Effects of GLP-1 Infusion Upon Whole-body Glucose Uptake and Skeletal Muscle Perfusion During Fed-state in Older Men.

INTRODUCTION: Ageing skeletal muscles become both insulin resistant and atrophic. The hormone glucagon-like peptide 1 (GLP-1) facilitates postprandial glucose uptake as well as augmenting muscle perfusion, independent of insulin action. We thus hypothesized exogenous GLP-1 infusions would enhance muscle perfusion and positively affect glucose metabolism during fed-state clamps in older people. METHODS: Eight men (71 ± 1 years) were studied in a randomized crossover trial. Basal blood samples were taken before postprandial (fed-state) insulin and glucose clamps, accompanied by amino acid infusions, for 3 hours. Reflecting this, following insertions of peripheral and femoral vessels cannulae and baseline measurements, peripheral IV infusions of octreotide, insulin (Actrapid), 20% glucose, and mixed amino acids; Vamin 14-EF with or without a femoral arterial GLP-1 infusion were started. GLP-1, insulin, and C-peptide were measured by ELISA. Muscle microvascular blood flow was assessed via contrast enhanced ultrasound. Whole-body glucose handling was assayed by assessing glucose infusion rate parameters. RESULTS: Skeletal muscle microvascular blood flow significantly increased in response to GLP-1 vs feeding alone (5.0 ± 2.1 vs 1.9 ± 0.7 fold-change from basal, respectively; P = 0.008), while also increasing whole-body glucose uptake (area under the curve 16.9 ± 1.7 vs 11.4 ± 1.8 mg/kg-1/180 minutes-1, P = 0.02 ± GLP, respectively). CONCLUSIONS: The beneficial effects of GLP-1 on whole-body glycemic control are evident with insulin clamped at fed-state levels. GLP-1 further enhances the effects of insulin on whole-body glucose uptake in older men, underlining its role as a therapeutic target. The effects of GLP-1 in enhancing microvascular flow likely also affects other glucose-regulatory organs, reflected by greater whole-body glucose uptake.

Glucagon-like peptide 1 infusions overcome anabolic resistance to feeding in older human muscle.

BACKGROUND: Despite its known insulin-independent effects, glucagon-like peptide-1 (GLP-1) role in muscle protein turnover has not been explored under fed-state conditions or in the context of older age, when declines in insulin sensitivity and protein anabolism, as well as losses of muscle mass and function, occur. METHODS: Eight older-aged men (71 ± 1 year, mean ± SEM) were studied in a crossover trial. Baseline measures were taken over 3 hr, prior to a 3 hr postprandial insulin (~30 mIU ml-1 ) and glucose (7-7.5 mM) clamp, alongside I.V. infusions of octreotide and Vamin 14 (±infusions of GLP-1). Four muscle biopsies were taken, and muscle protein turnover was quantified via incorporation of 13 C6 phenylalanine and arteriovenous balance kinetics, using mass spectrometry. Leg macro- and microvascular flow was assessed via ultrasound and anabolic signalling by immunoblotting. GLP-1 and insulin were measured by ELISA. RESULTS: GLP-1 augmented muscle protein synthesis (MPS; fasted: 0.058 ± 0.004% hr-1 vs. postprandial: 0.102 ± 0.005% hr-1 , p < 0.01), in comparison with non-GLP-1 trials. Muscle protein breakdown (MPB) was reduced throughout clamp period, while net protein balance across the leg became positive in both groups. Total femoral leg blood flow was unchanged by the clamp; however, muscle microvascular blood flow (MBF) was significantly elevated in both groups, and to a significantly greater extent in the GLP-1 group (MBF: 5 ± 2 vs. 1.9 ± 1 fold change +GLP-1 and -GLP-1, respectively, p < 0.01). Activation of the Akt-mTOR signalling was similar across both trials. CONCLUSION: GLP-1 infusion markedly enhanced postprandial microvascular perfusion and further stimulated muscle protein metabolism, primarily through increased MPS, during a postprandial insulin hyperaminoacidaemic clamp.

The effect of acute oral phosphatidic acid ingestion on myofibrillar protein synthesis and intracellular signaling in older males.

