Insulin secretion-independent effects of GLP-1 on canine liver glucose metabolism do not involve portal vein GLP-1 receptors

Whether glucagon-like peptide (GLP)-1 requires the hepatic portal vein to elicit its insulin secretion-independent effects on glucose disposal in vivo was assessed in conscious dogs using tracer and arteriovenous difference techniques. In study 1, six conscious overnight-fasted dogs underwent oral glucose tolerance testing (OGTT) to determine target GLP-1 concentrations during clamp studies. Peak arterial and portal values during OGTT ranged from 23 to 65 pM and from 46 to 113 pM, respectively. In study 2, we conducted hyperinsulinemic-hyperglycemic clamp experiments consisting of three periods (P1, P2, and P3) during which somatostatin, glucagon, insulin and glucose were infused. The control group received saline, the PePe group received GLP-1 (1 pmol.kg(-1).min(-1)) peripherally, the PePo group received GLP-1 (1 pmol.kg(-1).min(-1)) peripherally (P2) and then intraportally (P3), and the PeHa group received GLP-1 (1 pmol.kg(-1).min(-1)) peripherally (P2) and then through the hepatic artery (P3) to increase the hepatic GLP-1 load to the same extent as in P3 in the PePo group (n = 8 dogs/group). Arterial GLP-1 levels increased similarly in all groups during P2 ( approximately 50 pM), whereas portal GLP-1 levels were significantly increased (2-fold) in the PePo vs. PePe and PeHa groups during P3. During P2, net hepatic glucose uptake (NHGU) increased slightly but not significantly (vs. P1) in all groups. During P3, GLP-1 increased NHGU in the PePo and PeHa groups more than in the control and PePe groups (change of 10.8 +/- 1.3 and 10.6 +/- 1.0 vs. 5.7 +/- 1.0 and 5.4 +/- 0.8 micromol.kg(-1).min(-1), respectively, P < 0.05). In conclusion, physiological GLP-1 levels increase glucose disposal in the liver, and this effect does not involve GLP-1 receptors located in the portal vein.     

Oral antioxidant therapy improves endothelial function in Type 1 but not Type 2 diabetes mellitus

Oxidative stress decreases the bioavailability of endothelium-derived nitric oxide in diabetic patients. We investigated whether impaired endothelium-dependent vasodilation (EDV) in diabetes can be improved by long-term administration of oral antioxidants. Forty-nine diabetic subjects [26 Type 1 (T1) and 23 Type 2 (T2)] and 45 matched healthy control subjects were randomized to receive oral vitamin C (1,000 mg) and vitamin E (800 IU) daily or matching placebo for 6 mo. Vascular ultrasonography was used to determine brachial artery EDV and endothelium-independent vasodilation (EIV). EDV was decreased in both T1 (4.9 +/- 0.9%, P = 0.015) and T2 (4.1 +/- 1.0%, P < 0.01) subjects compared with control subjects (7.7 +/- 0.7%). EIV was decreased in T2 (15.0 +/- 1.2%, P < 0.01) but not T1 subjects (18.5 +/- 2.3%, P = 0.3) compared with controls (21.8 +/- 1.8%). Administration of antioxidant vitamins increased EDV in T1 (by 3.4 +/- 1.4%, P = 0.023) but not T2 subjects (by 0.5. +/- 0.4%, P = 0.3). Antioxidant therapy had no significant affect on EIV. Oral antioxidant therapy improves EDV in T1 but not T2 diabetes. These results are consistent with the lack of clinical benefit in studies that have included primarily T2 diabetic patients.     

