Rapid parallel measurements of macroautophagy and mitophagy in mammalian cells using a single fluorescent biosensor.

Mitochondrial dysfunction is implicated in many human diseases and occurs in normal aging. Mitochondrial health is maintained through organelle biogenesis and repair or turnover of existing mitochondria. Mitochondrial turnover is principally mediated by mitophagy, the trafficking of damaged mitochondria to lysosomes via macroautophagy (autophagy). Mitophagy requires autophagy, but is itself a selective process that relies on specific autophagy-targeting mechanisms, and thus can be dissociated from autophagy under certain circumstances. Therefore, it is important to assess autophagy and mitophagy together and separately. We sought to develop a robust, high-throughput, quantitative method for monitoring both processes in parallel. Here we report a flow cytometry-based assay capable of rapid parallel measurements of mitophagy and autophagy in mammalian cells using a single fluorescent protein biosensor. We demonstrate the ability of the assay to quantify Parkin-dependent selective mitophagy in CCCP-treated HeLa cells. In addition, we show the utility of the assay for measuring mitophagy in other cell lines, as well as for Parkin-independent mitophagy stimulated by deferiprone. The assay makes rapid measurements (10,000 cells per 6 seconds) and can be combined with other fluorescent indicators to monitor distinct cell populations, enabling design of high-throughput screening experiments to identify novel regulators of mitophagy in mammalian cells.

Whole grain wheat sourdough bread does not affect plasminogen activator inhibitor-1 in adults with normal or impaired carbohydrate metabolism.

BACKGROUND AND AIMS: Epidemiological studies suggest whole grain consumption is associated with a reduced risk of cardiovascular disease (CVD), possibly through alterations in glucose metabolism and subsequent effects on plasminogen activator inhibitor (PAI)-1, a novel biomarker for CVD. Our aim was to investigate the effect of 6 wk of whole grain wheat sourdough bread consumption versus refined white bread on PAI-1. METHODS AND RESULTS: Normoglycemic/normoinsulinemic (NGI; n = 14; age 53 ± 6 y; BMI 26.5 ± 2.9 kg/m(2)) and hyperglycemic/hyperinsulinemic (HGI; n = 14; age 57 ± 7 y; BMI 35.7 ± 5.7 kg/m(2)) adults incorporated whole grain wheat sourdough (162.5 g) or white (168.8 g) bread into their diet, for 6 wk in a randomized crossover study. Pre- and post-intervention, fasting blood samples were analyzed for PAI-1 (primary outcome), as well as glucose, insulin and glucagon (secondary outcomes) at fasting and postprandially after an oral glucose tolerance test (OGTT). Anthropometric measures, fasting glucose, insulin, glucagon and PAI-1 antigen and activity were not different between treatments in either NGI or HGI adults. Glucose incremental area under the curve (iAUC) was lower (19%, P = 0.02) after 6 wk consumption of whole grain wheat sourdough bread compared to white bread in the HGI group, with no differences in insulin or glucagon iAUC in either group. CONCLUSION: Our data showed decreased glucose iAUC after an OGTT following 6 wk whole grain wheat bread consumption in adults with differing glycemic/insulinemic status, but no improvements in PAI-1 or fasting glycemic parameters.

The regulation of muscle glycogen: the granule and its proteins.

Despite decades of studying muscle glycogen in many metabolic situations, surprisingly little is known regarding its regulation. Glycogen is a dynamic and vital metabolic fuel that has very limited energetic capacity. Thus its regulation is highly complex and multifaceted. The stores in muscle are not homogeneous and there appear to be various metabolic pools. Each granule is capable of independent regulation and fundamental aspects of the regulation appear to be associated with a complex set of proteins (some are enzymes and others serve scaffolding roles) that associate both with the granule and with each other in a dynamic fashion. The regulation includes altered phosphorylation status and often translocation as well. The understanding of the roles and the regulation of glycogenin, protein phosphatase 1, glycogen targeting proteins, laforin and malin are in their infancy. These various processes appear to be the mechanisms that give the glycogen granule precise, yet dynamic regulation.

