POST) portal vein DEN or sham surgery (CON, control). Insulin (30 pmol/kg·min) and glucose (variable) were infused to slowly decrease systemic glycemia to 50 mg/dL over 160 minutes. The average plasma glucose during clamp steady state was: 2.9 ± 0.1 mmol DEN-PRE, 2.9 ± 0.2 mmol DEN-POST, 2.9 ± 0.1 mmol CON-PRE, and 2.8 ± 0.0 mmol CON-POST. There were no significant differences in plasma insulin between DEN and CON, PRE and POST experiments. The epinephrine response to hypoglycemia was reduced by 62% in DEN but not in CON. Steady-state cortisol was 46% lower after DEN but not after CON. Our study shows, in a large animal model, that surgical disconnection of the portal vein from the afferent pathway of the hypoglycemic counterregulatory circuitry results in a substantial suppression of the epinephrine response and a significant impact on cortisol response. These findings directly demonstrate an essential role for the portal vein in sensing hypoglycemia and relating glycemic information to the central nervous system.

Argon beam coagulation versus fibrin sealant for hemostasis following liver resection: a randomized study in a porcine model.

BACKGROUND/AIMS: Bleeding from the raw liver surface represents a significant surgical complication after elective liver resection or hepatic trauma. The application of argon beam coagulation (ABC) has been proposed to improve hemostasis, but is associated with significant necrosis of the liver parenchyma. Topical hemostatic agents, i.e. fibrin sealant (FS), have also been recommended, yet the optimal management is under debate. This study compares the efficacy and safety of both methods following liver resection in an animal model. METHODOLOGY: Twenty pigs underwent liver resection, and were then randomized into ABC or FS group for treatment of raw liver surfaces. Intraoperative and postoperative parameters were studied. Animals were sacrificed at day 12, and extent of necrosis was assessed using a scoring system and morphometry. RESULTS: Intraoperative parameters did not show any significant difference between two groups except for shorter time of application in the FS group. Postoperatively, animals in the FS group showed significantly higher hemoglobin levels (p=0.0001). Histologically, FS showed a smaller depth of necrosis than ABC (p=0.022). CONCLUSIONS: The use of FS is superior to ABC for management of the raw liver surface after liver resection, in terms of application time, postoperative bleeding and the extent of liver tissue necrosis.

Simplified method to isolate highly pure canine pancreatic islets.

OBJECTIVES: The canine model has been used extensively to improve the human pancreatic islet isolation technique. At the functional level, dog islets show high similarity to human islets and thus can be a helpful tool for islet research. We describe and compare 2 manual isolation methods, M1 (initial) and M2 (modified), and analyze the variables associated with the outcomes, including islet yield, purity, and glucose-stimulated insulin secretion (GSIS). METHODS: Male mongrel dogs were used in the study. M2 (n = 7) included higher collagenase concentration, shorter digestion time, faster shaking speed, colder purification temperature, and higher differential density gradient than M1 (n = 7). RESULTS: Islet yield was similar between methods (3111.0 ± 309.1 and 3155.8 ± 644.5 islets/g, M1 and M2, respectively; P = 0.951). Pancreas weight and purity together were directly associated with the yield (adjusted R(2) = 0.61; P = 0.002). Purity was considerably improved with M2 (96.7% ± 1.2% vs 75.0% ± 6.3%; P = 0.006). M2 improved GSIS (P = 0.021). Independently, digestion time was inversely associated with GSIS. CONCLUSIONS: We describe an isolation method (M2) to obtain a highly pure yield of dog islets with adequate ß-cell glucose responsiveness. The isolation variables associated with the outcomes in our canine model confirm previous reports in other species, including humans.

Hyperspectral computed tomographic imaging spectroscopy of vascular oxygen gradients in the rabbit retina in vivo.

