A von Willebrand factor-derived heparin-binding peptide regulates cell--substrate adhesive strength and chemokinesis behavior

The ability of a soluble heparin-binding oligopeptide sequence derived from the von Willebrand factor (vWF) to modulate the adhesion and chemokinetic migration behavior of arterial smooth muscle cells was assessed using a novel glass microsphere centrifugation assay and automated time-lapse fluorescence videomicroscopy, respectively. Treatment of cells grown on fibronectin-coated substrates with the heparin-binding peptide resulted in the disassembly of focal adhesions, as assessed by immunohistochemical staining. These observations were consistent with six-fold decrease in cell--substrate adhesive strength (P<0.001), a biphasic effect on migration speed (P<0.05), as well as a dose-dependent reduction in the percentage of motile cells and the cell dispersion coefficient (mu=S(2)T/2). The specificity of this response to the vWF-derived heparin-binding peptide was supported by the absence of an observed effect in the presence of either a scrambled peptide or a consensus heparin-binding peptide sequence of similar heparin affinity. These data support the notion that competitive interactions between cell surface heparan sulfates with heparin-binding peptide domains located in soluble peptide fragments may modulate chemokinetic cell migration behavior and other adhesion-related processes.     

Acetylcholine and substance P stimulate bronchial epithelial cells to release eosinophil chemotactic activity

Weinvestigated a role of neuroregulation in the release of eosinophilchemotactic activity (ECA) from bovine bronchial epithelial cells(BBEC). BBEC were stimulated with acetylcholine (ACh) and substance P(SP), and the supernatant fluids were tested for ECA by a blind-wellchemotactic chamber technique. BBEC released ECA in response to ACh andSP in a dose- and time-dependent manner. Checkerboard analysis showedthat ECA in regard to ACh and SP was chemotactic rather thanchemokinetic. Partial characterization revealed that ECA involved bothlipids and peptides. The release of ECA in response to ACh and SP wasinhibited by nonspecific and 5-specific lipoxygenase inhibitors and bycycloheximide (P < 0.01). Molecular-sieve columnchromatography revealed that these mediators induced three molecularmass peaks (near 25 kDa, 9 kDa, and 400 Da, respectively). The lowestpeak, which represented the predominant activity, was blocked byleukotriene B₄-receptor antagonist (P < 0.01) but not by platelet-activating factor-receptor antagonist. The releaseof leukotriene B₄ in the supernatant fluids was increased in response to ACh and SP stimulation (P < 0.01).Platelet-activating factor was not detected. These results raise thepossibility of a role of neuroregulation for the elaboration of ECA inthe airway.

     

Myosin molecular motor dysfunction in dystrophic mouse diaphragm

Cross-bridge properties and myosin heavy chain (MHC) compositionwere investigated in isolated diaphragm from 6-mo-old control (n = 12) andmdx(n = 12) mice. Compared with control,peak tetanic tension fell by 50% inmdx mice(P < 0.001). The total number ofcross bridges per square millimeter(×10⁹), the elementaryforce per cross bridge, and the peak mechanical efficiency were lowerin mdx than in control mice (eachP < 0.001). The duration of thecycle and the rate constant for cross-bridge detachment weresignificantly lower in mdx than incontrol mice. In the overall population, there was a linearrelationship between peak tetanic tension and either total number ofcross bridges per square millimeter or elementary force per crossbridge (r = 0.996 andr = 0.667, respectively, eachP < 0.001). Themdx mice presented a higher proportionof type IIA MHC (P < 0.001) thancontrol mice and a reduction in type IIX MHC(P < 0.001) and slowmyosin isoforms (P < 0.01) comparedwith control mice. We concluded that, inmdx mice, impaired diaphragm strengthwas associated with qualitative and quantitative changes in myosin molecular motors. It is proposed that reduced force generated per crossbridge contributed to diaphragm weakness inmdx mice.

