Stimulation of Lactate Production in Human Granulosa Cells by Metformin and Potential Involvement of Adenosine 5′ Monophosphate-Activated Protein Kinase

Context: Production of 3-carbon units (as lactate) by granulosa cells (GCs) is important in follicular and oocyte development and may be modulated by metformin. Objective: The aim of the study was to examine the action of metformin on GC lactate production and potential mediation via AMP-activated protein kinase (AMPK). Design: GCs were prepared from follicular aspirates. After exposure to metformin and other potential modulators of AMPK in culture, aspects of cellular function were examined. Setting: The study was conducted in a private fertility clinic/university academic center. Patients: Women undergoing routine in vitro fertilization participated in the study. Interventions: All agents were added in culture. Main Outcome Measures: Lactate output of GCs was measured. Cell extracts were prepared after culture, and phosphorylated forms of AMPK and acetyl CoA carboxylase (ACC) were assayed using Western analysis. Results: Metformin led to a rapid increase in lactate production by GCs [minimum effective dose, 250 μm; maximum dose studied, 1 mm (1.22-fold; P < 0.01)]. This dose range of metformin was similar to that required for stimulation of phospho-AMPK in GCs [minimum effective dose, 250 μm; maximum effect, 500 μm (2.01-fold; P < 0.001)]. Increasing phospho-ACC, as a representative downstream target regulated by AMPK, was apparent over a lower range (minimum effective dose, 31 μm; maximum effect, 250 μm; P < 0.001). A level of metformin (125 μm) insufficient for the stimulation of lactate output when used alone potentiated the effects of suboptimal doses of insulin on lactate production. Adiponectin (2.5 μg/ml) had a small but significant effect on lactate output. Conclusions: Metformin activates AMPK in GCs, stimulating lactate production and increasing phospho-ACC. Metformin also enhances the action of suboptimal insulin concentrations to stimulate lactate production.

Hepatic lactate uptake versus leg lactate output during exercise in humans

The exponential rise in blood lactate with exercise intensity may be influenced by hepatic lactate uptake. We compared muscle-derived lactate to the hepatic elimination during 2 h prolonged cycling (62 ± 4% of maximal O2 uptake, V̇o2max) followed by incremental exercise in seven healthy men. Hepatic blood flow was assessed by indocyanine green dye elimination and leg blood flow by thermodilution. During prolonged exercise, the hepatic glucose output was lower than the leg glucose uptake (3.8 ± 0.5 vs. 6.5 ± 0.6 mmol/min; mean ± SE) and at an arterial lactate of 2.0 ± 0.2 mM, the leg lactate output of 3.0 ± 1.8 mmol/min was about fourfold higher than the hepatic lactate uptake (0.7 ± 0.3 mmol/min). During incremental exercise, the hepatic glucose output was about one-third of the leg glucose uptake (2.0 ± 0.4 vs. 6.2 ± 1.3 mmol/min) and the arterial lactate reached 6.0 ± 1.1 mM because the leg lactate output of 8.9 ± 2.7 mmol/min was markedly higher than the lactate taken up by the liver (1.1 ± 0.6 mmol/min). Compared with prolonged exercise, the hepatic lactate uptake increased during incremental exercise, but the relative hepatic lactate uptake decreased to about one-tenth of the lactate released by the legs. This drop in relative hepatic lactate extraction may contribute to the increase in arterial lactate during intense exercise.

Hindlimb glucose and lactate metabolism during umbilical cord compression and acute hypoxemia in the late-gestation ovine fetus

