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This study aims to investigate the vasorelaxation effects of a Rosa centifolia petal extract (ROSE CRYSTA®-70: ROSE-70) on the isolated aorta and the protective effect of ROSE-70 on human umbilical vein endothelial cells (HUVECs) dysfunction. ROSE-70 inhibited phenylephrine (PE) -induced contraction in an endothelium-dependent and endothelium-independent manner; however, this relaxation was lower in the endothelium-denuded aorta. ROSE-70-induced relaxation was attenuated by L-N

Chlorogenic acid (CGA) is a polyphenol found in coffee and medicinal herbs such as Lonicera japonica. In this study, the effect of CGA-induced relaxation on carbachol (CCh)-induced contraction of mouse urinary bladder was investigated. CGA (30–300 μg/ml) inhibited CCh- or U46619-induced contraction in a concentration-dependent manner. SQ22536 (adenylyl cyclase inhibitor) recovered CGA-induced relaxation of CCh-induced contraction; however, ODQ (guanylyl cyclase inhibitor) did not have the same effect. In addition, 3-isobutyl-1-methylxanthine (IBMX) enhanced CGA-induced relaxation; however, forskolin or sodium nitroprusside did not have the same effect. Moreover, Ro 20–1724, a selective phosphodiesterase (PDE) 4 inhibitor, enhanced CGA-induced relaxation, but vardenafil, a selective PDE5 inhibitor, did not have the same effect. In the presence of CCh, CGA increased cyclic adenosine monophosphate (cAMP) level, whereas SQ22536 inhibited the increase of cAMP levels. Moreover, higher cAMP levels were obtained with CGA plus IBMX treatment than the total cAMP levels obtained with separate CGA and IBMX treatments. In conclusion, these results suggest that CGA inhibited CCh-induced contraction of mouse urinary bladder by partly increasing cAMP levels via adenylyl cyclase activation.

Recent studies have shown that phloridzin, an inhibitor of sodium-glucose cotransporter (SGLT), strongly decreases high K+-induced contraction in phasic muscle, such as tenia coli, but slightly affects tonic muscle, such as trachea . In this study, we examined the inhibitory mechanism of phloridzin on high K+-induced muscle contraction in rat ileum, a phasic muscle. Phloridzin inhibited the high K+-induced contraction in the ileum and the aorta, and the relaxing effect of phloridzin at 1 mM in the ileum was approximately five-fold more potent than that in the aorta. The expression of SGLT1 mRNA in the ileum was higher than that of the aorta. Phloridzin significantly inhibited NADH/NAD ratio and phosphocreatine (PCr) content in the ileum; however, application of pyruvate recovered the inhibition of contraction and PCr content, but had no effect on ratio of NADH/NAD. High K+ increased 2-(N (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-2-deoxyglucose (2-NBDG) uptake in ileal smooth muscle cells, and phloridzin inhibited the increase in a concentration-dependent manner. These results suggest that phloridzin inhibits high K+-induced contraction because of the inhibition of energy metabolism via the inhibition of SGLT1.

The effects of a glycogen phosphorylase inhibitor (GPI) and metformin (MT) on hepatic glucose fluxes (μmol · kg(-1) · min(-1)) in the presence of basal and 4-fold basal levels of plasma glucagon were investigated in 18-h fasted conscious dogs. Compared with the vehicle treatment, GPI infusion suppressed net hepatic glucose output (NHGO) completely (-3.8 ± 1.3 versus 9.9 ± 2.8) despite increased glucose 6-phosphate (G-6-P) neogenesis from gluconeogenic precursors (8.1 ± 1.1 versus 5.5 ± 1.1). MT infusion did not alter those parameters. In response to a 4-fold rise in plasma glucagon levels, in the vehicle group, plasma glucose levels were increased 2-fold, and NHGO was increased (43.9 ± 5.7 at 10 min and 22.7 ± 3.4 at steady state) without altering G-6-P neogenesis (3.7 ± 1.5 and 5.5 ± 0.5, respectively). In the GPI group, there was no increase in NHGO due to decreased glucose-6-phosphatase flux associated with reduced G-6-P concentration. A lower G-6-P concentration was the result of increased net glycogenesis without altering G-6-P neogenesis. In the MT group, the increment in NHGO (22.2 ± 4.4 at 10 min and 12.1 ± 3.6 at steady state) was approximately half of that of the vehicle group. The lesser NHGO was associated with reduced glucose-6-phosphatase flux but a rise in G-6-P concentration and only a small incorporation of plasma glucose into glycogen. In conclusion, the inhibition of glycogen phosphorylase a activity decreases basal and glucagon-induced NHGO via redirecting glucose 6-phosphate flux from glucose toward glycogen, and MT decreases glucagon-induced NHGO by inhibiting glucose-6-phosphatase flux and thereby reducing glycogen breakdown.