     

Influence of diet and exercise on skeletal muscle and visceral adipose tissue in men

Ross, Robert, John Rissanen, Heather Pedwell, JenniferClifford, and Peter Shragge. Influence of diet and exercise onskeletal muscle and visceral adipose tissue in men. J. Appl. Physiol. 81(6): 2445-2455, 1996.Theeffects of diet only (DO) and diet combined with either aerobic (DA) orresistance (DR) exercise on subcutaneous adipose tissue (SAT), visceraladipose tissue (VAT), lean tissue (LT), and skeletal muscle (SM) tissue were evaluated in 33 obese men (DO, n = 11; DA, n = 11; DR,n = 11). All tissues were measured byusing a whole body multislice magnetic resonance imaging (MRI) model.Within each group, significant reductions were observed for bodyweight, SAT, and VAT (P < 0.05). Thereductions in body weight (~10%) and SAT (~25%) and VAT volume (~35%) were not different between groups(P > 0.05). For alltreatments, the relative reduction in VAT was greater than in SAT(P < 0.05). For the DA and DR groupsonly, the reduction in abdominal SAT (~27%) was greater(P < 0.05) than thatobserved for the gluteal-femoral region (~20%). Conversely, thereduction in VAT was uniform throughout the abdomen regardless oftreatment (P > 0.05). MRI-LT andMRI-SM decreased both in the upper and lower body regions for the DO group alone (P < 0.05). PeakO₂ uptake (liters) wassignificantly improved (~14%) in the DA group as was muscularstrength (~20%) in the DR group (P < 0.01). These findings indicate that DA and DR result in a greaterpreservation of MRI-SM, mobilization of SAT from the abdominal region,by comparison with the gluteal-femoral region, and improved functionalcapacity when compared with DO in obese men.

     

Plasticity in Skeletal, Cardiac, and Smooth Muscle: Selected Contribution: IGF-I antibody prevents increases in protein synthesis in epitrochlearis muscles from refed, diabetic rats

The purpose of this study was to examine whether immune neutralizationof muscle-produced insulin-like growth factor I (IGF-I) would preventan appropriate anabolic response to refeeding in diabetic rats. MaleSprague-Dawley rats were made diabetic by partial pancreatectomy andwere randomly assigned to be either control-fed, fasted, orfasted-refed (n = 7-8 per group). Diabetes decreased rates of protein synthesis and increased rates of protein degradation in incubated epitrochlearis muscles (P < 0.05). In both groups of rats, fasting lowered protein synthesis andincreased proteolysis and subsequent refeeding returned both parameters to near basal values (P < 0.05). Neutralization ofmuscle IGF-I by the addition of IGF-I antibody to the incubation mediumreduced protein synthesis an average of 22% for all groups(P < 0.05). However, rates of protein degradation werenot affected. In nondiabetic rats, refeeding increased proteinsynthesis in both control and antibody-treated muscles(P < 0.05). Refeeding also increased protein synthesisin the control muscles from diabetic rats (P < 0.01).In contrast, muscles from diabetic rats that were incubated withanti-IGF-I did not increase protein synthesis in response to refeeding.These data suggest that immune neutralization of muscle IGF-I inhypoinsulinemic rats negated the ability of endogenous IGF-I to promoteprotein synthesis and thereby prevented an appropriate anabolic response.

     

Mechanical loading: biphasic osteocyte survival and targeting of osteoclasts for bone destruction in rat cortical bone

Bone isremoved or replaced in defined locations by targeting osteoclasts andosteoblasts in response to its local history of mechanical loading.There is increasing evidence that osteocytes modulate this targeting bytheir apoptosis, which is associated with locally increasedbone resorption. To investigate the role of osteocytes in the controlof loading-related modeling or remodeling, we studied the effects onosteocyte viability of short periods of mechanical loading applied tothe ulnae of rats. Loading, which produced peak compressive strains of0.003 or 0.004, was associated with a 78% reduction in theresorption surface at the midshaft. The same loading regimen resultedin a 40% relative reduction in osteocyte apoptosis at the samesite 3 days after loading compared with the contralateral side(P = 0.01). The proportion of osteocytes that wereapoptotic was inversely related to the estimated local strain(P < 0.02). In contrast, a single short period ofloading resulting in strains of 0.008 engendered both tissuemicrodamage and subsequent bone remodeling and was associated with aneightfold increase in the proportion of apoptotic osteocytes(P = 0.02) at 7 days. This increase in osteocyteapoptosis was transient and preceded both intracorticalremodeling and death of half of the osteocytes (P < 0.01). The data suggest that osteocytes might use their U-shapedsurvival response to strain as a mechanism to influence boneremodeling. We hypothesize that this relationship reflects a causalmechanism by which osteocyte apoptosis regulates bone'sstructural architecture.

