Non-diabetic Rabbits Research Articles

VEGF‐A, VEGF‐D, VEGF receptor‐1, VEGF receptor‐2, NF‐KB, and RAGE in atherosclerotic lesions of diabetic Watanabe heritable hyperlipidemic rabbits

Plaque angiogenesis may be associated with the development of unstable and vulnerable plaques. Vascular endothelial growth factors (VEGFs) are potent angiogenic factors that can affect plaque neovascularization. Our objective was to determine the effect of diabetes on atherosclerosis and on the expression of angiogenesis-related genes in atherosclerotic lesions. Alloxan was used to induce diabetes in male Watanabe heritable hyperlipidemic (WHHL) rabbits that were sacrificed 2 and 6 months after the induction of diabetes. Nondiabetic WHHL rabbits served as controls. Blood glucose (Glc), serum-free fatty acids (FFA), and serum triglyceride levels were significantly higher in diabetic rabbits. Accelerated atherogenesis was observed in the diabetic WHHL rabbits together with increased intramyocellular lipids (IMCL), as determined by 1H-NMR spectroscopy. Atherosclerotic lesions in the diabetic rabbits had an increased content of macrophages and showed significant increases in immunostainings for vascular endothelial growth factor (VEGF)-A, VEGF-D, VEGF receptor-1, VEGF receptor-2, RAGE, and NF-kappaB. VEGF-A165 and VEGFR-2 mRNA levels were significantly increased in aortas of the diabetic rabbits, where a trend toward increased plaque vascularization was also observed. These results suggest that diabetes accelerates atherogenesis, up-regulates VEGF-A, VEGF-D, and VEGF receptor-2 expression, and increases NF-kappaB, RAGE, and inflammatory responses in atherosclerotic lesions in WHHL rabbits.

Impairment of endothelium- and nerve-mediated relaxation responses in the cavernosal smooth muscle of experimentally diabetic rabbits: role of weight loss and duration of diabetes

The effects of short- and long-term experimental diabetes on corporal nerve, endothelium and smooth-muscle responses were investigated, and the reasons for possible alterations in corporal smooth muscle responses such as hyperglycaemia, duration of experimental diabetes and/or altered tissue weight were evaluated. Rabbits were injected with alloxan (125 mg/kg) to induce diabetes. Age-matched non-diabetic and diabetic (3 and 9 weeks) and weight-matched non-diabetic groups (9 weeks) were used as control. In all groups, relaxation (carbachol, electrical field stimulation and sodiumnitroprusside) responses were examined. The relaxation responses were expressed as percentage of the precontraction to phenylephrine and as g response/g tissue weight. The effects of elevated glucose were also examined by incubating cavernosal strips in Krebs-Henseleit solution containing 44.4 mM glucose for 6 h. Cavernosal tissues of non-diabetic and 9-week diabetic rabbits were evaluated histologically. Sodiumnitroprusside (10(-7)-10(-4) M) responses were similar in all groups. Relaxation responses to electrical field stimulation (10 s train; amplitude 50 V; frequency 0.5-32 Hz; width 0.8 ms) were only attenuated in the 9-week diabetic group compared to the non-diabetic group. Carbachol (10(-8)-3 x 10(-5) M) responses were attenuated in both diabetic groups. When the relaxation responses expressed as g response/g tissue weight were evaluated, results were similar compared to those expressed as percentage of phenylephrine (10(-5) M). Neither carbachol nor electrical field stimulation mediated responses were impaired with glucose incubation. No morphological degenerations were observed in the endothelium. Diabetes may interfere with the synthesis and/or release of nitric oxide from both nerves and endothelium in corpus cavernosum, and alterations in endothelium-derived responses occur earlier than neurological disturbances. The sensitivity of cavernosal smooth muscle to nitric oxide did not alter in diabetes. Attenuation of responses was not due to decreased tissue weight caused by diabetes.