Glutamate availability is important in intramuscular amino acid metabolism and TCA cycle intermediates but does not affect peak oxidative metabolism.

Muscle glutamate is central to reactions producing 2-oxoglutarate, a tricarboxylic acid (TCA) cycle intermediate that essentially expands the TCA cycle intermediate pool during exercise. Paradoxically, muscle glutamate drops approximately 40-80% with the onset of exercise and 2-oxoglutarate declines in early exercise. To investigate the physiological relationship between glutamate, oxidative metabolism, and TCA cycle intermediates (i.e., fumarate, malate, 2-oxoglutarate), healthy subjects trained (T) the quadriceps of one thigh on the single-legged knee extensor ergometer (1 h/day at 70% maximum workload for 5 days/wk), while their contralateral quadriceps remained untrained (UT). After 5 wk of training, peak oxygen consumption (VO2peak) in the T thigh was greater than that in the UT thigh (P<0.05); VO2peak was not different between the T and UT thighs with glutamate infusion. Peak exercise under control conditions revealed a greater glutamate uptake in the T thigh compared with rest (7.3+/-3.7 vs. 1.0+/-0.1 micromol.min(-1).kg wet wt(-1), P<0.05) without increase in TCA cycle intermediates. In the UT thigh, peak exercise (vs. rest) induced an increase in fumarate (0.33+/-0.07 vs. 0.02+/-0.01 mmol/kg dry wt (dw), P<0.05) and malate (2.2+/-0.4 vs. 0.5+/-0.03 mmol/kg dw, P<0.05) and a decrease in 2-oxoglutarate (12.2+/-1.6 vs. 32.4+/-6.8 micromol/kg dw, P<0.05). Overall, glutamate infusion increased arterial glutamate (P<0.05) and maintained this increase. Glutamate infusion coincided with elevated fumarate and malate (P<0.05) and decreased 2-oxoglutarate (P<0.05) at peak exercise relative to rest in the T thigh; there were no further changes in the UT thigh. Although glutamate may have a role in the expansion of the TCA cycle, glutamate and TCA cycle intermediates do not directly affect VO2peak in either trained or untrained muscle.

High circulating levels of RBP4 and mRNA levels of aP2, PGC-1alpha and UCP-2 predict improvement in insulin sensitivity following pioglitazone treatment of drug-naïve type 2 diabetic subjects.

CONTEXT: High levels of circulating retinol-binding protein 4 (RBP4) and baseline expression of adipogenic genes correlate with subsequent improvement in insulin sensitivity following Thiazolidinedione (TZD) treatment. OBJECTIVE: The aim was to identify baseline characteristics and early changes related to TZD treatment that could predict a good treatment response. DESIGN: Subjects were examined with oral glucose tolerance test, intravenous glucose tolerance test, hyperinsulinaemic euglycaemic clamp, body composition and standard blood sampling at baseline and after 4 and 12 weeks treatment. Subcutaneous adipose tissue biopsies were taken from the abdominal region at baseline, after 3 days and 4 weeks treatment to examine the gene expression profile. SETTING: Research laboratory in a University hospital. PARTICIPANTS: Ten newly diagnosed and previously untreated type 2 diabetic subjects were treated with pioglitazone for 3 months. MAIN OUTCOME MEASURES: Baseline characteristics and early changes related to TZD treatment that could predict the response after 3 months. RESULTS: Pioglitazone improved insulin sensitivity after 4 weeks combined with lower glucose and insulin levels without any change in BMI. It was accompanied by lower circulating resistin and plasminogen activator inhibitor-1 levels rapidly increased levels of circulating total and high molecular weight adiponectin as well as adiponectin and adipocyte fatty acid-binding protein (aP2) mRNA expression in the adipose tissue. High levels of circulating RBP4 at baseline and adipose tissue expression of aP2, proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1alpha) and uncoupling protein 2 (UCP-2) predicted a good treatment response measured as improvement in insulin-stimulated whole-body glucose uptake after 3 months. CONCLUSIONS: Circulating levels of RBP4 as an index of insulin sensitivity and mRNA levels of adipogenic genes correlate with the subsequent improvement in insulin sensitivity following TZD treatment.