BACKGROUND: Age-related muscle loss (sarcopenia) may be driven by a diminished myofibrillar protein synthesis (MyoPS) response to anabolic stimuli (i.e. exercise and nutrition). Oral phosphatidic acid (PA) ingestion has been reported to stimulate resting muscle protein synthesis in rodents, and enhance resistance training-induced muscle remodelling in young humans. PURPOSE: This study examined the effects of acute oral PA ingestion on resting and exercise-induced MyoPS rates in older individuals. METHODS: Sixteen older males performed a bout of unilateral leg resistance exercise followed by oral ingestion of 750 mg of soy-derived PA or a rice-flour placebo (PL) over 60 min post-exercise. A primed-continuous infusion of l-[ring-13C6]-phenylalanine with serial muscle biopsies was used to determine MyoPS at rest and between 0-150 and 150-300 min post-exercise. RESULTS: Plasma [PA] concentrations were elevated above basal values from 180 to 300 min post-exercise in PA only (P = 0.02). Exercise increased MyoPS rates above basal values between 150 and 300 min post-exercise in PL (P = 0.001), but not PA (P = 0.83). Phosphorylation of p70S6K, rpS6, 4E-BP1 and Akt was elevated above basal levels in the exercised leg over 150-300 min post-exercise for PL only (P = 0.018, 0.007, 0.011 and 0.002, respectively), and were significantly greater than PA (P < 0.01 for all proteins). The effects of oral PA ingestion on proteolytic signaling markers were equivocal. CONCLUSIONS: Acute oral phosphatidic acid ingestion appears to interfere with resistance exercise-induced intramuscular anabolic signaling and MyoPS in older males and, therefore, may not be a viable treatment to counteract sarcopenia. Clinicaltials.gov registration no: NCT03446924.

Effects of leucine-enriched essential amino acid and whey protein bolus dosing upon skeletal muscle protein synthesis at rest and after exercise in older women.

BACKGROUND & AIMS: Impaired anabolic responses to nutrition and exercise contribute to loss of skeletal muscle mass with ageing (sarcopenia). Here, we tested responses of muscle protein synthesis (MPS), in the under represented group of older women, to leucine-enriched essential amino acids (EAA) in comparison to a large bolus of whey protein (WP). METHODS: Twenty-four older women (65 ± 1 y) received (N = 8/group) 1.5 g leucine-enriched EAA supplements (LEAA_1.5), 6 g LEAA (LEAA_6) in comparison to 40 g WP. A primed constant I.V infusion of 13C6-phenylalanine was used to determine MPS at baseline and in response to feeding (FED) and feeding-plus-exercise (FED-EX; 6 × 8 unilateral leg extensions; 75%1-RM). We quantified plasma insulin/AA concentrations, leg femoral blood flow (LBF)/muscle microvascular blood flow (MBF), and anabolic signalling via immunoblotting. RESULTS: Plasma insulineamia and EAAemia were greater and more prolonged with WP than LEAA, although LEAA_6 peaked at similar levels to WP. Neither LEAA or WP modified LBF or MBF. FED increased MPS similarly in the LEAA_1.5, LEAA_6 and WP (P < 0.05) groups over 0-2 h, with MPS significantly higher than basal in the LEAA_6 and WP groups only over 0-4 h. However, FED-EX increased MPS similarly across all the groups from 0 to 4 h (P < 0.05). Only p-p70S6K1 increased with WP at 2 h in FED (P < 0.05), and at 2/4 h in FED-EX (P < 0.05). CONCLUSIONS: In conclusion, LEAA_1.5, despite only providing 0.6 g of leucine, robustly (perhaps maximally) stimulated MPS, with negligible trophic advantage of greater doses of LEAA or even to 40 g WP. Highlighting that composition of EAA, in particular the presence of leucine rather than amount is most crucial for anabolism.

Fuel for the work required: a practical approach to amalgamating train-low paradigms for endurance athletes.

