Hiroharu Mifune

Exaggerated Blood Pressure Variability Superimposed on Hypertension Aggravates Cardiac Remodeling in Rats via Angiotensin II System-Mediated Chronic Inflammation

Hypertensive patients with large blood pressure variability (BPV) have aggravated end-organ damage. However, the pathogenesis remains unknown. We investigated whether exaggerated BPV aggravates hypertensive cardiac remodeling and function by activating inflammation and angiotensin II–mediated mechanisms. A model of exaggerated BPV superimposed on chronic hypertension was created by performing bilateral sinoaortic denervation (SAD) in spontaneously hypertensive rats (SHRs). SAD increased BPV to a similar extent in Wistar Kyoto rats and SHRs without significant changes in mean blood pressure. SAD aggravated left ventricular and myocyte hypertrophy and myocardial fibrosis to a greater extent and impaired left ventricular systolic function in SHRs. SAD induced monocyte chemoattractant protein-1, transforming growth factor-&bgr;, and angiotensinogen mRNA upregulations and macrophage infiltration of the heart in SHRs. The effects of SAD on cardiac remodeling and inflammation were much smaller in Wistar Kyoto rats compared with SHRs. Circulating levels of norepinephrine, the active form of renin, and inflammatory cytokines were not affected by SAD in Wistar Kyoto rats and SHRs. A subdepressor dose of candesartan abolished the SAD-induced left ventricular/myocyte hypertrophy, myocardial fibrosis, macrophage infiltration, and inductions of monocyte chemoattractant protein-1, transforming growth factor-&bgr;, and angiotensinogen and subsequently prevented systolic dysfunction in SHRs with SAD. These findings suggest that exaggerated BPV induces chronic myocardial inflammation and thereby aggravates cardiac remodeling and systolic function in hypertensive hearts. The cardiac angiotensin II system may play a role in the pathogenesis of cardiac remodeling and dysfunction induced by a combination of hypertension and exaggerated BPV.

Characterization and species differences in gastric ghrelin cells from mice, rats and hamsters

Ghrelin is a newly identified gastric peptide hormone that has various important functions, including growth‐hormone release and appetite stimulation. Ghrelin‐immunoreactive cells (ghrelin cells) are characterized by X‐type endocrine cells in the rat stomach. In the present study, we analysed ghrelin cells in fundi of stomach from ICR mice and Syrian hamsters immunohistochemically, immunoelectron microscopically and morphometrically, and compared the results with those from Wistar rats. Immunohistochemistry revealed that ghrelin cells were sparsely distributed in the proper gastric glands in all species. The number of ghrelin cells per unit area in hamsters was significantly lower than that in rats. Immunoelectron microscopy detected ghrelin immunolabelling in granules in the X‐type endocrine cells. However, the diameter of granules in the hamsters was significantly smaller than that in the mice and rats. Gastric ghrelin contents were determined by radioimmunoassay, and levels in the hamsters were significantly lower than those in mice and rats. The results from mice were identical to those from rats. In conclusion, gastric ghrelin cells in mice and hamsters are characterized by X‐type endocrine cells, as has been observed in rats. However, the data indicated that gastric ghrelin production was lower in hamster than in mouse or rat.

Natriuretic Peptides After Pulmonary Resection

BACKGROUND Little is known about alterations in the levels and influence of natriuretic peptide (NP) on cardiopulmonary function after pulmonary resection for lung cancer. This study was designed to investigate the patterns and activity of NP after pulmonary resection. METHODS We investigated changes in plasma A-type (atrial) NP and B-type (brain) NP (BNP) using radioimmunoassay, in lung cancer patients before and after lobectomy (n = 15) or pneumonectomy (n = 10). Patient characteristics, respiratory function, operative time, blood loss, intraoperative fluid administration, and intraoperative urine output in both groups were also compared. Pulmonary hemodynamic variables were monitored continuously. RESULTS Plasma concentrations of A-type NP and BNP did not differ between the two groups preoperatively. However, the group undergoing pneumonectomy exhibited higher concentrations of A-type NP and BNP than the group undergoing lobectomy on postoperative days 3 and 7. Alterations in A-type NP and BNP after pulmonary resection therefore differed according to the volume of lung tissue resected. Both mean pulmonary artery pressure and total pulmonary vascular resistance increased significantly in the pneumonectomy group. The total pulmonary vascular resistance on postoperative day 3 correlated with the plasma BNP concentration in the pneumonectomy group. CONCLUSIONS A-type NP and BNP effectively compensate for the right ventricular dysfunction noted after pulmonary resection, and this is more evident after pneumonectomy than after lobectomy. Changes in ventricular activity associated with changes in plasma BNP and total pulmonary vascular resistance are indicative of cardiopulmonary adjustments after pneumonectomy.

