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.     

Hyperphagia and obesity in OLETF rats lacking CCK-1 receptors

The brain-gut peptide cholecystokinin (CCK) inhibits food intake following peripheral or site directed central administration. Peripheral exogenous CCK inhibits food intake by reducing the size and duration of a meal. Antagonist studies have demonstrated that the actions of the exogenous peptide mimic those of endogenous CCK. Antagonist administration results in increased meal size and meal duration. The feeding inhibitory actions of CCK are mediated through interactions with CCK-1 receptors. The recent identification of the Otsuka-Long-Evans-Tokushima Fatty (OLETF) rat as a spontaneous CCK-1 receptor knockout model has allowed a more comprehensive evaluation of the feeding actions of CCK. OLETF rats become obese and develop non-insulin dependent diabetes mellitus (NIDDM). Consistent with the absence of CCK-1 receptors, OLETF rats do not respond to exogenous CCK. OLETF rats are hyperphagic and their increased food intake is characterized by a large increase in meal size with a decrease in meal frequency that is not sufficient to compensate for the meal size increase. Deficits in meal size control are evident in OLETF rats as young as 2 days of age. OLETF obesity is secondary to the increased food intake. Pair feeding to amounts consumed by intact control rats normalizes body weight, body fat and elevated insulin and glucose levels. Hypothalamic arcuate nucleus peptide mRNA expression in OLETF rats is appropriate to their obesity and is normalized by pair feeding. In contrast, pair fed and young pre-obese OLETF rats have greatly elevated dorsomedial hypothalamic (DMH) neuropeptide Y (NPY) mRNA expression. Elevated DMH NPY in OLETF rats appears to be a consequence of the absence of CCK-1 receptors. In intact rats NPY and CCK-1 receptors colocalize to neurons within the compact subregion of the DMH and local CCK administration reduces food intake and decreases DMH NPY mRNA expression. We have proposed that the absence of DMH CCK-1 receptors significantly contributes to the OLETF's inability to compensate for their meal size control deficit leading to their overall hyperphagia. Access to a running wheel and the resulting exercise normalizes food intake and body weight in OLETF rats. When given access to running wheels for 6 weeks shortly after weaning, OLETF rats do not gain weight to the same degree as sedentary OLETF rats and do not develop NIDDM. Exercise also prevents elevated levels of DMH NPY mRNA expression, suggesting that exercise exerts an alternative, non-CCK mediated, control on DMH NPY. The OLETF rat is a valuable model for characterizing actions of CCK in energy balance and has provided novel insights into interactions between exercise and food intake.     

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.     

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.     

Attenuated satiation response to intestinal nutrients in rats that do not express CCK-A receptors.

Pharmacological experiments suggest that satiation associated with intestinal infusion of several nutrients is mediated by CCK-A receptors. Otsuka Long-Evans Tokushima Fatty, (OLETF), rats do not express CCK-A receptors and are insensitive to the satiation-producing effects of exogenous CCK. To further evaluate the role of CCK-A receptors in satiation by intestinal nutrient infusion, we examined intake of solid (pelleted rat chow) or liquid (12.5% glucose) food intake, following intestinal infusions of fats (oleic acid or fat emulsion), sugars (maltotriose or glucose), or peptone in OLETF rats and Long Evans Tokushima Otsuka control rats (LETO). Intestinal infusion of glucose or maltotriose reduced solid food intake more in LETO than in OLETF rats from 30 min through 4 h post infusion. Reduction of solid food intake by intestinal infusions of fat or peptone did not differ between OLETF and LETO rats during the first 30 min post infusion, but reduction of intake by these infusates was attenuated in OLETF rats over the ensuing 4h post infusion. Intestinal infusion of glucose, oleate, fat emulsion and peptone reduced 30-min intake of 12.5% glucose more in LETO than OLETF rats. Furthermore, pretreatment with the CCK-A receptor antagonist, devazepide, attenuated intestinal nutrient-induced reduction of food intake only in LETO, but not OLETF rats. Our results confirm pharmacological results, indicating that CCK-A receptors participate in satiation by nutrients that elevate plasma CCK concentrations, as well as by nutrients that do not stimulate secretion of endocrine CCK. In addition, our results indicate: 1) that OLETF rats have deficits in the satiation response to a variety of intestinal nutrient infusions; 2) that the temporal pattern for CCK-A receptor participation in satiation by intestinal nutrients is different during ingestion of liquid and solid foods and 3) that intestinal nutrients provide some satiation signals that are CCK-A receptor mediated and some that are not.     