Using an amalgamation of previously studied "train-low" paradigms, we tested the effects of reduced carbohydrate (CHO) but high leucine availability on cell-signaling responses associated with exercise-induced regulation of mitochondrial biogenesis and muscle protein synthesis (MPS). In a repeated-measures crossover design, 11 males completed an exhaustive cycling protocol with high CHO availability before, during, and after exercise (HIGH) or alternatively, low CHO but high protein (leucine enriched) availability (LOW + LEU). Muscle glycogen was different (P < 0.05) pre-exercise (HIGH: 583 ± 158, LOW + LEU: 271 ± 85 mmol kg(-1) dw) but decreased (P < 0.05) to comparable levels at exhaustion (≈100 mmol kg(-1) dw). Despite differences (P < 0.05) in exercise capacity (HIGH: 158 ± 29, LOW + LEU: 100 ± 17 min), exercise induced (P < 0.05) comparable AMPKα2 (3-4-fold) activity, PGC-1α (13-fold), p53 (2-fold), Tfam (1.5-fold), SIRT1 (1.5-fold), Atrogin 1 (2-fold), and MuRF1 (5-fold) gene expression at 3 h post-exercise. Exhaustive exercise suppressed p70S6K activity to comparable levels immediately post-exercise (≈20 fmol min(-1) mg(-1)). Despite elevated leucine availability post-exercise, p70S6K activity remained suppressed (P < 0.05) 3 h post-exercise in LOW + LEU (28 ± 14 fmol min(-1) mg(-1)), whereas muscle glycogen resynthesis (40 mmol kg(-1) dw h(-1)) was associated with elevated (P < 0.05) p70S6K activity in HIGH (53 ± 30 fmol min(-1) mg(-1)). We conclude: (1) CHO restriction before and during exercise induces "work-efficient" mitochondrial-related cell signaling but; (2) post-exercise CHO and energy restriction maintains p70S6K activity at basal levels despite feeding leucine-enriched protein. Our data support the practical concept of "fuelling for the work required" as a potential strategy for which to amalgamate train-low paradigms into periodized training programs.

Acute cocoa flavanol supplementation improves muscle macro- and microvascular but not anabolic responses to amino acids in older men.

The anabolic effects of nutrition on skeletal muscle may depend on adequate skeletal muscle perfusion, which is impaired in older people. Cocoa flavanols have been shown to improve flow-mediated dilation, an established measure of endothelial function. However, their effect on muscle microvascular blood flow is currently unknown. Therefore, the objective of this study was to explore links between the consumption of cocoa flavanols, muscle microvascular blood flow, and muscle protein synthesis (MPS) in response to nutrition in older men. To achieve this objective, leg blood flow (LBF), muscle microvascular blood volume (MBV), and MPS were measured under postabsorptive and postprandial (intravenous Glamin (Fresenius Kabi, Germany), dextrose to sustain glucose ∼7.5 mmol·L(-1)) conditions in 20 older men. Ten of these men were studied with no cocoa flavanol intervention and a further 10 were studied with the addition of 350 mg of cocoa flavanols at the same time that nutrition began. Leg (femoral artery) blood flow was measured by Doppler ultrasound, muscle MBV by contrast-enhanced ultrasound using Definity (Lantheus Medical Imaging, Mass., USA) perflutren contrast agent and MPS using [1, 2-(13)C2]leucine tracer techniques. Our results show that although older individuals do not show an increase in LBF or MBV in response to feeding, these absent responses are apparent when cocoa flavanols are given acutely with nutrition. However, this restoration in vascular responsiveness is not associated with improved MPS responses to nutrition. We conclude that acute cocoa flavanol supplementation improves muscle macro- and microvascular responses to nutrition, independently of modifying muscle protein anabolism.

Intake of low-dose leucine-rich essential amino acids stimulates muscle anabolism equivalently to bolus whey protein in older women at rest and after exercise.

