Hepatic gluconeogenic fluxes and glycogen turnover during fasting in humans. A stable isotope study.

Fluxes through intrahepatic glucose-producing metabolic pathways were measured in normal humans during overnight or prolonged (60 h) fasting. The glucuronate probe was used to measure the turnover and sources of hepatic UDP-glucose; mass isotopomer distribution analysis from [2-13C1]glycerol for gluconeogenesis and UDP-gluconeogenesis; [U-13C6]glucose for glucose production (GP) and the direct UDP-glucose pathway; and [1-2H1]galactose for UDP-glucose flux and retention in hepatic glycogen. After overnight fasting, GP (fluxes in milligram per kilogram per minute) was 2.19+/-0.09, of which 0.79 (36%) was from gluconeogenesis, 1.40 was from glycogenolysis, 0.30 was retained in glycogen via UDP-gluconeogenesis, and 0.17 entered hepatic UDP-glucose by the direct pathway. Thus, total flux through the gluconeogenic pathway (1.09) represented 54% of extrahepatic glucose disposal (2.02) and the net hepatic glycogen depletion rate was 0.93 (46%). Prolonging [2-13C1]glycerol infusion slowly increased measured fractional gluconeogenesis. In response to prolonged fasting, GP was lower (1. 43+/-0.06) and fractional and absolute gluconeogenesis were higher (78+/-2% and 1.11+/-0.07, respectively). The small but nonzero glycogen input to plasma glucose (0.32+/-0.03) was completely balanced by retained UDP-gluconeogenesis (0.31+/-0.02). Total gluconeogenic pathway flux therefore accounted for 99+/-2% of GP, but with a glycogen cycle interposed. Prolonging isotope infusion to 10 h increased measured fractional gluconeogenesis and UDP-gluconeogenesis to 84-96%, implying replacement of glycogen by gluconeogenic-labeled glucose. Moreover, after glucagon administration, GP (1.65), recovery of [1-2H1]galactose label in plasma glucose (25%) and fractional gluconeogenesis (91%) increased, such that 78% (0.45/0.59) of glycogen released was labeled (i.e., of recent gluconeogenic origin). In conclusion, hepatic gluconeogenic flux into glycogen and glycogen turnover persist during fasting in humans, reconciling inconsistencies in the literature and interposing another locus of control in the normal pathway of GP.

Measurement of hepatic Ra UDP-glucose in vivo in rats: relation to glycogen deposition and labeling patterns.

We previously described an isotopic method for quantifying the rate of appearance of hepatic UDP-glucose (Ra UDP-Glc) and the direct entry of glucose into hepatic UDP-Glc in humans. Here, the method is tested in depth in rats. The basic principles are that dilution of labeled galactose in hepatic UDP-Glc, sampled noninvasively by the xenobiotic glucuronate (GlcUA) method, reveals Ra UDP-Glc. First, labeling patterns in secreted acetaminophen-GlcUA were compared with hepatic glycogen and plasma glucose by use of mass isotopomer distribution analysis from [2-(13)C]glycerol. Labeling was consistent with common precursor pools of glucose 6-phosphate and triose-phosphate for all end products studied in fasted and in intravenous glucose- and fructose-infused states. Next, [1-(3)H]galactose was administered. After a 24-h fast, Ra UDP-Glc was 25.0 +/- 1.7 mumol.kg body wt-1.min-1 and rose to 57.7 and 72.7 mumol.kg-1.min-1 at intravenous glucose infusion rates of 111 and 167-194 mumol.kg-1.min-1, respectively. Liver glycogen deposition correlated closely with Ra UDP-Glc (R2 = 0.76), although the turnover value was approximately 50% higher than the net deposition rate. In conclusion, the turnover of an intrahepatic metabolite, UDP-Glc, can be measured noninvasively, and Ra UDP-Glc correlates with liver glycogen deposition in rats.

Hepatic glucose-6-phosphatase flux and glucose phosphorylation, cycling, irreversible disposal, and net balance in vivo in rats. Measurement using the secreted glucuronate technique.