Interleukin-6 and its mRNA responses in exercise and recovery: relationship to muscle glycogen.

Increases in circulating interleukin-6 (IL-6) during exhaustive exercise have been suggested to be related to declining muscle glycogen. We addressed two hypotheses: (a) exhaustive exercise on two occasions will result in similar decreases in glycogen and increases in circulating IL-6 and its muscle mRNA; (b) increasing the rate of glycogen restoration via high-carbohydrate feeding in recovery will be associated with more rapid declines in muscle mRNA and circulating IL-6. Ten male subjects (22.6+/-0.8 year) cycled to exhaustion (65% VO(2 max)) on two occasions (117.8+/-2.9 min). Carbohydrate (1 g/kg bw) or water was ingested at exhaustion, 60, 120, 180, and 240 min post-exercise. Muscle biopsies were taken at rest, exhaustion, 30, 60, 120 and 300 min of recovery. Exercise resulted in a 14.5-fold increase (P<0.05) in IL-6 mRNA, 14.4-fold increase (P<0.05) in circulating IL-6, and a 80% decrease (P<0.05) in muscle glycogen from rest. The decline in glycogen was not correlated with the increase in IL-6 or IL-6 mRNA. During recovery, circulating IL-6 and its muscle mRNA decreased similarly in both trials; however, glycogen increased 150% (P<0.05) and 40% in the carbohydrate and water trials, respectively. Therefore, the declining IL-6 mRNA and IL-6 plasma concentrations during recovery were not related to carbohydrate availability or changes in glycogen.

Tissue-specific alterations of glucose transport and molecular mechanisms of intertissue communication in obesity and type 2 diabetes.

Insulin resistance plays a major role in the pathogenesis of type 2 diabetes. Insulin regulates blood glucose levels primarily by promoting glucose uptake from the blood into multiple tissues and by suppressing glucose production from the liver. The glucose transporter, GLUT4, mediates insulin-stimulated glucose uptake in muscle and adipose tissue. Decreased GLUT4 expression in adipose tissue is a common feature of many insulin resistant states. GLUT4 expression is preserved in skeletal muscle in many insulin resistant states. However, functional defects in the intracellular trafficking and plasma membrane translocation of GLUT4 result in impaired insulin-stimulated glucose uptake in muscle. Tissue-specific genetic knockout of GLUT4 expression in adipose tissue or muscle of mice has provided new insights into the pathogenesis of insulin resistance. We recently determined that the expression of serum retinol binding protein (RBP4) is induced in adipose tissue as a consequence of decreased GLUT4 expression. We found that RBP4 is elevated in the serum of insulin resistant humans and mice. Furthermore, we found that increasing serum RBP4 levels by transgenic overexpression or by injection of purified RBP4 protein into normal mice causes insulin resistance. Therefore, RBP4 appears to play an important role in mediating adipose tissue communication with other insulin target tissues in insulin resistant states.

Effects of chronic coffee consumption on glucose kinetics in the conscious rat.

Epidemiological studies indicate that regular coffee consumption reduces the risk of developing type 2 diabetes. Despite these findings, the biological mechanisms by which coffee consumption exerts these effects are unknown. The aim of this study was twofold: to develop a rat model that would further delineate the effects of regular coffee consumption on glucose kinetics, and to determine whether coffee, with or without caffeine, alters the actions of insulin on glucose kinetics in vivo. Male Sprague-Dawley rats were fed a high-fat diet for 4 weeks in combination with one of the following: (i) drinking water as placebo (PL), (ii) decaffeinated coffee (2 g/100 mL) (DC), or (iii) alkaloid caffeine (20 mg/100 mL) added to decaffeinated coffee (2 g/100 mL) (CAF). Catheters were chronically implanted in a carotid artery and jugular vein for sampling and infusions, respectively. Recovered animals (5 days postoperative) were fasted for 5 h before hyperinsulinemic-euglycemic clamps (2 mU x kg(-1) x min(-1)). Glucose was clamped at 6 mmol/L and isotopes (2-deoxy-[(14)C]glucose and [3-(3)H]glucose) were administered to obtain indices of whole-body and tissue-specific glucose kinetics. Glucose infusion rates and measures of whole-body metabolic clearance were greater in DC than in PL or CAF, indicating increased whole-body insulin sensitivity. As the only difference between DC and CAF was the addition of alkaloid caffeine, it can be concluded that caffeine antagonizes the beneficial effects of DC. Given these findings, decaffeinated coffee may represent a nutritional means of combating insulin resistance.