OBJECTIVE—We examined in 20-week-old Zucker diabetic fatty (ZDF) rats whether restoration of hepatic glucokinase (GK) expression would alter hepatic glucose flux and improve hyperglycemia. RESEARCH DESIGN AND METHODS—ZDF rats were treated at various doses with an adenovirus that directs the expression of rat liver GK (AdvCMV-GKL) dose dependently, and various metabolic parameters were compared with those of nondiabetic lean littermates (ZCL rats) before and during a hyperglycemic clamp. Viral infection per se did not affect hepatic GK activity, since expression of a catalytically inactive form of GK did not alter endogenous hepatic GK activity. RESULTS—ZDF rats compared with ZCL rats have lower hepatic GK activity (11.6 ± 1.9 vs. 32.5 ± 3.2 mU/mg protein), marked hyperglycemia (23.9 ± 1.2 vs. 7.4 ± 0.3 mmol/l), higher endogenous glucose production (80 ± 3 vs. 38 ± 3 μmol · kg−1 · min−1), increased glucose-6-phosphatase flux (150 ± 11 vs. 58 ± 8 μmol · kg−1 · min−1), and during a hyperglycemic clamp, a failure to suppress endogenous glucose production (80 ± 7 vs. −7 ± 4 μmol · kg−1 · min−1) and promote glucose incorporation into glycogen (15 ± 5 vs. 43 ± 3 μmol/g liver). Treatment of ZDF rats with different doses of AdvCMV-GKL, which restored hepatic GK activity to one to two times that of ZCL rats, normalized plasma glucose levels and endogenous glucose production. During a hyperglycemic clamp, glucose production was suppressed and glucose incorporation into glycogen was normal. CONCLUSIONS—Alteration of hepatic GK activity in ZDF rats has profound effects on plasma glucose and hepatic glucose flux.

Adiponectin is an adipokine that is specifically expressed in adipose tissues, directly sensitizes the body to insulin via specific receptors and its decreased plasma concentration is responsible for insulin resistance in obese humans. Diabetes is an important problem also in veterinary medicine, and feline diabetes is very similar to human type 2 diabetes, in which obesity is an important risk factor. In the present study, We obtained cDNA clones corresponding to feline adiponectin and adiponectin receptor 1 (AD-R1), whose nucleotide and deduced amino acid sequences were highly identical to those of other species, especially, the extra-cellular domain of feline AD-R1 was almost identical to that of human AD-R1. Adiponectin mRNA was exclusively detected in the adipose tissue, but AD-R1 was in all tissues tested in this study. Next, plasma samples were collected from 22 cats visiting veterinary practices. They were divided to 2 groups based on a five-point scale body condition score (BCS), such as normal group (BCS ranged from 2.5 through 3.5) and obese group (BCS ranged from 4.0 through 5.0). Plasma adiponectin in obese cats (7.2 +/- 1.5 microg/ml) was significantly lower than that of normal cats (18.0 +/- 3.2 microg/ml). These results suggest that adiponectin may be responsible for insulin function also in the cat, and it can be a target molecule for treatment of obesity and diabetes in cats.

This study examined the mechanism of vasorelaxation induced by dimethyl sulfoxide (DMSO) in endothelium-intact and -denuded rat aorta. DMSO (0.1-3%) inhibited phenylephrine (PE, 1 μmol/l)-induced contraction in a dose-dependent manner. However, this relaxation was lower in the absence of the endothelium. Increase in DMSO-induced relaxation in the presence of the endothelium was attenuated by preincubation in L-NG-nitroarginine methyl ester (L-NAME, 100 μmol/l) and by the removal of the endothelium. In the aorta with endothelium, DMSO (3%) and CCh (3 μmol/l) increased cGMP contents, significantly and L-NAME (100 μmol/l) inhibited the DMSO-induced increases of cGMP. In fura 2-loaded endothelium-denuded aorta, cumulative application of DMSO (1-3%) inhibited PE-induced muscle tension; however, this application did not affect the [Ca2+]i level. In PE-precontracted endothelium-denuded aorta, relaxation responses to fasudil were significantly less in the presence of DMSO compared to the control. These results suggest that DMSO causes relaxation by increasing the cGMP content in correlation with the release of NO from endothelial cells and by decreasing the Ca2+ sensitivity of contractile elements partly via inhibiting Rho-kinase in rat aorta.