Efeitos da metformina na disfunção endotelial da circulação renal induzida pela hiperglicemia em coelhos não diabéticos

To assess the effects of metformin (Met) on the endothelial dysfunction of the renal circulation of non-diabetic rabbits acutely induced by levels of glucose usually observed in diabetic patients in daily clinical practice. Isolated perfused kidneys from non-diabetic rabbits were exposed for 3 h to normal (100 mg/dl--control group) or high (270 mg/dl) D-glucose with or without Met (100 microM). The glucose levels used correspond to 2 h post-breakfast median values (272 mg/dl) obtained from a cohort of 780 type 2 diabetic (DM2) outpatients seen in our service. Vascular reactivity was evaluated with endothelium-dependent (ED) [acetylcholine-ACh] and independent (EI) [sodium nitroprusside-SNP] vasodilating agents. Kidneys perfused with high glucose had ED maximal vasodilation blunted in comparison to controls (respectively 25 +/- 3 and 41 +/- 3%; p < 0.01). A 3 h Met infusion restored the vasodilating effect of ACh in the renal circulation in the presence of high glucose, no different from controls (respectively 43 +/- 1.5 vs. 41 +/- 3% p > 0.05). Met did not affect maximum vasodilation induced by ACh in the presence of normal glucose levels. Finally, renal vasodilation induced by SNP was not modified by simultaneous infusion of glucose and Met. Acute hyperglycemia corresponding to the range observed in patients with DM2 induces endothelial dysfunction in the renal circulation of normal rabbits. Acute treatment with Met was able to protect the renal circulation against the effects of high glucose. The mechanisms involved in this protector effect deserve further investigation.

Metformin prevents the impairment of endothelium-dependent vascular relaxation induced by high glucose challenge in rabbit isolated perfused kidneys

High glucose concentrations are involved in the development of diabetic-associated vascular complications. We have previously reported that acute high glucose challenge, corresponding to post-prandial glycemia levels observed in patients with type 2 diabetes, blunts ACh-induced endothelium-dependent relaxation of the renal circulation of non-diabetic rabbits. Isolated perfused kidneys from non-diabetic rabbits were acutely exposed (3 h) to normal (5.5 mM--control group) or high (15 mM) D-glucose concentrations in the presence or absence of a continuous infusion of metformin (20 or 100 microM). Renal vascular reactivity was evaluated with endothelium-dependent (acetylcholine, ACh) and -independent (sodium nitroprusside, SNP) vasodilating agents. ACh-induced maximal renal vasodilation was reduced by high glucose infusion (15 mM) in comparison to the control group (25+/-3% and 41+/-3% respectively; P<0.01), being restored to 41+/-4% and 43+/-2% by a simultaneous 3-h infusion of 20 or 100 microM of metformin respectively (P>0.05). Perfusion of the kidneys with the angiotensin II-converting enzyme inhibitor captopril (10 microM) also significantly prevented the deleterious effects of high glucose challenge in the renal circulation. The use of a continuous infusion of N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM) did not affect the protective effect of metformin in the renal circulation (39+/-4%; P>0.05), while tetraethylammonium (TEA, 10 mM) partially blunted this effect (33+/-4, P<0.01). Renal vasodilation induced by SNP was not modified by simultaneous infusion of high glucose and/or metformin. It is concluded that the impairment of ACh-induced endothelium-dependent renal vasodilation observed after acute exposure to high glucose concentrations is abolished by metformin administration. These alterations of renal vascular reactivity can be accounted for, at least in part, by the activation of the renal renin-angiotensin system during hyperglycemia. The protective effects of metformin present some EDHF-dependent component and are not related to metabolic pathways dependent on nitric oxide.

Glucose levels observed in daily clinical practice induce endothelial dysfunction in the rabbit macro- and microcirculation.

