Subdepressor dose of benidipine ameliorates diabetic cardiac remodeling accompanied by normalization of upregulated endothelin system in rats

We investigated whether benidipine, a long-acting calcium channel blocker (CCB), can normalize cardiac expression profiles of the endothelin (ET)-1 system in insulin-resistant diabetes. Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of human Type 2 diabetes, were treated for 12 wk with vehicle or benidipine (3 mg.kg(-1).day(-1)). OLETF rats exhibited a significant increase in ET-1 in plasma and left ventricular (LV) tissues compared with nondiabetic controls. Expression of prepro-ET-1, ET-converting enzyme, and ET(A) and ET(B) receptors in LV tissues was also significantly higher in OLETF rats. The two MAPKs, JNK and p38MAPK, both of which are activated by ET-1, were more abundantly expressed in OLETF rat LV tissues. All these alterations were reversed to nondiabetic levels when OLETF rats were treated with the subdepressor dose of benidipine. Furthermore, benidipine therapy resulted in hindering cardiomyocyte hypertrophy and cardiac perivascular fibrosis in OLETF rats. The beneficial actions of benidipine at the subdepressor dose on cardiac remodeling in insulin-resistant diabetes may involve normalization of the upregulated ET-1 system.     

Cholecystokinin-8 (CCK-8) has no effect on heart rate in rats lacking CCK-A receptors.

Heart rate responses to i.v. administration of cholecystokinin-8 (CCK-8) were investigated in Otsuka Long-Evans Tokushima Fatty (OLETF) rats lacking CCK-A receptors and control Long-Evans Tokushima Otsuka (LETO) rats. The heart rate decreased after i.v. administration of 3 nmol.kg(-)(1) of CCK-8 in LETO rats, but not in OLETF rats. Bradycardia in the LETO rats disappeared after treatment with MK-329, but not after treatment with L-365,260. The expression of CCK-A receptor precursor mRNA was found exclusively in the atrium in LETO rats. These results suggest that CCK-8 decreases heart rate via CCK-A receptors located in the atrium of the rats.     

Cholecystokinin-8 increases Fos-like immunoreactivity in the brainstem and myenteric neurons of rats through CCK1 receptors

To examine the role of cholecystokinin1 receptor (CCK1) in the activation of brainstem and myenteric neurons by CCK, we compared the ability of exogenous CCK-8 to induce Fos-like immunoreactivity (Fos-LI) in these neurons in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, lacking CCK1 receptors, and Long-Evans Tokushima Otsuka (LETO) controls. Five groups (n=4 rats per group) of OLETF rats, and five LETO control groups, were injected intraperitoneally (IP) with 5, 10, 20, and 40 microg/kg CCK-8 or saline. Forty-micrometer brainstem sections containing the area postrema, nucleus of the solitary tract, and the dorsal motor nucleus of the vagus, and myenteric neurons of the duodenum, jejunum, and ileum underwent a diaminobenzidine reaction enhanced with nickel to reveal Fos-LI. CCK-8 did not increase Fos-LI in any of the tested neurons in the OLETF rats. CCK-8 increased Fos-LI in the brainstem of the LETO rats in a dose dependent manner. In the LETO rats only 40 microg/kg CCK-8 increased Fos-LI in the myenteric plexus of the jejunum. This study demonstrates that CCK-8 activates the brainstem and myenteric neurons through the CCK1 receptor.     

OLETF (Otsuka Long-Evans Tokushima Fatty) rats that lack the CCK 1 (A) receptor develop less behavioral sensitization to repeated cocaine treatment than wild type LETO (Long Evans Tokushima Otsuka) rats.

OLETF (Otsuka Long-Evans Tokushima Fatty) lacking the CCK 1 (A) receptor have similar spontaneous activity and locomotor response (horizontal and vertical activity) in response to a single injection of cocaine as the wild type LETO (Long Evans Tokushima Otsuka) rats. In contrast, the OLETF rats display more stereotypy in response to the first dose of cocaine than the LETO rats. Tested at 7 and 14 days after a one week daily treatment with cocaine, the LETO rats display robust behavioral sensitization to cocaine while the OLETF rats did not. These results support the hypothesis that endogenous CCK released by cocaine treatment and acting at CCK 1 receptors is required for the development and/or expression of this behavior.     