Diagnosis of retinal vascular diseases depends on ophthalmoscopic findings that most often occur after severe visual loss (as in vein occlusions) or chronic changes that are irreversible (as in diabetic retinopathy). Despite recent advances, diagnostic imaging currently reveals very little about the vascular function and local oxygen delivery. One potentially useful measure of vascular function is measurement of hemoglobin oxygen content. In this paper, we demonstrate a novel method of accurately, rapidly and easily measuring oxygen saturation within retinal vessels using in vivo imaging spectroscopy. This method uses a commercially available fundus camera coupled to two-dimensional diffracting optics that scatter the incident light onto a focal plane array in a calibrated pattern. Computed tomographic algorithms are used to reconstruct the diffracted spectral patterns into wavelength components of the original image. In this paper the spectral components of oxy- and deoxyhemoglobin are analyzed from the vessels within the image. Up to 76 spectral measurements can be made in only a few milliseconds and used to quantify the oxygen saturation within the retinal vessels over a 10-15 degree field. The method described here can acquire 10-fold more spectral data in much less time than conventional oximetry systems (while utilizing the commonly accepted fundus camera platform). Application of this method to animal models of retinal vascular disease and clinical subjects will provide useful and novel information about retinal vascular disease and physiology.

Diet-induced obesity prevents interstitial dispersion of insulin in skeletal muscle.

OBJECTIVE: Obesity causes insulin resistance, which has been interpreted as reduced downstream insulin signaling. However, changes in access of insulin to sensitive tissues such as skeletal muscle may also play a role. Insulin injected directly into skeletal muscle diffuses rapidly through the interstitial space to cause glucose uptake. When insulin resistance is induced by exogenous lipid infusion, this interstitial diffusion process is curtailed. Thus, the possibility exists that hyperlipidemia, such as that seen during obesity, may inhibit insulin action to muscle cells and exacerbate insulin resistance. Here we asked whether interstitial insulin diffusion is reduced in physiological obesity induced by a high-fat diet (HFD). RESEARCH DESIGN AND METHODS: Dogs were fed a regular diet (lean) or one supplemented with bacon grease for 9-12 weeks (HFD). Basal insulin (0.2 mU x min(-1) x kg(-1)) euglycemic clamps were performed on fat-fed animals (n = 6). During clamps performed under anesthesia, five sequential doses of insulin were injected into the vastus medialis of one hind limb (INJ); the contralateral limb (NINJ) served as a control. RESULTS: INJ lymph insulin showed an increase above NINJ in lean animals, but no change in HFD-fed animals. Muscle glucose uptake observed in lean animals did not occur in HFD-fed animals. CONCLUSIONS: Insulin resistance induced by HFD caused a failure of intramuscularly injected insulin to diffuse through the interstitial space and failure to cause glucose uptake, compared with normal animals. High-fat feeding prevents the appearance of injected insulin in the interstitial space, thus reducing binding to skeletal muscle cells and glucose uptake.

Novel canine models of obese prediabetes and mild type 2 diabetes.

Human type 2 diabetes mellitus (T2DM) is often characterized by obesity-associated insulin resistance (IR) and beta-cell function deficiency. Development of relevant large animal models to study T2DM is important and timely, because most existing models have dramatic reductions in pancreatic function and no associated obesity and IR, features that resemble more T1DM than T2DM. Our goal was to create a canine model of T2DM in which obesity-associated IR occurs first, followed by moderate reduction in beta-cell function, leading to mild diabetes or impaired glucose tolerance. Lean dogs (n = 12) received a high-fat diet that increased visceral (52%, P < 0.001) and subcutaneous (130%, P < 0.001) fat and resulted in a 31% reduction in insulin sensitivity (S(I)) (5.8 +/- 0.7 x 10(-4) to 4.1 +/- 0.5 x 10(-4) microU x ml(-1) x min(-1), P < 0.05). Animals then received a single low dose of streptozotocin (STZ; range 30-15 mg/kg). The decrease in beta-cell function was dose dependent and resulted in three diabetes models: 1) frank hyperglycemia (high STZ dose); 2) mild T2DM with normal or impaired fasting glucose (FG), 2-h glucose >200 mg/dl during OGTT and 77-93% AIR(g) reduction (intermediate dose); and 3) prediabetes with normal FG, normal 2-h glucose during OGTT and 17-74% AIR(g) reduction (low dose). Twelve weeks after STZ, animals without frank diabetes had 58% more body fat, decreased beta-cell function (17-93%), and 40% lower S(I). We conclude that high-fat feeding and variable-dose STZ in dog result in stable models of obesity, insulin resistance, and 1) overt diabetes, 2) mild T2DM, or 3) impaired glucose tolerance. These models open new avenues for studying the mechanism of compensatory changes that occur in T2DM and for evaluating new therapeutic strategies to prevent progression or to treat overt diabetes.