We investigated whether different concentrations of elevated glucose - corresponding to levels observed in patients with type 2 diabetes under routine care (post-prandial mean and maximum values) and those used for diagnosing diabetes - induce impairment of vascular reactivity of the macro- and microcirculation in non-diabetic rabbits. Aortic rings and isolated perfused kidneys from normal rabbits were acutely exposed (3 h) to normal (5.5 mm) or high (7-25 mM) D-glucose concentrations. Vascular reactivity was evaluated with endothelium-dependent [acetylcholine (ACh) and bradykinin (BK)] and independent [sodium nitroprusside (SNP)] vasodilating agents. Endothelium-dependent relaxation of the thoracic aorta induced by ACh or BK was significantly attenuated after a 3-h exposure to high D-glucose (15-25 mM) but not after corresponding increased osmolarity with mannitol solutions. Relaxation induced by SNP (endothelium-independent) was not affected by high D-glucose concentrations. Moreover, endothelium-dependent but not independent vasodilation of the isolated rabbit kidney was also impaired after 3-h perfusion with high D-glucose (11.1-25 mM). Perfusion with mannitol solutions (15-25 mM) partially blunted endothelium-dependent renal vasodilation. It is concluded that acute hyperglycemia corresponding to post-prandial levels in patients with type 2 diabetes induces endothelial dysfunction of conduit vessels as well as of the renal circulation of non-diabetic rabbits.

Defective chylomicron synthesis as a cause of delayed particle clearance in diabetes?

Chylomicron metabolism is abnormal in diabetes and the chylomicron particle may play a very important role in atherosclerosis. The aim of this study was to examine the effect of diabetes on the metabolism of chylomicrons in cholesterol-fed alloxan diabetic and nondiabetic rabbits. Five diabetic rabbits and 5 control rabbits were given [14C]linoleic acid and [3H]cholesterol by gavage. Lymph was collected following cannulation of the lymph duct and radiolabelled chylomicrons were isolated by ultracentrifugation. The chylomicrons from each animal were injected into paired control and diabetic recipients. Lymph apolipoprotein (apo) B48, apo B100, and apo E were measured using sodium dodecyl sulfate–polyacrylamide gradient gel electrophoresis. Mean blood sugar of the diabetic donors and diabetic recipients were 19.7 ± 2.3 and 17.2 ± 3.2 mmol/L. Diabetic rabbits had significantly raised plasma triglyceride (10.8 ± 13.9 versus 0.8 ± 0.5 mmol/L, P < 0.02). There was a large increase in apo B48 in lymph chylomicrons in the diabetic donor animals (0.19 ± 0.10 versus 0.04 ± 0.02 mg/h, P < 0.01) and apo B100 (0.22 ± 0.15 versus 0.07 ± 0.07 mg/h, P < 0.05) and a reduction in apo E on the lymph chylomicron particle (0.27 ± 0.01 versus 0.62 ± 0.07 mg/mg apo B, P < 0.001). Diabetic recipients cleared both control and diabetic chylomicron triglyceride significantly more slowly than control recipients (P < 0.05). Clearance of control chylomicron cholesterol was delayed when injected into diabetic recipients compared to when these chylomicrons were injected into control recipients (P < 0.005). Clearance of diabetic chylomicron cholesterol was significantly slower when injected into control animals compared to control chylomicron injected into control animals (P < 0.02). In this animal model of atherosclerosis, we have demonstrated that diabetes leads to the production of an increased number of lipid and apo E–deficient chylomicron particles. Chylomicron particles from the control animals were cleared more slowly by the diabetic recipient (both triglyceride and cholesterol). The chylomicron particles obtained from the diabetic animals were cleared even more slowly when injected into the diabetic recipient. Although there was an initial delay in clearance of chylomicron triglyceride from the diabetic particle when injected into the control animals, the clearance over the first 15 minutes was not significantly different when compared to the control chylomicron injected into the control animal. On the other hand, the cholesterol clearance was significantly delayed. Thus, diabetes resulted in the production of an increased number of lipid- and apo E–deficient chylomicron particles. These alterations account, in part, for the delay in clearance of these particles.

