A window into cellular metabolism: hepatic metabolism of (15)N-labelled substrates

It is now apparent that many of the subtleties of cellular metabolism are intrinsically associated with cell structure and that their physiological study requires techniques that respect the integrity of cells and organs. We have used 15N-substrates to examine urea synthesis in the intact perfused rat liver. This work permits us to determine the extent to which different amino acids donate nitrogen atoms to the two nitrogens of urea. It is apparent that alanine and the amino group of glutamine provide nitrogen for urea synthesis primarily via cytoplasmic aspartate, whereas mitochondrial ammonia is the preferred route of entry for nitrogen from pre-formed ammonia or from the amide nitrogen of glutamine. Most importantly, this methodology permits us to explore for the occurrence of metabolic channels in such a highly organised, physiological system. Our studies indicate that a metabolic channel does not exist between glutaminase and carbamoylphosphate synthetase 1.     

Hexokinase-mitochondria interaction mediated by Akt is required to inhibit apoptosis in the presence or absence of Bax and Bak

The serine/threonine kinase Akt inhibits mitochondrial cytochrome c release and apoptosis induced by a variety of proapoptotic stimuli. The antiapoptotic activity of Akt is coupled, at least in part, to its effects on cellular metabolism. Here, we provide genetic evidence that Akt is required to maintain hexokinase association with mitochondria. Targeted disruption of this association impairs the ability of growth factors and Akt to inhibit cytochrome c release and apoptosis. Targeted disruption of mitochondria-hexokinase (HK) interaction or exposure to proapoptotic stimuli that promote rapid dissociation of hexokinase from mitochondria potently induce cytochrome c release and apoptosis, even in the absence of Bax and Bak. These effects are inhibited by activated Akt, but not by Bcl-2, implying that changes in outer mitochondrial membrane (OMM) permeability leading to apoptosis can occur in the absence of Bax and Bak and that Akt inhibits these changes through maintenance of hexokinase association with mitochondria.     

Rapid method for determining the rate of DNA synthesis and cellular proliferation

A new method has been developed for determination of DNA synthesis during cell proliferation. The method is based on the metabolism of [U-(13)C(6)]glucose to deoxyribose (DR) and then incorporation of [U-(13)C(5)]DR into newly synthesized DNA. Extracted cellular DNA is subjected to HCl hydrolysis (2 h at 100 degrees C), which converts DR into levulinic acid. The (13)C enrichment in DR is determined in the trimethylsilyl derivative of levulinate using gas chromatography-mass spectrometry. The method is rapid and sensitive. It can precisely determine (13)C enrichment below 1 at.% excess in as little as 4 ng DNA. We have used this method to determine the rate of cell proliferation in vitro and the level of DR in a given amount of DNA. The current approach has significant advantages over previously described methods and overcomes several difficulties related to the determination of DNA synthesis both in vivo and in vitro.     

An autonomous system for identifying and governing a cell's state in yeast

We present an approach for an autonomous system that detects a particular state of interest in a living cell and can govern cell fate accordingly. Cell states could be better identified by the expression pattern of several genes than of a single one. Therefore, autonomous identification can be achieved by a system that measures the expression of these several genes and integrates their activities into a single output. We have constructed a system that diagnoses a unique state in yeast, in which two independent pathways, methionine anabolism and galactose catabolism, are active. Our design is based on modifications of the yeast two-hybrid system. We show that cells could autonomously report on their state, identify the state of interest, and inhibit their growth accordingly. The system's sensitivity is adjustable to detect states with limited dynamic range of inputs. The system's output depends only on the activity of input pathways, not on their identity; hence it is straightforward to diagnose any pair of inputs. A simple model is presented that accounts for the data and provides predictive power. We propose that such systems could handle real-life states-of-interest such as identification of aberrant versus normal growth.     

Studies of hepatic glutamine metabolism in the perfused rat liver with (15)N-labeled glutamine.

This study examines the role of glucagon and insulin in the incorporation of (15)N derived from (15)N-labeled glutamine into aspartate, citrulline and, thereby, [(15)N]urea isotopomers. Rat livers were perfused, in the nonrecirculating mode, with 0.3 mM NH(4)Cl and either 2-(15)N- or 5-(15)N-labeled glutamine (1 mM). The isotopic enrichment of the two nitrogenous precursor pools (ammonia and aspartate) involved in urea synthesis as well as the production of [(15)N]urea isotopomers were determined using gas chromatography-mass spectrometry. This information was used to examine the hypothesis that 5-N of glutamine is directly channeled to carbamyl phosphate (CP) synthesis. The results indicate that the predominant metabolic fate of [2-(15)N] and [5-(15)N]glutamine is incorporation into urea. Glucagon significantly stimulated the uptake of (15)N-labeled glutamine and its metabolism via phosphate-dependent glutaminase (PDG) to form U(m+1) and U(m+2) (urea containing one or two atoms of (15)N). However, insulin had little effect compared with control. The [5-(15)N]glutamine primarily entered into urea via ammonia incorporation into CP, whereas the [2-(15)N]glutamine was predominantly incorporated via aspartate. This is evident from the relative enrichments of aspartate and of citrulline generated from each substrate. Furthermore, the data indicate that the (15)NH(3) that was generated in the mitochondria by either PDG (from 5-(15)N) or glutamate dehydrogenase (from 2-(15)N) enjoys the same partition between incorporation into CP or exit from the mitochondria. Thus, there is no evidence for preferential access for ammonia that arises by the action of PDG to carbamyl-phosphate synthetase. To the contrary, we provide strong evidence that such ammonia is metabolized without any such metabolic channeling. The glucagon-induced increase in [(15)N]urea synthesis was associated with a significant elevation in hepatic N-acetylglutamate concentration. Therefore, the hormonal regulation of [(15)N]urea isotopomer production depends upon the coordinate action of the mitochondrial PDG pathway and the synthesis of N-acetylglutamate (an obligatory activator of CP). The current study may provide the theoretical and methodological foundations for in vivo investigations of the relationship between the hepatic urea cycle enzyme activities, the flux of (15)N-labeled glutamine into the urea cycle, and the production of urea isotopomers.     