     

Leukocyte adhesion molecule expression during intense resistance exercise

We hypothesized that expression of L-selectinand very late antigen-4 (VLA-4) integrin adhesionmolecules would influence cell type-specific redistribution duringexercise. Women subjects performed six sets of 10-repetition maximumsquats. L-selectin and VLA-4 integrin were measured by using flowcytometry pre- and postexercise on peripheral blood neutrophils andlymphocytes (n = 29 subjects) andlymphocyte subsets (n = 70 subjects),respectively. Neutrophil concentration increased 41.8%(P < 0.001), whereas the percentexpressing L-selectin was unchanged (79%). Lymphocyte concentrationincreased 61.8% (P < 0.001). Thepercent of T cells expressing L-selectin decreased from 73.5 ± 8.9 to 68.2 ± 11.4% (P < 0.001); the combined population of natural killer and B cells expressing L-selectin decreased from 80.4 ± 22.5 to 62.7 ± 25.8% (P < 0.001).VLA-4 integrin was expressed by nearly all lymphocytes both pre- andpostexercise. The proportional decrease in L-selectin positive cellscould have resulted from 1) sheddingof L-selectin, 2) selective entry ofL-selectin-negative subsets, or 3)selective removal of L-selectin-positive subsets.

     

Training intensity-dependent and tissue-specific increases in lactate uptake and MCT-1 in heart and muscle

We investigatedthe effects of 3 wk of moderate- (21 m/min, 8% grade) andhighintensity treadmill training (31 m/min, 15% grade) on1) monocarboxylate transporter 1 (MCT-1) content in rat hindlimb muscles and the heart and2) lactate uptake in isolated soleus(Sol) muscles and perfused hearts. In the moderately trained groupMCT-1 was not increased in any of the muscles [Sol, extensor digitorum longus (EDL), and red (RG) and white gastrocnemius(WG)] (P > 0.05). Similarly,lactate uptake in Sol strips was also not increased(P > 0.05). In contrast, in theheart, MCT-1 (+36%, P < 0.05) andlactate uptake (+72%, P < 0.05)were increased with moderate training. In the highly trained group,MCT-1 (+70%, P < 0.05) and lactateuptake (+79%, P < 0.05) wereincreased in Sol. MCT-1 was also increased in RG (+94%,P < 0.05) but not in WG and EDL(P > 0.05). In the highly trainedgroup, heart MCT-1 (+44%, P < 0.05)and lactate uptake (+173%, P < 0.05) were increased. In conclusion, it has been shown that1) in both heart and skeletal musclelactate uptake is increased only when MCT-1 is increased; 2) training-induced increases inMCT-1 occurred at a lower training intensity in the heart than inskeletal muscle; 3) in the heart, lactate uptake was increased much more after high-intensity training than after moderate-intensity training, despite similar increases inheart MCT-1 with these two training intensities; and4) the increases in MCT-1 occurredindependently of any changes in the heart's oxidative capacity (asmeasured by citrate synthase activity).

     

Effects of ovariectomy and hindlimb unloading on skeletal muscle

Female rats(7-8 mo old, n = 40) wererandomly placed into the intact control (Int) and ovariectomizedcontrol (Ovx) groups. Two weeks after ovariectomy, animals were furtherdivided into intact 2-wk hindlimb unloaded (Int-HU) and ovariectomizedhindlimb unloaded (Ovx-HU). We hypothesized that there would be greater hindlimb unloading-related atrophy in Ovx than in Int rats. In situcontractile tests were performed on soleus (Sol), plantaris (Plan),peroneus longus (Per), and extensor digitorum longus (EDL) muscles.Body weight and Sol mass were ~22% larger in Ovx than in Int groupand ~18% smaller in both HU groups than in Int rats (Ovx × HUinteraction, P < 0.05), and therewas a similar trend in Plan muscle (P < 0.07). There were main effects (P < 0.05) for both ovariectomy (growth) and hindlimb unloading(atrophy) on gastrocnemius mass. Mass of the Per and EDL muscles wasunaffected by either ovariectomy or hindlimb unloading. Time to peaktwitch tension for EDL and one-half relaxation times for Sol, Plan,Per, and EDL muscles were faster (P < 0.05) in Ovx than in Int animals. The results suggest that1) ovariectomy led to similarincreases of ~20% in body weight and plantar flexor mass;2) hindlimb unloading may haveprevented ovariectomy-related muscle growth;3) greater atrophy may have occurredin Sol and Plan of Ovx animals compared with controls; and4) removal of ovarian hormonalinfluence decreased skeletal muscle contraction times.