Differential body weight and feeding responses to high-fat diets in rats and mice lacking cholecystokinin 1 receptors

Prior data demonstrated differential roles for cholecystokinin (CCK)1 receptors in maintaining energy balance in rats and mice. CCK1 receptor deficiency results in hyperphagia and obesity of Otsuka Long-Evans Tokushima Fatty (OLETF) rats but not in mice. To ascertain the role of CCK1 receptors in high-fat-diet (HFD)-induced obesity, we compared alterations in food intake, body weight, fat mass, plasma glucose, and leptin levels, and patterns of hypothalamic gene expression in OLETF rats and mice lacking CCK1 receptors in response to a 10-wk exposure to HFD. Compared with Long-Evans Tokushima Otsuka (LETO) control rats, OLETF rats on HFD had sustained overconsumption over the 10-wk period. High fat feeding resulted in greater increases in body weight and plasma leptin levels in OLETF than in LETO rats. In situ hybridization determinations revealed that, while HFD reduced neuropeptide Y (NPY) mRNA expression in both the arcuate nucleus (Arc) and the dorsomedial hypothalamus (DMH) of LETO rats, HFD resulted in decreased NPY expression in the Arc but not in the DMH of OLETF rats. In contrast to these results in OLETF rats, HFD increased food intake and induced obesity to an equal degree in both wild-type and CCK1 receptor(-/-) mice. NPY gene expression was decreased in the Arc in response to HFD, but was not detectable in the DMH in both wild-type and CCK1 receptor(-/-) mice. Together, these data provide further evidence for differential roles of CCK1 receptors in the controls of food intake and body weight in rats and mice.     

Supplementation of alpha-tocopherol improves cardiovascular risk factors via the insulin signalling pathway and reduction of mitochondrial reactive oxygen species in type II diabetic rats

This study determined the effects of alpha- and gamma-tocopherol supplementation on metabolic control and oxidative stress in type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Blood glucose, haemoglobin A1c (HbA1c), urinary protein, plasma free fatty acid, triacylglycerol and plasminogen activator inhibitor-1 (PAI-1) levels in OLETF rats were significantly higher than in non-diabetic control Long-Evans Tokushima Otsuka (LETO) rats. Alpha-tocopherol inhibited the increase in urinary protein, blood glucose, HbA1c and PAI-1 levels, but gamma-tocopherol did not. Plasma and hepatic lipid peroxidation and hepatic steatosis were increased in OLETF rats. alpha-Tocopherol decreased lipid peroxidation. Mitochondrial reactive oxygen species production and uncoupling protein 2 (UCP2) expression were significantly increased in the heart and aorta of OLETF rats compared with LETO rats. Endothelial NO synthase and aortic nitrotyrosine were increased in OLETF rats. In contrast, the expression of phosphorylated vasodilator-stimulated phosphoprotein and glucose transporter 4 in the aorta was significantly decreased in OLETF rats. These abnormalities were reversed by alpha-tocopherol. These findings suggest that alpha-tocopherol may prevent cardiovascular tissues from oxidative stress and insulin signalling disorder resulting from diabetes mellitus.     

Preserved postischemic heart function in sucrose-fed type 2 diabetic OLETF rats

Cardiovascular disease is one of the most important causes of morbidity and mortality in diabetes mellitus, but there has been controversy over functional impairment of diabetic hearts and their tolerance to ischemia. We studied ischemic heart function in type 2 diabetic rats with different degrees of hyperglycemia and its relationship with cardiac norepinephrine release. Otsuka Long-Evans Tokushima Fatty rats (OLETF) and age-matched Long-Evans Tokushima Otsuka normal rats (LETO) were used. One group of OLETF rats was given 30% sucrose in drinking water (OLETF-S). Hearts were isolated and perfused in a working heart preparation and subjected to 30 min ischemia followed by 40 min reperfusion at age of 12 months. Hemodynamics and coronary norepinephrine overflow were examined. Fasting plasma glucose in OLETF increased markedly at 12 months and sucrose administration exacerbated hyperglycemia in diabetic rats (LETO 6.6 +/- 0.5, OLETF 8.3 +/- 0.7, OLETF-S 15.0 +/- 1.7 mmol/L, P < 0.01). Basic cardiac output in OLETF was decreased as compared with LETO and OLETF-S (LETO 29.4 +/- 2.5, OLETF 24.0 +/- 2.4, OLETF-S 27.0 +/- 0.9 ml/min/g, P < 0.05) and remained very low after ischemia, while in OLETF-S it was well preserved (OLETF 4.2 +/- 2.1, OLETF-S 13.7 +/- 2.6 ml/min/g, P < 0.01). Correspondently, cardiac norepinephrine released during ischemia and reperfusion was lower in OLETF-S (OLETF 2.3 +/- 1.0, OLETF-S 0.7 +/- 0.1 pmol/ml, P < 0.01). Thus, OLETF hearts were more vulnerable to ischemia but sucrose feeding rendered their hearts resistant to ischemia. Less norepinephrine release may play a role in preventing postischemic functional deterioration in sucrose-fed diabetic hearts.     

