Thesis (M. S.)--Biology, Georgia Institute of Technology, 2009. / Committee Chair: Merrill, Alfred; Committee Member: Bao, Gang; Committee Member: Chernoff, Yury; Committee Member: Lobachev, Kirill; Committee Member: Sewer, Marion.

Thesis (M. Sc.)--Carleton University, 2001. / Includes bibliographical references (p. 115-123). Also available in electronic format on the Internet.

In the human adrenal cortex, adrenocorticotropin (ACTH) activates steroid hormone biosynthesis by acutely increasing cholesterol delivery to the mitochondria and chronically up-regulating the transcription of steroidogenic genes (including CYP17). Sphingolipids are a diverse family of phospholipids and glycolipids that mediate a wide variety of cellular processes, including apoptosis, proliferation, and survival. Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate genes that are involved in cholesterol biosynthesis and fatty acid metabolism. In this study, we investigated the role of sphingolipids in ACTH-dependent steroidogenesis. H295R human adrenocortical cells were treated with ACTH or dibutyryl cAMP (Bt2cAMP) for various time periods and the content of several sphingolipid species was quantified by mass spectrometry. Both ACTH and Bt2cAMP decreased cellular amounts of sphingomyelin, ceramides, sphingosine (So) and sphingosine-1-phosphate (S1P). However, both ACTH and Bt2cAMP increased the activity of sphingosine kinase and the amounts of S1P released into the media. Both So and S1P increased CYP17 mRNA expression and increased cortisol biosynthesis. This increase in CYP17 transcription occurs by promoting SREBP binding to an SRE at -450/-436 basepairs upstream of the transcription initiation site. Furthermore, chromatin immunoprecipitation (ChIP) assays revealed that Bt2cAMP and S1P treatment results in an increase in acetylation of histone H3 and SREBP1 binding to CYP17 promoter. Additionally, transient transfection studies using wild type or mutated hCYP17 promoters and RNA interference (RNAi) assays confirmed the role of SREBP1 in mediating the stimulatory effect of S1P on CYP17 transcription. In summary, our studies demonstrate a link between sphingolipid metabolism and ACTH-dependent steroidogenesis which requires the activation of SREBP1 in human adrenal cortex.

An important branchpoint of mammalian sphingolipid metabolism occurs at the step where ceramides are glycosylated to glucosylceramide (GlcCer) versus galactosylceramide (GalCer), which are precursors of all mammalian glycosphingolipids. Relatively few studies have focused on this branchpoint because these monohexosylceramides are somewhat difficult to resolve chromatographically and because molecular biology tools have only recently become available to follow expression of these genes. The goal of this thesis is to better understand the mechanisms of cell regulation determining galactosylceramide synthesis.

We have studied the molecular and biological consequences of overexpression of serine palmiotyltransferase (SPT) using HEK293 cells stably transfected with SPTLC1 and SPTLC2 (termed "SPT1/2 cells"). The effects of the elevated SPT activity were analyzed by liquid chromatography, electrospray ionization tandem mass spectrometry. Most sphingolipid subspecies were elevated in SPT1/2 cells, with disproportionately higher dihydrosphingolipids and ceramides with stearic acid. Sphingomyelins were lower, however, which does not appear to be due to faster degradation, but possibly by substitution by dihydrosphingomyelins. Despite large increases in potentially growth inhibitory and lethal ceramides, SPT1/2 cells grow faster than HEK293 cells. We also noted by confocal microscopy that endogenous SPT1 is not only in the endoplasmic reticulum, but also in the nucleus and focal adhesions, which was confirmed by elimination of SPT1 using SPTLC1 siRNA and co-immunoprecipitation of SPT1 with vinculin. The appearance of SPT1 in focal adhesions is lost when cells reach confluence and reappears after a scratch assay to reinitiate migration; furthermore, SPTLC1 siRNA causes cell rounding. Thus, in addition to its "traditional" role in de novo sphingolipid biosynthesis in the ER, SPT1 is present in other cellular compartments and is required for normal cell morphology and migration. It is possible that some of the previously unnoticed properties of SPT1 are due to alternative isoforms because we have found at least one splice variant that is expressed in HEK293 cells.

