D-glucose overflow metabolism in an evolutionary engineered high-performance D-xylose consuming Saccharomyces cerevisiae strain

Co-consumption of D-xylose and D-glucose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. There is a need for improved sugar conversion rates to minimize fermentation times. Previously, we have employed evolutionary engineering to enhance D-xylose transport and metabolism in the presence of D-glucose in a xylose-fermenting S. cerevisiae strain devoid of hexokinases. Re-introduction of Hxk2 in the high performance xylose-consuming strains restored D-glucose utilization during D-xylose/D-glucose co-metabolism, but at rates lower than the non-evolved strain. In the absence of D-xylose, D-glucose consumption was similar to the parental strain. The evolved strains accumulated trehalose-6-phosphate during sugar co-metabolism, and showed an increased expression of trehalose pathway genes. Upon the deletion of TSL1, trehalose-6-phosphate levels were decreased and D-glucose consumption and growth on mixed sugars was improved. The data suggest that D-glucose/D-xylose co-consumption in high-performance D-xylose consuming strains causes the glycolytic flux to saturate. Excess D-glucose is phosphorylated enters the trehalose pathway resulting in glucose recycling and energy dissipation, accumulation of trehalose-6-phosphate which inhibits the hexokinase activity, and release of trehalose into the medium.

Autoregulation of glucose production in men with a glycerol load during rest and exercise

Related to hepatic autoregulation we evaluated hypotheses that 1) glucose production would be altered as a result of a glycerol load, 2) decreased glucose recycling rate (Rr) would result from increased glycerol uptake, and 3) the absolute rate of gluconeogenesis (GNG) from glycerol would be positively correlated to glycerol rate of disappearance (Rd) during a glycerol load. For these purposes, glucose and glycerol kinetics were determined in eight men during rest and during 90 min of leg cycle ergometry at 45 and 65% of peak O2 consumption (V˙o 2 peak). Trials were conducted after an overnight fast, with exercise commencing 12 h after the last meal. Subjects received a continuous infusion of [6,6-2H2]glucose, [1-13C]glucose, and [1,1,2,3,3-2H5]glycerol without (CON) or with an additional 1,000 mg (rest: 20 mg/min; exercise: 40 mg/min) of [2-13C]- or unlabeled glycerol added to the infusate (GLY). Infusion of glycerol dampened glucose Rr, calculated as the difference between [6,6-2H2]- and [1-13C]glucose rates of appearance (Ra), at rest [0.35 ± 0.12 (CON) vs. 0.12 ± 0.10 mg · kg−1 · min−1 (GLY), P < 0.05] and during exercise at both intensities [45%: 0.63 ± 0.14 (CON) vs. 0.04 ± 0.12 (GLY); 65%: 0.73 ± 0.14 (CON) vs. 0.04 ± 0.17 mg · kg−1 · min−1 (GLY), P < 0.05]. Glucose Ra and oxidation were not affected by glycerol infusion at rest or during exercise. Throughout rest and both exercise intensities, glycerol Rdwas greater in GLY vs. CON conditions (rest: 0.30 ± 0.04 vs. 0.58 ± 0.04; 45%: 0.57 ± 0.07 vs. 1.19 ± 0.04; 65%: 0.73 ± 0.06 vs. 1.27 ± 0.05 mg · kg−1 · min−1, CON vs. GLY, respectively). Differences in glycerol Rd(ΔRd) between protocols equaled the unlabeled glycerol infusion rate and correlated with plasma glycerol concentration ( r = 0.97). We conclude that infusion of a glycerol load during rest and exercise at 45 and 65% ofV˙o 2 peak 1) does not affect glucose Ra or Rd, 2) blocks glucose Rr, 3) increases whole body glycerol Rd in a dose-dependent manner, and 4) results in gluconeogenic rates from glycerol equivalent to CON glucose recycling rates.

Recycling of glucose and determination of the Cori Cycle and gluconeogenesis

We have derived equations, by employing [U-13C]glucose and mass isotopomer analysis, to determine the pathways of glycogen synthesis (J. Katz, W. P. Lee, P. A. Wals, and E. A. Bergner. J. Biol. Chem. 264: 12994–13004, 1989). More recently, by use of these methods we have derived equations to determine the rate of glucose recycling and of gluconeogenesis [Tayek and Katz. Am. J. Physiol.270 ( Endocrinol. Metab. 33): E709–E717, 1996 and 272 ( Endocrinol. Metab. 35): E476–E484, 1997, and Katz and Tayek. Am. J. Physiol. 275 ( Endocrinol. Metab. 38): E537–E542, 1988]. The former equations have been criticized and challenged by C. Des Rosiers, B. R. Landau, and H. Brunengraber [ Am. J. Physiol. 259 ( Endocrinol. Metab. 22): E757–E762, 1990], and the latter recently by B. R. Landau, J. Wahren, S. F. Previs, G. K. Ekberg, D. Yang, and H. Brunengraber [ Am. J. Physiol. 274 ( Endocrinol. Metab. 37): E954–E961, 1998]. Landau et al. claimed that our equations were in error and “corrected” them. Their analysis, and their values for recycling and gluconeogenesis (GNG) differ markedly from ours. We show here our equations and estimates of recycling and GNG to be correct. We present here a theoretical analysis of recycling and discuss the determination of the Cori Cycle and GNG. We illustrate by numerical examples the difference in parameters of glucose metabolism calculated by the methods of Katz and Landau. J. Radziuk and W. N. P. Lee [ Am. J. Physiol. 277 ( Endocrinol Metab. 40): E199–E207, 1999] and J. K. Kelleher [ Am. J. Physiol. 277 ( Endocrinol. Metab. 40): E395–E400, 1999] present a mathematical analysis that, although differing in some respects from Landau’s, supports his equation for GNG. We show in theappendix that their derivation of the equation for GNG is incorrect.