Caffeine's impairment of insulin-mediated glucose disposal cannot be solely attributed to adrenaline in humans.

Caffeine (CAF) impedes insulin-mediated glucose disposal (IMGD) and increases plasma adrenaline concentrations ([ADR]; 0.6 nm). While the antagonism of ADR abolishes the CAF effect, infusion of ADR (0.75 nm) has no effect on IMGD. We have now examined CAF and ADR in concert to determine whether or not they elicit an additive response on IMGD. We hypothesized that CAF + ADR would elicit a greater effect than either CAF or ADR alone (i.e. that CAF effects would not be solely attributed to ADR). Subjects (n = 8) completed four trials in a randomized manner. An isoglycaemic-hyperinsulinaemic clamp was performed 30 min after the following treatments were administered: (1) placebo capsules and saline infusion ([ADR] = 0.29 nm) (PL trial), (2) CAF capsules (dose = 5 mg kg(-1)) and saline infusion ([ADR] = 0.62 nm) (CAF trial), (3) PL capsules and ADR infusion ([ADR] = 1.19 nm) (ADR trial), and (4) CAF capsules (dose = 5 mg kg(-1)) and ADR infusion ([ADR] = 0.93 nm) (CAF + ADR trial). As expected, CAF, ADR and CAF + ADR decreased (P

Shortcomings in methodology complicate measurements of serum retinol binding protein (RBP4) in insulin-resistant human subjects.

AIMS/HYPOTHESIS: Levels of retinol binding protein (RBP4) are increased in the serum of insulin-resistant human subjects even before overt diabetes develops. RBP4 levels correlate with insulin resistance, BMI, WHR, dyslipidaemia and hypertension. Improvement of insulin sensitivity with exercise training is associated with reduction in serum RBP4 levels. Therefore serum RBP4 may be useful for early diagnosis of insulin resistance and for monitoring improvements in insulin sensitivity. We sought to determine the performance of assays for this application. SUBJECTS AND METHODS: We compared quantitative western blotting and three commercially available multiwell immunoassays in parallel measurements of RBP4 concentrations in serum from insulin-sensitive subjects and from insulin-resistant subjects with impaired glucose tolerance or type 2 diabetes. RESULTS: The assays yielded different absolute values and magnitudes of elevation of serum RBP4. Western blotting and a sandwich ELISA reported RBP4 concentrations that highly inversely correlated with insulin sensitivity measured by euglycaemic-hyperinsulinaemic clamp. However, western blotting yielded concentrations with a greater dynamic range and less overlap between control and insulin-resistant subjects. Two competitive enzyme-linked immunoassays undervalued serum RBP4 concentrations in insulin-resistant subjects, possibly due to assay saturation. Poor linearity of dilution also limited assay utility. All assays tested exhibited greater immunoreactivity with urinary (C-terminal proteolysed) RBP4 than with full-length RBP4, the predominant form in serum. CONCLUSIONS/INTERPRETATIONS: These findings support the use of quantitative western blotting standardised to full-length RBP4 protein as a 'gold standard' method for measuring serum RBP4 in insulin-resistant states. Other assays should use full-length RBP4 and be extensively cross-validated using other methods.

Quantitative assessment of human muscle glycogen granules size and number in subcellular locations during recovery from prolonged exercise.