Downregulation of the skeletal muscle pyruvate dehydrogenase complex in the Otsuka Long-Evans Tokushima Fatty rat both before and after the onset of diabetes mellitus

The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible oxidative decarboxylation of pyruvate in mitochondria. The PDC activity is regulated by a phosphorylation/dephosphorylation cycle catalyzed by specific kinases (PDK) and phosphatases (PDP). In this study, the regulatory mechanisms of PDC were examined in skeletal muscle of the spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rat before and after the onset of diabetes. The Long-Evans Tokushima Otsuka (LETO) rat was used as control. Plasma glucose and insulin concentrations were at normal levels in both groups at 8 weeks of age but were significantly higher in OLETF than in LETO rats at 25 weeks of age (1.2-fold for glucose and 15-fold for insulin), indicating development of diabetes in the former. Plasma free fatty acids were 1.6-fold concentrated and the skeletal muscle PDC activity state was significantly lower in OLETF than in LETO rats at both ages, suggesting suppression of pyruvate oxidation in OLETF rats even before the onset of diabetes. The PDK activity and the abundance of the PDK isoform 4 protein as well as mRNA were greater in OLETF rats at both ages. Conversely, the abundance of the PDP isoform 1 protein and mRNA was less in OLETF than in LETO rats at both ages. These results suggest that concomitant greater PDK4 and less PDP1 expression in skeletal muscle of OLETF rats before the onset of diabetes are responsible for the lowering of the PDC activity and may be related with the development of diabetes mellitus.     

Effect of leptin on insulin sensitivity in the Otsuka Long-Evans Tokushima Fatty rat

Leptin has been proposed to be a sensor of energy storage in adipose tissues, and is capable of mediating a feedback signal to the hypothalamus, which is involved in the regulation of energy homeostasis and body weight. In order to investigate the issue of whether resistance to the activity of leptin on insulin sensitivity is observed in young Otsuka Long-Evans Tokushima Fatty (OLETF) rats at 8 weeks of age, leptin (50 nmol/kg/h) was administered intravenously for 16 h to OLETF and Long-Evans Tokushima Otsuka (LETO) (lean controls) rats, followed by a measurement of insulin-stimulated glucose uptake in hindlimb muscles during hyperinsulinemic euglycemic clamp technique. In the case of LETO rats, the administration of leptin significantly decreased plasma insulin levels prior to the clamp test, but did not change plasma glucose levels. Furthermore, leptin led to an increase in insulin-stimulated glucose uptake in hindlimb muscles. However, in the case of OLETF rats, leptin administration changed neither plasma insulin levels nor insulin-stimulated glucose uptake. These data demonstrate that OLETF rats at 8 weeks of age have already become resistant to high concentration of peripheral leptin.     