Postprandial neuronal activation in the nucleus of the solitary tract is partly mediated by CCK-A receptors

CCK-A receptors and neurons of the nucleus of the solitary tract (NTS) are involved in the regulation of food intake, and in rats, there is evidence for involvement of an intestinal vagal afferent pathway. Studies investigating the role of CCK-A receptors in activation of NTS neurons using highly selective CCK-A receptor agonists and antagonists have yielded conflicting data. In the present study, we investigated CCK-induced and postprandial activation of NTS neurons, together with food intake studies, in CCK-A receptor-deficient Otsuka Long-Evans Tokushima fatty (OLETF) rats. Activated NTS neurons were detected using immunohistological staining for c-Fos protein. Exogenous CCK increased the number of c-Fos protein-positive cells in the NTS of Sprague-Dawley and CCK-A receptor-intact Long-Evans Tokushima Otsuka (LETO) rats but had no effect in CCK-A receptor-deficient OLETF rats. Food intake-induced c-Fos protein expression in NTS neurons was significantly reduced in CCK-A receptor-deficient OLETF rats compared with Sprague-Dawley or LETO rats. Postprandial c-Fos protein expression in the NTS was also significantly decreased after pretreatment with the CCK-A receptor antagonist MK329 after both short- and long-term fasting periods. Exogenous CCK decreased cumulative food intake in Sprague-Dawley and LETO rats but not in OLETF rats. These data demonstrate that CCK-A receptors are involved in the CCK- and food-induced c-Fos protein expression in the NTS. Taken together with the results of the food intake studies, this suggests that activation of CCK-A receptors is involved in the postprandial activation of NTS neurons and in the regulation of food intake.     

Role of dorsomedial hypothalamic neuropeptide Y in energy homeostasis

Hypothalamic neuropeptide Y (NPY) is primarily expressed in the arcuate nucleus (Arc) and the dorsomedial hypothalamus (DMH). Although Arc NPY gene expression is responsive to circulating leptin, the regulation of DMH NPY expression is leptin-independent. DMH NPY expression is increased in response to chronic food restriction, but not acute food deprivation. DMH NPY expression is elevated in pair-fed OLETF rats lacking cholecystokinin (CCK)-1 receptors. A role for CCK in controlling DMH NPY expression is demonstrated by the down-regulation of DMH NPY by parenchymal DMH CCK administration in intact rats. Moreover, access to running wheels normalizes body weight and prevents altered DMH NPY expression of OLETF rats. Together, these data suggest that DMH NPY plays an important role in feeding and body weight control.     

Altered taste sensitivity in obese, prediabetic OLETF rats lacking CCK-1 receptors

Otsuka Long-Evans Tokushima fatty (OLETF) rats lack the CCK-1 receptor, are hyperphagic, progressively become obese, and develop type-2 diabetes. We recently demonstrated an increased preference for both real and sham feeding of sucrose in this strain, suggesting altered orosensory sensitivity. To investigate taste functions, we used an automated gustometer with 10-s access to different concentrations of various sapid stimuli. Tests were repeated at 10 and 18 wk of age to assess the early and advanced stages of prediabetes, respectively. Compared with age-matched, nonmutant controls, the OLETF rats showed higher avidity for sucrose at both ages. This difference increased as a function of age and tastant concentration. An exaggerated response also occurred for saccharin, alanine, and fructose, but not for Polycose. Similarly, OLETF rats consumed monosodium-glutamate more at the lower concentrations compared with controls, an effect that age also accentuated. In contrast, there was no statistical strain or age differences in responses to NaCl, MgCl2, citric acid, quinine-HCl, and the trigeminal stimulus capsaicin. These findings demonstrate that compared with controls, OLETF rats differ in their gustatory functions with an overall augmented sensitivity for sweet that progresses during prediabetes. This effect explains their overconsumption of sweet solutions and may contribute to the overall hyperphagia and obesity in this strain.     