Thesis (Ph. D.)--University of California, San Diego, and San Diego State University, 2004. / Vita. Includes bibliographical references (leaves 150-167).

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 84-106).

Acute kidney injury (AKI), resulting from chemotherapeutic agents such as cisplatin, remains an obstacle in the treatment of cancer. Cisplatin-induced AKI involves apoptotic and necrotic cell death, pathways regulated by sphingolipids such as ceramide and glucosylceramide. Results from this study indicate that C57BL/6J mice treated with cisplatin had increased ceramide and hexosylceramide levels in the renal cortex 72 h following cisplatin treatment. Pretreatment of mice with inhibitors of acid sphingomyelinase and de novo ceramide synthesis (amitriptyline and myriocin, respectively) prevented accumulation of ceramides and hexosylceramide in the renal cortex and protected from cisplatin-induced AKI. To determine the role of ceramide metabolism to hexosylceramides in kidney injury, we treated mice with a potent and highly specific inhibitor of glucosylceramide synthase, the enzyme responsible for catalyzing the glycosylation of ceramides to form glucosylceramides. Inhibition of glucosylceramide synthase attenuated the accumulation of the hexosylceramides and exacerbated ceramide accumulation in the renal cortex following treatment of mice with cisplatin. Increasing ceramides and decreasing glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI according to markers of kidney function, kidney injury, inflammation, cell stress, and apoptosis. Under conditions of high ceramide generation, data suggest that metabolism of ceramides to glucosylceramides buffers kidney ceramides and helps attenuate kidney injury.-Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Sharp, D. Siow, J. Megyesi, J. Shayman, A.Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury.

Thesis (Ph.D)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Physiology. Title from title-page of electronic thesis. Includes bibliographical references.

Sphingolipids have been implicated as potential atherogenic lipids. Inhibition of hepatic serine palmitoyl transferase (SPT) reduces plasma sphingomyelin (SM) levels leading to a reduction of atherosclerosis in apolipoprotein-E gene knockout (apoE-/-) mice. In my thesis I have investigated the possibility that the reduced atherosclerosis resulting from SPT inhibition is associated with decreases in plasma glycosphingolipids (GSL). Furthermore, I examined whether SPT inhibition can lead to regression of atherosclerotic lesions. This thesis shows the SPT inhibitor myriocin inhibits atherosclerosis in apoE-/- mice fed a high fat diet. Lesion inhibition was most pronounced at the aortic arch and distal sites of the thoracic and abdominal aorta. There was also a trend towards a reduction in lesion area at the aortic root. Myriocin treatment resulted in significant reductions in both plasma SM and GSL concentrations. Moreover, it was demonstrated that myriocin significantly inhibited the progression of established atherosclerosis. Although the inhibition of lesion progression was observed mainly in the distal regions of the aorta, regression of lesion size was not detected. In addition, this thesis demonstrated for the first time that selective inhibition of GSL synthesis by the GSL synthesis inhibitor, d-threo-1- ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4) had no significant impact on lesion area in apoE-deficient mice. Thus, despite the previously observed positive correlations between plasma and aortic GSL concentrations and the development of atherosclerosis, this thesis indicates that inhibition of GSL synthesis does not inhibit atherosclerosis in vivo. In other studies we assessed the possibility that myriocin may also be acting to increase hepatic apoA-I production via the inhibition of ERK phosphorylation. To address this, HepG2 cells and primary mouse hepatocytes were treated with myriocin. This significantly increased apoA-I mRNA, and protein levels. It also increased apoA-I secrection, and decreased ERK phosphorylation. These in vitro data indicate that ERK phosphorylation plays a role in modulating apoA-I expression, and that myriocin???s mechanism of action is linked to this pathway. Overall, this thesis has expanded the current literature regarding the role of sphingolipid synthesis inhibition and atherosclerosis.