Experimental hyperlipidemia dramatically reduces access of insulin to canine skeletal muscle.

A complex sequence of steps is required for insulin to cause glucose uptake. Impairment of any one of these steps can contribute to insulin resistance. We observed the effect of insulin resistance induced by hyperlipidemia on the dynamics of insulin injected into skeletal muscle. Basal insulin euglycemic clamps (0.2 mU/min/kg) with or without lipid infusions (20% at 1.5 ml/min) were done on anesthetized dogs. Sequential insulin doses were administered by intramuscular injection directly into the vastus medialis of one hindlimb, using the contralateral leg for comparison. Intramuscular insulin injection in normal animals caused a clear dose-dependent increment in interstitial insulin levels, as well as dose-dependent increase in leg glucose uptake. In a second group of animals, lipid was infused before and during intramuscular insulin injection to cause systemic increase in free fatty acids (FFAs). In sharp contrast, systemic lipid infusion caused insulin resistance, indicated by reduced glucose infusion required to maintain euglycemia, and prevented injection-induced increase in lymphatic insulin and leg glucose uptake observed without lipid. The injected insulin was instead detected in the venous outflow from the leg. Lipid infusion caused intramuscular insulin to be diverted from interstitium into the capillary circulation, preventing a rise in interstitial insulin and any increase in local leg glucose uptake. The diversion of insulin from the interstitium under hyperlipidemic conditions may play a role in the insulin resistance observed coincident with elevated nocturnal FFAs as is observed in obesity.

Greater omentectomy improves insulin sensitivity in nonobese dogs.

Visceral adiposity is strongly associated with insulin resistance; however, little evidence directly demonstrates that visceral fat per se impairs insulin action. Here, we examine the effects of the surgical removal of the greater omentum and its occupying visceral fat, an omentectomy (OM), on insulin sensitivity (S(I)) and beta-cell function in nonobese dogs. Thirteen male mongrel dogs were used in this research study; animals were randomly assigned to surgical treatment with either OM (n = 7), or sham-surgery (SHAM) (n = 6). OM failed to generate measurable changes in body weight (+2%; P = 0.1), or subcutaneous adiposity (+3%; P = 0.83) as assessed by magnetic resonance imaging (MRI). The removal of the greater omentum did not significantly reduce total visceral adipose volume (-7.3 +/- 6.4%; P = 0.29); although primary analysis showed a trend for OM to increase S(I) when compared to sham operated animals (P = 0.078), further statistical analysis revealed that this minor reduction in visceral fat alleviated insulin resistance by augmenting S(I) of the periphery (+67.7 +/- 35.2%; P = 0.03), as determined by the euglycemic-hyperinsulinemic clamp. Insulin secretory response during the hyperglycemic step clamp was not directly influenced by omental fat removal (presurgery 6.82 +/- 1.4 vs. postsurgery: 6.7 +/- 1.2 pmol/l/mg/dl, P = 0.9). These findings provide new evidence for the deleterious role of visceral fat in insulin resistance, and suggest that a greater OM procedure may effectively improve insulin sensitivity.

