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.

The mammalian target of rapamycin complex 1 (mTORC1), a master regulator of cell growth and proliferation, is aberrantly activated in cancer, genetic tumor syndromes and obesity. Much progress has been made to understand the upstream pathways that regulate mTORC1, most of which converge upon its negative regulator, the Tuberous Sclerosis Complex (TSC) 1-TSC2 complex. However, the cell intrinsic consequences of aberrant mTORC1 activation remain poorly characterized. Using systems in which mTORC1 is constitutively activated by genetic loss of TSC1 or TSC2 and pharmacologically inhibited by treatment with an mTORC1-specific inhibitor rapamycin, we have identified that mTORC1 controls specific aspects of cellular metabolism, including glycolysis, the pentose phosphate pathway, and de novo lipogenesis. Induction of the pentose phosphate pathway and de novo lipogenesis is achieved by activation of a transcriptional program affecting metabolic gene targets of sterol regulatory element-binding protein (SREBP). We have demonstrated that mTORC1 stimulates the accumulation of processed, active SREBP, although details of the molecular mechanism remain to be elucidated. To understand the physiological and pathological relevance of mTORC1-dependent activation of SREBPs and lipogenesis, we explored these findings in the liver and in cancer. While we find that the induction of hepatic SREBP1c and lipogenesis by insulin requires mTORC1, mTORC1 activation is not sufficient to stimulate hepatic SREBP1c in the absence of Akt signaling, revealing the existence of an additional downstream pathway also required for this induction. We demonstrate that this mTORC1-independent pathway involves Akt-mediated suppression of Insig2a, a liver-specific transcript encoding the SREBP1c inhibitor INSIG2. In cancer, our initial findings demonstrate that mTORC1 plays a role downstream of TSC-deficiency and oncogenic PIK3CA and K-Ras to activate lipogenic SREBP targets and de novo lipogenesis. Further studies of the connection between mTORC1 and SREBPs in disease may offer insights into novel therapeutic approaches.

The goal of this research project is to understand how a high sugar diet may affect pancreatic beta cell function. High glucose concentrations lead to an increase in lipid droplets and TORC1 in beta cells, which promote high basal secretion of insulin (Erion K.A. et al., JBC, 2015). SREBP is a key regulator of cholesterol and lipid synthesis and depends on TORC1 activity. The active form of SREBP is located in the nucleus. Does glucose-induced lipid synthesis in beta cells increase via SREBP? To answer this question, we propose: 1) To test the effect of high glucose (11mM) on nuclear SREBP in INS-1 cells in comparison to physiological glucose (4mM). 2) To determine if nuclear SREBP is affected when PIP4Kgamma (a regulator of TORC1) is suppressed. SREBP translocation from the cytosol to the nucleus was measured by immunofluorescence. SREBP processing was measured by western blot. SREBP1 activation increased in response to prolonged exposure to excess glucose after at least 48hrs. Both translocation and processing increased in 11mM glucose compared to 4mM glucose. When PIP4Kgamma was suppressed in INS-1 cells, SREBP translocation was inhibited. Lipid droplet accumulation was measured by nile red staining and it was found that de novo lipid synthesis only contributes to a small fraction of total lipid droplets. In conclusion, SREBP is activated in beta cells when in excess glucose. This may allow for lipid accumulation and basal hypersecretion of insulin due to over nutrition.

京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第13373号 / 論医科博第6号 / 新制||医科||8(附属図書館) / (主査)教授 長船 健二, 教授 稲垣 暢也, 教授 渡邊 直樹 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Plasma membranes have a structural property, commonly referred to as membrane fluidity, that is compositionally regulated. The two main features of plasma membrane lipid composition that determine membrane fluidity are the ratio of cholesterol to phospholipids and the ratio of saturated to unsaturated fatty acids that are incorporated into the phospholipids. These ratios are determined, at least in part, by regulation of membrane lipid biosynthesis-particularly that of cholesterol and oleate. It now appears that cholesterol and oleate biosynthesis are feedback regulated by a common transcriptional mechanism which is governed by the maturation of the SREBP transcription factors. In this article, we briefly review our current understanding of transcriptional regulation of plasma membrane lipid biosynthesis by sterols and oleate. We also discuss studies related to the mechanism by which the physical state of membrane lipids signals the transcriptional regulatory machinery to control the rates of synthesis of these structural components of the lipid bilayer.