Maternal attenuation of hypothalamic paraventricular nucleus norepinephrine switches avoidance learning to preference learning in preweanling rat pups

Infant rats learn to prefer stimuli paired with pain, presumably due to the importance of learning to prefer the caregiver to receive protection and food. With maturity, a more 'adult-like' learning system emerges that includes the amygdala and avoidance/fear learning. The attachment and 'adult-like' systems appear to co-exist in older pups with maternal presence engaging the attachment system by lowering corticosterone (CORT). Specifically, odor-shock conditioning (11 odor-0.5 mA shock trials) in 12-day-old pups results in an odor aversion, although an odor preference is learned if the mother is present during conditioning. Here, we propose a mechanism to explain pups ability to 'switch' between the dual learning systems by exploring the effect of maternal presence on hypothalamic paraventricular nucleus (PVN) neural activity, norepinephrine (NE) levels and learning. Maternal presence attenuates both PVN neural activity and PVN NE levels during odor-shock conditioning. Intra-PVN NE receptor antagonist infusion blocked the odor aversion learning with maternal absence, while intra-PVN NE receptor agonist infusion permitted odor aversion learning with maternal presence. These data suggest maternal control over pup learning acts through attenuation of PVN NE to reduce the CORT required for pup odor aversion learning. Moreover, these data also represent pups' continued maternal dependence for nursing, while enabling aversion learning outside the nest to prepare for pups future independent living.     

Alcohol-induced increase in BMP levels promotes fatty liver disease in male prediabetic stage Otsuka Long-Evans Tokushima Fatty rats

Alcohol consumption exacerbates liver abnormalities in animal models, but whether it increases the severity of liver disease in early diabetic or prediabetic rats is unclear. To investigate the molecular mechanisms underlying alcohol-induced liver steatosis or hepatitis, we used a prediabetic animal model. Otsuka Long-Evans Tokushima Fatty (OLETF) and Long-Evans Tokushima Fatty (LETO) rats were pair-fed with an ethanol-containing liquid diet for 6 weeks. Compared with controls, OLETF and LETO rats displayed more pronounced liver steatosis and higher plasma levels of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamate pyruvate transaminase (SPGT), indicating liver injury. Ethanol-fed LETO (Pd-L-E) rats showed mild liver steatosis and no inflammation compared with ethanol-fed OLETF (Pd-O-E) rats. Although precursor and active SREBP-1 levels in the liver of ethanol-fed OLETF rats significantly increased compared with control diet-fed OLETF rats (Pd-O-C), those of Pd-L-E rats did not. Bone morphogenetic protein (BMP) and TGF-β1 balance in Pd-O-E rats was significantly altered because BMP signaling was upregulated by inducing BMP2, BMP4, BMP7, BMP9, Smad1, and Smad4, whereas TGF-β1, Smad3, and Erk were downregulated. Activation of TGF-β/Smad signaling inhibited BMP2 and BMP9 expression and increased epithelial-mesenchymal transition (EMT) marker levels (Hepcidin, Snail, and Twist) in the liver of LETO rats. Livers of ethanol-fed OLETF rats showed increased levels of vimentin, FSP-1, α-SMA, MMP1, MMP13, and collagen III compared with rats of other groups, whereas EMT marker levels did not change. Thus, BMP exerted anti- and/or pro-fibrotic effects in ethanol-fed rats. Therefore, BMP and TGF-β, two key members of the TGF-β superfamily, play important but diverse roles in liver steatosis in young LETO and OLETF rats.     

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.     

Neonatal overnutrition programming impairs cholecystokinin effects in adultmale rats

Overfeeding and rapid weight gain during early life are risk factors for the development of obesity in adulthood. This metabolic malprogramming may be mediated by endocrine disturbances during critical periods of development. Cholecystokinin (CCK) acts on the central nervous system by elevating thermogenesis and the activity of anorectic neurons, modulating overall energy balance. Therefore, we tested the hypothesis that postnatal overfeeding impaired CCK effects. Pups were raised in either a litter of three (neonatal overnutrition/small litter group) or 12 (controls/normal litter group) pups per dam to study the effects of postnatal overfeeding on the central and peripheral CCK systems in adulthood. Rats raised in small litters became overweight during lactation and remained overweight as adults, with increased adiposity and plasma levels of lipids, glucose, insulin, and leptin. Neonatally over-nourished rats showed attenuation of gastric emptying and anorexigenic response to CCK, suggesting that offspring from the SL group may present CCK resistance as adult male rats. Consistent with this idea, overweight rats displayed impaired central response in c-Fos immunoreactivity on the nucleus tractus solitarius, area postrema, paraventricular nucleus, central amygdala, arcuate nucleus, and dorsomedial hypothalamus in response to peripheral CCK at adulthood. The small litter group of adult male rats also exhibited reduced norepinephrine- and CCK-stimulated thermogenesis. Unresponsiveness to the effects of CCK may contribute to overweight and metabolic dysfunctions observed in postnatally over-nourished adult rats. Thus, the involvement of an impaired CCK system, among other neurohormonal failures, may contribute to the development of obesity.     