     

Metabolic effects of physical training in ovariectomized and hyperestrogenic rats

This study was undertaken to evaluate theeffects of regular endurance-type exercise on glucose tolerance andglucose-stimulated insulin response (GSIR) in ovariectomized (OVX) ratswith and without estrogen replacement. To do that, OVX Sprague-Dawleyrats were compared with an OVX estradiol-treated group (OVXE2) and asham-operated (Sham) group. Each of these groups was subdivided into asedentary and a treadmill-trained (8 wk) group. Intravenous glucosetolerance tests (0.5 g/kg) were conducted in all rats 48 h afterthe last training session. Plasma levels of 17-estradiol and theuterus weight were significantly (P < 0.05) lower inOVX compared with results in Sham and significantly (P < 0.01) higher in OVXE2 (hyperestrogenic) compared with results inSham. Body weights were significantly (P < 0.01)different among groups, in the following decreasing order: OVX, Sham,and OVXE2. The average daily food intake was significantly(P < 0.01) increased in OVX rats compared with Sham,whereas estradiol treatment diminished this effect (P < 0.01). Exercise training was found to alter none of theabove-mentioned variables in all three experimental conditions. Although the mean integrated area under the glucose and insulin curveswas not affected by OVX, training induced a significant (P < 0.01) reduction in the mean integrated area underthe insulin curve in all three experimental conditions. It is concludedthat the positive effects of physical training on improving GSIR in OVXand hyperestrogenic animals are similar to what has been found in Sham.

     

Mechanical strain regulates syndecan-4 expression and shedding in smooth muscle cells through differential activation of MAP kinase signaling pathways

Syndecan-4 (S4) belongs to a family of transmembrane proteoglycans, acts as a coreceptor for growth factor binding as well as cell-matrix and cell-cell interactions, and is induced in neointimal smooth muscle cells (SMCs) after balloon catheter injury. We investigated S4 expression in SMCs in response to several force profiles and the role of MAP kinase signaling pathways in regulating these responses. S4 mRNA expression increased in response to 5% and 10% cyclic strain (4 h: 200 ± 34% and 182 ± 17%, respectively; P < 0.05) before returning to basal levels by 24 h. Notably, the SMC mechanosensor mechanism was reset after an initial 24-h "preconditioning" period, as evident by an increase in S4 gene expression following a change in cyclic stress from 10% to 20% (28 h: 181 ± 1%; P < 0.05). Mechanical stress induced a late decrease in cell-associated S4 protein levels (24 h: 70 ± 6%; P < 0.05), with an associated increase in S4 shedding (24 h: 537 ± 109%; P < 0.05). To examine the role of MAP kinases, cells were treated with U-0126 (ERK1/2 inhibitor), SB-203580 (p38 inhibitor), or JNKI I (JNK/SAPK inhibitor). Late reduction in cell-associated S4 levels was attributed to ERK1/2 and p38 signaling. In contrast, accelerated S4 shedding required both ERK1/2 (5-fold reduction in accelerated shedding; P < 0.05) and JNK/SAPK (4-fold reduction; P < 0.05) signaling. Given the varied functions of S4, stress-induced effects on SMC S4 expression and shedding may represent an additional component of the proinflammatory, growth-stimulating pathways that are activated in response to changes in the mechanical microenvironment of the vascular wall. heparan sulfate proteoglycan     

Enhanced brain natriuretic peptide response to peak exercise in heart transplant recipients

We investigatedthe atrial (ANP) and brain natriuretic peptides (BNP), catecholamines,heart rate, and blood pressure responses to graded upright maximalcycling exercise of eight matched healthy subjects andcardiac-denervated heart transplant recipients (HTR). Baseline heart rate and diastolic blood pressure, together with ANP(15.2 ± 3.7 vs. 4.4 ± 0.8 pmol/l;P < 0.01) and BNP (14.3 ± 2.6 vs. 7.4 ± 0.6 pmol/l; P < 0.01), were elevated in HTR, but catecholamine levels were similarin both groups. Peak exercise O₂uptake and heart rate were lower in HTR. Exercise-inducedmaximal ANP increase was similar in both groups (167 ± 34 vs. 216 ± 47%). Enhanced BNP increase was significant only in HTR (37 ± 8 vs. 16 ± 8%; P < 0.05).Similar norepinephrine but lower peak epinephrine levels were observedin HTR. ANP and heart rate changes from rest to 75% peak exercise werenegatively correlated (r = 0.76, P < 0.05),and BNP increase was correlated with left ventricular mass index(r = 0.83, P < 0.01) after hearttransplantation. Although ANP increase was notexaggerated, these data support the idea that the chronotropiclimitation secondary to sinus node denervation might stimulate ANPrelease during early exercise in HTR. Furthermore, the BNPresponse to maximal exercise, which is related to the left ventricularmass index of HTR, is enhanced after heart transplantation.