Attenuation of lipopolysaccharide anorexia by antagonism of caudal brain stem but not forebrain GLP-1-R

The central glucagon-like peptide-1 (GLP-1) system has been implicated in the control of feeding behavior. Here we explore GLP-1 mediation of the anorexic response to administration of systemic LPS and address the relative importance of caudal brain stem and forebrain GLP-1 receptor (GLP-1-R) for the mediation of the response. Fourth-intracerebroventricular delivery of the GLP-1-R antagonist exendin-(9-39) (10 microg) did not itself affect food intake in the 24 h after injection but significantly attenuated the otherwise robust (approximately 60%) reduction in food intake obtained after LPS (100 microg/kg) treatment. This result highlights a role for caudal brain stem GLP-1-R in the mediation of LPS anorexia but does not rule out the possibility that forebrain receptors also contribute to the response. Forebrain contribution was addressed by delivery of the GLP-1-R antagonist to the third ventricle with the caudal flow of cerebrospinal fluid blocked by occlusion of the cerebral aqueduct. Exendin-(9-39) delivery thus limited to forebrain did not attenuate the anorexic response to LPS. These data suggest that LPS anorexia is mediated, in part, by release of the native peptide acting on GLP-1-R within the caudal brain stem.     

GLP-1 and GLP-2 act in concert to inhibit fasted,but not fed,small bowel motility in the rat

Small bowel motility was studied in rats at increasing (1-20 pmol/kg/min) intravenous doses of either glucagon-like peptide-1 (GLP-1) or glucagon-like peptide-2 (GLP-2) alone, or in combination in the fasted and fed state. There was a dose-dependent inhibitory action of GLP-1 on the migrating myoelectric complex (MMC), where the dose of 5 pmol/kg/min induced an increased MMC cycle length. No effect was seen with GLP-2 alone, but the combination of GLP-1 and GLP-2 induced a more pronounced inhibitory effect, with significant increase of the MMC cycle length from a dose of 2 pmol/kg/min. During fed motility, infusion of GLP-1 resulted in an inhibition of spiking activity compared to control. In contrast, infusion of GLP-2 only numerically increased spiking activity compared to control, while the combination of GLP-1 and GLP-2 resulted in no change compared to control. In summary, this study demonstrates an additive effect of peripheral administration of GLP-1 and GLP-2 on fasted small bowel motility. In the fed state, GLP-1 and GLP-2 seem to display counter-balancing effects on motility of the small intestine.     

Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) differentially regulates orthosteric but not allosteric agonist binding and function

The glucagon-like peptide-1 receptor (GLP-1R) is a prototypical family B G protein-coupled receptor that exhibits physiologically important pleiotropic coupling and ligand-dependent signal bias. In our accompanying article (Koole, C., Wootten, D., Simms, J., Miller, L. J., Christopoulos, A., and Sexton, P. M. (2012) J. Biol. Chem. 287, 3642-3658), we demonstrate, through alanine-scanning mutagenesis, a key role for extracellular loop (ECL) 2 of the receptor in propagating activation transition mediated by GLP-1 peptides that occurs in a peptide- and pathway-dependent manner for cAMP formation, intracellular (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). In this study, we examine the effect of ECL2 mutations on the binding and signaling of the peptide mimetics, exendin-4 and oxyntomodulin, as well as small molecule allosteric agonist 6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline (compound 2). Lys-288, Cys-296, Trp-297, and Asn-300 were globally important for peptide signaling and also had critical roles in governing signal bias of the receptor. Peptide-specific effects on relative efficacy and signal bias were most commonly observed for residues 301-305, although R299A mutation also caused significantly different effects for individual peptides. Met-303 was more important for exendin-4 and oxyntomodulin action than those of GLP-1 peptides. Globally, ECL2 mutation was more detrimental to exendin-4-mediated Ca(2+)i release than GLP-1(7-36)-NH(2), providing additional evidence for subtle differences in receptor activation by these two peptides. Unlike peptide activation of the GLP-1R, ECL2 mutations had only limited impact on compound 2 mediated cAMP and pERK responses, consistent with this ligand having a distinct mechanism for receptor activation. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition of the receptor by peptide agonists.     