The insulinotropic activity of a Nepalese medicinal plant Biophytum sensitivum: preliminary experimental study

The effect of the leaf extract of Biophytum sensitivum, an annual herb used in traditional Nepalese folk medicine for the treatment of hyperglycemic patients, was studied on glucose homeostasis in rabbits. In the first set of experiments, an acute effect of the extract on fasting plasma glucose (fpg) levels and serum insulin response was examined in non-diabetic and alloxan-diabetic rabbits. Initial dose–response studies showed that a dose of 200 mg/kg body weight (b.w.) was optimum for hypoglycemia. A single administration of this dose to 16-h fasted non-diabetic rabbits brought about a 16.1% fall in fpg at the end of 1 and 2 h, and the hypoglycemic effect persisted at the end of 6 h (13.8% fall). Serum insulin levels showed a significant rise in the treated animals, which suggested a pancreatic mode of action (i.e. insulinotropic effect) of B. sensitivum. The study of an acute effect of the extract in alloxan-diabetic rabbits, however, showed that it failed to produce such hypoglycemic or serum insulin response. In another set of experiments, administration of the above dose of the leaf extract attenuated the plasma glucose response to oral administration of 3 g/kg b.w. glucose load. The serum insulin levels in the treated animals showed a rise at the end of 2 (13.7% rise) and 6 h (12.6% rise). Taken together, these observations suggest that the hypoglycemic response of B. sensitivum may be mediated through stimulating the synthesis/release of insulin from the beta cells of Langerhans.

Cisplatin Pharmacokinetics and Its Nephrotoxicity in Diabetic Rabbits

Background: This study was designed to investigate the relationship between the attenuation of cisplatin (CDDP)-induced nephrotoxicity in experimental diabetic rabbits and the plasma pharmacokinetics of the free ultrafilterable and total plasma platinum (Pt) levels. Methods: Two groups of age-matched male New Zealand white rabbits were used; the first group consisted of rabbits with streptozotocin-induced diabetes (single i.v. bolus dose of streptozotocin of 65 mg/kg in citrate buffer, pH 4.6), and the second group of nondiabetic rabbits treated with the same volume of citrate buffer. Both groups were treated with CDDP (5 mg/kg, single i.v. bolus dose) 3 days after induction of the diabetic state in the first group. The plasma Pt levels were followed for 4 h after CDDP administration, in which the free ultrafilterable and total plasma Pt concentrations were determined by atomic absorption spectrometry. The pharmacokinetic parameters of free ultrafilterable plasma Pt were determined using a noncompartment pharmacokinetic model of analysis. Results: The total plasma Pt levels declined in a biphasic manner and were adequately described by a two-compartment model. No significant change was observed in the pharmacokinetics of either the free ultrafilterable or total plasma Pt levels in the diabetic group in comparison with the control nondiabetic group (p > 0.05). However, 4 h after CDDP administration, the total plasma Pt level of the nondiabetic group was significantly higher than that of the diabetic rabbits (p < 0.001). Indices of nephrotoxicity were determined 7 days after CDDP administration. The results revealed that the diabetic state protected against CDDP-induced nephrotoxicity. The nondiabetic rabbits exhibited highly significant elevations in the serum creatinine and urea levels and a decrease in the serum albumin level (p < 0.001) in comparison with the diabetic group. Conclusions: These findings might suggest that the reduction in CDDP-induced nephrotoxicity in diabetic rabbits is not due to a change in the plasma pharmacokinetic profile within the drug follow-up period. It could be anticipated that the rapid decline in the total plasma Pt level after CDDP administration to diabetic rabbits, as well as the reduction in the terminal elimination half-life of the total plasma Pt level might be responsible for the reduction in CDDP-induced nephrotoxicity. Also, alterations in the how kidneys of diabetics deal with the renal excretion of Pt and reduction of its accumulation in kidney tissue are not excluded.

Streptozotocin- and alloxan-induced diabetes modifies total plasma and lipoprotein lipid concentration and composition without altering cholesteryl ester transfer activity.