Although data relating to muscle glycogen are interpreted as showing it is homogenous when quantified biochemically, it is actually in granules in specific subcellular locations. We hypothesized that postexercise restoration of muscle glycogen would occur initially by an increase in granule number followed by an increase in size, and also that restoration would differ in various subcellular locations. Five men performed prolonged exercise and had muscle biopsies taken at 0, 4, 24 and 48 h of recovery. We quantified granule number and size as well as the total volume of glycogen in the subsarcolemmal and the intra- and intermyofibrillar regions, using transmission electron microscopy. Muscle glycogen was reduced to 36 +/- 8.3 mmol glucosyl units (kg dry weight)(-1) at exhaustion, and was preferentially depleted and subsequently repleted in the intramyofibrillar space. The repletion rate was greatest in the first 4 h; this was associated with a 186% increase in number (P < or = 0.05) and no change in particle size (P > or = 0.05). From 4 h to 48 h, there was an increase in particle size (P < or = 0.05) but not number (P > or = 0.05). Net rate of G volume synthesis per unit area was 50% greater (P < or = 0.05) in the subsarcolemmal than the myofibrillar compartment. Conversely, the net rate of single-particle volume synthesis was greater (P < or = 0.05) in the myofibrillar than the subsarcolemmal compartment. Glycogen granules varied in size and number depending on location, and in all compartments resynthesis of glycogen was characterized initially by an increase in granule number and later by an increase in size.

Acute caffeine ingestion does not impair glucose tolerance in persons with tetraplegia.

Acute caffeine (Caf) ingestion impairs glucose tolerance in able-bodied humans during an oral glucose tolerance test (OGTT). The mechanism responsible for this effect remains unclear, however, it is suggested to be due to the accompanying increase in epinephrine concentration. We examined whether or not Caf would elicit a glucose intolerance in persons with tetraplegia (TP) who do not exhibit an increased epinephrine response following Caf ingestion. All TP [n = 14; 9 incomplete (Inc) lesion, 5 complete (Com) lesion] completed two OGTT 1 h after consuming either gelatin (Pl) or Caf capsules (dose = 4 mg/kg). Blood samples were collected at baseline (time = 0 min), 1 h after capsule ingestion (time = 60 min), and every 30 min during the OGTT (time = 90-180 min). Glucose, insulin, proinsulin, and C-peptide responses were similar (P > 0.05) between treatments, demonstrating no effect of Caf on glucose tolerance. This lack of a Caf effect may be due to the low epinephrine concentration that remained unchanged (P > 0.05) throughout all experiments. Interestingly, the Com exhibited a 50% higher glucose response (P 0.05) lower insulin response (vs. Inc), suggesting a more pronounced glucose intolerance within this subgroup. Furthermore, nine TP (5 Com, 4 Inc) had glucose levels of >or= 7.8 mM at the end of the OGTT (time = 180 min), classifying them as glucose intolerant. In summary, acute Caf ingestion does not increase epinephrine concentration or impair glucose tolerance in TP.

Hemodynamics and O2 uptake during maximal knee extensor exercise in untrained and trained human quadriceps muscle: effects of hyperoxia.

To elucidate the potential limitations on maximal human quadriceps O2 capacity, six subjects trained (T) one quadriceps on the single-legged knee extensor ergometer (1 h/day at 70% maximum workload for 5 days/wk), while their contralateral quadriceps remained untrained (UT). Following 5 wk of training, subjects underwent incremental knee extensor tests under normoxic (inspired O2 fraction = 21%) and hyperoxic (inspired O2 fraction = 60%) conditions with the T and UT quadriceps. Training increased quadriceps muscle mass (2.9 +/- 0.2 to 3.1 +/- 0.2 kg), but did not change fiber-type composition or capillary density. The T quadriceps performed at a greater peak power output than UT, under both normoxia (101 +/- 10 vs. 80 +/- 7 W; P < 0.05) and hyperoxia (97 +/- 11 vs. 81 +/- 7 W; P < 0.05) without further increases with hyperoxia. Similarly, thigh peak O2 consumption, blood flow, vascular conductance, and O2 delivery were greater in the T vs. the UT thigh (1.4 +/- 0.2 vs. 1.1 +/- 0.1 l/min, 8.4 +/- 0.8 vs. 7.2 +/- 0.8 l/min, 42 +/- 6 vs. 35 +/- 4 ml x min(-1) x mmHg(-1), 1.71 +/- 0.18 vs. 1.51 +/- 0.15 l/min, respectively) but were not enhanced with hyperoxia. Oxygen extraction was elevated in the T vs. the UT thigh, whereas arteriovenous O2 difference tended to be higher (78 +/- 2 vs. 72 +/- 4%, P < 0.05; 160 +/- 8 vs. 154 +/- 11 ml/l, respectively; P = 0.098) but again were unaltered with hyperoxia. In conclusion, the present results demonstrate that the increase in quadriceps muscle O2 uptake with training is largely associated with increases in blood flow and O2 delivery, with smaller contribution from increases in O2 extraction. Furthermore, the elevation in peak muscle blood flow and vascular conductance with endurance training seems to be related to an enhanced vasodilatory capacity of the vasculature perfusing the quadriceps muscle that is unaltered by moderate hyperoxia.