The objective of these studies was to investigate the milk fat synthesis regulation by transcription factors. In the first study, bovine mammary epithelial (MAC-T) cells were treated with sterol regulatory element binding protein-1 (SREBP-1) specific siRNA. The mRNA and protein expression of SREBP-1 were decreased by more than 90% by siRNA. Fatty acid (FA) synthesis, uptake, and selected lipogenic enzyme expression were reduced in cells treated with SREBP-1 siRNA. Therefore, SREBP-1 plays an important role in integrated regulation of lipid synthesis in MAC-T cells through regulation of key enzymes. In the second study, MAC-T cells treated with hormones or FA were transfected with luciferase reporter constructs containing response elements for SREBP-1, peroxisome proliferator-activated receptor γ (PPARγ), or liver X receptor (LXR). The activation of PPARγ and SREBP-1 were stimulated by insulin and insulin combined with leptin, respectively. Trans-10, cis-12 conjugated linoleic acid (CLA) inhibited SREBP-1 activation, and this inhibition was not attenuated by insulin and leptin. Neither trans-10 nor cis-12 double bond inhibited SREBP-1 activation. Taken together, trans-10 and cis-12 double bonds need to be conjugated in CLA to reduce SREBP-1 activation and this inhibition cannot be overcome by insulin and leptin combination in MAC-T cells. In the third study, lactating dairy cows were intravenously infused with 0.625 g/h trans-10, cis-12 CLA for 14 h. We confirmed the appearance of trans-10, cis-12 CLA in the milk of CLA treated cows. Milk and component yield were not affected by the CLA treatment. The desaturation of stearic acid was reduced by CLA. The mRNA and protein expression of transcription factors or lipogenic enzymes were not affected by trans-10, cis-12 CLA. DNA-binding activities for PPARγ and LXR and the activation of SREBP-1 to its mature form were not changed by the treatment. The infusion time in this study was probably too short to induce any changes in transcription factors and lipogenic enzymes. We confirmed DNA-binding activities of PPARγ and LXR in bovine mammary gland. Overall, a prominent role for SREBP-1 in mammary epithelial cell lipid synthetic pathways was described and regulation of transcription factor activation by trans-10, cis-12 CLA was specific to SREBP-1. / Ph. D.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19600号 / 医博第4107号 / 新制||医||1014(附属図書館) / 32636 / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 清水 章, 教授 川上 浩司, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20287号 / 医科博第78号 / 新制||医科||5(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 岩井 一宏, 教授 萩原 正敏, 教授 横出 正之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Diabetic nephropathy represents the leading cause of end stage renal disease worldwide and requires a kidney transplant or dialysis to survive. The number of patients suffering from diabetes is expected to increase, thus the number of patients with diabetic nephropathy is expected to concomitantly increase. Current treatment for diabetic nephropathy is not sufficient to prevent disease progression in most patients thus there is a need to develop novel therapies to treat diabetic nephropathy. The earliest changes that occur during the pathogenesis of diabetes occur in the glomerulus. The mesangial cells are a subpopulation of cells in the glomerulus that are responsible for coordinating responses with other nearby cell types. Transforming growth factor (TGF)-β1 is a cytokine that mesangial cells secrete, and has been identified as a profibrotic factor during the pathogenesis of diabetic nephropathy. Concerns have been raised in the use of direct anti-TGF-β1 therapy due to adverse events (such as dyspepsia and diarrhea) and lack of efficacy of anti-TGF-β1 monoclonal antibody LY2382770 in patients with diabetic nephropathy. Thus, therapy aimed at modulating TGF-β1 expression or activity may be efficacious in the treatment of diabetic nephropathy while avoiding potential adverse effects. The hypothesis of this thesis is that SREBP-1 and cell surface GRP78 are novel regulators of TGF-β1 signaling in mesangial cells. Our first study aims to define a novel pathway by which SREBP-1 regulates TGF-β1 signaling in kidney mesangial cells. Our results indicate that SREBP-1 regulates the expression of the type I TGF-β1 receptor through its secretion in exosomes. Our second study expands on these findings and aims to determine if inhibition of SREBP in vivo with the inhibitor fatostatin may prevent diabetic nephropathy. Our results indicate that treatment with fatostatin does not prevent diabetic nephropathy, but accentuates kidney injury in non-diabetic mice. Preliminary results from our lab have indicated that under diabetic conditions, GRP78 is upregulated at the cell surface and may contribute to the activation of SREBP-1 in an ER-stress dependent mechanism. Our third study thus aims to characterize the expression of cell surface GRP78 in diabetic conditions, and to determine its pathological relevance in the development of diabetic nephropathy. Our results have established novel pathways by which TGF-β1 signaling is regulated in mesangial cells. This will assist in identification of novel therapeutic targets that may be of use in the treatment of diabetic nephropathy. / Thesis / Doctor of Philosophy (PhD) / Diabetic kidney disease is the leading cause of end stage renal disease and represents an important risk factor for mortality. The goal of this thesis is to understand and describe the pathways and mechanisms that contribute to the development of diabetic kidney disease in order to identify novel therapeutic targets. This thesis has identified the protein sterol regulatory element binding protein (SREBP)-1 and the cell surface presentation of another protein, the 78 kDa glucose regulated protein (GRP78), as contributors to diabetic kidney disease. Furthermore, this thesis has demonstrated that anti-SREBP therapy with the drug fatostatin did not prevent diabetic kidney disease. These studies show that while inhibiting SREBP-1 and cell surface GRP78 may be effective in the treatment of diabetic kidney disease, the drug fatostatin should not be used for treatment.

