(505f) Flux and Transcriptome Alterations in Mammalian Glycerol Kinase Disorders

We will present flux and transcriptome alterations due to
glycerol kinase (GK) overexpression or deficiency in mammalian cells, to
understand the role played by flux and systems dynamics in glycerol kinase
deficiency (GKD), an X-linked, single-gene inherited disorder of metabolism.

Glycerol kinase (GK) is an important lipogenic enzyme in
mammalian liver, adipose tissue, and other organs. It also performs several
activities unrelated to its biochemical function of phosphorylating glycerol
[1]. For instance, GK functions as ATP-stimulated translocation protein (ASTP),
which enhances the nuclear binding of the activated glucocorticoid-receptor
complex, a transcription factor [2]. Studies have also suggested links between
GK and improved insulin sensitivity ? GK is overexpressed in response to
thiazolidinediones [3], a common drug to treat type 2 diabetes, and this
overexpression relieves insulin resistance [4]. The hereditary disorder GKD
exhibits complexities that are not trivially explained by lack of the biochemical
activity of GK. Thus far, there has been no correlation between genotype and
phenotype in patients with this disorder. We have previously hypothesized that
systems dynamics, including flux through metabolic pathways, can play a
significant role in imparting a phenotype that is not easily deduced from the
genotype [5]. In this presentation we will report flux, microarray, and network
component analysis of tissue cell lines overexpressing GK or that are deficient
in GK, to investigate the function of flux and systems dynamics in GKD.

We cultured wild type and GK-overexpressing H4IIE rat
hepatoma cells on a mathematically designed mixture of U-¹³C, 1-¹³C
and naturally abundant glucose. Gas chromatography-mass spectrometry was used
to measure mass isotopomer abundances in protein hydrolysates from the cells.
Fluxes were evaluated from the isotopomer data by using a mathematical
metabolic pathway model that incorporated isotopomer balancing. The
GK-overexpressing cell lines exhibited significantly reduced cell growth and
lactate production rates compared to the wild type, while consuming carbon
sources at nearly the same rates as the wild type. Unsupervised principal
component analysis of these measurements and mass isotopomer abundances
revealed that fluxes of carbohydrate metabolism differ between the
GK-overexpressing cell lines and the wild type. Flux evaluation by
comprehensive isotopomer balancing and global optimization revealed that the
GK-overexpressing cell lines exhibited a substantially higher flux through the
pentose phosphate pathway. This is likely due to increased NADPH requirement in
the cytosol, and may be mediated by glycerol kinase or its network partners.

Furthermore, we will report a microarray analysis of the
wild type and GK-overexpressing cell lines, followed by a network component
analysis (NCA) of the microarray data. This is expected to reveal the
transcription factor activities altered due to GK overexpression, and together
with the flux results, would shed light on network interactions involving GK
and the role of systems dynamics in GKD.

The results in this presentation will highlight the
multifaceted role of glycerol kinase, and such investigations can be valuable
toward dissecting the biochemistry and elucidating the pathology of this and
other metabolic disorders.

Keywords: Glycerol kinase, glycerol kinase deficiency,
microarray, network component analysis, metabolic flux, pentose phosphate
pathway, isotopomer, moonlighting enzyme.

References

1. Sriram, G., J.A. Martinez, E.R.B. McCabe, J.C. Liao, and
K.M. Dipple, Single-gene disorders: What role could moonlighting enzymes play? Am.
J. Hum. Genet., 2005. 76: 911-924.

2. Huq, A.H., R.S. Lovell, M.J. Sampson, W.K. Decker, M.B.
Dinulos, C.M. Disteche, and W.J. Craigen, Isolation, mapping, and functional
expression of the mouse X chromosome glycerol kinase gene. Genomics,
1996. 36: 530-534.

3. Lee, D.H., D.B. Park, Y.K. Lee, C.S. An, Y.S. Oh, J.S.
Kang, S.H. Kang, and M.Y. Chung, The effects of thiazolidinedione treatment on
the regulations of aquaglyceroporins and glycerol kinase in OLETF rats. Metabolism,
2005. 54: 1282-1289.

4. Guan, H.P., Y. Li, M.V. Jensen, C.B. Newgard, C.M.
Steppan, and M.A. Lazar, A futile metabolic cycle activated in adipocytes by
antidiabetic agents. Nat. Med., 2002. 8: 1122-1128.

5. Dipple, K.M., J.K. Phelan, and E.R. McCabe, Consequences
of complexity within biological networks: robustness and health, or
vulnerability and disease. Mol. Genet. Metab., 2001. 74: 45-50.