Measurement of hepatic glucose production (HGP) by standard isotope dilution reveals only the net release of glucose from the liver, not the flux across glucose-6-phosphatase ([G6Pase] or total hepatic glucose output), hepatic glucose cycling (HGC), irreversible glucose disposal into glycogen in the liver (hepatic Rd), or net hepatic glucose balance. We describe two independent isotopic techniques for measuring these parameters in vivo, both of which use secreted glucuronate (GlcUA). HGC can be quantified by measuring a correction factor for glucose label retained in hepatic glucose-6-phosphate (G6P), sampled as GlcUA. A complementary technique for measuring total hepatic glucose output is also described (reverse dilution), requiring administration of no labeled glucose but instead a labeled gluconeogenic precursor and unlabeled glucose. Hepatic Rd is calculated by multiplying the rate of appearance (Ra) of hepatic UDP-glucose ([UDP-glc] based on dilution of labeled galactose in GlcUA) times the direct entry of glucose into hepatic UDP-glc and the fraction of labeled UDP-glc retained in the liver. The sum of hepatic Rd plus HGC represents the total hepatic glucose phosphorylation rate. Rats received intravenous (i.v.) glucose infusions at a rate of 15 to 30 mg/kg/min after a 24-hour fast. Despite a suppression of net HGP more than 50%, total hepatic glucose output was not significantly decreased, because of increased HGC. Total hepatic glucose output calculated by reverse dilution yielded similar results during i.v. glucose infusions at 15 mg/kg/min, although values were higher than obtained by the correction-factor method at 30 mg/kg/min. The fraction of labeled UDP-glc released into blood glucose, representing a hepatic glycogen cycle, decreased from 35% (fasted) to nearly 0% (i.v. glucose 30 mg/kg/min). Hepatic Rd was 1.4, 4.6, and 7.5 mg/kg/min (fasted and i.v. glucose 15 and 30 mg/kg/min, respectively); total hepatic glucose phosphorylation increased substantially (from 4.2 to 8.5 to 12.7 mg/kg/min) and net hepatic glucose balance changed from negative to positive during i.v. glucose. In conclusion, hepatic G6Pase flux, glucose phosphorylation, HGC, disposal of glucose into glycogen, and net glucose balance can be measured noninvasively in vivo under various metabolic conditions by techniques involving the GlcUA probe.

Gluconeogenesis and intrahepatic triose phosphate flux in response to fasting or substrate loads. Application of the mass isotopomer distribution analysis technique with testing of assumptions and potential problems.

We measured gluconeogenesis (GNG) in rats by mass isotopomer distribution analysis, which allows enrichment of the true biosynthetic precursor pool (hepatic cytosolic triose phosphates) to be determined. Fractional GNG from infused [3-13C]lactate, [1-13C]lactate, and [2-13C]glycerol was 88 +/- 2, 89 +/- 3, and 87 +/- 2%, respectively, after 48 h of fasting. [2-13C]Glycerol was the most efficient label and allowed measurement of rate of appearance of intrahepatic triose phosphate (Ra triose-P), by dilution. IV fructose (10-15 mg/kg/min) increased absolute GNG by 81-147%. Ra triose-P increased proportionately, but endogenous Ra triose-P was almost completely suppressed, suggesting feedback control. Interestingly, 15-17% of fructose was directly converted to glucose without entering hepatic triose-P. IV glucose reduced GNG and Ra triose-P. 24-h fasting reduced hepatic glucose production by half, but absolute GNG was unchanged due to increased fractional GNG (51-87%). Reduced hepatic glucose production was entirely due to decreased glycogen input, from 7.3 +/- 1.8 to 1.1 +/- 0.2 mg/kg/min. Ra triose-P fell during fasting, but efficiency of triose-P disposal into GNG increased, maintaining GNG constant. Secreted glucuronyl conjugates and plasma glucose results correlated closely. In summary, GNG and intrahepatic triose-P flux can be measured by mass isotopomer distribution analysis with [2-13C]glycerol.