Expression of two-pore channels in mammalian primary cells and tissues, and their role in adipose tissue formation and function
<p>Two-pore channels (TPCs, gene name <em>Tpcn</em>) have recently been identified as endolysosomal cation channels modulated by the potent calcium (Ca<sup>2+</sup>) releasing messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Gene knockout (KO) and RNA k...
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| Format: | Thesis |
| Language: | English |
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2012
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| _version_ | 1797074076479520768 |
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| author | Tunn, R |
| author2 | Parrington, J |
| author_facet | Parrington, J Tunn, R |
| author_sort | Tunn, R |
| collection | OXFORD |
| description | <p>Two-pore channels (TPCs, gene name <em>Tpcn</em>) have recently been identified as endolysosomal cation channels modulated by the potent calcium (Ca<sup>2+</sup>) releasing messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Gene knockout (KO) and RNA knockdown studies have implicated TPCs in fundamental cellular processes, including secretion, of insulin in pancreatic islets, and differentiation, of skeletal myoblasts and osteoclasts.</p> <p>Investigations of <em>Tpcn1</em> and <em>Tpcn2</em> mRNA expression have indicated widespread tissue distribution, but a lack of suitable antibodies has impeded study of the endogenous proteins. In this study, an anti-TPC1 antibody was purified from immune sera and used in immunoblotting investigations to demonstrate TPC1 protein expression in a wide range of mouse tissues, with highest expression levels observed in kidney, liver and adipose tissue. Endogenous mouse TPC1 was demonstrated to be glycosylated, with apparent differences in the extent of glycosylation in different tissues based on the indicated molecular weight before and after treatment with a deglycosylating enzyme, which may have implications for the functional regulation of channel activity.</p> <p>Given the increasing prevalence of type 2 diabetes and obesity, an understanding of the molecular basis of glucose homeostasis and adipose tissue formation and function is an important scientific goal. <em>Tpcn</em> KO mice have been developed; in both <em>Tpcn1</em> KOs and <em>Tpcn2</em> KOs, impaired pancreatic β-cell Ca<sup>2+</sup> signalling and reduced insulin secretion from the whole pancreas were demonstrated. However, the whole-animal phenotype has not been extensively researched. In this study, intraperitoneal glucose tolerance tests were conducted in <em>Tpcn</em> KO mice. These indicated that glucose homeostasis was not significantly affected in <em>Tpcn2</em> KOs or <em>Tpcn1/2</em> double KOs (DKOs), and only mildly impaired in <em>Tpcn1</em> KOs, despite the defects previously observed at the cellular and tissue level. In addition, body composition was investigated in <em>Tpcn1</em> KO, <em>Tpcn2</em> KO and <em>Tpcn1/2</em> DKO animals using magnetic resonance spectroscopy and time domain-nuclear magnetic resonance. Single <em>Tpcn</em> KOs were found to have lower adipose tissue levels as a percentage of body composition, while <em>Tpcn1/2</em> DKOs were shown to have increased bodyweight but normal body composition.</p> <p>To investigate potential roles for TPCs in adipose tissue formation, Tpcn expression during adipogenesis was investigated using an <em>in vitro</em> multipotent mesenchymal stem cell line model of adipogenic differentiation. <em>Tpcn2</em> mRNA levels were demonstrated by quantitative PCR to be transiently increased during the early stages of adipogenic differentiation, and cyclic AMP (cAMP) was identified as the factor that induced this upregulation. Lentiviruses were developed to express fluorescently-tagged TPCs, and overexpression of TPC2 was demonstrated to partially overcome the requirement for the cAMP-inducing agent in the medium used for the induction of adipogenesis. Collectively, these data suggest that TPCs may play a role in the formation and/or function of adipose tissue.</p> |
| first_indexed | 2024-03-06T23:31:05Z |
| format | Thesis |
| id | oxford-uuid:6c0b970d-6133-4752-987a-e21f6e2dc69c |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T23:31:05Z |
| publishDate | 2012 |
| record_format | dspace |