GLP-1类似物蛋白结构及功能研究进展

基于胰高血糖素样肽-1(glucagon-like peptide-1,GLP-1)结构改变得到的GLP-1类似物,能迅速、高效、持久地降低血糖及糖化血红蛋白,具有改善胰岛β细胞功能、调节收缩压、保护心血管、降低血脂、减轻体重、延迟胃排空、增加饱腹感等作用,成为近年来2型糖尿病治疗领域研究的热点。本文对GLP-1及其类似物的结构、功能、不良反应、蛋白质结构研究的方法作一综述。     

Physically active lifestyle enhances vagal-cardiac function but not central autonomic neural interaction in elderly humans

The cause of the age-related impairment of arterial baroreflex function remains ill-defined; moreover, it is unknown whether this impairment results from aging per se or from an inactive lifestyle associated with aging. In this study, we sought to: 1) determine whether elderly individuals who maintained an active lifestyle had an enhanced carotid baroreflex function as compared with their sedentary counterparts; and 2) determine whether this difference was due in part to altered function of the arterial baroreceptor and/or altered central modulation. Eight healthy, sedentary (SED, 68+/-2 yr) and eight physically active (ACT, 68+/-1 yr) elderly men with peak O(2) consumption 25.5+/-1.2 vs 35.7+/-2.4 ml/min/kg (P<0.01), respectively, were assessed with carotid baroreceptor (CBR) function using 5s pulses of neck pressure or suction (ranging from +40 to -80 Torr) delivered to the carotid sinus region at rest and during lower body negative pressure (LBNP) of -15 and -40 Torr. Changes in heart rate (HR) and mean arterial pressure (MAP) were assessed for CBR-HR and CBR-MAP gains, respectively. Overall CBR-HR gains in a range of approximately 120 mmHg of carotid sinus pressure were greater (P<0.01) in ACT than SED at rest and during LBNP. The derived peak CBR-HR slopes between ACT and SED at rest were -0.32+/-0.07 vs -0.11+/-0.02 bpm/mmHg (P=0.007), respectively. However, there was no statistical difference (P=0.37) in CBR-MAP gains between the groups. Neither CBR-MAP (P=0.08) nor CBR-HR (P=0.41) gain was augmented by LBNP in the elderly. CONCLUSION: Active lifestyle enhances the CBR-HR reflex sensitivity as a result of the improved vagal-cardiac function in elderly people. Aging is associated with an absence of central autonomic interaction in the control of blood pressure regardless of physical fitness.     

Immunologic function of endothelial cells: guinea pig aortic endothelial cells support mitogen-induced T lymphocyte activation, but do not function as antigen-presenting cells

The possibility that vascular endothelial cells (EC), like macrophages (M phi), can function as accessory cells necessary for mitogen- and antigen-induced T cell activation was examined. EC were enzymatically detached from the luminal surfaces of guinea pig aortas and then propagated in culture. Lymph node T lymphocytes were rigorously depleted of adherent cells, such that they completely lost the capacity to respond to mitogenic stimulation with phytohemagglutinin or concanavalin A. In this system, EC restored mitogen-induced T cell DNA synthesis as effectively as did M phi. This effect could not be explained by a facilitation of residual accessory cell activity within the responding T cell population, because EC restored mitogen responsiveness to T cells that had been treated with anti-Ia antibody and complement. Support of mitogen responsiveness could not be accounted for by secreted products of M phi or EC in the absence of intact accessory cells. In addition to the capacity to serve as fully sufficient accessory cells for the induction of mitogen-stimulated T cell proliferation, EC exerted a number of modulatory influences on T lymphocyte responses in cultures supported by M phi. When such cultures were supplemented with small numbers of EC, responses were dramatically augmented; larger numbers of EC resulted in marked suppression. At least part of these immunomodulatory effects could be accounted for by the activity of secreted products of EC. EC did not express detectable Ia antigens assayed either by indirect immunofluorescence, with the use of the fluorescence-activated cell sorter, or by complement-mediated cytotoxicity. Moreover, treating the EC population with anti-Ia antibody and complement had no effect on its capacity to support mitogen-induced T cell DNA synthesis. As would be expected from the lack of Ia antigen expression, EC were incapable of presenting antigen to primed T cells. They did, however, carry enough antigen into the cultures such that effective antigen presentation could occur when the cultures were supplemented with M phi that were syngeneic but not allogeneic to the responding T cells. Moreover, EC were capable of dramatically augmenting antigen-specific responses stimulated by antigen-pulsed M phi. There was no genetic restriction for this EC-mediated augmentation of antigen responsiveness. These results indicate that EC are capable of functioning as completely sufficient accessory cells for mitogen-induced T cell DNA synthesis and, in addition, are able to modulate ongoing M phi-supported T lymphocyte responses in both a positive and negative manner.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of interleukin-1 therapy on peripheral blood granulocyte function in humans

