UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells.

It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O(2) at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F(1)F(0) ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.

Turnover of nonessential fatty acids in cardiolipin from the rat heart.

Cardiolipin (CL) is a unique phospholipid (PL) found in the mitochondria of mammalian cells. CL remodeling is accompanied by turnover of its fatty acid acyl groups. Abnormalities in CL remodeling have been found in Barth's syndrome, diabetes, and obesity. The objective of this study was to determine nonessential fatty acid turnover in CL and phosphatidylethanolamine (PE) in the rat heart in vivo. Sprague-Dawley rats were fed either a regular chow or a high-fat diet for 15 weeks, and consumed 6% deuterium-enriched drinking water as a tracer for 14 days. CL and PE were extracted from cardiac tissue and isolated by TLC. Fatty acids from CL, PE, and plasma were analyzed by GC/MS for deuterium incorporation. Results showed oleate and vaccenate turnover were the highest in CL whereas palmitate and stearate turnover were low. Among the nonessential fatty acids in PE, turnover of stearate and vaccenate were the highest. The high turnover rate in vaccenate was unexpected, because vaccenate previously had no known metabolic or physiologic function. In conclusion, the similarly high turnover rates of both oleate and vaccenate readily suggest that remodeling is an important functional aspect of PL metabolism in CL.

Stearoyl-CoA desaturase enzyme 1 inhibition reduces glucose utilization for de novo fatty acid synthesis and cell proliferation in 3T3-L1 adipocytes.

Stearoyl-CoA desaturase enzyme 1 (SCD1) is a lipogenic enzyme that is upregulated in obesity, insulin resistance, and cancer. Since glucose is a substrate for both de novo fatty acid synthesis and deoxyribose synthesis, we hypothesized that SCD1 affects these multiple synthetic pathways through changes in glucose utilization. This study determined glucose utilization for fatty acid synthesis and cell proliferation in 3T3-L1 preadipocytes during SCD1 inhibition. The effects of SCD1 on cellular metabolism as mediated by its monounstaurated fatty acid products (palmitoleate and oleate) were also observed. 3T3-L1 preadipocytes underwent differentiation induction in conjunction with one of the following treatments for 4 days: (A) no treatment, (B) SCD1 inhibitor CGX0290, (C) CGX0290 + palmitoleate, or (D) CGX0290 + oleate. All cells received medium with 50 % [U13C]-glucose. Cells were harvested on day 7 for studies of fatty acid metabolism, tricarboxylic acid (TCA) cycle activities, and deoxyribose synthesis. CGX0290 decreased fatty acid desaturation, glucose utilization for fatty acid synthesis (acetyl-CoA enrichment), and de novo synthesis. CGX0290 treatment also led to decreased cell density through increased cell death. Further analysis showed that deoxyribose new synthesis and oxidative pentose phosphate pathway activity were unchanged, while non-oxidative transketolase pathway activity was stimulated. Palmitoleate and oleate supplementation each partially ameliorated the effects of CGX0290. In 3T3-L1 cells, SCD1 promotes glucose utilization for fatty acid synthesis. In cell proliferation, SCD1 may promote cell survival, but does not impact the oxidative pathway of deoxyribose production. These effects may be mediated through the production of palmitoleate and oleate.

Tracer-based metabolomics: concepts and practices.

Tracer-based metabolomics is a systems biology tool that combines advances in tracer methodology for physiological studies, high throughput "-omics" technologies and constraint based modeling of metabolic networks. It is different from the commonly known metabolomics or metabonomics in that it is a targeted approach based on a metabolic network model in cells. Because of its complexity, it is the least understood among the various "-omics." In this review, the development of concepts and practices of tracer-based metabolomics is traced from the early application of radioactive isotopes in metabolic studies to the recent application of stable isotopes and isotopomer analysis using mass spectrometry; and from the modeling of biochemical reactions using flux analysis to the recent theoretical formulation of the constraint based modeling. How these newer experimental methods and concepts of constraint-based modeling approaches can be applied to metabolic studies is illustrated by examples of studies in determining metabolic responses of cells to pharmacological agents and nutrient environment changes.

Measurement of glucose and fructose in clinical samples using gas chromatography/mass spectrometry.

OBJECTIVE: The impact of increased fructose consumption on carbohydrate metabolism is a topic of current interest, but determination of serum level has been hindered due to low concentration and interference from serum glucose. We are reporting a method for the quantification of glucose and fructose in clinical samples using gas chromatography/mass spectrometry (GC/MS). The accuracy and precision of GC/MS and an enzymatic assay were compared. DESIGN AND METHODS: Mass spectrometry fragmentation patterns of methyloxime peracetate derivatized aldose and ketose were determined. Unique fragments for glucose and fructose were used for quantitative analysis using isotope labeled recovery standards. RESULTS: Methyloxime peracetate derivatives of glucose and fructose showed characteristic loss of acetate (M-60) or ketene (M-42) under chemical ionization (CI). Under electron impact (EI) ionization, a unique C1-C2 fragment of glucose was formed, while a C1-C3 fragment was formed from keto-hexoses. These unique fragments were used in the quantitative assay of glucose and fructose in clinical samples. In clinical samples, the GC/MS assay has a lower limit of detection than that of the enzymatic assay. In plasma samples from patients evaluated for diabetes the average serum glucose and fructose were 6.19+/-2.72 mM and 46+/- 25.22 microM. Fructose concentrations in many of these samples were below the limit of detection of the enzymatic method. CONCLUSION: Derivatization of aldose and ketose monosaccharides to their respective O-methyloxime acetates for GC/MS analysis is a facile method for determination of serum/plasma glucose and fructose samples.

Improvement of metal and tissue adhesion on surface-modified parylene C.

A general method for chemical surface functionalization of parylene C [PC, (para-CH2-C6H3Cl-CH2-)n] films is reported. Friedel-Crafts acylation is used to activate the surface of the PC film, and the resulting carbonyl groups are then used to form a range of different organic functional groups to the surface of the parylene film, including alcohol, imine, thiol, phthalimide, amine, and maleimide. The presence of these functional groups on the parylene surface was confirmed by Fourier transform infrared spectroscopy. Static water drop contact angle measurements were also used to demonstrate the changes in hydrophilicity of the PC film surface, consistent with each of the surface modifications. Enhanced metal (gold) adhesion was achieved by anchoring a thiol group onto the acylated surface of PC film. Acylation of parylene with 2-chloropropionyl chloride gave a surface bound chloropropionyl group. Grafting of poly-N-isopropylacrylamide (pNIPAM) onto the chloropropionyl substituted PC film via atom transfer radical polymerization (ATRP) was carried out. The grafted pNIPAM on the parylene surface leads to temperature-dependent cellular tissue adhesion on the PC film.