| spelling | oxford-uuid:6c0b970d-6133-4752-987a-e21f6e2dc69c2022-03-26T19:08:14ZExpression of two-pore channels in mammalian primary cells and tissues, and their role in adipose tissue formation and functionThesishttp://purl.org/coar/resource_type/c_db06uuid:6c0b970d-6133-4752-987a-e21f6e2dc69cPharmacologyPhysiologyCell Biology (see also Plant sciences)Genetics (life sciences)EnglishOxford University Research Archive - Valet2012Tunn, RParrington, J <p>Two-pore channels (TPCs, gene name <em>Tpcn</em>) have recently been identified as endolysosomal cation channels modulated by the potent calcium (Ca<sup>2+</sup>) releasing messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Gene knockout (KO) and RNA knockdown studies have implicated TPCs in fundamental cellular processes, including secretion, of insulin in pancreatic islets, and differentiation, of skeletal myoblasts and osteoclasts.</p> <p>Investigations of <em>Tpcn1</em> and <em>Tpcn2</em> mRNA expression have indicated widespread tissue distribution, but a lack of suitable antibodies has impeded study of the endogenous proteins. In this study, an anti-TPC1 antibody was purified from immune sera and used in immunoblotting investigations to demonstrate TPC1 protein expression in a wide range of mouse tissues, with highest expression levels observed in kidney, liver and adipose tissue. Endogenous mouse TPC1 was demonstrated to be glycosylated, with apparent differences in the extent of glycosylation in different tissues based on the indicated molecular weight before and after treatment with a deglycosylating enzyme, which may have implications for the functional regulation of channel activity.</p> <p>Given the increasing prevalence of type 2 diabetes and obesity, an understanding of the molecular basis of glucose homeostasis and adipose tissue formation and function is an important scientific goal. <em>Tpcn</em> KO mice have been developed; in both <em>Tpcn1</em> KOs and <em>Tpcn2</em> KOs, impaired pancreatic β-cell Ca<sup>2+</sup> signalling and reduced insulin secretion from the whole pancreas were demonstrated. However, the whole-animal phenotype has not been extensively researched. In this study, intraperitoneal glucose tolerance tests were conducted in <em>Tpcn</em> KO mice. These indicated that glucose homeostasis was not significantly affected in <em>Tpcn2</em> KOs or <em>Tpcn1/2</em> double KOs (DKOs), and only mildly impaired in <em>Tpcn1</em> KOs, despite the defects previously observed at the cellular and tissue level. In addition, body composition was investigated in <em>Tpcn1</em> KO, <em>Tpcn2</em> KO and <em>Tpcn1/2</em> DKO animals using magnetic resonance spectroscopy and time domain-nuclear magnetic resonance. Single <em>Tpcn</em> KOs were found to have lower adipose tissue levels as a percentage of body composition, while <em>Tpcn1/2</em> DKOs were shown to have increased bodyweight but normal body composition.</p> <p>To investigate potential roles for TPCs in adipose tissue formation, Tpcn expression during adipogenesis was investigated using an <em>in vitro</em> multipotent mesenchymal stem cell line model of adipogenic differentiation. <em>Tpcn2</em> mRNA levels were demonstrated by quantitative PCR to be transiently increased during the early stages of adipogenic differentiation, and cyclic AMP (cAMP) was identified as the factor that induced this upregulation. Lentiviruses were developed to express fluorescently-tagged TPCs, and overexpression of TPC2 was demonstrated to partially overcome the requirement for the cAMP-inducing agent in the medium used for the induction of adipogenesis. Collectively, these data suggest that TPCs may play a role in the formation and/or function of adipose tissue.</p> |
| spellingShingle | Pharmacology Physiology Cell Biology (see also Plant sciences) Genetics (life sciences) Tunn, R Expression of two-pore channels in mammalian primary cells and tissues, and their role in adipose tissue formation and function |
| title | Expression of two-pore channels in mammalian primary cells and tissues, and their role in adipose tissue formation and function |
| title_full | Expression of two-pore channels in mammalian primary cells and tissues, and their role in adipose tissue formation and function |
| title_fullStr | Expression of two-pore channels in mammalian primary cells and tissues, and their role in adipose tissue formation and function |
| title_full_unstemmed | Expression of two-pore channels in mammalian primary cells and tissues, and their role in adipose tissue formation and function |
| title_short | Expression of two-pore channels in mammalian primary cells and tissues, and their role in adipose tissue formation and function |
| title_sort | expression of two pore channels in mammalian primary cells and tissues and their role in adipose tissue formation and function |
| topic | Pharmacology Physiology Cell Biology (see also Plant sciences) Genetics (life sciences) |
| work_keys_str_mv | AT tunnr expressionoftwoporechannelsinmammalianprimarycellsandtissuesandtheirroleinadiposetissueformationandfunction |