W. N. Paul Lee

Dynamic profiling of the glucose metabolic network in fasted rat hepatocytes using [1,2-13C2]glucose

Recent studies in metabolic profiling have underscored the importance of the concept of a metabolic network of pathways with special functional characteristics that differ from those of simple reaction sequences. The characterization of metabolic functions requires the simultaneous measurement of substrate fluxes of interconnecting pathways. Here we present a novel stable isotope method by which the forward and reverse fluxes of the futile cycles of the hepatic glucose metabolic network are simultaneously determined. Unlike previous radio-isotope methods, a single tracer [1,2-13C2]D-glucose and mass isotopomer analysis is used. Changes in fluxes of substrate cycles, in response to several gluconeogenic substrates, in isolated fasted hepatocytes from male Wistar rats were measured simultaneously. Incubation with these substrates resulted in a change in glucose-6-phosphatase/glucokinase and glycolytic/gluconeogenic flux ratios. Different net redistributions of intermediates in the glucose network were observed, resulting in distinct metabolic phenotypes of the fasted hepatocytes in response to each substrate condition. Our experimental observations show that the constraints of concentrations of shared intermediates, and enzyme kinetics of intersecting pathways of the metabolic network determine substrate redistribution throughout the network when it is perturbed. These results support the systems-biology notion that network analysis provides an integrated view of the physiological state. Interaction between metabolic intermediates and glycolytic/gluconeogenic pathways is a basic element of cross-talk in hepatocytes, and may explain some of the difficulties in genotype and phenotype correlation.

Determination of a glucose-dependent futile recycling rate constant from an intraperitoneal glucose tolerance test

Increased glucose cycling between glucose and glucose-6-phosphate is characteristic of insulin resistance and hyperglycemia seen with Type II diabetes. Traditionally, glucose cycling is determined by the difference between hepatic glucose output measured with separate [2-3H]glucose and [6-3H]glucose infusions. We demonstrate a novel method for determining hepatic glucose recycling from an intraperitoneal glucose tolerance test (IPGTT). A single tracer, [1, 2-13C(2)]glucose (a M2 glucose isotopomer), was administered at 1mg/g body weight to 4-month-old C57BL/6 mice. Hepatic glucose recycling was monitored by the appearance of a plasma M1 isotopomer of glucose, which is produced by the action of the pentose cycle on the M2 glucose isotopomer in the liver. The initial M2 enrichment was 56% and decreased to 13% at the end of 3 h, and the M1 enrichment peaked at 2 h. The ratio of plasma M1/M2 glucose increased linearly with time to approximately 25%, and the regression of the M1/M2 ratio against time gives a slope, termed the in vivo glucose-dependent futile recycling rate constant k(HR). k(HR) estimates glucose/glucose-6-phosphate futile cycling, along with glucose recycling through the pentose cycle. These observations demonstrate complex substrate cycling during an IPGTT using a single stable isotope tracer.

Glucose intolerance and lipid metabolic adaptations in response to intrauterine and postnatal calorie restriction in male adult rats