Regional distribution and the dynamics of n-decanoyl ghrelin, another acyl-form of ghrelin, upon fasting in rodents.

n-Decanoyl ghrelin (D-ghrelin), a member of ghrelin-derived peptides, is found in plasma and the stomach; however, there have so far been no studies describing its dynamics. A D-ghrelin-specific radioimmunoassay was established to examine the tissue distribution and the kinetics of D-ghrelin in mice. The effect of D-ghrelin on food intake was also examined and compared to n-octanoyl ghrelin (O-ghrelin). D-ghrelin was detected throughout the gastrointestinal tissue and plasma with highest level in the stomach. An immunofluorescent study revealed the co-localization of D- and O-ghrelin in the same stomach cells. Upon fasting, the levels of D-ghrelin in the stomach and plasma significantly increased, while that of O-ghrelin in the stomach declined. D-ghrelin increased the 2 h food consumption in mice as O-ghrelin does. These findings indicate that D-ghrelin is mainly produced in the stomach to work in concert with O-ghrelin. The different kinetics of D- and O-ghrelin in the stomach upon fasting implies the possibility of D-ghrelin-specific bioregulation.

Cardiac Musculature of the Cranial Vena cava in the Rat

The distribution and morphological features of cardiac musculature in rat cranial venae cavae were examined by light and transmission electron microscopy. Cardiac myocytes are encountered from the right atrium to the root of the subclavian vein. The musculature consists of several well-developed circular or spiral myocyte layers. The ultrastructure of myocytes in the cranial venae cavae exhibits a similar structure to that of atrial myocytes. Abundant myofibrils and mitochondria are detected within the cytoplasm of these myocytes, suggesting an active contraction of the musculature. Characteristic caveolae are accumulated near the sarcolemma of cardiac myocytes in the cranial venae cavae showing their high pinocytotic activity. Immunohistochemical analysis reveals the presence of an atrial natriuretic polypeptide-like substance in the cranial vena cava and the proximal portion of the subclavian vein. Ultrastructural studies also demonstrate the distribution of atrial granules within the musculature. This musculature in the vena cava may be considered part of the endocrine atrium.

Atrial natriuretic peptide (ANP): a study of ANP and its mRNA in cardiocytes, and of plasma ANP levels in non-obese diabetic mice.

SummaryAtrial natriuretic peptide (ANP) levels in cardiocytes and plasma were examined by using immunohistochemistry, electron microscopy, and radioimmunoassay in non-obese diabetic mice (NOD). Cardiocyte ANP mRNA expression was measured by the polymerase chain reaction method. ANP immunoreactivity in the auricular cardiocytes was more prominent in hyperglycemic mice (NOD-h) than in normoglycemic mice (NOD-n). Ultrastructural examination showed that auricular cardiocytes of the NOD-h group contained more cytoplasmic granules than cells of the NOD-n group. Ultrastructural morphometry indicated that the number of granules per auricular cardiocyte was significantly larger in the NOD-h group than in the NOD-n group. (P<0.01), whereas the granule diameter was significantly smaller in the NOD-h group (P<0.01). Radioimmunoassay showed that ANP levels in the NOD-h auricular cardiocytes were significantly higher than those in the NOD-n cardiocytes (P<0.01); the opposite was true in plasma. Cardiocyte ANP mRNA expression was lower in the NOD-h group than in the NOD-n group.

Beneficial effects of metformin on energy metabolism and visceral fat volume through a possible mechanism of fatty acid oxidation in human subjects and rats

Objective Metformin is known to have a beneficial effect on body weight and body composition, although the precise mechanism has not been elucidated yet. The aim of this study is to investigate the effects of metformin on energy metabolism and anthropometric factors in both human subjects and rats. Methods In human studies, metformin (1500mg/day) was administered to 23 healthy subjects and 18 patients with type 2 diabetes for 2 weeks. Metabolic parameters and energy metabolism were measured during a meal tolerance test in the morning before and after the treatment of metformin. In animal studies, 13 weeks old SD rats were fed 25–26 g of standard chow only during 12-hours dark phase with either treated by metformin (2.5mg/ml in drinking water) or not for 2 weeks, and metabolic parameters, anthropometric factors and energy metabolism together with expressions related to fat oxidation and adaptive thermogenesis were measured either in fasting or post-prandial state at 15 weeks old. Results Post-prandial plasma lactate concentration was significantly increased after the metformin treatment in both healthy subjects and diabetic patients. Although energy expenditure (EE) did not change, baseline respiratory quotient (RQ) was significantly decreased and post-prandial RQ was significantly increased vice versa following the metformin treatment in both groups. By the administration of metformin to SD rats for 2 weeks, plasma levels of lactate and pyruvate were significantly increased in both fasting and post-prandial states. RQ during a fasting state was significantly decreased in metformin-treated rats compared to controls with no effect on EE. Metformin treatment brought about a significant reduction of visceral fat mass compared to controls accompanied by an up-regulation of fat oxidation-related enzyme in the liver, UCP-1 in the brown adipose tissue and UCP-3 in the skeletal muscle. Conclusion From the results obtained, beneficial effects of metformin on visceral fat reduction has been demonstrated probably through a mechanism for a potential shift of fuel resource into fat oxidation and an upregulation of adaptive thermogenesis independent of an anorexigenic effect of this drug.