Dysregulated anabolic responses to nutrition/exercise may contribute to sarcopenia; however, these characteristics are poorly defined in female populations. We determined the effects of two feeding regimes in older women (66 ± 2.5 yr; n = 8/group): bolus whey protein (WP-20 g) or novel low-dose leucine-enriched essential amino acids (EAA) [LEAA; 3 g (40% leucine)]. Using [(13)C6]phenylalanine infusions, we quantified muscle (MPS) and albumin (APS) protein synthesis at baseline and in response to both feeding (FED) and feeding plus exercise (FED-EX; 6 × 8 knee extensions at 75% 1-repetition maximum). We also quantified plasma insulin/AA concentrations, whole leg (LBF)/muscle microvascular blood flow (MBF), and muscle anabolic signaling by phosphoimmunoblotting. Plasma insulinemia and EAA/aemia were markedly greater after WP than LEAA (P < 0.001). Neither LEAA nor WP modified LBF in response to FED or FED-EX, whereas MBF increased to a similar extent in both groups only after FED-EX (P < 0.05). In response to FED, both WP and LEAA equally stimulated MPS 0-2 h (P < 0.05), abating thereafter (0-4 h, P > 0.05). In contrast, after FED-EX, MPS increased at 0-2 h and remained elevated at 0-4 h (P < 0.05) with both WP and LEAA. No anabolic signals quantifiably increased after FED, but p70 S6K1 Thr(389) increased after FED-EX (2 h, P < 0.05). APS increased similarly after WP and LEAA. Older women remain subtly responsive to nutrition ± exercise. Intriguingly though, bolus WP offers no trophic advantage over LEAA.

The effects of resistance exercise training on macro- and micro-circulatory responses to feeding and skeletal muscle protein anabolism in older men.

KEY POINTS: Increases in limb blood flow in response to nutrition are reduced in older age. Muscle microvascular blood flow (MBF) in response to nutrition is also reduced with advancing age and this may contribute to age-related 'anabolic resistance'. Resistance exercise training (RET) can rejuvenate limb blood flow responses to nutrition in older individuals. We report here that 20 weeks of RET also restores muscle MBF in older individuals. Restoration of MBF does not, however, enhance muscle anabolic responses to nutrition. ABSTRACT: The anabolic effects of dietary protein on skeletal muscle depend on adequate skeletal muscle perfusion, which is impaired in older people. This study explores fed state muscle microvascular blood flow, protein metabolism and exercise training status in older men. We measured leg blood flow (LBF), muscle microvascular blood volume (MBV) and muscle protein turnover under post-absorptive and fed state (i.v. Glamin to double amino acids, dextrose to sustain glucose ∼7-7.5 mmol l(-1) ) conditions in two groups: 10 untrained men (72.3 ± 1.4 years; body mass index (BMI) 26.5 ± 1.15 kg m(2) ) and 10 men who had undertaken 20 weeks of fully supervised, whole-body resistance exercise training (RET) (72.8 ± 1.4 years; BMI 26.3 ± 1.2 kg m(2) ). We measured LBF by Doppler ultrasound and muscle MBV by contrast-enhanced ultrasound. Muscle protein synthesis (MPS) was measured using [1, 2-(13) C2 ] leucine with breakdown (MPB) and net protein balance (NPB) by ring-[D5 ] phenylalanine tracers. Plasma insulin was measured via ELISA and indices of anabolic signalling (e.g. Akt/mTORC1) by immunoblotting from muscle biopsies. Whereas older untrained men did not exhibit fed-state increases in LBF or MBV, the RET group exhibited increases in both LBF and MBV. Despite our hypothesis that enhanced fed-state circulatory responses would improve anabolic responses to nutrition, fed-state increases in MPS (∼50-75%; P < 0.001) were identical in both groups. Finally, whereas only the RET group exhibited fed-state suppression of MPB (∼-38%; P < 0.05), positive NPB achieved was similar in both groups. We conclude that RET enhances fed-state LBF and MBV and restores nutrient-dependent attenuation of MPB without robustly enhancing MPS or NPB.

Leucine-enriched protein feeding does not impair exercise-induced free fatty acid availability and lipid oxidation: beneficial implications for training in carbohydrate-restricted states.