Dexras1 inhibits adenylyl cyclase.

Dexras1 is a steroid hormone-induced Ras family G protein that acts as a receptor-independent activator of signaling by Gi/o family heterotrimeric G proteins. We examined the effects of Dexras1 on the activity of adenylyl cylase, a target of inhibitory regulation by Gialpha x GTP. Constitutively active Gsalpha (Q227L) increased cAMP levels 43-fold above baseline, and Dexras1 expression inhibited cAMP levels by 61% (P < 0.01). Dexras1 mediated inhibition of adenylyl cyclase was blocked by treatment pertussis toxin or by co-expression of RGS4, but was not inhibited by with dominant-interfering (G203T or G204A) mutants of Gi alpha2. Dexras1 decreased forskolin-stimulated CREB activation (P < 0.01) and this activity was also inhibited by co-expression of RGS4. These findings indicate that Dexras1 expression leads to ligand-independent activation of both Gialpha- and G(beta)gamma-dependent arms of the Gi signaling cascade, and suggest that Dexras1 may exert physiologically relevant inhibitory effects on the cAMP-PKA-CREB.

Caffeine ingestion does not impede the resynthesis of proglycogen and macroglycogen after prolonged exercise and carbohydrate supplementation in humans.

The purpose of this study was to examine the effects of caffeine (Caf) ingestion on pro- (PG) and macroglycogen (MG) resynthesis in 10 healthy men. Subjects completed two trials, consisting of a glycogen-depleting exercise, while ingesting either Caf or placebo capsules. Throughout recovery, biopsies were taken at 0 (exhaustion), 30, 120, and 300 min, and 75 g of carbohydrate were ingested at 0, 60, 120, 180, and 240 min. Whereas Caf ingestion resulted in a higher blood glucose concentration and decreased glycogen synthase fractional velocity (P

Quantification of subcellular glycogen in resting human muscle: granule size, number, and location.

A few qualitative investigations suggested that location of muscle glycogen (G) granules in specific sites may be associated with distinct metabolic roles. Similarly, it has been suggested that the acid-soluble and -insoluble G fractions (macro- and proglycogen, respectively) are different metabolic pools and also could exist as separate entities. We employed a transmission electron microscopic technique to quantify subcellular G particle size, number, and location in human vastus lateralis biopsies of 11 resting men. The intra- and interobserver variability for the various measures was generally <4%. Granule size and number were quantified in subcellular compartments (subsarcolemmal, intra- and intermyofibrillar). Subcellular location was critical: G was more densely concentrated in the subsarcolemmal than in the myofibrillar space, whereas the single-particle volume was greater in the latter. Single-particle diameter ranged from 10 to 44 etam and followed a continuous, normal distribution. This implies that proglycogen is not a distinct entity, but rather that pro- and macroglycogen are divisions of smaller and larger molecules. These results demonstrate a compartmentalized pattern of subcellular G deposition in human skeletal muscle for both the size and density of granules.

Effects of exercise and thermal stress on caffeine pharmacokinetics in men and eumenorrheic women.