During a phase I trial of interleukin-1 (IL-1) in patients with ovarian carcinomas, the effects of this treatment on blood granulocyte respiratory burst and locomotive responses were examined. Differences in baseline granulocyte function in patients as well as dose-related effects of IL-1 treatment were observed. Patients enrolled early in the trial (low-dose patients) had significantly lower locomotive responses before treatment than their paired controls; these low responses normalized after 5 days of continuous-infusion IL-1 treatment. Patients enrolled later (high-dose patients) had normal locomotive responses before treatment and IL-1 treatment was associated with suppression of responses to selected stimuli at the end of treatment. Pretreatment respiratory burst responses in both low-and high-dose patient groups were essentially normal, but the rates of granulocyte H₂O₂ production following phorbol myristate acetate stimulation became significantly less than control values at the end of treatment. In vitro exposure of either patient or control cells to 150 U/ml IL-1 did not alter their locomotive or respiratory burst responses, suggesting the observed in vivo effects were not mediated directly by IL-1. Treatment with IL-1 is associated with changes in ex vivo granulocyte function that are related to the IL-1 dose. Treatment with low doses of IL-1 may provide a means of normalizing abnormal polymorphonuclear leukocyte function in some patients with ovarian malignancies.     

Sitagliptin augments protective effects of GLP-1 against advanced glycation end product receptor axis in endothelial cells

Sitagliptin is a stable inhibitor of dipeptidyl peptidase-IV, a responsible enzyme that mainly inactivates glucagon-like peptide-1 (GLP-1), and now one of the widely used agents for the treatment of diabetes. However, effects of sitagliptin on vascular injury are largely unknown. Since advanced glycation end products (AGEs) and their receptor (RAGE) axis contribute to vascular damage in diabetes, we investigated here whether sitagliptin inhibits the AGE-RAGE-induced endothelial cell damage in vitro. Although effects of 10?pM GLP-1 or 0.5?μM sitagliptin monotherapy on RAGE gene and protein expression were modest, combination therapy completely blocked the AGE-induced increase in RAGE mRNA and protein levels in human umbilical vein endothelial cells (HUVEC). AGEs induced reactive oxygen species (ROS) generation and reduced endothelial nitric oxide synthase (eNOS) mRNA level in HUVEC, both of which were also completely blocked by the treatment with 10?pM GLP-1 and 0.5?μM sitagliptin, but not with GLP-1 or sitagliptin monotherapy. Further, anti-RAGE antibody restored the decrease in eNOS mRNA level in AGE-exposed HUVEC. The present study suggests that sitagliptin augments the effects of GLP-1 on eNOS mRNA level in AGE-exposed HUVEC by suppressing RAGE expression and subsequent ROS generation. Sitagliptin may work as a vasoprotecitve agent in diabetes by blocking the AGE-RAGE axis.     

