Tissue-specific expression and circulating concentrations of nesfatin-1 in domestic animals.

Nesfatin-1 is a naturally occurring 82-amino acid protein encoded in the precursor nucleobindin-2 (NUCB2) and has been implicated in multiple physiological functions, including food intake and blood glucose regulation. This study aimed to characterize nesfatin-1 in domestic species, especially cats (Felis catus), dogs (Canis lupus familiaris), and pigs (Sus scrofa). Our in silico analysis demonstrated that the NUCB2/nesfatin-1 amino acid sequence, especially the bioactive core region of the peptide, is very highly conserved (more than 90% identity) in domestic animals. Expression of mRNAs encoding NUCB2/nesfatin-1 was detected in the cat, dog, and pig stomach and pancreas. Immunohistochemistry revealed the presence of nesfatin-1 in the gastric mucosa of the stomach of dogs, cats, and pigs, and in the pancreatic islet ß-cells of dogs and pigs. No nesfatin-1 immunoreactivity was found in the cat pancreas. Nesfatin-1 was detected in the serum of dog, cat, pig, bison, cow, horse, sheep, and chicken. Circulating nesfatin-1 in male and female dogs remained unchanged at 60 min after glucose administration, suggesting a lack of meal responsiveness in nesfatin-1 secretion in this species. The presence of nesfatin-1 in the gastric and endocrine pancreatic tissues suggests possible roles for this peptide in the metabolism of domestic animals. Future research should focus on elucidating the species-specific functions and mechanisms of action of nesfatin-1 in health and disease of domestic animals.

Effect of an extruded pea or rice diet on postprandial insulin and cardiovascular responses in dogs.

Peas are increasing in popularity as a source of carbohydrate, protein and fibre in extruded canine diets. The aim of this study was to test the health effects of two canine diets with identical macronutrient profiles, but containing either yellow field peas or white rice as the carbohydrate source on metabolism, cardiovascular outcomes and adiposity. First, the acute glycemic, insulinemic and cardiovascular responses to the pea- or rice-based diets were determined in normal weight beagles (n = 7 dogs). The glycemic index did not differ between the pea diet (56 ± 12) and rice diet (63 ± 9). Next, obese beagles (n = 9) were fed the yellow field pea diet or white rice diet ad libitum for 12 weeks in a crossover study. Adiposity (measured using computed tomography), metabolic (oral glucose tolerance test, plasma leptin, adiponectin, C-reactive protein) and cardiovascular assessments (echocardiography and blood pressure) were performed before and after each crossover study period. After 12 weeks on each diet, peak insulin (p = 0.05) and area under the curve (AUC) for insulin after a 10 g oral glucose tolerance test (p = 0.05) were lower with the pea than the rice diet. Diet did not show a significant effect on body weight, fat distribution, cardiovascular variables, adiponectin or leptin. In conclusion, a diet containing yellow field peas reduced the postprandial insulin response after glucose challenge in dogs despite continued obesity, indicating improved metabolic health.

Effect of beta-naphthoflavone and dimethylbenz[a]anthracene on apoptosis and HSP70 expression in juvenile channel catfish (Ictalurus punctatus) ovary.