La maladie de Parkinson (MP) est caractérisée par une perte progressive des terminaisons présynaptiques dopaminergiques et reste actuellement incurable. Néanmoins, dans les études épidémiologiques, il a été montré que l’utilisation des statines, médicaments hypocholestérolémiants, diminue le risque de développer une MP. Les statines sont également capables d'inhiber les effets neurodégénératifs dans les modèles précliniques in-vitro et in-vivo de la MP. Cependant, les mécanismes moléculaires à l’origine de ces effets neuroprotecteurs ne sont pas encore complétement élucidés. Ainsi, nous avons étudié les effets potentiels des statines sur l'expression des marqueurs synaptiques et sur le transport de la dopamine. Dans nos études, les statines induisent la croissance des neurites dans les cellules dopaminergiques et déclenchent une augmentation de l’expression des protéines synaptiques dopaminergiques telles que le transporteur vésiculaire des monoamines (VMAT2) et le transporteur de la dopamine. Les statines induisent une diminution de la recapture de la dopamine cellulaire et des changements d’affinités aux niveaux des sites de liaison des inhibiteurs sélectifs du VMAT2. L’activation du facteur de transcription nucléaire protéine-1 se liant à l'élément de régulation des stérols (SREBP-1), cholestérol-dépendent, serait l’élément inducteur de la surexpression des marqueurs dopaminergiques présynaptiques induite par les statines. En outre, ces résultats soutiennent un potentiel thérapeutique neuroprotecteur et/ou neurorestaurateur des statines précédemment proposées dans la MP et permettent de mettre en évidence de nouvelles cibles thérapeutiques comme le facteur SREBP. / Parkinson disease (PD) is characterized by a progressive loss of dopaminergic presynaptic terminals and remains incurable. However in epidemiological studies, it has been shown that the use of statins, which are hypocholesterolemic drugs, diminishes the risk to develop a PD. Statins are able to inhibit the neurodegenerative effects in in-vitro and in-vivo models of PD. However, the molecular mechanisms driving neuroprotective effects are not yet fully understood. Consequently, we investigated the potential effects of statins on the synaptic expression and dopamine transport function in the dopaminergic system. In our studies, statins enhance the neurite outgrowth in the dopaminergic cells and trigger an increase in the expression levels of presynaptic dopaminergic proteins such as vesicular monoamine transporter 2 (VMAT2) and dopamine transporter. Statins induce a reduction of dopamine cellular uptake and modulate the binding-affinity of the specific inhibitors for VMAT2. The activation of the nuclear transcriptional factor sterol regulatory element-binding protein 1 (SREBP-1), cholesterol-dependent, could be the key element of the overexpression of dopaminergic presynaptic markers induced by the statins. Furthermore, these findings highlight the therapeutic neuroprotective and/or neurorestorative potentials of statins previously proposed in PD and allow to bring out new potential therapeutic targets such as SREBP factor.