The objectives of this study were to determine the total plasma and lipoprotein lipid concentration and composition and cholesteryl ester transfer activity in two diabetic animal models (alloxan-induced diabetes in rabbits and streptozotocin-induced diabetes in rats). Furthermore, we wanted to determine if the severity of diabetes influences lipoprotein lipid profiles and cholesteryl ester transfer activity. Rats and rabbits were randomly divided into non-diabetic and diabetic groups. Rats were administered either 55 mg/kg or 100 mg/kg of streptozotocin intravenously through the tail vein, while rabbits were administered 100 mg/kg or 200 mg/kg of alloxan intravenously through the marginal ear vein under light anesthesia. Hyperglycaemia was tested for at 48 hr following the doses. Total and lipoprotein cholesterol and triglyceride concentrations using enzymatic kits and cholesteryl ester transfer activity from low-density lipoproteins to high-density lipoproteins using 3H-cholesteryl ester incorporated into low-density lipoproteins were determined. Elevations in both total and lipoprotein cholesterol and triglyceride concentrations and alterations in lipoprotein lipid composition are observed following the onset of drug-induced diabetes in rats and rabbits compared to non-diabetics. However, these findings were observed only in animals administered the higher streptozotocin and alloxan dose. Furthermore, cholesteryl ester transfer from low-density lipoproteins to high-density lipoproteins is not significantly different in drug-induced diabetic compared to non-diabetic rats and rabbits, regardless of which streptozotocin and alloxan dose was used. These findings suggest that difference in lipoprotein lipid concentration and composition as a result of drug-induced diabetes is independent of cholesteryl ester transfer activity in both rats and rabbits. Furthermore, diabetic severity may influence lipoprotein metabolism in these animal models.

Improved Myocardial Tolerance to Ischaemia in the Diabetic Rabbit

Because cardiac complications after myocardial infarction are more frequent in diabetics, we tested whether experimentally-induced diabetes may increase ischaemic myocardial injury in 23 rabbits. Diabetes was induced in randomized rabbits with the alloxan method. After 2 months, diabetic rabbits underwent a 30-min coronary occlusion followed by 3-h reperfusion and were compared with controls. Collateral flow was measured by the radioactive microsphere technique and infarct size by tetrazolium staining. Infarct size represented 28.6±4% of area-at-risk in controls and 16.5±3% in diabetics (P<0.05). Collateral flow (0.06±0.03 ml/min/g in controls and 0.014±0.004 ml/min/g in diabetics) and area-at-risk (50.2±4.2% of left ventricle in controls and 53.9±5.4% in diabetics) were similar in both groups. There was a significant positive correlation between area-at-risk and infarct size in both groups (r=0.60 and 0.70, respectively) and for a given area-at-risk, diabetic rabbits developed smaller myocardial infarction than controls (covariance analysis,P<0.01). In additional experiments, hyperglycemia induced by intravenous glucose infusion in non-diabetic rabbits did not protect the ischaemic myocardium (infarct size: 37.9±12.5%). In conclusion, diabetes in the rabbit induces a chronic and metabolic form of preconditioning. Further studies are needed to explore the mechanism and time course of this protection.

Hypoglycaemic Effect ofMoringa stenopetala Aqueous Extract in Rabbits

The hypoglycaemic effect of Moringa stenopetala extract was assessed in nondiabetic rabbits by blood glucose analysis. In vivo experiments were carried out in rabbits that received the test material and the standard, glibenclamide. The plant extract, although less potent than glibenclamide, was found to lower blood glucose concentration. The hypoglycaemic effect was observed to increase with time and with an increase in the dose of the extract. © 1997 by John Wiley & Sons, Ltd.

Diabetes and insulin-induced stimulation of L-arginine transport and nitric oxide synthesis in rabbit isolated gastric glands.