Antiapoptotic effects of GLP-1 in murine HL-1 cardiomyocytes

Activation of apoptosis contributes to cardiomyocyte dysfunction and death in diabetic cardiomyopathy. The peptide glucagon-like peptide-1 (GLP-1), a hormone that is the basis of emerging therapy for type 2 diabetic patients, has cytoprotective actions in different cellular models. We investigated whether GLP-1 inhibits apoptosis in HL-1 cardiomyocytes stimulated with staurosporine, palmitate, and ceramide. Studies were performed in HL-1 cardiomyocytes. Apoptosis was induced by incubating HL-1 cells with staurosporine (175 nM), palmitate (135 μM), or ceramide (15 μM) for 24 h. In staurosporine-stimulated HL-1 cardiomyocytes, phosphatidylserine exposure, Bax-to-Bcl-2 ratio, Bad phosphorylation (Ser(136)), BNIP3 expression, mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, DNA fragmentation, and mammalian target of rapamycin (mTOR)/p70S6K phosphorylation (Ser(2448) and Thr(389), respectively) were assessed. Apoptotic hallmarks were also measured in the absence or presence of low (5 mM) and high (10 mM) concentrations of glucose. In addition, phosphatidylserine exposure and DNA fragmentation were analyzed in palmitate- and ceramide-stimulated cells. Staurosporine increased apoptosis in HL-1 cardiomyocytes. GLP-1 (100 nM) partially inhibited staurosporine-induced mitochondrial membrane depolarization and completely blocked the rest of the staurosporine-induced apoptotic changes. This cytoprotective effect was mainly mediated by phosphatidylinositol 3-kinase (PI3K) and partially dependent on ERK1/2. Increasing concentrations of glucose did not influence GLP-1-induced protection against staurosporine. Furthermore, GLP-1 inhibited palmitate- and ceramide-induced phosphatidylserine exposure and DNA fragmentation. Incretin GLP-1 protects HL-1 cardiomyocytes against activation of apoptosis. This cytoprotective ability is mediated mainly by the PI3K pathway and partially by the ERK1/2 pathway and seems to be glucose independent. It is proposed that therapies based on GLP-1 may contribute to prevent cardiomyocyte apoptosis.     

Intravenous CCK-8, but not GLP-1, suppresses ghrelin and stimulates PYY release in healthy men

We have investigated the effects of exogenous CCK-8 and GLP-1, alone and in combination, on ghrelin and PYY secretion. Nine healthy males were studied on four occasions. Plasma ghrelin and PYY concentrations were measured during 150 min intravenous infusions of: (i) isotonic saline, (ii) CCK-8 at 1.8 pmol/kg/min, (iii) GLP-1 at 0.9 pmol/kg/min or (iv) CCK-8 and GLP-1 combined. CCK-8 markedly suppressed ghrelin and stimulated PYY when compared with control between t=0-120 min (P<0.001 for both). GLP-1 had no effect on ghrelin, but decreased PYY slightly at 120 min (P<0.05). During infusion of CCK-8+GLP-1, there was comparable suppression of ghrelin (P<0.001), but the stimulation of PYY was less (P<0.001), than that induced by CCK-8, between t=20-120 min. In conclusion, in healthy subjects, in the doses evaluated, exogenous CCK-8 suppresses ghrelin and stimulates PYY, and exogenous GLP-1 has no effect on ghrelin and attenuates the effect of CCK-8 on PYY.     

Beneficial effects of resistance exercise on glycemic control are not further improved by protein ingestion

Purpose

To investigate the mechanisms underpinning modifications in glucose homeostasis and insulin sensitivity 24 h after a bout of resistance exercise (RE) with or without protein ingestion.

Methods

Twenty-four healthy males were assigned to a control (CON; n = 8), exercise (EX; n = 8) or exercise plus protein condition (EX+PRO; n = 8). Muscle biopsy and blood samples were obtained at rest for all groups and immediately post-RE (75% 1RM, 8×10 repetitions of leg-press and extension exercise) for EX and EX+PRO only. At 24 h post-RE (or post-resting biopsy for CON), a further muscle biopsy was obtained. Participants then consumed an oral glucose load (OGTT) containing 2 g of [U-¹³C] glucose during an infusion of 6, 6-[²H₂] glucose. Blood samples were obtained every 10 min for 2 h to determine glucose kinetics. EX+PRO ingested an additional 25 g of intact whey protein with the OGTT. A final biopsy sample was obtained at the end of the OGTT.

Results

Fasted plasma glucose and insulin were similar for all groups and were not different immediately post- and 24 h post-RE. Following RE, muscle glycogen was 26±8 and 19±6% lower in EX and EX+PRO, respectively. During OGTT, plasma glucose AUC was lower for EX and EX+PRO (75.1±2.7 and 75.3±2.8 mmol·L⁻¹∶120 min, respectively) compared with CON (90.6±4.1 mmol·L⁻¹∶120 min). Plasma insulin response was 13±2 and 21±4% lower for EX and CON, respectively, compared with EX+PRO. Glucose disappearance from the circulation was ∼12% greater in EX and EX+PRO compared with CON. Basal 24 h post-RE and insulin-stimulated PAS-AS160/TBC1D4 phosphorylation was greater for EX and EX+PRO.