Detailed Localization of Augmented Angiotensinogen mRNA and Protein in Proximal Tubule Segments of Diabetic Kidneys in Rats and Humans

In the intrarenal renin-angiotensin system, angiotensinogen levels are well known to be increased in diabetes, and these enhanced intrarenal angiotensinogen levels may initiate the development and accelerate the progression of diabetic nephropathy. However, the specific localization of the augmented angiotensinogen in proximal tubule segments in diabetes is still unknown. We investigated the detailed localization of angiotensinogen in 3 proximal tubule segments in the diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats and the control Long-Evans Tokushima Otsuka (LETO) rats. We also prepared OLETF rats treated with angiotensin II type 1 receptor blocker, olmesartan or with a combination of vasodilator agents. Moreover, biopsied samples of human kidney cortex were used to confirm the results of animal studies. We examined the co-localization of angiotensinogen with segment-specific markers by double staining using fluorescence in situ hybridization and/or immunofluorescence. Angiotensinogen mRNA expression was barely detectable in segment 1. In segment 3, the area of angiotensinogen mRNA expression was augmented in the OLETF rats compared with the LETO rats. Angiotensinogen protein expression areas in segments 1 and 3 were also increased in the OLETF rats compared with the LETO rats. Chronic treatment with olmesartan ameliorated these areas of augmented angiotensinogen expression. Biopsied human kidney samples showed similar results. These data suggest that the augmented angiotensinogen mRNA levels in segment 3 and angiotensinogen protein levels in segments 1 and 3 may contribute to the progression of diabetic nephropathy.     

Combined effect of ACE inhibitor and exercise training on insulin resistance in type 2 diabetic rats

The aim of this study was to investigate whether a combined treatment of ACE inhibitor and exercise training is more effective than either treatment alone in alleviating the insulin resistant states in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model of type 2 diabetes. OLETF rats (25 weeks old) were randomly divided into 5 groups; sedentary control, exercise-trained, temocapril (ACE inhibitor; 2 mg/kg/day)-treated, with and without exercise, and losartan (AT1 receptor antagonist; 1 mg/kg/day)-treated. Long-Evans Tokushima Otsuka rats were used as a non-diabetic control. Body weight, the amount of abdominal fat and blood pressure were higher for OLETF rats than for control rats. However, glucose infusion rate (GIR), an index of insulin resistance, was decreased greatly in OLETF rats. The fasting levels of blood glucose, insulin and lipids were also increased in the diabetic strain. In OLETF rats, both temocapril and losartan reversed hypertensive states significantly, whereas GIR and hyperlipidemia were improved when rats were treated with ACE inhibitors, but not with the AT1 receptor antagonist. Exercise training decreased body weight and the amount of abdominal fat, and also increased GIR in parallel with improved dislipidemia. The combination of the ACE inhibitor with exercise training also improved obesity, hyperinsulinemia, dislipidemia and fasting level of blood glucose, and this combination resulted in the greatest improvement of insulin resistance. These results suggest that the combination of ACE inhibitor and exercise training may be a beneficial treatment for mixed diabetic and hypertensive conditions.     

Independent ingestion and microstructure of feeding patterns in infant rats lacking CCK-1 receptors

Otsuka Long-Evans Tokushima fatty (OLETF) rats are a strain of Long-Evans Tokushima Otsuka (LETO) rats that do not express CCK-1 receptors, developing in adulthood, hyperphagia, obesity, and non-insulin-dependent diabetes mellitus (NIDDM). We examined weight gain and meal patterns during a 30-min independent ingestion test on postnatal days 2-4 and again on days 9-11 in OLETF and LETO rat pups. OLETF pups were significantly heavier compared with their LETO controls at both ages, and they consumed significantly more of the sweet milk diet. The difference in intake can be attributed to a significant increase in meal size and duration. Number of clusters and bursts of licking within a meal were greater in OLETF rat pups, with no difference between strains in burst and cluster size. Interlick interval (ILI) was not significantly different between OLETF and LETO pups. This measure decreased on days 9-11 compared with days 2-4 in both strains. Latency to start feeding was significantly shorter on days 2-4 in OLETF vs. LETO pups, but this difference disappeared at the second test at the older age. Two- to four-day-old OLETF pups consumed a larger volume of milk during the first minute of feeding, and their initial lick rate and decay of lick rate were significantly larger compared with their LETO controls. Lack of CCK-1 receptors, or other OLETF-related abnormalities, therefore, resulted in a satiation deficit, leading to increased meal size, hyperphagia, and increased weight gain as early as 2-4 postnatal days.     