When rearing chicks, Leach’s storm-petrels (Oceanodroma leucorhoa) commute between foraging areas and breeding colonies with heavy food loads. At this time they should maximize the size of energy-supplying organs in response to increased energy expenditure but minimize total body mass to decrease the energetic cost of flight. Nineteen storm-petrels were killed to examine the changes in body composition and the masses of energy-supplying organs in birds that were incubating and rearing chicks. Parents lost a mean of 7.95 g in body mass between the stages of incubation and chick-rearing mainly via a loss of skin including subcutaneous adipose tissue, and a small fraction of heart and digestive organs, which are considered energy-supplying organs. This mass loss actually enables them to decrease flight cost by 14.4%. The benefits of decreasing flight costs by reducing total body mass are greater than if the energy-supplying organs of birds are enlarged only.

It is known that in rodents and humans the beta 3-adrenergic receptor (beta 3-AR) is present primarily in adipocytes and plays a significant role in the adrenergic stimulation of lipolysis. We examined the expression of beta 3-AR mRNA in the dog and the lipomobilizing effects of beta 3-AR-selective agonists in vivo. Reverse transcription polymerase chain reaction of RNA extracted from dog adipose tissue produced a cDNA fragment, the nucleotide sequence of which was highly homologous to the corresponding regions of human (86.4%) and mouse (79.5%) beta 3-AR cDNA. The beta 3-AR mRNA was present at high levels in subcutaneous and visceral adipose tissues, but undetectable in other organs. When a selective beta 3-AR agonist, CL316,243, was infused intravenously into beagle dogs, the plasma level of free fatty acid increased in 30 min and persisted at higher levels for several hours. ICI D7114, another beta 3-AR agonist, also showed a similar lipomobilizing effect, but with lower potency. beta 3-AR agonist infusion also increased the plasma insulin level. These results suggested that functional beta 3-AR is present in adipose tissues of the dog and that it is effective for in vivo lipomobilization.

Retinol-binding protein 4 (RBP4) is a specific transporter of retinol and was recently identified as an adipokine potentially involved in type 2 diabetes in humans and rodents. However, the function and structure of feline RBP4 have not been reported. In this study, we describe the molecular cloning and expression analysis of feline RBP4. The complete feline RBP4 cDNA encodes a precursor protein comprising an 18 amino acid signal peptide and a 183 amino acid mature protein. Feline RBP4 was mapped to chromosome D2. Mature feline RBP4 is 83–94% homologous to the RBPs of humans, cows and rodents. RT-PCR analysis revealed feline RBP4 expression in liver and adipose tissues.

To elucidate the role of glycogen in the contraction of tracheal smooth muscle, we investigated the changes in the glycogen contents of the bovine trachea during contractions induced by high K(+) and hypoxia (achieved by bubbling N(2) instead of O(2)), either in a glucose-free condition or in the presence of iodoacetic acid (IAA), an inhibitor of glycolysis. Hyperosmotic addition of 65 mM KCl (H-65 K(+)) induced a sustained contraction. A glucose-free condition did not affect H-65 K(+)-induced contraction. However, hypoxia slightly inhibited the contraction, and glucose-free PSS with hypoxia or IAA remarkably inhibited the H-65 K(+)-induced contraction. H-65 K(+) induced a sustained increase in reduced pyridine nucleotide (PNred) fluorescence, representing glycolysis activity. Hypoxia alone slightly enhanced PNred fluorescence, and when combined with a glucose-free condition, it remarkably enhanced the H-65 K(+)-induced PNred fluorescence. IAA inhibited PNred fluorescence. In the presence of H-65 K(+), a glucose-free condition, hypoxia and the combination of glucose-free PSS and hypoxia decreased the glycogen contents. However, IAA had no effect on glycogen contents. Although hypoxia or glucose-free PSS did not affect PCr and ATP contents, the combination of hypoxia and glucose-free PSS or IAA induced a gradual decrease of PCr content. In conclusion, we suggest that endogenous glycogen was utilized to increase the activity of glycolysis for maintaining high K(+)-induced contraction of the bovine trachea in the glucose -free and/or hypoxic condition.