Conclusions

Prior RE improves glycemic control and insulin sensitivity through an increase in the rate at which glucose is disposed from the circulation. However, co-ingesting protein during a high-glucose load does not augment this response at 24 h post-exercise in healthy, insulin-sensitive individuals.     

Differential effects of oxyntomodulin and GLP-1 on glucose metabolism

Glucagon-like peptide-1 (GLP-1) and oxyntomodulin (OXM) are peptide hormones secreted postprandially from the gut that stimulate insulin secretion in a glucose-dependent manner. OXM activates both the GLP-1 receptor (GLP1R) and the glucagon receptor (GCGR). It has been suggested that OXM acutely modulates glucose metabolism solely through GLP1R agonism. Because OXM activates the GLP1R with lower affinity than GLP-1, we generated a peptide analog (Q→E, OXMQ3E) that does not exhibit glucagon receptor agonist activity but retains the same affinity as OXM for GLP1R. We compared the effects of OXM and OXMQ3E in a glucose tolerance test and, to better characterize the effect on glucose metabolism, we performed controlled infusions of OXM or OXMQ3E during a hyperglycemic clamp performed in wild-type, Glp1r(-/-), and Gcgr(-/-) mice. Our findings show that OXM, but not OXMQ3E, activates the GCGR in vivo. Second, OXM and OXMQ3E improve glucose tolerance following an acute glucose challenge and during a hyperglycemic clamp in mice. Finally, OXM infusion during a glucose clamp reduces the glucose infusion rate (GIR) despite a simultaneous increase in insulin levels in Glp1r(-/-) mice, whereas OXM and OXMQ3E increase GIR to a similar extent in Gcgr(-/-) mice. In conclusion, activation of the GCGR seems to partially attenuate the acute beneficial effects on glucose and contributes to the insulinotropic action of oxyntomodulin.     

Nitric oxide mediates the mitogenic effects of insulin and vascular endothelial growth factor but not of leptin in endothelial cells

The regulation of vascular wall homeostasis by nitric oxide (NO) generated by endothelium is being intensively studied. In the present paper, the involvement of NO in the vascular endothelial growth factor (VEGF), insulin or leptin-stimulated proliferation of human endothelial cells (HUVEC) was measured by [3H]thymidine or bromodeoxyuridine incorporation. VEGF and insulin, but not leptin, increased NO generation in HUVEC, as detected with ISO-NO electrode. Proliferation of HUVEC induced by leptin was not changed or was higher in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME) a nitric oxide synthase (NOS) inhibitor. In contrast, L-NAME blunted the proproliferative effect of VEGF and insulin. Furthermore, we demonstrated that, in human arterial smooth muscle cells (hASMC) transfected with endothelial NOS (eNOS) gene, the generation of biologically active VEGF protein was NO-dependent. Inhibition of NO generation by L-NAME decreased the synthesis of VEGF protein and attenuated HUVEC proliferation induced by conditioned media from transfected hASMC. Endothelium-derived NO seems to participate in VEGF and insulin, but not leptin, mitogenic activity. Additionally, the small amounts of NO released from endothelial cells, as mimicked by eNOS transfection into hASMC, may activate generation of VEGF in sub-endothelial smooth muscle cells, leading to increased synthesis of VEGF protein necessary for turnover and restitution of endothelial cells.     

Rac3, but not Rac1, promotes ox-LDL induced endothelial dysfunction by downregulating autophagy