The metabolic adaptation of the hindlimb in the fetus to a reversible period of adverse intrauterine conditions and, subsequently, to a further episode of acute hypoxemia has been examined. Sixteen sheep fetuses were chronically instrumented with vascular catheters and transit-time flow probes. In nine of these fetuses, umbilical blood flow was reversibly reduced by 30% from baseline for 3 days (umbilical cord compression), while the remaining fetuses acted as sham-operated, age-matched controls. Acute hypoxemia was subsequently induced in all fetuses by reducing maternal fractional inspired oxygen concentration for 1 h. Paired hindlimb arteriovenous blood samples were taken at appropriate intervals during cord compression and acute hypoxemia, and by using femoral blood flow and the Fick principle, substrate delivery, uptake, and output were calculated. Umbilical cord compression reduced blood oxygen content and delivery to the hindlimb and increased hindlimb oxygen extraction and blood glucose and lactate concentration in the fetus. However, hindlimb glucose and oxygen consumption were unaltered during umbilical cord compression. In contrast, hindlimb oxygen delivery and uptake were significantly reduced in all fetuses during subsequent acute hypoxemia, but glucose extraction, oxygen extraction, and hindlimb lactate output significantly increased in sham-operated control fetuses only. Preexposure of the fetus to a temporary period of adverse intrauterine conditions alters the metabolic response of the fetal hindlimb to subsequent acute stress. Additional data suggest that circulating blood lactate may be derived from sources other than the fetal hindlimb under these circumstances. The lack of hindlimb lactate output during acute hypoxemia in umbilical cord-compressed fetuses, despite a significant fall in oxygen delivery to and uptake by the hindlimb, suggests that the fetal hindlimb may not respire anaerobically after exposure to adverse intrauterine conditions.

Hepatic intralobular mapping of fructose metabolism in the rat liver

Detailed mapping of glucose and lactate metabolism along the radius of the hepatic lobule was performed in situ in rat livers perfused with 1.5 mM lactate before and during the addition of 5 mM fructose. The majority of fructose uptake occurred in the periportal region; 45% of fructose taken up in the periportal half of the lobular volume being converted into glucose. Periportal lactate uptake was markedly decreased by addition of fructose. Basal perivenous lactate output, which was derived from glucose synthesized periportally, was increased in the presence of fructose. During fructose infusion there was a small decrease in cell pH periportally, but acidification of up to 0.5 pH units perivenously. The evidence suggests that in situ the apparent direct conversion of fructose into lactate represents, to a substantial extent, the result of periportal conversion of fructose into glucose and the subsequent uptake and glycolysis to lactate in the perivenous zone of some of that glucose. 31P NMR spectroscopy showed that the cellular concentration of phosphomonoesters changes very little periportally during fructose infusion, but there was an approximate twofold increase perivenously, presumably due to the accumulation of fructose 1-phosphate. It may be inferred that fructokinase activity is expressed throughout the hepatic lobule.

Reversal of permeability transition during recovery of hearts from ischemia and its enhancement by pyruvate

We have used mitochondrial entrapment of 2-deoxy-d-[3H]glucose (2-DG) to demonstrate that recovery of Langendorff-perfused rat hearts from ischemia is accompanied by reversal of the mitochondrial permeability transition (MPT). In hearts loaded with 2-DG before 40 min of ischemia and 25 min of reperfusion, 2-DG entrapment [expressed as 105 × (mitochondrial 2-[3H]DG dpm per unit citrate synthase)/(total heart 2-[3H]DG dpm/g wet wt)] increased from 11.1 ± 1.3 (no ischemia, n = 4) to 32.5 ± 1.9 ( n = 6; P < 0.001). In other experiments, 2-DG was loaded after 25 min of reperfusion to determine whether some mitochondria that had undergone the MPT during the initial phase of reperfusion subsequently “resealed” and thus no longer took up 2-DG. The reduction of 2-DG entrapment to 20.6 ± 2.4 units ( n = 5) confirmed that this was the case. Pyruvate (10 mM) in the perfusion medium increased recovery of left ventricular developed pressure from 57.2 ± 10.3 to 98.9 ± 10.8% ( n = 6; P < 0.05) and reduced entrapment of 2-DG loaded preischemically and postischemically to 23.5 ± 1.5 ( n = 4; P < 0.001) and 10.5 ± 0.5 ( n = 4; P < 0.01) units, respectively. The presence of pyruvate increased tissue lactate content at the end of ischemia and decreased the effluent pH during the initial phase of reperfusion concomitant with an increase in lactate output. We suggest that pyruvate may inhibit the MPT by decreasing pHi and scavenging free radicals, thus protecting hearts from reperfusion injury.