Direct administration of insulin into skeletal muscle reveals that the transport of insulin across the capillary endothelium limits the time course of insulin to activate glucose disposal.

OBJECTIVE: Intravenous insulin infusion rapidly increases plasma insulin, yet glucose disposal occurs at a much slower rate. This delay in insulin's action may be related to the protracted time for insulin to traverse the capillary endothelium. An increased delay may be associated with the development of insulin resistance. The purpose of the present study was to investigate whether bypassing the transendothelial insulin transport step and injecting insulin directly into the interstitial space would moderate the delay in glucose uptake observed with intravenous administration of the hormone. RESEARCH DESIGN AND METHODS: Intramuscular injections of saline (n = 3) or insulin (n = 10) were administered directly into the vastus medialis of anesthetized dogs. Injections of 0.3, 0.5, 0.7, 1.0, and 3.0 units insulin were administered hourly during a basal insulin euglycemic glucose clamp (0.2mU x min(-1) x kg(-1)). RESULTS: Unlike the saline group, each incremental insulin injection caused interstitial (lymph) insulin to rise within 10 min, indicating rapid diffusion of the hormone within the interstitial matrix. Delay in insulin action was virtually eliminated, indicated by immediate dose-dependent increments in hindlimb glucose uptake. Additionally, bypassing insulin transport by direct injection into muscle revealed a fourfold greater sensitivity to insulin of in vivo muscle tissue than previously reported from intravenous insulin administration. CONCLUSIONS: Our results indicate that the transport of insulin to skeletal muscle is a rate-limiting step for insulin to activate glucose disposal. Based on these results, we speculate that defects in insulin transport across the endothelial layer of skeletal muscle will contribute to insulin resistance.

Beta-cell "rest" accompanies reduced first-pass hepatic insulin extraction in the insulin-resistant, fat-fed canine model.

During insulin resistance, glucose homeostasis is maintained by an increase in plasma insulin via increased secretion and/or decreased first-pass hepatic insulin extraction. However, the relative importance of insulin secretion vs. clearance to compensate for insulin resistance in obesity has yet to be determined. This study utilizes the fat-fed dog model to examine longitudinal changes in insulin secretion and first-pass hepatic insulin extraction during development of obesity and insulin resistance. Six dogs were fed an isocaloric diet with an approximately 8% increase in fat calories for 12 wk and evaluated at weeks 0, 6, and 12 for changes in 1) insulin sensitivity by euglycemic-hyperinsulinemic clamp, 2) first-pass hepatic insulin extraction by direct assessment, and 3) glucose-stimulated insulin secretory response by hyperglycemic clamp. We found that 12 wk of a fat diet increased subcutaneous and visceral fat as assessed by MR imaging. Consistent with increased body fat, the dogs exhibited a approximately 30% decrease in insulin sensitivity and fasting hyperinsulinemia. Although insulin secretion was substantially increased at week 6, beta-cell sensitivity returned to prediet levels by week 12. However, peripheral hyperinsulinemia was maintained because of a significant decrease in first-pass hepatic insulin extraction, thus maintaining hyperinsulinemia, despite changes in insulin release. Our results indicate that when obesity and insulin resistance are induced by an isocaloric, increased-fat diet, an initial increase in insulin secretion by the beta-cells is followed by a decrease in first-pass hepatic insulin extraction. This may provide a secondary physiological mechanism to preserve pancreatic beta-cell function during insulin resistance.

Reduced access to insulin-sensitive tissues in dogs with obesity secondary to increased fat intake.