Role of ANG II in coronary capillary angiogenesis at the insulin-resistant stage of a NIDDM rat model

With the use of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of human non-insulin-dependent diabetes mellitus (NIDDM), we assessed whether ANG II is involved in coronary capillary angiogenesis at the insulin-resistant stage of NIDDM (20 wk of age). In OLETF rats, ANG II labeling and angiotensin type 1 (AT(1)) receptor expression in coronary vessels were increased more than in nondiabetic controls. A marked increase in vascular expression of vascular endothelial growth factor (VEGF) at both mRNA and protein levels was found in OLETF rats. The increased expression level of VEGF was associated with accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) activated by increased advanced glycation end products (AGEs). Morphometric analysis showed a significantly increased total coronary capillary density, which was a result of arterialization of the venular capillary portion in OLETF rats. Treatment of OLETF rats with candesartan, an AT(1) receptor blocker, inhibited vascular expressions of VEGF, HIF-1alpha, and AGEs, and ameliorated the morphometric changes. These results suggest a key role of ANG II in the pathogenesis of the coronary capillary remodeling in this NIDDM model.     

Trypsin inhibitor and maternal reunion increase plasma cholecystokinin in neonatal rats.

Intragastric soybean trypsin inhibitor increased plasma CCK bioactivity by 87% in nondeprived, 9-12-day-old rat pups. Reunion with the dam for 1 h after overnight maternal deprivation also increased plasma CCK significantly. These results demonstrate that CCK can be released from the small intestine of rats as early as postnatal day 9.     

Calorie restriction improves whole-body glucose disposal and insulin resistance in association with the increased adipocyte-specific GLUT4 expression in Otsuka Long-Evans Tokushima fatty rats

Calorie restriction (CR) has been shown to improve peripheral insulin resistance and type 2 diabetes in animal models. However, the exact mechanism of CR on GLUT4 expression and translocation in insulin-sensitive tissues has not been well elucidated. In the present study, we examine the effect of CR on the expression of glucose transporter 4 (GLUT4), GLUT4 translocation, and glucose transport activity in adipose tissue from Otsuka Long-Evans Tokushima Fatty (OLETF) rat and control (LETO) rats. CR (70% of satiated group) ameliorated hyperglycemia and improved impaired glucose tolerance (IGT) in OLETF rats. In skeletal muscle, the expression levels of GLUT4 and GLUT1 were not significantly different between LETO and OLETF rats, and were not affected by CR. By contrast, the expression level of GLUT4 was markedly decreased in the adipose tissue of OLETF rats, but was dramatically increased by CR. The GLUT4 recruitment stimulated by insulin was also improved in OLETF rat adipocytes by CR. The insulin-stimulated 2-deoxyglucose (2DG) uptake was significantly increased in adipocytes from the CR OLETF rats, as compared with the satiated OLETF rats. Taken together, these results suggest that CR improves whole body glucose disposal and insulin resistance in OLETF rats, and that these effects may associate with the increased adipocyte-specific GLUT4 expression.     

Examining the role of cholecystokinin in appetitive learning in the infant rat.

The role of Cholecystokinin (CCK), a gut hormone and neuropeptide, in early learning was examined. Pairing a novel odor (presented away from the nest) with exogenously administered CCK (0.25 & 0.5 microg/kg IP) has been shown to produce a conditioned-odor preference in infant rats (Weller, A.; Blass, E.M. Behav. Neurosci. 104:199-206; 1990). This suggests that CCK can act as a positive unconditioned stimulus (UCS). In the present study the possibility that CCK mediates learning was examined in 12-day-old rats, using rewards that represent aspects of the dam and the nest. In Experiments 1 and 2, pups received the selective CCK1 receptor antagonist devazepide (600 microg/kg), the selective CCK2 receptor antagonist L365,260 (600 microg/kg), or vehicle. In a series of training trials, choosing a particular floor texture was rewarded by 20 sec. on a rug texture (experiment 1) or with maternal (feces) odor (experiment 2). In experiment 3, after administering devazepide (0, 600, or 1000 microg/kg) a novel odor was paired once with reunion of the pup with its dam. The dependent measure in all studies was the pup's relative preference toward the (tactile or olfactory) conditioned stimulus (CS), determined in preference tests. Conditioned preferences were evident in all experiments. The CCK receptor antagonists did not increase conditioned preference levels. L365, 260 (experiment 2) and devazepide (experiment 1) clearly blocked the appearance of the conditioned effect in one of the experiments. In addition, devazepide treatment eliminated the conditioned effect in the two other experiments, by increasing preference levels in the control groups. In summary, the results suggest that endogenous CCK mediates some aspects of the infant's acquisition of new associations. The role of the two receptor-subtypes appears to be different, depending on the context and the nature of the rewarding stimulus.     