Dietary troglitazone decreases oxidative stress in early stage type II diabetic rats

Oxidative stress is involved in the initiation and development of atherosclerosis in diabetes. We tested the hypothesis that oxidative stress is already increased in early stage type II diabetes, and that troglitazone may prevent the increase. Three groups of 20 week old rats were studied: untreated Otsuka Long-Evans Tokushima Fatty (OLETF) rats, as an animal model of type II diabetes, OLETF rats treated with troglitazone, and control Long-Evans Tokushima Otsuka (LETO) rats. Plasma lipid hydroperoxides (LOOH) concentration, as an indication of lipid peroxidation, and superoxide dismutase (SOD) activity in the thoracic aorta were measured. Plasma LOOH concentration was significantly higher in non-treated OLETF rats compared to LETO rats and treatment with troglitazone completely prevented this increase. SOD activity was significantly decreased in non-treated OLETF rats compared to LETO rats and troglitazone attenuated the diminution of it. These observations demonstrate oxidative stress is already increased in the early stage of type II diabetes and we confirmed troglitazone has the effect of an antioxidant in vivo.     

Altered basal and stimulated accumbens dopamine release in obese OLETF rats as a function of age and diabetic status

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat lacking the CCK-1 receptor is hyperphagic, prefers palatable and high-calorie meals, and gradually develops obesity and type 2 diabetes. To determine dopamine levels in this strain, we used in vivo quantitative (no net flux) microdialysis at three different ages representing nondiabetic (8 wk), prediabetic (18 wk), and diabetic (56 wk) stages in OLETF and age-matched lean Long-Evans Tokushima Otsuka (LETO) controls. Results showed significantly elevated basal dopamine levels in the caudomedial nucleus accumbens of OLETF rats compared with LETO at younger ages (8 wk: 20.10 +/- 5.61 nM vs. 15.85 +/- 5.63 nM; 18 wk: 7.37 +/- 3.71 nM vs. 4.75 +/- 1.25 nM, means +/- SD). In contrast, at 56 wk of age, a profound decline in extracellular dopamine concentrations was seen in both strains with a tendency for a greater effect in OLETF rats (1.78 +/- 0.40 nM vs. 2.39 +/- 0.42 nM). Further, extracellular fraction, an index for reuptake, was higher in 56-wk-old OLETF compared with LETO (0.648 +/- 0.049 vs. 0.526 +/- 0.057). Potassium-stimulated dopamine efflux revealed an increased capacity of vesicular pool in OLETF rats compared with LETO across all age groups with an accentuated strain difference at 56 wk. These findings demonstrate altered striatal dopamine functions (i.e., increased stimulated release and uptake) in obese OLETF rat. This could be due to the lack of functional CCK-1 receptors, or metabolic and hormonal factors associated with the development of obesity and insulin resistance, or both.     

Imbalance between endothelium-derived relaxing and contracting factors in mesenteric arteries from aged OLETF rats, a model of Type 2 diabetes

We investigated whether the balance between endothelium-derived relaxing factors (EDRFs) and endothelium-derived contracting factors (EDCFs) might be altered in mesenteric arteries from aged Otsuka Long-Evans Tokushima Fatty (OLETF) rats (a Type 2 diabetic model) [vs. age-matched control Long-Evans Tokushima Otsuka (LETO) rats]. ACh-induced relaxation was impaired in the OLETF group, and a tendency for the relaxation to reverse at high ACh concentrations was observed in both groups. This tendency was abolished by indomethacin. Nitric oxide- and/or endothelium-derived hypolarizing factor-mediated relaxation and the protein expressions of phospho-endothelial nitric oxide synthase (Ser1177) and extracellular superoxide dismutase were also reduced in OLETF. An ACh-induced contraction was observed at higher ACh concentrations in the presence of N(G)-nitro-L-arginine (L-NNA) but was greater in OLETF rats. This contraction in OLETF rats was reduced by cyclooxygenase (COX) inhibitors and by prostanoid-receptor antagonists. The ACh-induced productions of thromboxane A(2) and PGE(2) were greater in OLETF than LETO rats, as were the mesenteric artery COX-1 and COX-2 protein expressions. Moreover, tert-butyl hydroperoxide (t-BOOH) (membrane-permeant oxidant) induced a concentration-dependent contraction that was greater in OLETF rats. The t-BOOH-mediated contraction was increased both by L-NNA and by endothelium removal in LETO but not OLETF rats, suggesting that a negative modulatory role of the endothelium was lost in OLETF rats. These results suggest that an imbalance between EDRFs and EDCFs may be implicated in the endothelial dysfunction seen in aged OLETF mesenteric arteries, and may be attributable to increased oxidative stress.     

