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Glycerol kinase (GK) is a multifunctional enzyme located at the interface of carbohydrate and fat metabolism. It contributes to both central carbon metabolism and adipogenesis; specifically, through its role as the ATP-stimulated translocation promoter (ASTP). GK overexpression leads to increased ASTP activity and increased fat storage in H4IIE cells. We performed metabolic flux analysis in human GK-overexpressing H4IIE cells and found that overexpressing cells had significantly altered fluxes through central carbon and lipid metabolism including increased flux through the pentose phosphate pathway and increased production of lipids. We also observed an equal contribution of glycerol to carbohydrate metabolism in all cell lines, suggesting that GK's alternate functions rather than its enzymatic function are important for these processes. To further elucidate the contributions of the enzymatic (phosphorylation) and alternative (ASTP) functions of GK in adipogenesis, we performed experiments on mammalian GK and E. coli GK. We determined that the ASTP function of GK (which is absent in E. coli GK) plays a greater role than the enzymatic activity in these processes. These studies further emphasize GK's diverse functionality and provides fundamental insights into the multiple protein functions of glycerol kinase., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mathematical modeling approaches have been commonly used in complex signaling pathway studies such as the insulin signal transduction pathway. Our expanded mathematical model of the insulin signal transduction pathway was previously shown to effectively predict glucose clearance rates using mRNA levels of key components of the pathway in a mouse model. In this study, we re-optimized and applied our expanded model to study insulin sensitivity in other species and tissues (human skeletal muscle) with altered protein activities of insulin signal transduction pathway components. The model has now been optimized to predict the effect of short term exercise on insulin sensitivity for human test subjects with obesity or type II diabetes mellitus. A comparison between our extended model and the original model showed that our model better simulates the GLUT4 translocation events of the insulin signal transduction pathway and glucose uptake as a clinically relevant model output. Results from our extended model correlate with O'Gorman's published in-vivo results. This study demonstrates the ability to adapt this model to study insulin sensitivity to many biological systems (human skeletal muscle and mouse liver) with minimal changes in the model parameters., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mathematical models of biological pathways facilitate a systems biology approach to medicine. However, these models need to be updated to reflect the latest available knowledge of the underlying pathways. We developed a mathematical model of the insulin signal transduction pathway by expanding the last major previously reported model and incorporating pathway components elucidated since the original model was reported. Furthermore, we show that inputting gene expression data of key components of the insulin signal transduction pathway leads to sensible predictions of glucose clearance rates in agreement with reported clinical measurements. In one set of simulations, our model predicted that glycerol kinase knockout mice have reduced GLUT4 translocation, and consequently, reduced glucose uptake. Additionally, a comparison of our extended model with the original model showed that the added pathway components improve simulations of glucose clearance rates. We anticipate this expanded model to be a useful tool for predicting insulin sensitivity in mammalian tissues with altered expression protein phosphorylation or mRNA levels of insulin signal transduction pathway components., (Copyright © 2014 Elsevier Inc. All rights reserved.)

To examine glycerol homeostasis and metabolism is essential for understanding of pathogenesis and evaluation of treatment efficacy in disorders of glycerol metabolism. In this study, we designed the intraperitoneal glycerol tolerance test (IPGlyTT) and studied glycerol tolerance in vivo using glycerol kinase (Gyk) carrier (C) and wild type (WT) mice. Serum glycerol concentrations in WT almost normalized at 90 min after injection, whereas Gyk C mice retained high serum glycerol concentrations at least until 180 min after injection. These results showed that glycerol tolerance was impaired in Gyk C mice compared to WT mice. The IPGlyTT is useful in accessing glycerol homeostasis and metabolism in animal models such as Gyk C mice and will be valuable in assessing therapeutic interventions in Gyk KO mice., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Glycerol kinase (GK) is an enzyme with diverse (moonlighting) cellular functions. GK overexpression affects central metabolic fluxes substantially; therefore, to elucidate the mechanism underlying these changes, we employed a systems-level evaluation of GK overexpression in H4IIE rat hepatoma cells. Microarray analysis revealed altered expression of genes in metabolism (central carbon and lipid), which correlated with previous flux analysis, and of genes regulated by the glucocorticoid receptor (GR). Oil Red O staining showed that GK overexpression leads to increased fat storage in H4IIE cells. Network component analysis revealed that activities of peroxisome proliferator-activated receptor alpha, GR, and seven other transcription factors were altered by GK overexpression. The increased activity of GR was experimentally verified by quantitative RT-PCR of GR-responsive genes in the presence and absence of the glucocorticoid agonist, dexamethasone. This systems biology approach further emphasizes GK's essential role in central and lipid metabolism and experimentally verifies GK's alternative (moonlighting) function of affecting GR transcription factor activity., (Copyright 2010 Elsevier Inc. All rights reserved.)

Symptomatic glycerol kinase deficiency (GKD) is associated with episodic metabolic and central nervous system deterioration. We report here the first application of weighted gene co-expression network analysis (WGCNA) to investigate a knockout (KO) murine model of a human genetic disease. WGCNA identified networks and key hub transcripts from liver mRNA of glycerol kinase (Gyk) KO and wild-type (WT) mice. Day of life 1 (dol1) samples from KO mice contained a network module enriched for organic acid metabolism before Gyk KO mice develop organic acidemia and die on dol3-4. Furthermore, the module containing Gyk was enriched with apoptotic genes. We used causal testing to elucidate the causal relationships between intramodular hub genes Acot, Psat and Plk3. Important causal relationships are confirmed in cell cultures. We provide evidence that GK may have an apoptotic moonlighting role that is lost in GKD. This first application of WGCNA to mouse knockout data provides insights into the molecular mechanisms of GKD pathogenesis. The resulting systems-genetic gene screening method identifies candidate biomarkers for GKD.