Complex environmental mixtures such as pulp mill effluents and crude oil have been shown to increase ovarian cell apoptosis and affect heat shock protein (HSP) expression in fish. We hypothesize that polyaromatic hydrocarbons (PAH) mediate these effects. To test this hypothesis, we exposed juvenile channel catfish (Ictalurus punctatus) acutely to the aryl hydrocarbon receptor (AhR) agonists, beta-naphthoflavone (BNF; 75 mg/kg) or the model PAH, dimethylbenz[a]anthracene (DMBA; 50 mg/kg) via intraperitoneal injection. Apoptotic DNA fragmentation and HSP70 expression were determined in ovary and liver. Hepatic cytochrome P450 1A (CYP1A) was significantly induced, confirming that BNF and DMBA had distributed to internal organs and stimulated AhR. At 96 h post-injection, BNF and DMBA significantly increased apoptosis and decreased HSP70 expression in juvenile catfish ovaries. Although primary oocytes underwent the greatest rates of apoptosis compared to early or late vitellogenic follicles in all treatment groups, the cell type undergoing increased rates of apoptosis after BNF or DMBA exposure was not clear using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyUTP nick end labeling (TUNEL). There was a significant negative relationship between expression of HSP70 and apoptosis in juvenile channel catfish ovaries. This differed from liver of these fish which did not exhibit increased apoptosis and instead increased hepatic HSP70 expression at 96 h post-injection. However, DMBA had no effect on apoptosis or HSP70 levels in more reproductively mature juvenile fish that were housed at a lower water temperature. This may be due to a developmental or temperature-dependent component to these responses. We propose that the decrease in ovarian HSP70 expression in response to BNF and DMBA may be causally related to the increase in ovarian cell apoptosis. Further experiments using a full time course, dose-response and methods to confirm that AhR is a direct mediator of these effects are required.

Effect of bile salts, lipid, and humic acids on absorption of benzo[a]pyrene by isolated channel catfish (Ictalurus punctatus) intestine segments.

Dietary absorption of lipophilic contaminants may be a significant route of exposure in aquatic organisms. Bile salts, lipids, and humic acids are important factors that may influence the intestinal absorption of a contaminant such as benzo[a]pyrene (BaP). We hypothesized that bile salts, monoglycerides, and free fatty acids would increase BaP intestinal absorption, while triglycerides, humic acids, and sediment would decrease BaP intestinal absorption. We have established and validated an in vitro model to examine modification of 3H-BaP absorption in everted intestinal segments from channel catfish (Ictalurus punctatus). Uptake of BaP into the everted intestinal segments continued to increase over the times examined in this study (60 min) and apparently occurs passively; thus, fugacity-based models of uptake are supported. Absorption of BaP into intestinal cells was significantly decreased by the addition of monoglycerides and free fatty acids to bile salts in the incubation media. Addition of triglycerides decreased BaP absorption even further. Humic acids may have decreased BaP intestinal absorption, while natural sediment may have increased BaP absorption. The results of this study suggest that all lipids may decrease intestinal uptake of lipophilic contaminants if they remain in unabsorbable excess in the intestinal lumen by retaining BaP in lipid/bile micelles. In contrast, if triglycerides are hydrolyzed into monoglycerides/free fatty acids prior to absorption, lipophilic contaminant uptake will likely be facilitated. Thus, it may be the hydrolytic state of lipids that determines its effects on BaP absorption. Humic acids alone may decrease dietary uptake of BaP, but our results suggest that other components in natural sediment may counteract this effect to cause a slight enhancement of BaP uptake. Further studies are needed to determine the dietary conditions necessary for bioaccumulation to contribute significantly to lipophilic contaminant body burdens in benthivorous fish. Finally, the everted intestinal segment technique has the potential to be used in other species and with different contaminants.

The involvement of protein kinase C in myosin phosphorylation and force development in rat tail arterial smooth muscle.