Obese OLETF rats exhibit increased operant performance for palatable sucrose solutions and differential sensitivity to D2 receptor antagonism

CCK-1-receptor-deficient Otsuka Long-Evans Tokushima fatty (OLETF) rats are hyperphagic and exhibit a greater preference for sucrose compared with lean controls [Long-Evans Tokushima Otsuka (LETO)]. To directly assess motivation to work for sucrose reward in this model of obesity and type 2 diabetes, we examined the operant performance of OLETF rats at nondiabetic and prediabetic stages (14 and 24 wk of age, respectively) on fixed-ratio (FR) and progressive-ratio (PR) schedules of reinforcement. To evaluate the involvement of dopamine systems, the effects of the D1 receptor antagonist SCH23390 (100 and 200 nmol/kg ip) and the D2 receptor antagonist raclopride (200 and 400 nmol/kg ip), were also tested on PR responding for sucrose. Compared with age-matched LETO rats, 14-wk-old OLETF rats emitted more licks on the "active" empty spout operant on the FR-10 schedule of reinforcement to obtain 0.01 M and 0.3 M sucrose and completed higher ratio requirements on the PR schedule to gain access to 0.3 M and 1.0 M sucrose. At 24 wk, this effect was limited to 1.0 M sucrose. Both antagonists were potent in reducing operant responding to 0.3 M sucrose in both strains at both ages, and there was no strain effect to SCH23390 at either age. OLETF rats, on the other hand, showed an increased sensitivity to the higher dose of raclopride, resulting in reduced responding to sucrose reinforcement at 24 wk. Taken together, these findings provide the first direct evidence for an increased motivation for sucrose reward in the OLETF rats and suggest altered D2 receptor regulation with the progression of obesity and prediabetes.     

Increased sensitivity of serotonin on the voltage-dependent K channels in mesenteric arterial smooth muscle cells of OLETF rats

This study examined the mechanisms of hypertension in diabetes. We investigated the effects of serotonin (5-HT) on voltage-dependent K⁺ (Kv) channel activity, vasoconstriction, 5-HT receptor expression levels, and the involvement of protein kinase C (PKC) in mesenteric arteries of Otsuka Long-Evans Tokushima fatty (OLETF) rats compared with Long-Evans Tokushima Otsuka (LETO) rats. Blood pressure, body weight, blood glucose level, and mesenteric arterial wall thickness were greater in OLETF rats. The 5-HT-induced vasoconstriction of mesenteric arteries was greater in OLETF rats than in LETO rats and inhibited by the 5-HT_2A inhibitor inhibitor, ketanserin. The Kv currents in mesenteric arterial smooth muscle cells (MASMCs), determined using a perforated patch clamp technique, was inhibited by 1 mM 4-AP (42.5 ± 4.1% vs. 63.5 ± 2.3% in LETO vs. OLETF rats at +40 mV), but was insensitive to 1 mM TEA and 100 nM iberiotoxin. The inhibition of Kv current by 1 μM 5-HT in MASMCs was greater in OLETF rats than in LETO rats (17.1 ± 2.2% vs. 33.2 ± 2.7% in LETO vs. OLETF rats at +40 mV), and the inhibition was prevented by treatment with the PKCα- and β- selective inhibitor, Gö6976. The expression level of 5-HT_2A, but not 5-HT_2B, receptor and the expression levels of total PKC, PKCβ, and PKCε, but not PKCα, were higher in the mesenteric arteries of OLETF rats compared with LETO rats. The enhanced expression of 5-HT_2A receptor together with PKCβ may promote mesenteric vasoconstriction and increase vascular resistance in OLETF rats.     