Enhanced de novo lipogenesis (DNL), an adult hepatic adaption, is seen with high carbohydrate or low-fat diets. We hypothesized that ad libitum intake after prenatal calorie restriction will result in adult-onset glucose intolerance and enhanced DNL with modified lipid metabolic gene expression profile. Stable isotopes were used in 15-month-old adult male rat offspring exposed to prenatal (IUGR), pre- and postnatal (IPGR), or postnatal (PNGR) caloric restriction vs. controls (CON). IUGR vs. CON were heavier with hepatomegaly but unchanged visceral white adipose tissue (WAT), glucose intolerant with reduced glucose-stimulated insulin secretion (GSIS), pancreatic β-cell mass, and total glucose clearance rate but unsuppressed hepatic glucose production. Liver glucose transporter (Glut) 1 and DNL increased with decreased hepatic acetyl-CoA carboxylase (ACC) and fatty acid synthase but increased WAT fatty acid transport protein-1 and peroxisomal proliferator-activated receptor-γ, resistin, and visfatin gene expression. In contrast, PNGR and IPGR were lighter, had reduced visceral WAT, and were glucose tolerant with unchanged hepatic glucose production but with increased GSIS, β-cell mass, glucose clearance rate, and WAT insulin receptor. Hepatic Glut1 and DNL were also increased in lean IPGR and PNGR with increased hepatic ACC, phosphorylated ACC, and pAMPK and reduced WAT fatty acid transport protein-1, peroxisomal proliferator-activated receptor-γ, and ACCα. We conclude the following: 1) the heavy, glucose-intolerant and insulin-resistant IUGR adult phenotype is ameliorated by postnatal caloric restriction; 2) increased DNL paralleling hepatic Glut1 is a biomarker of exposure to early caloric restriction rather than the adult metabolic status; 3) hepatic lipid enzyme expression reflects GSIS rather than DNL; and 4) WAT gene expression reflects an obesogenic vs. lean phenotype.

Hepatic de Novo Lipogenesis in Stable Low-Birth-Weight Infants During Exclusive Breast Milk Feedings and During Parenteral Nutrition

BACKGROUND Low-birth-weight (LBW) infants have high energy requirements and are dependent on high fat intake to maintain adequate postnatal growth. Fat energy is transported in plasma as triglycerides, which are either derived from the diet or from de novo lipogenesis (DNL). It is our hypothesis that DNL plays an important physiologic role in adapting to exclusive breast milk (EBM) feeding or to parenteral nutrition (PN). METHODS We studied hepatic de novo lipogenesis in 14 LBW (<34-week gestation) appropriate for gestational age and receiving either EBM feedings or full PN support. Stable isotope tracer [2-(13)C] acetate was administered for 72 hours to achieve an estimated 10% enrichment of daily fat intake. Fatty acids were extracted from plasma for gas chromatography-mass spectrometry analyses. RESULTS Percent new synthesis of palmitate was 13.1% +/- 2.5% in the EBM group and 14.9% +/- 0.7% in the PN group (NS), stearate was 11.1% +/- 2.7% in the EBM group and 10.6% +/- 14% in the PN group (NS) and cholesterol was 12.7% +/- 2.1% in the EBM group and 17.4% +/- 4.6% in the PN group (NS) after 72 hours of tracer administration (mean +/- SEM). The plasma lipid fatty acid composition in palmitate, oleate, and stearate with intake of 3.6 +/- 0.6 g/kg/d of IV lipids (ILs) was similar to EBM-feeding infants taking 6.3 +/- 0.13 g/kg/d of fat. CONCLUSIONS De novo lipogenesis is active in stable LBW infants maintaining standard postnatal growth. Hepatic DNL permits newborn infants to meet the fat energy needs of peripheral tissues for growth and storage and to maintain plasma fatty acid composition in adaptation to different dietary fat intake.

Calibration of isotope ratio mass spectrometry working standard for 2H/1H ratio analysis

A method is described for the calibration of an isotope ratio mass spectrometry working standard for 2H/1H analysis using highly purified (> 99.99% enriched) deuterium oxide (2H2O). Serial dilution of such a compound can be used to construct a standard curve for calibration purposes using the relationship [formula: see text] where APE is the atom percent excess. It was found that the determined rWR had a precision of +/- 1%. Most of the variation came from sample handling, which includes the serial dilution of the weighed deuterium oxide, the reduction of water to hydrogen gas and the introduction of the hydrogen gas into the mass spectrometer. The use of this calibration procedure allows the direct determination of the isotope ratio rSA, and APE from a standard curve over a wide range of enrichment. Unlike the reference standards V-SMOW and SLAP, the highly purified deuterium oxide is non-exhaustible and readily available. The highly purified deuterium oxide should be used in place of secondary standards to calibrate working references for 2H/1H analysis in biological studies.