Browsing by Subject "mTORC1"
Now showing items 1-4 of 4
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(2017)Obesity and insulin resistance (IR) are key factors lead to equine metabolic syndrome and laminitis. Diet may play an important role in eliciting obesity by affecting insulin dynamics. Insulin-pathway signaling and mTORC1 genes may contribute to incred IR. The first objective of this study was to find and validate internal control genes for quantitative PCR method for adipose tissues in Finnhorse mares. The second aim was to quantitate the expression of mTORC1 and insulin-pathway associated genes after pasture season in two different treatment groups of Finnhorse mares and compare gene expression differences between treatment groups. In addition, gene expression differences were compared between two different adipose tissues. Twenty-two mares were equally divided into eleven equal pairs, the two mares of each group were randomly grazed either on cultivated high-yielding pasture (CG) or on semi-natural grassland (NG) from the end of May to the beginning of September. Eight pairs of Finnhorse mares were selected for gene expression profiling. Subcutaneous adipose tissue (SAT) samples were collected from two groups of Finnhorse mares after pasture season. Gene expression of neck and tailhead SAT were determined with quantitative Real-Time PCR method (qPCR). The selected internal control genes were actin beta (ACTB), glucuronidase beta (GUSB) and mitochondrial ribosomal protein L39 (MRPL39). Candidate genes were mechanistic target of rapamycin (MTOR), sterol regulatory element binding transcription factor 1 (SREBF1), sterol regulatory element binding transcription factor 2 (SREBF2), TBC1 domain family member 7 (TBC1D7), leptin (LEP), glucose transporter type 4 (GLUT4), monocyte chemoattractant protein-1 (MCP-1), retinol binding protein 4 (RBP4), tuberous sclerosis 1 (TSC1), tuberous sclerosis 2 (TSC2). There were no distinct gene expression differences between NG and CG groups in both neck and tailhead SAT. However, RBP4 had significantly (P=0.035) higher and GLUT4 had a trend (P=0.064) to higher mRNA expression in CG group in neck SAT. TSC1 had a trend (P=0.071) of higher expression in CG group in tailhead SAT. Gene expression differences were observed between tailhead and neck SAT. SREBF1 and GLUT4 had significantly (P=0.007 and P=0.026, respectively) higher expression levels in tailhead SAT compared to neck SAT. RBP4 had a trend (P=0.066) to higher expression in neck SAT compared to tailhead SAT. Minor differences in gene expression between NG and CG groups indicate that pasture-associated fat depositionmaynotconsiderably affect expressionof insulin-pathway and mTORC1 genes associated to obesity and IR in studied subcutaneous adipose tissues. These results also provide additional evidence to our hypothesis that fattening resulting on unrestricted grazing on cultivated high-yielding pasture does not increase the risk of metabolic diseases in Finnhorse mares when they have normal body condition at the beginning of the grazing season.
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(2017)Obesity and insulin resistance (IR) are key factors lead to equine metabolic syndrome and laminitis. Diet may play an important role in eliciting obesity by affecting insulin dynamics. Insulin-pathway signaling and mTORC1 genes may contribute to incred IR. The first objective of this study was to find and validate internal control genes for quantitative PCR method for adipose tissues in Finnhorse mares. The second aim was to quantitate the expression of mTORC1 and insulin-pathway associated genes after pasture season in two different treatment groups of Finnhorse mares and compare gene expression differences between treatment groups. In addition, gene expression differences were compared between two different adipose tissues. Twenty-two mares were equally divided into eleven equal pairs, the two mares of each group were randomly grazed either on cultivated high-yielding pasture (CG) or on semi-natural grassland (NG) from the end of May to the beginning of September. Eight pairs of Finnhorse mares were selected for gene expression profiling. Subcutaneous adipose tissue (SAT) samples were collected from two groups of Finnhorse mares after pasture season. Gene expression of neck and tailhead SAT were determined with quantitative Real-Time PCR method (qPCR). The selected internal control genes were actin beta (ACTB), glucuronidase beta (GUSB) and mitochondrial ribosomal protein L39 (MRPL39). Candidate genes were mechanistic target of rapamycin (MTOR), sterol regulatory element binding transcription factor 1 (SREBF1), sterol regulatory element binding transcription factor 2 (SREBF2), TBC1 domain family member 7 (TBC1D7), leptin (LEP), glucose transporter type 4 (GLUT4), monocyte chemoattractant protein-1 (MCP-1), retinol binding protein 4 (RBP4), tuberous sclerosis 1 (TSC1), tuberous sclerosis 2 (TSC2). There were no distinct gene expression differences between NG and CG groups in both neck and tailhead SAT. However, RBP4 had significantly (P=0.035) higher and GLUT4 had a trend (P=0.064) to higher mRNA expression in CG group in neck SAT. TSC1 had a trend (P=0.071) of higher expression in CG group in tailhead SAT. Gene expression differences were observed between tailhead and neck SAT. SREBF1 and GLUT4 had significantly (P=0.007 and P=0.026, respectively) higher expression levels in tailhead SAT compared to neck SAT. RBP4 had a trend (P=0.066) to higher expression in neck SAT compared to tailhead SAT. Minor differences in gene expression between NG and CG groups indicate that pasture-associated fat depositionmaynotconsiderably affect expressionof insulin-pathway and mTORC1 genes associated to obesity and IR in studied subcutaneous adipose tissues. These results also provide additional evidence to our hypothesis that fattening resulting on unrestricted grazing on cultivated high-yielding pasture does not increase the risk of metabolic diseases in Finnhorse mares when they have normal body condition at the beginning of the grazing season.