Myosin light-chain phosphorylation is the primary mechanism for activating smooth-muscle contraction and occurs principally at Ser-19 of the 20 kDa light chains of myosin (LC(20)). In some circumstances, Thr-18 phosphorylation may also occur. Protein kinase C (PKC) can regulate LC(20) phosphorylation indirectly via signalling pathways leading to inhibition of myosin light-chain phosphatase. The goal of this study was to determine the relative importance of myosin light-chain kinase (MLCK) and PKC in basal and stimulated LC(20) phosphorylation in rat tail arterial smooth-muscle strips (RTA). Two MLCK inhibitors (ML-9 and wortmannin) and two PKC inhibitors (chelerythrine and calphostin C) that have different mechanisms of action were used. Results showed the following: (i) basal LC(20) phosphorylation in intact RTA is mediated by MLCK; (ii) alpha(1)-adrenoceptor stimulation increases LC(20) phosphorylation via MLCK and PKC; (iii) Ca(2+)-induced LC(20) phosphorylation in Triton X-100-demembranated RTA is catalysed exclusively by MLCK, consistent with the quantitative loss of PKCs alpha and beta following detergent treatment; (iv) very little LC(20) diphosphorylation (i.e. Thr-18 phosphorylation) occurs in intact or demembranated RTA at rest or in response to contractile stimuli; and (v) the level of LC(20) phosphorylation correlates with contraction in intact and demembranated RTA, although the steady-state tension-LC(20) phosphorylation relationship is markedly different between the two preparations such that the basal level of LC(20) phosphorylation in intact muscles is sufficient to generate maximal force in demembranated preparations. This may be due, in part, to differences in the phosphatase/kinase activity ratio, resulting from disruption of a signalling pathway leading to myosin light-chain phosphatase inhibition following detergent treatment.

Ca2+-independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments.

1. Smooth muscle contraction is activated primarily by the Ca2+-calmodulin (CaM)-dependent phosphorylation of the 20 kDa light chains (LC20) of myosin. Activation can also occur in some instances without a change in intracellular free [Ca2+] or indeed in a Ca2+-independent manner. These signalling pathways often involve inhibition of myosin light chain phosphatase and unmasking of basal kinase activity leading to LC20 phosphorylation and contraction. 2. We have used demembranated rat caudal arterial smooth muscle strips and isolated chicken gizzard myofilaments in conjunction with the phosphatase inhibitor microcystin-LR to investigate the mechanism of Ca2+-independent phosphorylation of LC20 and contraction. 3. Treatment of Triton X-100-demembranated rat caudal arterial smooth muscle strips with microcystin at pCa 9 triggered a concentration-dependent contraction that was slower than that induced by pCa 4.5 or 6 but reached comparable steady-state levels of tension. 4. This Ca2+-independent, microcystin-induced contraction correlated with phosphorylation of LC20 at serine-19 and threonine-18. 5. Whereas Ca2+-dependent LC20 phosphorylation and contraction were inhibited by a synthetic peptide (AV25) based on the autoinhibitory domain of myosin light chain kinase (MLCK), Ca2+-independent, microcystin-induced LC20 phosphorylation and contraction were resistant to AV25. 6. Ca2+-independent LC20 kinase activity was also detected in chicken gizzard smooth muscle myofilaments and catalysed phosphorylation of endogenous myosin LC20 at serine-19 and/or threonine-18. This is in contrast to MLCK which phosphorylates threonine-18 only after prior phosphorylation of serine-19. 7. Gizzard Ca2+-independent LC20 kinase could be separated from MLCK by differential extraction from myofilaments and by CaM affinity chromatography. Its activity was resistant to AV25. 8. We conclude that inhibition of smooth muscle myosin light chain phosphatase (MLCP) unmasks the activity of a Ca2+-independent LC20 kinase associated with the myofilaments and distinct from MLCK. This kinase, therefore, probably plays a role in Ca2+ sensitization and Ca2+-independent contraction of smooth muscle in response to stimuli that act via Ca2+-independent pathways, leading to inhibition of MLCP.

Influence of streptozotocin diabetes on the alpha-1 adrenoceptor and associated G proteins in rat arteries.