Physiological hyperinsulinemia provokes hemodynamic actions and augments access of macromolecules to insulin-sensitive tissues. We investigated whether induction of insulin resistance by a hypercaloric high-fat diet has an effect on the extracellular distribution of macromolecules to insulin-sensitive tissues. Male mongrel dogs were randomly selected into two groups: seven dogs were fed an isocaloric control diet ( approximately 3,900 kcal, 35% from fat), and six dogs were fed a hypercaloric high-fat diet ( approximately 5,300 kcal, 54% from fat) for a period of 12 weeks. During hyperinsulinemic-euglycemic clamps, we determined transport parameters and distribution volumes of [(14)C]inulin by applying a three-compartment model to the plasma clearance data of intravenously injected [(14)C]inulin (0.8 microCi/kg). In another study with direct cannulation of the hindlimb skeletal muscle lymphatics, we investigated the effect of physiological hyperinsulinemia on the appearance of intravenously injected [(14)C]inulin in skeletal muscle interstitial fluid and compared the effect of insulin between control and high-fat diet groups. The hypercaloric high-fat diet resulted in significant weight gain (18%; P<0.001) associated with marked increases of subcutaneous (140%; P<0.001) and omental (83%; P<0.001) fat depots, as well as peripheral insulin resistance, measured as a significant reduction of insulin-stimulated glucose uptake during clamps (-35%; P<0.05). Concomitantly, we observed a significant reduction of the peripheral distribution volume of [(14)C]inulin (-26%; P<0.05), whereas the vascular distribution volume and transport and clearance parameters did not change as a cause of the diet. The second study directly confirmed our findings, suggesting a marked reduction of insulin action to stimulate access of macromolecules to insulin-sensitive tissues (control diet 32%, P<0.01; high-fat diet 18%, NS). The present results indicate that access of macromolecules to insulin-sensitive tissues is impaired during diet-induced insulin resistance and suggest that the ability of insulin itself to stimulate tissue access is diminished. We speculate that the observed diet-induced defects in stimulation of tissue perfusion contribute to the development of peripheral insulin resistance.

Physiological hyperinsulinemia in dogs augments access of macromolecules to insulin-sensitive tissues.

Pharmacological doses of insulin increase limb blood flow and enhance tissue recruitment for small solutes such as glucose. We investigated whether elevating insulin within the physiological range (68 +/- 6 vs. 425 +/- 27 pmol/l) can influence tissue recruitment of [(14)C]inulin, an inert diffusionary marker of molecular weight similar to that of insulin itself. During hyperinsulinemic-euglycemic clamps, transport parameters and distribution volumes of [(14)C]inulin were determined in conscious dogs by applying a three-compartment model to the plasma clearance data of intravenously injected [(14)C]inulin (0.8 microCi/kg). In a second set of experiments in anesthetized dogs with direct cannulation of the hindlimb skeletal muscle lymphatics, we measured a possible effect of physiological hyperinsulinemia on the response of the interstitial fluid of skeletal muscle to intravenously injected [(14)C]inulin and compared this response with the model prediction from plasma data. Physiological hyperinsulinemia caused a 48 +/- 10% (P < 0.005) and a 35 +/- 15% (P < 0.05) increase of peripheral and splanchnic interstitial distribution volumes for [(14)C]inulin. Hindlimb lymph measurements directly confirmed the ability of insulin to enhance the access of macromolecules to the peripheral interstitial fluid compartment. The present results show that physiological hyperinsulinemia will enhance the delivery of a substance of similar molecular size to insulin to previously less intensively perfused regions of insulin-sensitive tissues. Our data suggest that the delivery of insulin itself to insulin-sensitive tissues could be a mechanism of insulin action on cellular glucose uptake independent of and possibly synergistic with either enhanced blood flow distribution or GLUT4 transporter recruitment to enhance glucose utilization. Because of the differences between inulin and insulin itself, whether delivery of the bioactive hormone is increased remains speculative.

Investigation of the effect of acepromazine on intravenous glucose tolerance tests in dogs.