1. The properties of L-arginine transport have been characterized and correlated with cGMP production (index of nitric oxide (NO)) in whole gastric glands isolated from non-diabetic and alloxan-diabetic rabbits. 2. In non-diabetic and diabetic glands, transport of L-arginine was stereoselective, Na+ and pH independent and inhibited by other cationic amino acids. L-Arginine transport was slightly inhibited by L-leucine and L-phenylalanine, but unaffected by other neutral amino acids. 3. Diabetes enhanced the Vmax for saturable L-arginine transport from 10.7 +/- 1.0 to 17.7 +/- 0.5 pmol (mg protein)-1 s-1, with negligible changes in K(m). 4. Accumulation of the membrane potential-sensitive probe tetra[3H]phenylphosphonium (TPP+) was increased 2-fold in diabetic compared with non-diabetic gastric glands, suggesting a membrane hyperpolarization. 5. Basal intracellular cGMP levels were elevated 2-fold in diabetic gastric glands, and in non-diabetic glands histamine, vasoactive intestinal peptide, and bradykinin increased cGMP levels. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (100 microM) abolished basal cGMP accumulation. 6. Addition of extracellular L-arginine induced a concentration-dependent increase in cGMP levels in gastric glands isolated from non-diabetic rabbits, but had no effect on elevated cGMP levels in diabetic glands. 7. Insulin induced a rapid (5 min) concentration-dependent increase in cGMP levels in non-diabetic gastric glands, but reduced elevated cGMP levels in diabetic gastric glands. 8. The present study has identified a specific transport system for L-arginine in gastric glands which resembles the classical system y+. Our findings also provide the first direct evidence that diabetes increases the basal activity of system y+ and NO synthase in gastric glands. The differential modulation of L-arginine transport by insulin and L-arginine identified in non-diabetic and diabetic glands, may be of importance in protecting the gastric mucosa from injuries associated with diabetes.

Evidence for a major role for glucagon in regulation of plasma glucose in conscious, nondiabetic, and alloxan-induced diabetic rabbits

Evidence for a major role for glucagon in regulation of plasma glucose in conscious, nondiabetic, and alloxan-induced diabetic rabbits

Evidence for a major role for glucagon in regulation of plasma glucose in conscious, nondiabetic, and alloxan-induced diabetic rabbits.

Effects of glucagon immunoneutralization on plasma glucose, insulin, and glucagon were studied 2-4 h after intravenous injection of a high-affinity, monoclonal glucagon antibody into normal as well as moderately and severely alloxan (ALX)-induced diabetic rabbits (n = 5-7). A monoclonal trinitrophenyl antibody was used in control studies. Endogenous glucagon was completely neutralized as evidenced by undetectable levels of free glucagon and high plasma glucagon-binding capacities. In postabsorbtive normal rabbits, glucagon neutralization decreased plasma glucose by 2.2 +/- 0.3 mmol/l, and the resulting plasma levels of insulin and glucagon (indirectly measured) were 8 +/- 3 and 640 +/- 129% of baseline, respectively. However, when euglycemia was maintained by means of glucose infusion (steady-state plasma glucose and glucose infusion rate: 6.6 +/- 0.1 mmol/l and 3.0 +/- 0.4 mg.kg-1.min-1), both plasma insulin and glucagon remained unaltered. Thus, the glucose infusion rate accurately reflects glucagon's contribution to postabsorbtive glucose production. In both moderately and severely diabetic rabbits, immunoneutralization of glucagon decreased plasma glucose by approximately 8 mmol/l, leading to euglycemia (7.3 +/- 1.1 mmol/l) and reduced hyperinsulinemia (41 +/- 9% of baseline) in the former and to partial restoration of euglycemia (12.7 +/- 1.8 mmol/l) and unchanged insulin levels in the latter group of diabetic rabbits (P < 0.05 vs. controls in all studies). No significant changes were observed in control studies. In conclusion, glucagon is an important regulator of postabsorbtive glucose production in normal rabbits and plays an important role in the maintenance of hyperglycemia in ALX-induced diabetic rabbits.

Diabetes mellitus induces accelerated growth of aortic smooth muscle cells: association with overexpression of PDGF β‐receptors

The mechanism of diabetic macroangiopathy was studied from the view point of phenotypic change of aortic smooth muscle cells (SMC). The growth rates of cultured SMC of diabetic rats or rabbits were higher than those of non-diabetic animals (controls). This difference of the growth responses was observed specifically with platelet-derived growth factor (PDGF). Of the three PDGF dimers, PDGF-AB heterodimer (PDGF-AB) and PDGF-BB homodimer (PDGF-BB) stimulated growth of diabetic SMC more than that of control SMC but PDGF-AA homodimer (PDGF-AA) did not. The binding of 125I-PDGF to the diabetic SMC was greater than that to control SMC. This was due to increase in the number of cell surface receptors for PDGF. On in vitro culture, SMC from diabetic rats expressed more PDGF beta-receptor mRNA than SMC from non-diabetic rats. Moreover, in vivo, the aortic media of diabetic rabbits expressed PDGF beta-receptor mRNA, but that from non-diabetic rabbits did not. Thus diabetic SMC over-react on PDGF stimulation through over-expression of the PDGF beta-receptor gene. The significance of this fact in development of diabetic macroangiopathy is discussed.