Intracellular lactate controls adenosine output from dog gracilis muscle during moderate systemic hypoxia

The influence of systemic hypoxia on lactate and adenosine output from isolated constant-flow-perfused gracilis muscle was determined in anesthetized dogs. The lactate transport inhibitor alpha-cyano-4-hydroxycinnamic acid (CHCA) was employed to distinguish the direct effects of hypoxia on adenosine output from the effects produced indirectly by a change in lactate concentration. Reduction of arterial PO2 from 135 +/- 4 to 39 +/- 2 mmHg raised arterial lactate from 1.26 +/- 0.32 to 2.22 +/- 0.45 mM but decreased venoarterial lactate difference from 0.53 +/- 0.09 to -0.13 +/- 0.19 mM, indicating that lactate output from the muscle was abolished. Arterial adenosine did not change, but venoarterial adenosine difference increased from 20.6 +/- 10.1 to 76.5 +/- 14.4 nM. CHCA infusion during hypoxia abolished adenosine output from gracilis muscle (venoarterial adenosine difference = -20.5 +/- 40.6 nM). In isolated rat soleus muscle fibers, intracellular pH increased from 6.96 +/- 0.04 to 7.71 +/- 0.14 in response to a reduction of PO2 from 459 +/- 28 to 53 +/- 3 mmHg. Correspondingly, adenosine output decreased from 3.71 +/- 0.15 to 3.04 +/- 0.27 nM. These data suggest that hypoxia did not directly stimulate adenosine output from red oxidative skeletal muscle, but rather systemic hypoxia increased lactate delivery and the resulting increase in intracellular lactate decreased intracellular pH, which stimulated adenosine output.

Lactate and acid-base exchange during brief intense contractions of skeletal muscle in situ

Our goal was to design a stimulation-contraction paradigm using an isolated in situ dog gastrocnemius muscle preparation that would provide an experimental model for brief intense intermittent (IC) exercise in humans. Second, acid-base and ion exchanges across the muscle were investigated using four 30-s bouts of isotonic tetanic contractions (2 s-1, 100-ms train, 50 impulses/s) with 4 min of rest between bouts. During the bouts, peak power output (W) was 18.2 mW/g in the first bout; it declined by 4.4% by the fourth bout and by 12–16% in each bout. Compared with repetitive continuous contractions (CC) at maximal O2 uptake (VO2), W was greater and VO2 (approximately 3.5 mumol.g-1.min-1) and CO2 production (approximately 4.5 mumol.g-1.min-1) were less with IC. Venous-arterial (v-a) differences and lactate output peaked immediately after each bout and were not different from the values reported for CC at maximal VO2. Thus, with IC, VO2/W was lower and the CO2 production/VO2 and lactate output/VO2 ratios were greater than those seen with CC. These differences suggest that this stimulation-contraction paradigm may be an appropriate model for brief intense exercise. The v-a [H+] difference was a direct result of the v-a PCO2 difference. The venous strong ion difference was always greater than or equal to the arterial strong ion difference because the v-a [Cl-] difference was opposite and greater than the v-a lactate concentration difference, whereas the v-a [Na+] and [K+] differences were small.(ABSTRACT TRUNCATED AT 250 WORDS)

Gut and muscle tissue PO2 in endotoxemic dogs during shock and resuscitation

There is indirect evidence that tissue hypoxia occurs in human sepsis and surface measures of muscle tissue PO2 (PtiO2) in hypodynamic endotoxic animals are decreased. This study assessed systemic and regional tissue oxygenation in a more relevant model of hyperdynamic endotoxicosis. We isolated venous outflow from the left hindlimb and a segment of ileum in six anesthetized dogs to measure muscle and gut O2 delivery and uptake (VO2) and lactate flux, gut intramucosal pH (pHi) by tonometry, and PtiO2 by multi-point surface electrodes placed on mucosal and serosal surfaces of gut and on muscle. We then infused Escherichia coli lipopolysaccharide (LPS; 2 mg/kg) over 1 h followed by a 2-h infusion of dextran (0.5 ml.kg-1.min-1). LPS infusion significantly decreased systemic and gut VO2, cardiac output (Q), and blood pressure and increased arterial lactate and gut lactate flux. Resuscitation increased Q to above baseline and restored systemic VO2. In response to LPS and then resuscitation, muscle PtiO2 distribution did not change, suggesting little microcirculatory disturbance, although mean PtiO2 first decreased and then increased. In contrast, gut VO2 and pHi remained low and lactate output remained high, despite restoration of gut blood flow. Gut VO2, lactate flux, pHi, and PtiO2 histograms were consistent with a marked redistribution of blood flow within the gut wall, away from the mucosa and toward the muscularis. These data show that, in hyperdynamic acute endotoxemia, skeletal muscle PtiO2 and VO2 are well maintained, but blood flow within the gut is significantly disturbed with mucosal hypoxia.