Acute alcohol administration attenuates insulin-mediated glucose use by skeletal muscle

The aim of the present work was to test the effect of acute in vivo alcohol administration (180–190 mg/dl plasma for 3 h) on glucose utilization by tissues under basal conditions or after a hyperinsulinemic (100–130 microU/ml) euglycemic clamp in fasted rats. In vivo glucose use by individual tissues was assessed by the tracer 2-deoxy-D-glucose technique. Alcohol administration to saline-infused rats markedly inhibited glucose use by skeletal muscles, including the soleus, white and red quadriceps, and gastrocnemius, as well as by the heart. Ethanol infusion, however, had no effect on glucose use by the diaphragm, lung, liver, skin, ileum, brain, and adipose tissue. The insulin-stimulated glucose use was also inhibited by alcohol selectively in the muscles, with no effect on other tissues tested, except a moderate inhibition in the brain. Ethanol inhibited muscle glucose use by an average of approximately 50% under both basal and insulin-stimulated conditions. However, because insulin treatment more than doubled basal glucose use by these muscles, the 50% inhibition by ethanol treatment represents a greater inhibition of absolute glucose use under insulin-stimulated rather than under basal conditions. Our data demonstrate that acute alcohol intake attenuates basal and hormone-induced glucose utilization in a tissue-specific fashion. The inhibitory effect of alcohol on skeletal muscle glucose use could contribute to the previously observed decreased glucose recycling in humans after acute alcohol intake.

Glucose turnover after a mixed meal in dogs: glucoregulation without change in arterial glycemia

Because the dog can respond to a mixed-meal challenge with little or no change in plasma glucose concentration, we used kinetic techniques to quantify the magnitude and duration of changes in glucoregulation. Glucose turnover was measured using [3-3H]glucose and [U-14C]glucose over two 19-h periods in healthy dogs, first during a fast (n = 6) and then throughout the postprandial state (n = 6) after a single mixed meal. Mean arterial glycemia remained constant in the fasted state (7.5 +/- 0.2 mM) and in the fed state (7.6 +/- 0.3 mM). Glucose appearance (Ra), however, increased slowly after the meal from 38 +/- 2 mg/min to a maximum of 79 +/- 8 mg/min after 6 h and stayed elevated until 12 h (P < 0.001). In parallel, glucose disappearance (Rd) rose from 35 +/- 3 to 83 +/- 7 mg/min, closely matching the corresponding Ra. Glucose recycling rose from 25 +/- 8% at baseline to a maximum of 53 +/- 15% (P < 0.05) at 14 h in fed dogs, whereas levels for fasted dogs stayed between 19 +/- 7% at 0 h and 27 +/- 12% at 6 h. Insulin levels rose significantly 30 min after the meal from 67 +/- 7 pM to a peak of 208 +/- 54 pM at 6 h but remained elevated for 12 h. We conclude that 1) the dog was able to maintain postprandial glucoregulation by very precise matching of Ra and Rd such as to maintain glycemia constant.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of physiological hyperinsulinemia on the intracellular metabolic partition of plasma glucose

Methodology for assessing the glycolytic and oxidative fluxes from plasma glucose, by measuring 3H2O and 14CO2 rates of production during [3-3H]- and [U-14C]glucose infusion, was tested in healthy subjects. In study 1, during staircase 3H2O infusion in six subjects, calculated rates of 3H2O appearance agreed closely with 3H2O infusion rates. In study 2, when [2-3H]glucose and NaH14CO3 were infused in four subjects in the basal state and during a 4-h euglycemic insulin (approximately 70 microU/ml) clamp, accurate estimates of the rates of [2-3H]glucose detritiation were obtained (94-97% of the expected values), and the recovery factor of NaH14CO3 did not change during hyperinsulinemia. In study 3, 11 subjects underwent a 4-h euglycemic insulin (approximately 70 microU/ml) clamp with [3-3H]- and [U-14C]glucose infusion and measurement of gaseous exchanges by indirect calorimetry to estimate the rates of total glycolysis, glycogen synthesis, glucose oxidation, nonoxidative glycolysis, hepatic glucose production, glucose recycling, and glucose conversion to fat. Hyperinsulinemia stimulated glycogen synthesis above baseline more than glycolysis [increment of 4.78 +/- 0.37 vs. 2.0 +/- 0.17 mg.min-1 x kg-1 of lean body mass (LBM), respectively, P < 0.01] and incompletely suppressed (approximately 87%) hepatic glucose production. The major component of nonoxidative glycolysis shifted from glucose recycling in the postabsorptive state (approximately 57% of nonoxidative glycolysis) to glucose conversion to fat during hyperinsulinemia (approximately 59% of nonoxidative glycolysis). Lipid oxidation during the insulin clamp was negatively correlated with both isotopic glucose oxidation (r = -0.822, P < 0.002) and glycolysis (r = -0.582, P < 0.07). In conclusion, in healthy subjects, glycogen synthesis plays a greater role than glycolysis and glucose oxidation in determining insulin-mediated glucose disposal. Part of insulin-mediated increase in glycolysis/oxidation might be secondary to the relief of the competition between fat and glucose for oxidation.