Leptin is a protein synthesized and secreted primarily by adipose tissue. The blood leptin concentration is known to reflect body fat content in rodents, humans and dogs, and thereby is useful for quantitative assessment of obesity. In the present study, we produced recombinant feline leptin in Escherichia coli transfected with feline leptin cDNA we cloned previously. The recombinant feline leptin with a molecular weight of 16 kDa induced phosphorylation of the signal transducers and activators of transcription 3 (STAT3) protein in the cells expressing rat leptin receptor. The anti-feline leptin antibody raised in rabbits reacted well to feline and human leptin and less to rodents' leptin in Western blot analysis. Sandwich enzyme-linked immunosorbent assay (ELISA) was developed, using rabbit anti-feline leptin antibody and recombinant feline leptin as a standard. In this ELISA system, cross-reactivity to human, rat and mouse leptin was 30.7%, 69.5% and 66.6%, respectively. The plasma leptin levels of 24 healthy cats were in a range from 0.3 to 29.7 ng/ml with the mean +/- SEM of 4.5 +/- 1.3 ng/ml, being positively proportional to body fat content. These results indicate that our ELISA system may be useful for assessment of obesity in cats.

Uncoupling proteins (UCPs) are members of the mitochondrial transporter family that dissipate the proton gradient as heat more than via ATP synthesis. In the present study, nucleotide and amino acid sequences of UCPs 1, 2 and 3 of a dog were determined, and their mRNA expression in various peripheral tissues was examined. The sequences were highly (76–97%) homologous to those of other species. Although lower homologies (60–74%) were found when compared among the three canine UCPs, their deduced amino acid sequences had some common domains, such as three mitochondrial carrier protein motifs, six transmembrane α-helix domains, and putative purine nucleotide binding domains. By Northern blot analyses, UCP1 mRNA was not detected in any tissues examined. UCP2 mRNA was expressed in most tissues, particularly abundantly in adipose tissue, spleen and lung. Two sizes of UCP3 mRNA were found exclusively in heart and skeletal muscle. These results suggest that canine UCPs have uncoupling activity, and are involved in the regulation of metabolic heat production and/or energy expenditure, as do those of other species.

Leptin, the product of the obese (ob) gene, is an adipocyte-derived hormone involved in regulating food intake and energy expenditure in humans and rodents. To determine the primary structure of feline leptin, we cloned the feline leptin cDNA using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of complementary DNA (cDNA) ends (RACE) methods. The full-length feline leptin cDNA was 2935 bp with a 501 bp open reading frame encoding the precursor peptide of 167 amino acids including 21 residues of signal peptide. The sequence of a 146-amino acid mature leptin was 81.5-91.8% homologous to those of other species. RT-PCR analysis revealed that the leptin mRNA was expressed in adipose tissues and not detected in liver, heart, kidney, lung, pancreas, brain and skeletal muscle. These data show that feline leptin is highly homologous to leptins of other species, and expressed in adipose tissues in cats.

Leptin is the ob gene product secreted from adipocytes in mammals, and thereby its plasma level reflects body fat content. To establish an assay method for leptin in the dog, rabbit anti-canine leptin antibody was obtained using canine recombinant leptin as an antigen. This antibody reacted to canine leptin much stronger than mouse, rat and human leptins. Sandwich enzyme-linked immunosorbent assay (ELISA) using this antibody was developed. The serum leptin levels of 13 healthy dogs were in a range from 1.4 to 5.6 ng/ml with the mean +/- SEM of 3.0 +/- 0.3 ng/ml.

The aim of this study was to evaluate the effectiveness of beta 3-adrenergic agonists for the treatment and prevention of obesity in the dog. When a selective beta 3-adrenergic agonist, CL316,243 (0.1 mg/kg), was given orally to adult beagles every day for 5-7 weeks, body weight and girth were decreased compared with control placebo-treated dogs. Gross anatomical examinations revealed no noticeable abnormalities in CL316,243-treated dogs, except an apparent decrease in abdominal fat. Immunohistochemical examination of perirenal adipose tissue showed a remarkable increase in brown adipocytes expressing a thermogenic protein, uncoupling protein (UCP). The increased expression of UCP and its mRNA in CL316,243-treated dogs was also confirmed by Western blot and reverse transcription polymerase chain reaction analyses. It was concluded that treatment with a beta 3-adrenergic agonist stimulates UCP expression, which may lead to an increase in energy expenditure, and thereby is useful for the treatment and prevention of obesity in the dog.