Given that the enhanced oxidative adaptations observed when training in carbohydrate (CHO)-restricted states is potentially regulated through free fatty acid (FFA)-mediated signalling and that leucine-rich protein elevates muscle protein synthesis, the present study aimed to test the hypothesis that leucine-enriched protein feeding enhances circulating leucine concentration but does not impair FFA availability or whole body lipid oxidation during exercise. Nine males cycled for 2 h at 70% VO2peak when fasted (PLACEBO) or having consumed a whey protein solution (WHEY) or a leucine-enriched whey protein gel (GEL), administered as 22 g 1 h pre-exercise, 11 g/h during and 22 g 30 min post-exercise. Total leucine administration was 14.4 g and 6.3 in GEL and WHEY, respectively. Mean plasma leucine concentrations were elevated in GEL (P = 0.001) compared with WHEY and PLACEBO (375 ± 100, 272 ± 51, 146 ± 14 µmol L(-1), respectively). No differences (P = 0.153) in plasma FFA (WHEY 0.53 ± 0.30, GEL 0.45 ± 0.25, PLACEBO 0.65 ± 0.30, mmol L(-1)) or whole body lipid oxidation during exercise (WHEY 0.37 ± 0.26, GEL 0.36 ± 0.24, PLACEBO 0.34 ± 0.24 g/min) were apparent between trials, despite elevated (P = 0.001) insulin in WHEY and GEL compared with PLACEBO (38 ± 16, 35 ± 16, 22 ± 11 pmol L(-1), respectively). We conclude that leucine-enriched protein feeding does not impair FFA availability or whole body lipid oxidation during exercise, thus having practical applications for athletes who deliberately train in CHO-restricted states to promote skeletal muscle adaptations.

Pharmacological enhancement of leg and muscle microvascular blood flow does not augment anabolic responses in skeletal muscle of young men under fed conditions.

Skeletal muscle anabolism associated with postprandial plasma aminoacidemia and insulinemia is contingent upon amino acids (AA) and insulin crossing the microcirculation-myocyte interface. In this study, we hypothesized that increasing muscle microvascular blood volume (flow) would enhance fed-state anabolic responses in muscle protein turnover. We studied 10 young men (23.2 ± 2.1 yr) under postabsorptive and fed [iv Glamin (∼10 g AA), glucose ∼7.5 mmol/l] conditions. Methacholine was infused into the femoral artery of one leg to determine, via bilateral comparison, the effects of feeding alone vs. feeding plus pharmacological vasodilation. We measured leg blood flow (LBF; femoral artery) by Doppler ultrasound, muscle microvascular blood volume (MBV) by contrast-enhanced ultrasound (CEUS), muscle protein synthesis (MPS) and breakdown (MPB; a-v balance modeling), and net protein balance (NPB) using [1,2-(13)C2]leucine and [(2)H5]phenylalanine tracers via gas chromatography-mass spectrometry (GC-MS). Indexes of anabolic signaling/endothelial activation (e.g., Akt/mTORC1/NOS) were assessed using immunoblotting techniques. Under fed conditions, LBF (+12 ± 5%, P < 0.05), MBV (+25 ± 10%, P < 0.05), and MPS (+129 ± 33%, P < 0.05) increased. Infusion of methacholine further enhanced LBF (+126 ± 12%, P < 0.05) and MBV (+79 ± 30%, P < 0.05). Despite these radically different blood flow conditions, neither increases in MPS in response to feeding (0.04 ± 0.004 vs. 0.08 ± 0.01%/h, P < 0.05) nor improvements in NPB (-4.4 ± 2.4 vs. 16.4 ± 5.7 nmol Phe·100 ml leg(-1)·min(-1), P < 0.05) were affected by methacholine infusion (MPS 0.07 ± 0.01%/h; NPB 24.0 ± 7.7 nmol Phe·100 ml leg(-1)·min(-1)), whereas MPB was unaltered by either feeding or infusion of methacholine. Thus, enhancing LBF/MBV above that occurring naturally with feeding alone does not improve muscle anabolism.

Acute skeletal muscle wasting in critical illness.