Stepwise differentiation of human embryonic stem cells into early endoderm derivatives and their molecular characterization

Human embryonic stem cells have the potential to differentiate into all human cell types and therefore hold a great therapeutic promise. Differentiation into the embryonic endoderm and its derivatives is of special interest since it can provide a cure for severe widespread clinical conditions such as diabetes and hepatic failure. In this work we established a unique experimental outline that enables the study of early human endoderm development and can help improve and create new differentiation protocols. To this end we started with mesendoderm cells and separated them into early endoderm and mesoderm progenitor cells using CXCR4 and PDGFRA cell surface markers. We molecularly characterized the different lineages, and demonstrated the importance of the TGFβ pathway in definitive endoderm initiation. The endoderm progenitor cells were then purified creating an endodermal differentiation niche that is not affected by other cell populations. We followed the differentiation of these cells at different time points, and demonstrated an up regulation of genes indicative to differentiation into both foregut and hindgut. Surprisingly, upon continued culture, there was significant down regulation of the hepatic gene signature. This down regulation could be rescued with FGF2 treatment demonstrating its importance in hepatic cell maintenance. In conclusion, we suggest that isolating endoderm progenitor cells is crucial for the analysis of their fate, and enables the identification of factors involved in their differentiation and maintenance.     

Inhibition of cholesterol transport into skin cells in cultures by phytosterol-loaded microemulsion

Cholesterol and plant phytosterols are lipophilic compounds solubilized by intestinal micelles in a competitive manner. In this work, we used radioactive cholesterol- and phytosterol-loaded oil-in-water microemulsions to follow their incorporation and mutual competition in HaCaT keratinocytes, SZ95 sebocytes, and skin pieces in cultures. Dynamic light scattering showed homogenous nanostructures of 10.5+/-1.5 nm diameter and cryo-transmission electron microscopy confirmed the presence of uniform spherical droplets of 7.0+/-1.0 nm diameter. Up to 320 nmol/ml of cholesterol can be solubilized and transported into cells with minimal toxic effect by 0.5 wt% nanodroplets in a cell medium. Phytosterols inhibit incorporation of cholesterol into cells, in vitro, at molar ratios (phytosterols/cholesterol) of 4 and above. The loaded nanodroplets accumulate in intracellular vesicles (presumably endosomes). No metabolic conversion of cholesterol or phytosterols was found in these cells, in vitro, after 24 h, at 37 degrees C.     

The role of mitochondrially bound arginase in the regulation of urea synthesis: studies with [U-15N4]arginine, isolated mitochondria, and perfused rat liver

The main goal of the current study was to elucidate the role of mitochondrial arginine metabolism in the regulation of N-acetylglutamate and urea synthesis. We hypothesized that arginine catabolism via mitochondrially bound arginase augments ureagenesis by supplying ornithine for net synthesis of citrulline, glutamate, N-acetylglutamate, and aspartate. [U-(15)N(4)]arginine was used as precursor and isolated mitochondria or liver perfusion as a model system to monitor arginine catabolism and the incorporation of (15)N into various intermediate metabolites of the urea cycle. The results indicate that approximately 8% of total mitochondrial arginase activity is located in the matrix, and 90% is located in the outer membrane. Experiments with isolated mitochondria showed that approximately 60-70% of external [U-(15)N(4)]arginine catabolism was recovered as (15)N-labeled ornithine, glutamate, N-acetylglutamate, citrulline, and aspartate. The production of (15)N-labeled metabolites was time- and dose-dependent. During liver perfusion, urea containing one (U(m+1)) or two (U(m+2)) (15)N was generated from perfusate [U-(15)N(4)]arginine. The output of U(m+2) was between 3 and 8% of total urea, consistent with the percentage of activity of matrix arginase. U(m+1) was formed following mitochondrial production of [(15)N]glutamate from [alpha,delta-(15)N(2)]ornithine and transamination of [(15)N]glutamate to [(15)N]aspartate. The latter is transported to cytosol and incorporated into argininosuccinate. Approximately 70, 75, 7, and 5% of hepatic ornithine, citrulline, N-acetylglutamate, and aspartate, respectively, were derived from perfusate [U-(15)N(4)]arginine. The results substantiate the hypothesis that intramitochondrial arginase, presumably the arginase-II isozyme, may play an important role in the regulation of hepatic ureagenesis by furnishing ornithine for net synthesis of N-acetylglutamate, citrulline, and aspartate.