Exogenous advanced glycosylation end products induce complex vascular dysfunction in normal animals: a model for diabetic and aging complications.

Advanced glycosylation end products (AGEs) have been implicated in many of the complications of diabetes and normal aging. Markedly elevated vascular tissue and circulating AGEs were linked recently to the accelerated vasculopathy of end-stage diabetic renal disease. To determine the pathogenic role of AGEs in vivo, AGE-modified albumin was administered to healthy nondiabetic rats and rabbits alone or in combination with the AGE-crosslink inhibitor aminoguanidine. Within 2-4 weeks of AGE treatment, the AGE content of aortic tissue samples rose to six times the amount found in controls (P < 0.001). Cotreatment with aminoguanidine limited tissue AGE accumulation to levels two times that of control. AGE administration was associated with a significant increase in vascular permeability, as assessed by 125I label tracer methods. This alteration was absent in animals that received aminoguanidine in addition to AGE. Significant mononuclear cell migratory activity was observed in subendothelial and periarteriolar spaces in various tissues from AGE-treated rats compared to normal cellularity noted in tissues from animals treated with aminoguanidine. Blood pressure studies of AGE-treated rats and rabbits revealed markedly defective vasodilatory responses to acetylcholine and nitroglycerin compared to controls (P < 0.001), consistent with marked NO. inactivation; aminoguanidine treatment significantly prevented this defect. These in vivo data demonstrate directly that AGEs, independent of metabolic or genetic factors, can induce complex vascular alterations resembling those seen in diabetes or aging. AGE administration represents an animal model system for the study of diabetic and aging complications as well as for assessing the efficacy of newly emerging therapies aimed at inhibiting advanced glycosylation.

A new approach for insulin delivery via the pulmonary route: Design and pharmacokinetics in non-diabetic rabbits

A mini-mist medication air compressor fitted nebulizer was used to deliver insulin in the form of a fine mist via the rabbit's pulmonary system. An insulin immune test was first performed in order to ensure the rabbit's respiratory compliance and then the bioavailability was examined as a function of insulin dose concentration and dose volume. An increase in dose concentration from 2 to 5 U kg − rbw was found to reduce the percentage minimum plasma glucose concentration (%MPGC) from 71 to 38% without affecting the time T required to attain these values (i.e., T%MPGC = 20 min). The values of the percentage total reduction in plasma glucose from 0 to 3 h (%TRPG 0–3h) were also found to increase from 15 to 37% as insulin concentration increased. On the other hand, an increase in dose volume from 1 to 3 ml did not lead to marked changes in both %MPGC and %TRPG(in0–3h) but did increase the value of T%MPGC from 20 to 50 min. Comparative study between pulmonary administered insulin (PAI and subcutaneously administered insulin (SAI) showed that the T%MPGC for PAI was about 1 5 of that for SAI with an acceptable duration for maintaining the lowering of plasma glucose. The apparent values of %MPGC and %TRPC 0–3h for PAI were about 36% of those for SAI and related to incomplete dose consumption as part of PAI was always retained inside the nebulizer. The corrected bioavailability for PAI was therefore calculated and found to be greater than 50% of that for SAI.