     

Effects of resistance training and chromium picolinate on body composition and skeletal muscle in older men

The effects of chromium picolinate (CrPic)supplementation and resistance training (RT) on skeletal muscle size,strength, and power and whole body composition were examined in 18 men(age range 56-69 yr). The men were randomly assigned(double-blind) to groups (n = 9) thatconsumed either 17.8 µmol Cr/day (924 µg Cr/day) as CrPic or alow-Cr placebo for 12 wk while participating twice weekly in ahigh-intensity RT program. CrPic increased urinary Cr excretion~50-fold (P < 0.001). RT-inducedincreases in muscle strength (P < 0.001) were not enhanced by CrPic. Arm-pull muscle power increased withRT at 20% (P = 0.016) but not at 40, 60, or 80% of the one repetition maximum, independent of CrPic.Knee-extension muscle power increased with RT at 20, 40, and 60%(P < 0.001) but not at 80% of onerepetition maximum, and the placebo group gained more muscle power thandid the CrPic group (RT by supplemental interaction,P < 0.05). Fat-free mass(P < 0.001), whole body muscle mass(P < 0.001), and vastus lateralistype II fiber area (P < 0.05)increased with RT in these body-weight-stable men, independent ofCrPic. In conclusion, high-dose CrPic supplementation did not enhancemuscle size, strength, or power development or lean body mass accretionin older men during a RT program, which had significant, independenteffects on these measurements.

     

Influence of voluntary exercise on hypothalamic norepinephrine

We combined hypothalamic tissue and plasma determinations ofnorepinephrine, dihydroxyphenylalanine, and dihydroxyphenylglycol withmeasurements of abdominal fat in voluntary running rats to examine therelationship among exercise training, hypothalamic and sympatheticnervous function, and body fat stores. The hypothalamic concentrationsof norepinephrine, dihydroxyphenylalanine, and dihydroxyphenylglycolwere reduced after exercise training(P < 0.01), with the amount ofnorepinephrine being strongly associated with the plasma norepinephrine(r = 0.58, P < 0.05) and dihydroxyphenylglycol (r = 0.65, P = 0.01) concentrations. Exercisetraining resulted in a diminution in abdominal fat mass(P < 0.01). A strongrelationship existed between fat mass and hypothalamic norepinephrinecontent (r = 0.83, P < 0.001). The presence of apositive relationship between the arterial and hypothalamicnorepinephrine levels provides presumptive evidence of an associationbetween noradrenergic neuronal activity of the hypothalamus andsympathetic nervous function. The observation that abdominal fat massis linked with norepinephrine in the hypothalamus raises thepossibility that alterations in body fat stores provide an afferentsignal linking hypothalamic function and the activity of thesympathetic nervous system.

     

Neuromuscular fatigue after maximal stretch-shortening cycle exercise

Strojnik, V., and P. V. Komi. Neuromuscular fatigueafter maximal stretch-shortening cycle exercise. J. Appl. Physiol. 84(1): 344-350, 1998.To examinesome possible sites of fatigue during short-lasting maximally intensivestretch-shortening cycle exercise, drop jumps on an inclined sledgeapparatus were analyzed. Twelve healthy volunteers performed jumpsuntil they were unable to maintain jumping height >90% of theirmaximum. After the workout, the increases in the blood lactateconcentration and serum creatine kinase activation were statisticallysignificant (P < 0.001 and P < 0.05, respectively) but rathersmall in physiological terms. The major changes after the workout wereas follows: the single twitch was characterized by smaller peak torque(P < 0.05) and shorter time to peak(P < 0.05) and half-relaxation time(P < 0.01). The double-twitch torqueremained at the same level (P > 0.05), but with a steeper maximal slope of torque rise(P < 0.05); during 20- and 100-Hzstimulation the torque declined (both P < 0.01) and the maximal voluntarytorque changed nonsignificantly but with a smaller maximal slope oftorque rise (P < 0.01) and a higheractivation level (P < 0.05),accompanied by an increased electromyogram amplitude. These findingsindicate that the muscle response after the short-lasting consecutivemaximum jumps on the sledge apparatus may involve two distinctmechanisms acting in opposite directions:1) The contractile mechanism seemsto be potentiated through a shorterCa²⁺ transient and fastercross-bridge cycling, as implied by twitch changes.2) High-frequency action potentialpropagation shows an impairment, which is suggested as the possibledominant reason for fatigue in exercise of this type.