Absence of the cholecystokinin-A receptor deteriorates homeostasis of body temperature in response to changes in ambient temperature

The circadian rhythm of the body core temperature (T(c)) and the effects of changes in ambient temperatures on the homeostasis of T(c) in Otsuka Long Evans Tokushima Fatty (OLETF) rats, which are naturally occurring cholecystokinin (CCK)-A receptor (CCK-AR) gene knockout (-/-) rats, were examined. In addition, the peripheral responses to warming or cooling of the preoptic and anterior hypothalamic region (PO/AH) were determined. The circadian rhythm of T(c) in OLETF rats was similar to that in Long-Evans Tokushima (LETO) rats; this rhythm was characterized by a higher T(c) during the dark period and a lower T(c) during the light period. When the ambient temperature was changed within the limits of 0 degrees C to 30 degrees C, the changes in T(c) of LETO rats were associated with the changes in ambient temperature, whereas those in OLETF rats were dissociated from the temperature changes. The OLETF rats showed a large hysteresis. The peripheral responses to warming or cooling of PO/AH, including shivering of the neck muscle and changes in skin temperature of the tail and footpad, were similar in OLETF and LETO rats. To confirm the role of CCK-AR in the regulation of body temperature, the values of T(c) in the CCK-AR(-/-) mice were compared with those in CCK-B receptor (CCK-BR) (-/-), CCK-AR(-/-)BR(-/-), and wild-type mice. In the mice, the circadian rhythms of T(c) were the same, regardless of the genotype. Mice without CCK-AR showed larger hysteresis than mice with CCK-AR. From these results, we conclude that the lack of CCK-AR causes homeostasis of T(c) in rats and mice to deteriorate.     

Effect of AOB, a fermented-grain food supplement, on oxidative stress in type 2 diabetic rats

Reactive oxygen species (ROS) play an important role in the pathogenesis of diabetic complications. Antioxidant Biofactor (AOB) is a mixture of commercially available fermented grain foods and has strong antioxidant activity. This study investigated the effect of AOB supplementation of standard rat food on markers of oxidative stress and inflammation in Otsuka Long-Evans Tokushima Fatty (OLETF) rats with type 2 diabetes. Blood glucose, hemoglobin A1c, plasma free fatty acid, triacylglycerol and plasminogen activator inhibitor-1 (PAI-1) were significantly higher in OLETF rats than in non-diabetic control Long-Evans Tokushima Otsuka (LETO) rats at 29 weeks. AOB (6.5% of diet) was given to rats during 29-33 weeks of diabetic phase in OLETF rats. OLETF rats with AOB supplementation showed decreased blood glucose, hemoglobin A1c, triacyglycerol, low density lipoprotein, cholesterol and PAI-1. Mitochondrial ROS production was significantly increased in heart, aorta, liver and renal artery of OLETF rats. Uncoupling protein 2 (UCP2) is known to regulate ROS production. We found aortic UCP2 protein expression increased in OLETF rats, and AOB returned UCP2 expression to normal. Aortic endothelial NO synthase (eNOS) was also increased in OLETF rats more than in LETO rats at 33 weeks. In contrast, phosphorylated vasodilator-stimulated phosphoprotein, an index of the NO-cGMP pathway, was significantly diminished. AOB increased eNOS proteins in LETO and OLETF rats. In conclusion, AOB significantly improved the NO-cGMP pathway via normalizing ROS generation in OLETF rats. The data suggest that dietary supplementation with AOB contributes to nutritional strategies for the prevention and treatment of type 2 diabetes mellitus.     

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.