Previous studies from this laboratory have demonstrated an enhancement in both the contractile and signaling response to stimulation of either alpha-1 adrenoceptors or guanine nucleotide binding proteins (G proteins) in arteries from male Wistar rats with 12 to 14 weeks of streptozotocin-induced diabetes. The purpose of the present investigation was to determine whether changes in arterial alpha-1 adrenoceptors or the G proteins coupled to them are associated with the enhanced responsiveness of the diabetic arteries. No difference in affinity was detected between control and diabetic aorta or caudal artery membranes in saturation binding of [3H]prazosin to alpha-1 adrenoceptors. However, the alpha-1 adrenoceptor number was significantly decreased in caudal artery but not aorta from diabetic rats. In competition binding experiments, a low-affinity and a high-affinity binding site for norepinephrine were detected in the absence of guanine nucleotides and NaCl in control arteries, whereas only the low-affinity site was detected in diabetic arteries, suggesting that coupling of the alpha-1 adrenoceptor to G proteins is impaired in diabetic aorta and caudal artery. The levels of immunoreactive Gi2,3alpha and Gq/11alpha were not different between control and diabetic aorta or caudal artery. Thus, not only do changes in the number and coupling of the alpha-1 adrenoceptor or level of G proteins not explain the enhanced contractile responses of diabetic arteries to norepinephrine, but also the changes in alpha-1 adrenoceptor binding would counteract the enhancement. Instead, an increase in the activity of the G proteins or phospholipase C-beta coupled to the alpha-1 adrenoceptor may be mediating the enhanced responsiveness elicited by alpha-1 adrenoceptor stimulation in diabetic arteries.

Noradrenaline stimulation of high-affinity GTPase activity in membranes from rat aorta and caudal artery.

The ability of noradrenaline (NA) to stimulate increases in high-affinity GTPase activity in sarcolemma-enriched rat aorta and caudal artery membranes was examined in the present study. In aortic membranes, NA significantly (P < 0.05; N = 5) increased the Vmax from a basal value of 103 +/- 29 to 156 +/- 38 pmol Pi/min/mg protein, but did not affect the Km which was 0.32 +/- 0.08 microM in the absence and 0.58 +/- 0.16 microM in the presence of NA. However, in caudal artery membranes, NA significantly (P < 0.05; N = 6) increased both the Vmax and the Km from basal values of 69 +/- 12 pmol Pi/min/mg protein and 0.24 +/- 0.05 microM, respectively, to 205 +/- 54 pmol Pi/min/mg protein and 1.01 +/- 0.25 microM, respectively. Removing the endothelium from both artery preparations did not alter significantly basal GTPase activity or the magnitude of the increase stimulated by NA. Prazosin significantly inhibited NA-stimulated increases in GTPase activity in membranes from endothelium-denuded caudal artery and aorta, and in endothelium-intact caudal artery membranes. However, yohimbine significantly inhibited NA-stimulated increases in GTPase activity only in preparations from endothelium-intact caudal arteries. Therefore, in endothelium-intact caudal artery membranes, NA stimulated increases in GTPase activity that were apparently mediated by both alpha 1-adrenoceptors and alpha 2-adrenoceptors, while in endothelium-denuded aortic and caudal artery membranes this increase was mediated solely by alpha 1-adrenoceptors. Western blotting of these arteries confirmed the presence of both Gi alpha 2,3 and Gq/11 alpha, which are candidates for mediating the alpha 1-adrenoceptor-stimulated increases in GTPase activity.

Enhanced contractile responses of arteries from streptozotocin diabetic rats to sodium fluoride.