     

Aerosol probes of lung injury in a 28-wk longitudinal study of mild experimental emphysema in dogs

After baseline measurements of lung mechanics,effective air space diameter (EAD), and aerosol dispersion (AD), threedogs were exposed to two treatments of aerosolized papain (3 ml of a4% solution), and measurements were repeated during a 28-wk follow-upperiod. EAD and AD were measured with boluses of 0.7-µm particles ofdi-2-ethylhexl sebacate, with Pen (i.e., volumetric boluspenetration/total lung capacity) between 0.1 and 0.4. After papainexposure, EAD increased a mean of 28%(P < 0.0001) and AD (Pen = 0.3, 0.4)increased 4-7% (P < 0.03). Theprogression of injury was indicated by increasing trends in total lungcapacity (P < 0.05), residual volume(P < 0.05), and EAD(P = 0.06) through week 18. There was no evidence ofdisease progression between weeks 18 and 28, whereas some of the data forindividual dogs suggested partial recovery from lung injury atweek 28. The results show that aerosolprobes can detect and characterize mild lung injury in experimental emphysema.     

Effect of mechanical deformation on structure and function of polymorphonuclear leukocytes

Kitagawa, Yuko, Stephan F. Van Eeden, Darlene M. Redenbach,Maleki Daya, Blair A. M. Walker, Maria E. Klut, Barry R. Wiggs, andJames C. Hogg. Effect of mechanical deformation on structure andfunction of polymorphonuclear leukocytes. J. Appl.Physiol. 82(5): 1397-1405, 1997.The presentstudies were designed to test the hypothesis that mechanicaldeformation of polymorphonuclear leukocytes (PMN) leads to functionalchanges that might influence their transit in the pulmonarycapillaries. Human leukocytes were passed through 5- or 3-µm-porepolycarbonate filters under controlled conditions. Morphometricanalysis showed that the majority of PMN were deformed and that thisdeformation persisted longer after filtration through 3-µm filtersthan through 5-µm filters (P < 0.05) but did not result in the cytoskeletal polarizationcharacteristic of migrating cells. Flow cytometric studies of thefiltered PMN showed that there was a transient increase in thecytosolic free Ca²⁺ concentrationafter both 3- and 5-µm filtration (P < 0.01) with an increase in F-actin content after 3-µm filtration(P < 0.05). AlthoughL-selectin expression on PMN wasnot changed by either 5- or 3-µm filtration, CD18 and CD11b wereincreased by 3-µm filtration (P < 0.05). Priming of the PMN withN-formyl-methionyl-leucyl-phenylalanine (0.5 nM) before filtration resulted in an increase of CD11b by both 5 (P < 0.05)- and 3-µm(P < 0.01) filtration. Neither 5- nor 3-µm filtration induced hydrogen peroxide production. We conclude that mechanical deformation of PMN, similar to what occurs in thepulmonary microvessels, induces both structural and functional changesin the cells, which might influence their passage through the pulmonarycapillary bed.

     