Conditioned preference for sweet stimuli in OLETF rat: effects of food deprivation

The Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an outbred strain of Long- Evans Tokushima Otsuka rat (LETO) that lacks CCK-1 receptor expression, is hyperphagic and develops obesity and type-2 diabetes. The present study sought to assess how OLETF rats alter intake, preference, and conditioned preference of palatable solutions after acute food deprivation. Our results show that after 24 h chow restriction, LETO rats increase both sucrose intake and two-bottle sucrose preference relative to their free-fed baseline, whereas OLETF rats do not increase sucrose intake (0.3 M or 1.0 M sucrose) or preference (1.0 M vs. 0.3 M sucrose) when they are food deprived. In contrast, OLETF rats exhibit a higher conditioned flavor preference when sucrose is used as unconditioned stimulus (US) relative to LETO rats, whether overnight food restricted (81% vs. 71% for OLETF and LETO rats, respectively) or free fed (82% vs. 54% for OLETF and LETO rats, respectively) during the test. When a noncaloric saccharin solution is used as US, OLETF rats show a higher preference for the saccharin-associated flavor relative to LETO rats when nondeprived (76% vs. 58% for OLETF and LETO rats, respectively); however, neither strain shows differential conditioned flavor preference for saccharin in the deprivation state during the test. These findings suggest that OLETF rats fail to integrate postabsorptive and orosensory effects of sucrose in a conditioning setting to influence intake. Thus, it appears that OLETF rats form preferences for sucrose based largely on orosensory and hedonic properties of the solution, rather than caloric value.     

Coronary capillary network remodeling and hypofibrinolysis in aged obese diabetic rats: Implications for increased myocardial vulnerability to ischemia

Despite the known abnormalities of cardiac function in patients with overt non-insulin dependent diabetes mellitus (NIDDM) the temporal changes of coronary capillary network remodeling leading to potential microcirculatory dysfunction have not been elucidated. To this end, left ventricular subendocardial capillary network of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, characterized by hypertension, obesity, hyperglycemia, hyperinsulinemia and mild NIDDM, and control Long-Evans Tokushima (LETO) rats were investigated. Total capillary density in OLETF was significantly higher than that in LETO at 20 weeks, suggesting compensatory improvement of O₂ transport at early stages of NIDDM. The increase in capillary density in OLETF was lost at 40 and 60 weeks due to the decreases of intermediate capillary portions and venular capillary portions. Although capillary domain area (area innervated by single capillary) in OLETF was lower than that in LETO at 20 weeks, the values were similar between OLETF and LETO at 40 and 60 weeks, suggesting that adaptive improvement in the capacity for O₂ transport with a high perfusion was lost in late stages of NIDDM. Activity of plasma plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of proteo(fibrino)lysis, in OLETF was higher than that in LETO at 40 and 60 weeks, suggesting that increase of PAI-1 may downregulate compensatory adaptive capillary network remodeling by inhibiting proteolysis and angiogenesis in the cardiac interstitium. Loss of adaptive myocardial microcirculation may therefore contribute to increased vulnerability in ischemic injury and to cardiac dysfunction in NIDDM.     

Neuronal nitric oxide synthase and cyclooxygenase-2 in diabetic nephropathy of type 2 diabetic OLETF rats.

Neuronal nitric oxide synthase (nNOS) and cyclooxygenase-2 (COX-2) regulate the tubuloglomerular feedback (TGF) and renin-angiotensin system (RAS) in the kidney. In type 1 diabetic rats, renal overproduction of these enzymes and their relationship to the pathogenesis of diabetic nephropathy has been demonstrated. In the present study, we histologically and immunohistochemically investigated the kidneys of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, as a model of type 2 diabetes, at 62 weeks of age (chronic phase of diabetes). The kidneys of OLETF rats showed typical diabetic nephropathy. Quantitative scores for glomerulosclerosis and interstitial fibrosis in OLETF rats were significantly higher than those of age-matched control Long-Evans Tokushima Otsuka (LETO) rats. nNOS- and COX-2-positive immunoreactions were observed in the distal tubules and collecting ducts. These reactions appeared to be more widely distributed in OLETF, and the number of nNOS-and COX-2-positive sites in the OLETF were significantly more than those in LETO rats. Expression of renin, angiotensin II, and inducible nitric oxide synthase (iNOS) were also examined immunohistochemically, and no differences between OLETF and LETO rats were observed in the distributions and the number of immunoreactive-sites. In conclusion, the overproduction of nNOS and COX-2 in the kidney of OLETF rats was confirmed, suggesting that the overproduction of nNOS and/or COX-2 does not affect the intrarenal RAS or iNOS production but does affect TGF.