Glycerol kinase (GK) is at the interface of fat and carbohydrate metabolism and has been linked to obesity and type 2 diabetes mellitus (T2DM). The purpose of this study was to investigate the role of GK in fat metabolism and insulin signaling in skeletal muscle (an important end organ tissue in T2DM). Microarray analysis determined that there were 525 genes that were differentially expressed (1.2-fold, p value<0.05) between knockout (KO) and wild-type (WT) mice. Quantitative PCR (qPCR) confirmed the differential expression of genes including glycerol kinase (Gyk), phosphatidylinositol 3-kinase regulatory subunit, polypeptide 1 (p85 alpha) (Pik3r1), insulin-like growth factor 1 (Igf1), and growth factor receptor bound protein 2-associated protein 1 (Gab1). Network component analysis demonstrated that transcription factor activities of myogenic differentiation 1 (MYOD), myogenic regulatory factor 5 (MYF5), myogenin (MYOG), nuclear receptor subfamily 4, group A, member 1 (NUR77) are decreased in the Gyk KO whereas the activity of paired box 3 (PAX3) is increased. The activity of MYOD was confirmed using a DNA binding assay. In addition, myoblasts from Gyk KO had less ability to differentiate into myotubes compared to WT myoblasts. These findings support our previous studies in brown adipose tissue and demonstrate that the role of Gyk in muscle is due in part to its non-metabolic (moonlighting) activities.

Glycerol kinase has several diverse activities in mammalian cells. Glycerol kinase deficiency is a complex, single-gene, inborn error of metabolism wherein no genotype-phenotype correlation has been established. Since glycerol kinase has been suggested to exhibit additional activities than glycerol phosphorylation, expression level perturbation in this enzyme may affect cellular physiology globally. To investigate this possibility, we conducted metabolic investigations of wild-type and two glycerol kinase-overexpressing H4IIE rat hepatoma cell lines constructed in this study. The glycerol kinase-overexpressing cell lines exhibited a significantly higher consumption of carbon sources per cell, suggesting excess carbon expenditure. Furthermore, we quantified intracellular metabolic fluxes by employing stable isotope 13C labeling with a mathematically designed substrate mixture, gas chromatography-mass spectrometry, and comprehensive isotopomer balancing. This flux analysis revealed that the pentose phosphate pathway flux in the glycerol kinase-overexpressing cell lines was 2-fold higher than that in the wild-type, in addition to subtler flux changes in other pathways of carbohydrate metabolism. Furthermore, the activity and transcript level of the lipogenic enzyme glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the pentose phosphate pathway, were also about 2-fold higher than that of the wild-type; these data corroborate the flux analysis results. This study shows that glycerol kinase affects carbon metabolism globally, possibly through its additional functions, and highlights glycerol kinase's multifaceted role in cellular physiology.

Glycerol kinase (GK) is an enzyme at the interface of carbohydrate and fat metabolism. Mutations in the GK gene result in a rare inborn error of metabolism, GK deficiency (GKD), and at least one of these mutations (N288D) is associated with insulin resistance and diabetes mellitus. In an attempt to identify potential modifiers of the GKD phenotype, and to elucidate better the relationship between GKD and diabetes mellitus, we investigated the GK promoter. We examined the GK promoter using in silico methods, transient transfections of GK promoter-luciferase constructs in HepG2 hepatocellular carcinoma cells, and gel shift assays using liver nuclear extracts. We determined that the first 100 bp of the GK 5(') upstream region was sufficient for basal levels of transcription and that there was a functional HNF-4alpha binding site in the first 500 bp of the 5(') upstream region that was important for increased levels of GK expression in vitro. The involvement of both GK and HNF-4alpha in the etiology of diabetes mellitus is intriguing, and we speculate that HNF-4alpha represents a potential modifier of the GKD phenotype.

Clones containing the rat class I alcohol dehydrogenase (ADH) gene were isolated from a Charon 4A genomic library. The gene spans approximately 13 kb and comprises nine exons and eight introns. The upstream 436 bp contain canonical TATA and CCAAT sequences, an inverted CACCC box, a TG3 box found in mouse and human ADH promoters, and regions of homology to glucocorticoid response elements. The 5'-untranslated region of the ADH transcript has the potential to form a stable stem-loop structure. The first intron contains an unusual stretch of alternating purines and pyrimidines similar to that found in the same location in the mouse ADH gene. The amino acid insertion found in rat alcohol dehydrogenase results from a shift in the 3' splice junction of the fourth intron which adds an extra three base pairs to the fifth exon. Intron-exon boundaries are otherwise identical to those in mouse and human ADH genes. H4IIE cells stably transfected with plasmids containing the chloramphenicol acetyltransferase (CAT) gene fused behind the first 436 bp of the promoter region express CAT, but the CAT activity is not inducible by dexamethasone. The elements responsible for glucocorticoid stimulation of ADH gene transcription appear to reside outside of this region.