Metabolic effects of low cortisol during exercise in humans

This studyexamined the physiological effect of reduced plasma cortisol (C) duringprolonged exercise in humans. The effects of normal C (NC) werecompared with metyrapone-induced low C (LC) on plasma substrateavailability and the respiratory exchange ratio during 2 h of exerciseat ~60% peak O₂ consumption innine subjects. The C responses were compared with preexercise (Pre) levels and with a rest day (Con). At rest, C was attenuated by ~70%for LC compared with NC. At rest, plasma glucose, lactate, glycerol,-hydroxybutyrate, alanine, branched-chain amino acids, insulin,glucagon, growth hormone, epinephrine, and norepinephrine were similarunder LC and NC (P > 0.05). Duringexercise under NC, plasma C increased compared with Pre, whereas itremained unchanged during LC. During NC, plasma C was elevated at 90 min (compared with Con) and at 120 min (compared with Con and Pre). During exercise, plasma glucose decreased to the same extent and lactate was similar under both conditions, whereas plasma glycerol, -hydroxybutyrate, alanine, and branched-chain amino acids were higher (P < 0.01) under NC. Plasmainsulin declined (P = 0.01) to agreater extent under LC, whereas growth hormone, epinephrine, andnorepinephrine tended to be higher (0.05  P  0.10). Plasma glucagon increasedunder both conditions (P < 0.01).The respiratory exchange ratio did not differ between conditions. Weconclude that, during exercise, 1) Caccelerates lipolysis, ketogenesis, and proteolysis;2) under LC, glucoregulatory hormoneadjustments maintain glucose homeostasis; and3) LC does not alter whole body substrate utilization or the ability to complete 2 h of moderate exercise.

     

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation

Tight junctions serve as the rate-limiting barrier to passivemovement of hydrophilic solutes across intestinal epithelia. Afteractivation of Na⁺-glucosecotransport, the permeability of intestinal tight junctions isincreased. Because previous analyses of this physiological tightjunction regulation have been restricted to intact mucosae, dissectionof the mechanisms underlying this process has been limited. Tocharacterize this process, we have developed a reductionist modelconsisting of Caco-2 intestinal epithelial cells transfected with theintestinal Na⁺-glucosecotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstratephysiological Na⁺-glucosecotransport. Activation of SGLT1 results in a 22 ± 5% fall intransepithelial resistance (TER) (P < 0.001). Similarly, inactivation of SGLT1 by addition of phloridzinincreases TER by 24 ± 2% (P < 0.001). The increased tight junction permeability is size selective,with increased flux of small nutrient-sized molecules, e.g., mannitol,but not of larger molecules, e.g., inulin. SGLT1-dependent increases intight junction permeability are inhibited by myosin light-chain kinaseinhibitors (20 µM ML-7 or 40 µM ML-9), suggesting that myosinregulatory light-chain (MLC) phosphorylation is involved in tightjunction regulation. Analysis of MLC phosphorylation showed a 2.08-foldincrease after activation of SGLT1 (P < 0.01), which was inhibited by ML-9(P < 0.01). Thus monolayersincubated with glucose and myosin light-chain kinase inhibitors arecomparable to monolayers incubated with phloridzin. ML-9 also inhibitsSGLT1-mediated tight junction regulation in small intestinal mucosa(P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junctionregulation subsequent to SGLT1 activation. The intimate relationshipbetween tight junction regulation and MLC phosphorylation suggests thata critical step in regulation of epithelial tight junction permeabilitymay be myosin ATPase-mediated contraction of the perijunctionalactomyosin ring and subsequent physical tension on the tight junction.

     

High glucose and endothelial cell growth: novel effects independent of autocrine TGF-beta 1 and hyperosmolarity

Human endothelial cells wereexposed to 5 mM glucose (control), 25 mM (high) glucose, or osmoticcontrol for 72 h. TGF-1 production, cell growth, death, andcell cycle progression, and the effects of TGF-1 and TGF-neutralization on these parameters were studied. High glucose andhyperosmolarity increased endothelial TGF-1 secretion(P < 0.0001) and bioactivity (P < 0.0001). However, high glucose had a greater effect on reducingendothelial cell number (P < 0.001) and increasingcellular protein content (P < 0.001) than the osmoticcontrol. TGF- antibody only reversed the antiproliferative andhypertrophic effects of high glucose. High glucose altered cell cycleprogression and cyclin-dependent kinase inhibitor expressionindependently of hyperosmolarity. High glucose increased endothelialcell apoptosis (P < 0.01), whereashyperosmolarity induced endothelial cell necrosis (P < 0.001). TGF- antibody did not reverse the apoptotic effectsobserved with high glucose. Exogenous TGF-1 mimicked the increased Sphase delay but not endoreduplication observed with high glucose. High glucose altered endothelial cell growth, apoptosis, and cellcycle progression. These growth effects occurred principally via aTGF-1 autocrine pathway. In contrast, apoptosis andendoreduplication occurred independently of this cytokine and hyperosmolarity.