The endothelial dysfunction induced by oxidized low-density lipoprotein (ox-LDL) plays an important role in the pathogenesis of atherosclerosis, which can lead to oxidative stress and inflammation. The role of autophagy in the process of atherosclerosis has drawn increasing attention. The human umbilical vein endothelial cells (HUVECs), whose Ras-related C3 botulinum toxin substrate 1 (Rac1) and Rac3 was knockdown, were used to detect whether the possible molecular mechanisms of Rac1 and Rac3 for anti-inflammatory in endothelial cells was effected by downregulation of autophagy. The HUVECs were incubated with ox-LDL. The inflammatory factors and autophagy proteins were evaluated to ascertain and compare the effect of Rac1 and Rac3 on autophagy. Then, 3-methyladenine (3-MA) as an inhibiter of autophagy was used to detect whether the effect of Rac1 and Rac3 was related to autophagy. ox-LDL-induced cell dysfunction in HUVECs was determined by testing the formation of foam cells, the expression of nuclear factor (NF)-κB and nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 and NF-κB p65 and other inflammatory factors, the release of reactive oxygen species by oxidative stress and the dysfunction of the cytomembrane. And ApoE^−/− mice on a high-fat diet were used as an animal model to detect the effect of Rac1 and Rac3 in vivo. The results showed that when Rac1 and Rac3 were decreased in HUVECs, the cell dysfunction caused by ox-LDL was inhibited. If 3-MA was used to inhibit autophagy in Rac1 and Rac3 knockdown cells, the injury induced by ox-LDL on the cells was recovered. These results indicated that the effect of Rac1 and Rac3 was combined with ox-LDL, which was related to inhibition of autophagy. The effect of Rac3 was more significant than that of Rac1.     

Beneficial effects of GH/IGF-1 on skeletal muscle atrophy and function in experimental heart failure

Muscle atrophy is a determinant of exercise capacity in heart failure (CHF). Myocyte apoptosis, triggered by tumor necrosis factor- (TNF-) or its second messenger sphingosine (SPH), is one of the causes of atrophy. Growth hormone (GH) improves hemodynamic and cardiac trophism in several experimental models of CHF, but its effect on skeletal muscle in CHF is not yet clear. We tested the hypothesis that GH can prevent skeletal muscle apoptosis in rats with CHF. CHF was induced by injecting monocrotaline. After 2 wk, 2 groups of rats were treated with GH (0.2 mg·kg^–1·day^–1 and 1.0 mg·kg^–1·day^–1) subcutaneously. A third group of controls had saline. After 2 additional weeks, rats were killed. Tibialis anterior cross-sectional area, myosin heavy chain (MHC) composition, and a study on myocyte apoptosis and serum levels of TNF- and SPH were carried out. The number of apoptotic nuclei, muscle atrophy, and serum levels of TNF- and SPH were decreased with GH at high but not at low doses compared with CHF rats. Bcl-2 was increased, whereas activated caspases and bax were decreased. The MHC pattern in GH-treated animals was similar to that of controls. Monocrotaline slowed down both contraction and relaxation but did not affect specific tetanic force, whereas absolute force was decreased. GH treatment restored contraction and relaxation to control values and brought muscle mass and absolute twitch and tetanic tension to normal levels. These findings may provide an insight into the therapeutic strategy of GH given to patients with CHF to improve exercise capacity. apoptosis; cytokines; myosin heavy chains     

Secretion of endothelin-1 in human endothelial cell line but not in B cell line by transfection of preproendothelin-1 cDNA.

Stable transformants with preproendothelin-1 (preproET-1) cDNA were established for the study of the regulation of endothelin-1 (ET-1) biosynthesis in human cells. ET-1, a potent vasoconstrictor peptide, is produced by endothelial cells and is secreted into the blood at a low level. Human preproET-1 cDNA was introduced into two immortal human cell lines, t-HUE2, an endothelial cell line, and Raji, a B cell line, with Ecogpt selection. Several stable transformants of t-HUE2 expressed extraordinarily high levels of preproET-1 specific mRNA and secreted ET-1 into serum-free culture medium, while the transformants of Raji cells expressed high levels of ET-1 mRNA, but secreted a negligible amount of ET-1. Immunocytochemical studies of intracellular ET-1 content revealed that there were some defects in the translation or processing of preproET-1 in the B cell line transformants. In addition, the ratio of ET-1 to ET-1 precursor (big ET-1) was much higher in the t-HUE2 transformants than in normal endothelial cells, suggesting that t-HUE2 transformants (for example t-HUE2-1) possess high levels of endothelin converting enzyme (ECE). The establishment of stable transformants producing high levels of ET-1 in serum-free medium will be useful for the study of cell-type-specific translation and processing to mature ET-1, and of the regulatory factors of ECE.