The influence of gender, exercise, and thermal stress on caffeine pharmacokinetics is unclear. We hypothesized that these factors would not have an effect on the metabolism of caffeine. Eight women participated in four 8-h trials and six men participated in two 8-h trials after the ingestion of 6 mg/kg caffeine. The women performed two resting trials (1 in the follicular phase and 1 in the luteal phase of the menstrual cycle) and two exercise trials (90 min of cycling exercise at 65% of maximal O(2) uptake, 1 h after caffeine ingestion) in the follicular phase (1 without and 1 with an additional thermal stress). The men performed one exercise and one resting trial. Menstrual cycle, gender, and exercise, with or without an additional thermal stress, had no effect on the pharmacokinetic measurements or urine caffeine. There was a trend for higher plasma caffeine and lower plasma paraxanthine concentrations in the women. These results confirm that gender, exercise, and thermal stress have no effect on caffeine pharmacokinetics in men and women.

Caffeine and exercise: metabolism, endurance and performance.

Caffeine is a common substance in the diets of most athletes and it is now appearing in many new products, including energy drinks, sport gels, alcoholic beverages and diet aids. It can be a powerful ergogenic aid at levels that are considerably lower than the acceptable limit of the International Olympic Committee and could be beneficial in training and in competition. Caffeine does not improve maximal oxygen capacity directly, but could permit the athlete to train at a greater power output and/or to train longer. It has also been shown to increase speed and/or power output in simulated race conditions. These effects have been found in activities that last as little as 60 seconds or as long as 2 hours. There is less information about the effects of caffeine on strength; however, recent work suggests no effect on maximal ability, but enhanced endurance or resistance to fatigue. There is no evidence that caffeine ingestion before exercise leads to dehydration, ion imbalance, or any other adverse effects. The ingestion of caffeine as coffee appears to be ineffective compared to doping with pure caffeine. Related compounds such as theophylline are also potent ergogenic aids. Caffeine may act synergistically with other drugs including ephedrine and anti-inflammatory agents. It appears that male and female athletes have similar caffeine pharmacokinetics, i.e., for a given dose of caffeine, the time course and absolute plasma concentrations of caffeine and its metabolites are the same. In addition, exercise or dehydration does not affect caffeine pharmacokinetics. The limited information available suggests that caffeine non-users and users respond similarly and that withdrawal from caffeine may not be important. The mechanism(s) by which caffeine elicits its ergogenic effects are unknown, but the popular theory that it enhances fat oxidation and spares muscle glycogen has very little support and is an incomplete explanation at best. Caffeine may work, in part, by creating a more favourable intracellular ionic environment in active muscle. This could facilitate force production by each motor unit.

Caffeine ingestion elevates plasma insulin response in humans during an oral glucose tolerance test.

We tested the hypothesis that caffeine ingestion results in an exaggerated response in blood glucose and (or) insulin during an oral glucose tolerance test (OGTT). Young, fit adult males (n = 18) underwent 2 OGTT. The subjects ingested caffeine (5 mg/kg) or placebo (double blind) and 1 h later ingested 75 g of dextrose. There were no differences between the fasted levels of serum insulin, C peptide, blood glucose, or lactate and there were no differences within or between trials in these measures prior to the OGTT. Following the OGTT, all of these parameters increased (P < or = 0.05) for the duration of the OGTT. Caffeine ingestion resulted in an increase (P < or = 0.05) in serum fatty acids, glycerol, and plasma epinephrine prior to the OGTT. During the OGTT, these parameters decreased to match those of the placebo trial. In the caffeine trial the serum insulin and C peptide concentrations were significantly greater (P < or = 0.001) than for placebo for the last 90 min of the OGTT and the area under the curve (AUC) for both measures were 60 and 37% greater (P < or = 0.001), respectively. This prolonged, increased elevation in insulin did not result in a lower blood glucose level; in fact, the AUC for blood glucose was 24% greater (P = 0.20) in the caffeine treatment group. The data support our hypothesis that caffeine ingestion results in a greater increase in insulin concentration during an OGTT. This, together with a trend towards a greater rather than a more modest response in blood glucose, suggests that caffeine ingestion may have resulted in insulin resistance.