OBJECTIVE: To investigate the effects of administration of acepromazine on IV glucose tolerance tests (IVGTTs) in dogs. ANIMALS: 8 male mixed-breed dogs. PROCEDURE: With a 1-week interval between tests, each dog underwent (in random order) an IVGTT with or without pretest administration of acepromazine maleate (0.1 mg/kg, SC, 30 minutes prior to the start of the IVGTT). Food was withheld from the dogs for 14 hours prior to each test. Blood samples were obtained at 20, 10, and 1 minute prior to and at 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 19, 22, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, and 180 minutes after administration of glucose. RESULTS: There were no significant differences in the baseline (ie, after food was withheld) plasma glucose, lactate, and insulin concentrations between dogs undergoing the IVGTT and acepromazine-IVGTT; however, lower baseline free fatty acid concentration was observed in acepromazine-treated dogs. Analysis of data via the application of Bergman's minimal model of glucose kinetics revealed no differences in insulin sensitivity, acute insulin response to glucose, disposition index, or glucose effectiveness between dogs treated or not treated with acepromazine before testing. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that in dogs undergoing IV glucose tolerance testing, pretest administration of small doses of acepromazine can be used as a means of chemical restraint without interfering with results of the glucose metabolism assessment.

NK cells regulate CD4 responses prior to antigen encounter.

NK cells not only respond rapidly to infection, shaping subsequent adaptive immunity, but also play a role in regulating autoimmune disease. The ability of NK cells to influence adaptive immunity before Ag exposure was examined in a gender-dependent model of preferential Th1 and Th2 activation. The inability of young adult male SJL mice to activate Th1 cells was reversed via depletion of NK1.1(+) cells, whereas the presence or the absence of NK1.1(+) cells did not alter responses in age-matched females. Consistent with a gender-dependent role in regulating adaptive immunity, significantly more NK1.1(+) cells were present in males compared with females, and this difference was reversed by castration. In contrast to NK1.1(+) cells derived from C57BL/6 mice, no spontaneous cytokine secretion was detected in NK1.1(+) cells derived from either male or female SJL mice, although an increased frequency of IL-10-secreting NK1.1(+) cells was observed in males vs females following in vitro stimulation. Direct evidence that NK1.1(+) cells in males influence CD4(+) T cell activation before Ag exposure was demonstrated via the adoptive transfer of APC from control and NK1.1-depleted males. The absence of a functional NK T cell population in SJL mice suggests that NK cells influence adaptive immunity before Ag exposure via alterations in APC activity.

Mechanism of action in dogs of slow-acting insulin analog O346.

We compared metabolic effects as well as plasma and interstitial fluid kinetics of fatty acid-acylated insulin, Lys(B29)(N(epsilon)-omega-carboxynonadecanoyl)-des(B30) human insulin (O346), with previously determined kinetics of native insulin and insulin detemir. Euglycemic clamps with iv injection of O346 (90 pmol/kg) or saline control were performed in 10 male mongrel dogs under inhalant anesthesia. The t(1/2) for the clearance of O346 from plasma was 375.7 +/- 26.7 min; the t(1/2) for the appearance of O346 in interstitial fluid was 137 +/- 20 min (mean +/- SEM). Glucose disposal with O346 injection was increased 4-fold (t = 480 min, 8.3 +/- 1.42 mg/min/kg) compared with preinjection (t = 0 min, 2.1 +/- 0.13 mg/min/kg; P < 0.05) or saline control (t = 480 min, 2.09 +/- 0.22 mg/min/kg; P < 0.05). O346 plasma elimination and transendothelial transport were 0.3% and 3.5% of regular insulin and 3% and 50% of insulin detemir, respectively. Combination of in vivo results and compartmental modeling suggests that the duration of action of O346 after iv injection is about 25-fold and 10-fold longer compared with regular human insulin and insulin detemir, respectively. This study demonstrates that O346 stimulates glucose disposal very slowly, but when injected iv, its effect may be maintained for as long as 48 h as estimated from simulation analysis. The data suggest that O346 bound to albumin in plasma acts as a storage compartment for O346 from which the analog is slowly released to insulin-sensitive tissues. Reduced liver clearance of O346 is suggested to be the major mechanism for the protracted action.