1. Previous studies from this laboratory have demonstrated that alpha 1-adrenoceptor-mediated increases in tension and phosphoinositide metabolism are enhanced in the aorta and mesenteric arteries from diabetic rats. The purpose of the present investigation was to determine whether contractile responses to sodium fluoride (NaF), which directly stimulates GTP-binding proteins (G-proteins), are also enhanced in diabetic arteries. 2. NaF (1-20 mM) in the presence of 10 microM aluminium chloride produced slowly developing, concentration-dependent contractions in mesenteric arteries from three month streptozotocin-diabetic (60 mg kg-1, i.v.) male Wistar rats and age-matched control rats. The maximum contractile response but not the sensitivity to NaF was significantly greater in mesenteric arteries from diabetic than from control rats, as was the response to noradrenaline (NA). Maximum contractile responses of aorta and caudal artery from diabetic rats to NaF were also significantly enhanced. 3. Removal of the endothelium and denervation with 6-hydroxydopamine did not significantly alter the maximum contractile response of mesenteric arteries from either control or diabetic rats to NaF. Similarly, NaF had no effect on cyclic AMP levels in aorta, and no difference in cyclic AMP levels, either basally or in the presence of NaF, was detected between control and diabetic rat aorta. 4. Contractile responses of mesenteric arteries from both control and diabetic rats to NaF were diminished in calcium-free Krebs solution, but the NaF response remained significantly elevated in mesenteric arteries from diabetic rats compared to control. 5. Ryanodine (30 microM) which depletes intracellular calcium stores, nifedipine (3 microM) which blocks dihydropyridine-sensitive calcium channels and calphostin C (0.5 microM) which selectively inhibits protein kinase C, all significantly inhibited maximum contractile responses of mesenteric arteries from control and diabetic rats to NaF. There were no significant differences between control and diabetic arteries in the relative magnitude of the inhibition produce by the three antagonist. 6. These data suggest that there may be increased activation of the same signalling processes that mediate NA-stimulated vasoconstriction, perhaps contraction-associated G-proteins or the effectors coupled to these G-proteins, in response to NaF in mesenteric arteries from diabetic rats. This may also be responsible for the enhanced contractile responses of these arteries to alpha 1-adrenoceptor stimulation.

Pharmacological investigation of signaling mechanisms contributing to phasic and tonic components of the contractile response of rat arteries to noradrenaline.

The mechanisms contributing to the contractile responses to two different concentrations of noradrenaline (NA) in rat aorta and mesenteric artery were compared using nifedipine, which inhibits calcium influx through dihydropyridine-sensitive channels, ryanodine, which depletes intracellular calcium stores, and calphostin C, which inhibits protein kinase C (PKC). Both submaximal and maximal concentrations of NA induced a biphasic response in aorta and mesenteric artery, with an early fast phase and a later sustained tonic component. Calcium release from intracellular stores contributed to the phasic component of contraction to both concentrations of NA in aorta, although to a greater extent to the submaximal concentration. In aorta, inhibiting both intracellular calcium release and calcium influx through dihydropyridine-sensitive channels simultaneously or inhibiting PKC activity alone significantly reduced the tonic response to a maximal concentration of NA. However, the tonic response to a submaximal concentration of NA in aorta was significantly inhibited only when intracellular calcium stores were depleted with ryanodine. In mesenteric artery, the phasic response to a maximal concentration of NA was significantly depressed only when both calcium influx and intracellular calcium release were inhibited simultaneously. However, the phasic response to a submaximal concentration of NA was significantly inhibited by blocking calcium influx alone, but not by blocking intracellular calcium release alone. The tonic component of the contractile response to both concentrations of NA in mesenteric artery appeared to be mediated in part by calcium from both intracellular and extracellular sources.(ABSTRACT TRUNCATED AT 250 WORDS)

Contractile responses of caudal arteries from diabetic rats to adrenergic nerve stimulation.

The effect of 12-14 weeks streptozotocin-induced diabetes on contractile responses of the highly innervated caudal artery to adrenergic nerve stimulation was studied. The maximum contractile response and sensitivity (as reflected by the pD2 value) to noradrenaline (NA) was significantly greater in diabetic than in control caudal arteries. The maximum contractile response, but not sensitivity, to tyramine was significantly enhanced in diabetic compared with control caudal arteries when expressed as stress (mN/mm2), but not when expressed as a percentage of the maximum NA response obtained between the responses of control and diabetic caudal arteries to electrical field stimulation of adrenergic nerves, when expressed either as stress or as a percentage of the corresponding NA response. Pretreatment with phentolamine markedly inhibited the contractile responses to field stimulation of both diabetic and control caudal arteries, while 6-hydroxydopamine essentially abolished responses to electrical field stimulation and tyramine in both tissues. Finally, pretreatment with hydrocortisone, timolol, and desipramine increased sensitivity to NA in control and diabetic caudal arteries by a similar magnitude. Thus, the enhanced contractile responses of caudal arteries from diabetic rats to NA do not appear to be associated with the development of autonomic neuropathy.