IMPORTANCE: Survivors of critical illness demonstrate skeletal muscle wasting with associated functional impairment. OBJECTIVE: To perform a comprehensive prospective characterization of skeletal muscle wasting, defining the pathogenic roles of altered protein synthesis and breakdown. DESIGN, SETTING, AND PARTICIPANTS: Sixty-three critically ill patients (59% male; mean age: 54.7 years [95% CI, 50.0-59.6 years]) with an Acute Physiology and Chronic Health Evaluation II score of 23.5 (95% CI, 21.9-25.2) were prospectively recruited within 24 hours following intensive care unit (ICU) admission from August 2009 to April 2011 at a university teaching and a community hospital in England. Patients were recruited if older than 18 years and were anticipated to be intubated for longer than 48 hours, to spend more than 7 days in critical care, and to survive ICU stay. MAIN OUTCOMES AND MEASURES: Muscle loss was determined through serial ultrasound measurement of the rectus femoris cross-sectional area (CSA) on days 1, 3, 7, and 10. In a subset of patients, the fiber CSA area was quantified along with the ratio of protein to DNA on days 1 and 7. Histopathological analysis was performed. In addition, muscle protein synthesis, breakdown rates, and respective signaling pathways were characterized. RESULTS: There were significant reductions in the rectus femoris CSA observed at day 10 (−17.7% [95% CI, −25.9% to 8.1%]; P < .001). In the 28 patients assessed by all 3 measurement methods on days 1 and 7, the rectus femoris CSA decreased by 10.3% (95% CI, 6.1% to 14.5%), the fiber CSA by 17.5% (95% CI, 5.8% to 29.3%), and the ratio of protein to DNA by 29.5% (95% CI, 13.4% to 45.6%). Decrease in the rectus femoris CSA was greater in patients who experienced multiorgan failure by day 7 (−15.7%; 95% CI, −27.7% to 11.4%) compared with single organ failure (−3.0%; 95% CI, −5.3% to 2.1%) (P < .001), even by day 3 (−8.7% [95% CI, −59.3% to 50.6%] vs −1.8% [95% CI, −12.3% to 10.5%], respectively; P = .03). Myofiber necrosis occurred in 20 of 37 patients (54.1%). Protein synthesis measured by the muscle protein fractional synthetic rate was depressed in patients on day 1 (0.035%/hour; 95% CI, 0.023% to 0.047%/hour) compared with rates observed in fasted healthy controls (0.039%/hour; 95% CI, 0.029% to 0.048%/hour) (P = .57) and increased by day 7 (0.076% [95% CI, 0.032%-0.120%/hour]; P = .03) to rates associated with fed controls (0.065%/hour [95% CI, 0.049% to 0.080%/hour]; P = .30), independent of nutritional load. Leg protein breakdown remained elevated throughout the study (8.5 [95% CI, 4.7 to 12.3] to 10.6 [95% CI, 6.8 to 14.4] µmol of phenylalanine/min/ideal body weight × 100; P = .40). The pattern of intracellular signaling supported increased breakdown (n = 9, r = −0.83, P = .005) and decreased synthesis (n = 9, r = −0.69, P = .04). CONCLUSIONS AND RELEVANCE: Among these critically ill patients, muscle wasting occurred early and rapidly during the first week of critical illness and was more severe among those with multiorgan failure compared with single organ failure. These findings may provide insights into skeletal muscle wasting in critical illness.

Naloxone as part of a prolonged release oxycodone/naloxone combination reduces oxycodone-induced slowing of gastrointestinal transit in healthy volunteers.

OBJECTIVES: This exploratory study in healthy volunteers investigated the effect of single doses of oxycodone on gastrointestinal (GI) transit time and the degree to which a single dose of naloxone reverses the oxycodone-induced effect. METHODS: Fifteen healthy male volunteers received: oxycodone 10 and2 0 mg, oxycodone/naloxone 10/5 and 20/10 mg (all as prolonged release tablets) and placebo. Each dose was radiolabelled and administered with a capsule containing radiolabelled resin (surrogate for GI contents). RESULTS: Scintigraphic analysis showed that 20 mg oxycodone significantly increased colon arrival time (mean 7.19 vs 5.15 h for placebo, p = 0.0159). Mean colon arrival time for oxycodone/naloxone 20/10 mg (5.16 h) was similar to placebo, although the difference between oxycodone/naloxone 20/10 mg versus oxycodone 20 mg was not significant (p = 0.0653). Colonic geometric centre analysis showed a significant increase in mean time for the resin to reach the colon following oxycodone 10 and 20 mg compared with placebo (increases of 5.3 and 8.8 h). There was no significant effect of naloxone at the lower dose; however, oxycodone/naloxone 20/10 mg significantly reduced mean colonic transit time by 2.1 h (p = 0.0376). CONCLUSION: A single dose of oxycodone 20 mg significantly prolonged GI transit time but this effect was reduced by co-administration of naloxone.