Voluntary exercise contributed to an amelioration of abnormal feeding behavior, locomotor activity and ghrelin production concomitantly with a weight reduction in high fat diet-induced obese rats.

In the present study, effects of voluntary exercise in an obese animal model were investigated in relation to the rhythm of daily activity and ghrelin production. Male Sprague-Dawley rats were fed either a high fat diet (HFD) or a chow diet (CD) from four to 16 weeks old. They were further subdivided into either an exercise group (HFD-Ex, CD-Ex) with a running wheel for three days of every other week or sedentary group (HFD-Se, CD-Se). At 16 weeks old, marked increases in body weight and visceral fat were observed in the HFD-Se group, together with disrupted rhythms of feeding and locomotor activity. The induction of voluntary exercise brought about an effective reduction of weight and fat, and ameliorated abnormal rhythms of activity and feeding in the HFD-Ex rats. Wheel counts as voluntary exercise was greater in HFD-Ex rats than those in CD-Ex rats. The HFD-obese had exhibited a deterioration of ghrelin production, which was restored by the induction of voluntary exercise. These findings demonstrated that abnormal rhythms of feeding and locomotor activity in HFD-obese rats were restored by infrequent voluntary exercise with a concomitant amelioration of the ghrelin production and weight reduction. Because ghrelin is related to food anticipatory activity, it is plausible that ghrelin participates in the circadian rhythm of daily activity including eating behavior. A beneficial effect of voluntary exercise has now been confirmed in terms of the amelioration of the daily rhythms in eating behavior and physical activity in an animal model of obesity.

Changes in Subcellular Distribution of n-Octanoyl or n-Decanoyl Ghrelin in Ghrelin-Producing Cells

Background: The enzyme ghrelin O-acyltransferase (GOAT) catalyzes the acylation of ghrelin. The molecular form of GOAT, together with its reaction in vitro, has been reported previously. However, the subcellular processes governing the acylation of ghrelin remain to be elucidated. Methods: Double immunoelectron microscopy was used to examine changes in the relative proportions of secretory granules containing n-octanoyl ghrelin (C8-ghrelin) or n-decanoyl ghrelin (C10-ghrelin) in ghrelin-producing cells of mouse stomachs. The dynamics of C8-type (possessing C8-ghrelin exclusively), C10-type (possessing C10-ghrelin only), and mixed-type secretory granules (possessing both C8- and C10-ghrelin) were investigated after fasting for 48 h or after 2 weeks feeding with chow containing glyceryl-tri-octanoate (C8-MCT) or glyceryl-tri-decanoate (C10-MCT). The dynamics of C8- or C10-ghrelin-immunoreactivity (ir-C8- or ir-C10-ghrelin) within the mixed-type granules were also investigated. Results: Immunoelectron microscopic analysis revealed the co-existence of C8- and C10-ghrelin within the same secretory granules (mixed-type) in ghrelin-producing cells. Compared to control mice fed standard chow, the ratio of C10-type secretory granules increased significantly after ingestion of C10-MCT, whereas that of C8-type granules declined significantly under the same treatment. After ingestion of C8-MCT, the proportion of C8-type secretory granules increased significantly. Within the mixed-type granules the ratio of ir-C10-ghrelin increased significantly and that of ir-C8-ghrelin decreased significantly upon fasting. Conclusion: These findings confirmed that C10-ghrelin, another acyl-form of active ghrelin, is stored within the same secretory granules as C8-ghrelin, and suggested that the types of medium-chain acyl-molecules surrounding and available to the ghrelin-GOAT system may affect the physiological processes of ghrelin acylation.

Ghrelin Acylation by Ingestion of Medium-Chain Fatty Acids

We found in a primary study that ingestion of medium-chain fatty acids (MCFAs) or medium-chain triacylglycerols (MCTs) increased the stomach contents of acyl ghrelin, and we further showed that the carbon-chain length of the acyl groups that modified the nascent ghrelin peptides corresponded to that of the ingested MCFAs or MCTs. These findings clearly demonstrated that the ingested MCFAs are directly used for the acyl-modification of ghrelin. Before the discovery of ghrelin-O-acyltransferase (GOAT), our in vivo study suggested that the putative GOAT preferred MCTs (composed of C6:0 to C10:0 FFAs) to either short- or long-chain triglycerides. In another study, we suggested that MCFAs or MCTs might represent a potential therapeutic modality for the clinical manipulation of energy metabolism through the modulation of ghrelin activity. After the discovery of GOAT, many studies have been done on the acylation of ghrelin using MCFAs, MCTs, or their derivatives; however, results and interpretations have been inconsistent, largely due to the differences in experimental conditions. This chapter describes detailed methods for the analysis of ghrelin acylation in vivo to facilitate future research in this field.