Metabolism of arachidonic acid in neutrophils from alloxan-diabetic rabbits

The production of 5-lipoxygenase products from arachidonic acid was investigated in polymorphonuclear leukocytes (PMNL) isolated from non-diabetic and alloxan-induced diabetic rabbits: (i) production of 5-hydroxyeicosatetraenoic acid, leukotriene B 4, and the two 6-trans-leukotriene B 4 isomers were significantly decreased in the PMNL of diabetic rabbits when compared to non-diabetic rabbits; (ii) production of LTB 4 and 5-HETE from diabetic PMNL required the addition of Ca 2+ and A23187 to a greater degree than control incubations; and (iii) the availability of substrate in the PMNL of diabetics was not a limiting factor for 5-lipoxygenase product formation. Alternative pathways of arachidonic acid metabolism were also evaluated: the recovery of exogenous leukotriene B 4 and 5-hydroxyeicosatetraenoic acid were identical using PMNL from control and diabetic rabbits and peptido-leukotrienes were not detected by radioimmunoassay. The data suggest that the activity of 5-lipoxygenase and the production of 5-hydroperoxyeicosatetraenoic acid in the diabetic PMNL may be limiting factors since the formation of leukotriene B 4, leukotriene B 4 isomers, and 5-hydroxyeicosatetraenoic acid are depressed in PMNL of diabetic rabbits. Alternative pathways do not account for the conversion of arachidonic acid to other products nor are the elimination pathways for LTB 4 and 5-HETE different. Decreased formation of 5-hydroxyeicosatetraenoic acid and leukotriene B 4 could predispose diabetic subjects to infection due to a decrease in mediators leading to the local accumulation of PMNL in the inflammatory response.

A defect in sodium-dependent amino acid uptake in diabetic rabbit peripheral nerve. Correction by an aldose reductase inhibitor or myo-inositol administration.

A myo-inositol-related defect in nerve sodium-potassium ATPase activity in experimental diabetes has been suggested as a possible pathogenetic factor in diabetic neuropathy. Because the sodium-potassium ATPase is essential for other sodium-cotransport systems, and because myo-inositol-derived phosphoinositide metabolites regulate multiple membrane transport processes, sodium gradient-dependent amino acid uptake was examined in vitro in endoneurial preparations derived from nondiabetic and 14-d alloxan diabetic rabbits. Untreated alloxan diabetes reduced endoneurial sodium-gradient dependent uptake of the nonmetabolized amino acid 2-aminoisobutyric acid by greater than 50%. Administration of an aldose reductase inhibitor prevented reductions in both nerve myo-inositol content and endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Myo-inositol supplementation that produced a transient pharmacological elevation in plasma myo-inositol concentration, but did not raise nerve myo-inositol content, reproduced the effect of the aldose reductase inhibitor on endoneurial sodium-dependent 2-aminoisobutyric acid uptake. Phorbol myristate acetate, which acutely normalizes sodium-potassium ATPase activity in diabetic nerve, did not acutely correct 2-aminoisobutyric uptake when added in vitro. These data suggest that depletion of a small myo-inositol pool may be implicated in the pathogenesis of defects in amino acid uptake in diabetic nerve and that rapid correction of sodium-potassium ATPase activity with protein kinase C agonists in vitro does not acutely normalize sodium-dependent 2-aminoisobutyric acid uptake.

Absence of Angiogenesis-lnhibitory Activity in Aqueous Humor of Diabetic Rabbits

We studied inhibitory activity of angiogenesis in the eye to explore the formation of new blood vessels in diabetic microvascular diseases. When we examined the effects of extracts from various ocular tissues of nondiabetic rabbits on the proliferation of bovine aortic endothelial cells, potent inhibitory activity was found in lens and aqueous humor. Aqueous humor inhibited both angiogenesis on chorioallantoic membrane in vivo and promotion of endothelial cell growth in vitro, which were induced by retinal extracts. However, in diabetic rabbits, aqueous humor lost the ability to inhibit the growth of endothelial cells. The loss of activity became evident 1 mo after the alloxan injection and lasted while the animals were hyperglycemic. In addition, diabetic aqueous humor failed to suppress retinal extract-induced angiogenesis promotion and endothelial cell growth. The absence of the inhibitory activity of angiogenesis in diabetic aqueous humor might be involved in promotion of neovascularization in diabetic ocular disease.