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(2022)Tiivistelmä – Referat – Abstract The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase is a master regulator of cell growth. In the presence of environmental cues, such as nutrients and growth factor, mTORC1 is transported to the lysosome where it is activated by a small GTPase Rheb. Dysregulation of mTORC1 has been linked to several diseases such as cancer and neurodegeneration. Despite our growing understanding of the nutrient-driven activation mechanism of mTORC1, we still do not fully understand how nutrients are transported out of the lysosome or how nutrient sensing is connected to nutrient transport. Recently, SLC38A9, a small lysosomal transmembrane protein, was identified as a mediator of the efflux of essential amino acids from the lysosome to the cytosol. It also acts as an amino acid sensor for mTORC1, playing a role in its activation. Due to poorly vascularized tumor cores, cancers such as pancreatic ductal adenocarcinoma, have access to very scarce amounts of free nutrients. Consequently, they rely on scavenging of protein macromolecules from the extracellular environment, followed by digestion inside lysosomes. The digested nutrients are released to the cytosol via transporters such as SLC38A9 and activate the mTORC1 pathway which carries out the growth processes. In fact, recent studies in mouse xenograft models have shown a severely slowed down growth of PDAC tumors with SLC38A9 knocked out. Blocking of SLC38A9 activity with pharmacologics or biologics would prevent the release of digested amino acids from the lysosomes, starving cancer cells of nutrients, while sparing normal cells that do not feed on extracellular proteins. However, SLC38A9 is still poorly understood, and development of selective inhibitors first requires mechanistic understanding of the protein and knowing what its binding pockets look like. In order to obtain this information, we aimed to determine the three-dimensional structure of SLC38A9 through cryogenic electron microscopy (cryo-EM). However, two significant challenges hindered our ability to obtain high-resolution images of this membrane protein: (i) its small size, and (ii) its constant conformational changes. To address this, I proceeded to develop a set of nanobodies that would bind SLC38A9 with high affinity and specificity. Nanobodies allow for locking of target proteins in specific conformational states, and they can also serve as chaperones for visualizing proteins in cryo-EM. To obtain these nanobodies, I used a library of 100 million unique nanobodies, displayed on the surface of yeast cells. Specific SLC38A9 binder nanobodies were obtained through multiple rounds of selection and sorting, using decreasing concentrations of fluorescently- labeled SLC38A9. After the final selection round, single colonies were picked and the strength of binding to SLC38A9 was evaluated. High-throughput screening results showed that we were able to obtain specific SLC38A9 binders and that there was variation in binding strength among the selected nanobodies. These nanobodies will enable the determination of the cryo-EM structure of SLC38A9 and also serve as tools to further dissect the function and mechanisms of SLC38A9 in amino-acid efflux from lysosomes to cytosol, providing further insights for the development of novel cancer therapeutics.
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(2022)Tiivistelmä – Referat – Abstract The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase is a master regulator of cell growth. In the presence of environmental cues, such as nutrients and growth factor, mTORC1 is transported to the lysosome where it is activated by a small GTPase Rheb. Dysregulation of mTORC1 has been linked to several diseases such as cancer and neurodegeneration. Despite our growing understanding of the nutrient-driven activation mechanism of mTORC1, we still do not fully understand how nutrients are transported out of the lysosome or how nutrient sensing is connected to nutrient transport. Recently, SLC38A9, a small lysosomal transmembrane protein, was identified as a mediator of the efflux of essential amino acids from the lysosome to the cytosol. It also acts as an amino acid sensor for mTORC1, playing a role in its activation. Due to poorly vascularized tumor cores, cancers such as pancreatic ductal adenocarcinoma, have access to very scarce amounts of free nutrients. Consequently, they rely on scavenging of protein macromolecules from the extracellular environment, followed by digestion inside lysosomes. The digested nutrients are released to the cytosol via transporters such as SLC38A9 and activate the mTORC1 pathway which carries out the growth processes. In fact, recent studies in mouse xenograft models have shown a severely slowed down growth of PDAC tumors with SLC38A9 knocked out. Blocking of SLC38A9 activity with pharmacologics or biologics would prevent the release of digested amino acids from the lysosomes, starving cancer cells of nutrients, while sparing normal cells that do not feed on extracellular proteins. However, SLC38A9 is still poorly understood, and development of selective inhibitors first requires mechanistic understanding of the protein and knowing what its binding pockets look like. In order to obtain this information, we aimed to determine the three-dimensional structure of SLC38A9 through cryogenic electron microscopy (cryo-EM). However, two significant challenges hindered our ability to obtain high-resolution images of this membrane protein: (i) its small size, and (ii) its constant conformational changes. To address this, I proceeded to develop a set of nanobodies that would bind SLC38A9 with high affinity and specificity. Nanobodies allow for locking of target proteins in specific conformational states, and they can also serve as chaperones for visualizing proteins in cryo-EM. To obtain these nanobodies, I used a library of 100 million unique nanobodies, displayed on the surface of yeast cells. Specific SLC38A9 binder nanobodies were obtained through multiple rounds of selection and sorting, using decreasing concentrations of fluorescently- labeled SLC38A9. After the final selection round, single colonies were picked and the strength of binding to SLC38A9 was evaluated. High-throughput screening results showed that we were able to obtain specific SLC38A9 binders and that there was variation in binding strength among the selected nanobodies. These nanobodies will enable the determination of the cryo-EM structure of SLC38A9 and also serve as tools to further dissect the function and mechanisms of SLC38A9 in amino-acid efflux from lysosomes to cytosol, providing further insights for the development of novel cancer therapeutics.
Now showing items 1-4 of 4