Toxigenic Helicobacter pylori infection precedes gastric hypochlorhydria in cancer relatives, and H. pylori virulence evolves in these families.

PURPOSE: Helicobacter pylori infection by virulent strains is associated with gastric adenocarcinoma. We aimed to determine whether infection with virulent H. pylori preceded precancerous gastric hypochlorhydria and atrophy in gastric cancer relatives and quantify the extent of virulence factor evolution. EXPERIMENTAL DESIGN: H. pylori strains from 51 Scottish gastric cancer relatives were characterized by genetic fingerprinting and typing the vacuolating cytotoxin gene (vacA), the cytotoxin-associated gene (cagA), and housekeeping genes. We phenotyped strains by coculture with gastric epithelial cells and assessing vacuolation (microscopy), CagA tyrosine phosphorylation (immunoblot), and interleukin-8 secretion (ELISA). RESULTS: Toxigenic (vacA type s1/m1) H. pylori was associated with precancerous gastric hypochlorhydria (P<0.01). Adult family members with this type of H. pylori had the same strain as currently noncohabiting adult family members in 68% cases, implying acquisition during childhood from each other or a common source. We analyzed different isolates of the same strain within families and showed that H. pylori commonly microevolved to change virulence: this occurred in 22% individuals and a striking 44% cases where the strain was shared within families. Microevolution in vacA occurred by extragenomic recombination and in cagA by this or duplication/deletion. Microevolution led to phenotypic changes in virulence. Passage of microevolved strains could be tracked within families. CONCLUSIONS: Toxigenic H. pylori infection precedes and so likely causes gastric hypochlorhydria, suggesting that virulent H. pylori increases cancer risk by causing this condition. Microevolution of virulence genes is common within families of gastric cancer patients and changes H. pylori virulence.

Determinants and consequences of different levels of CagA phosphorylation for clinical isolates of Helicobacter pylori.

BACKGROUND & AIMS: The Helicobacter pylori cag pathogenicity island encodes a secretory system that translocates CagA into epithelial cells, where it becomes tyrosine phosphorylated and induces cytoskeletal rearrangements. Strains with more CagA tyrosine phosphorylation motifs are most closely associated with gastric cancer. Here we assess whether clinical strains can deliver CagA, whether strains with different numbers of CagA phosphorylation motifs have CagA phosphorylated to different degrees, and whether this induces different amounts of epithelial cytoskeletal change. METHODS: Forty-four H. pylori strains from South African patients, all cagA gene positive, were cocultured with the gastric adenocarcinoma cell line AGS. CagA expression and phosphorylation were determined by Western blot and interleukin-8 secretion by enzyme-linked immunosorbent assay. The cagA 3' variable regions of 22 strains were sequenced and shown to possess 3-6 phosphorylation motifs. These strains were used to quantify CagA phosphorylation and cytoskeletal rearrangements. RESULTS: cagA genotype and typing of cag pathogenicity island genes were poorly predictive of phenotype. Thirty-four of 44 strains expressed CagA protein that could be delivered to and phosphorylated within AGS cells. Only these 34 strains induced interleukin-8 secretion from AGS cells. Among those strains, the number of CagA tyrosine phosphorylation motifs determined the degree of CagA phosphorylation and the level of biologic activity in terms of degree and extent of AGS cell elongation. CONCLUSIONS: H. pylori strains that deliver CagA with more phosphorylation motifs induce higher levels of CagA phosphorylation in epithelial cells, induce more cytoskeletal changes, and are more likely to be associated with gastric cancer.