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- Sharoyko, Vladimir, et al.
(författare)
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Loss of TFB1M results in mitochondrial dysfunction that leads to impaired insulin secretion and diabetes.
- 2014
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Ingår i: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 23:21, s. 5733-5749
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Tidskriftsartikel (refereegranskat)abstract
- We have previously identified Transcription Factor B1 Mitochondrial (TFB1M) as a Type 2 Diabetes (T2D) risk gene, using human and mouse genetics. To further understand the function of TFB1M and how it is associated with T2D we created a β-cell specific knockout of Tfb1 m, which gradually developed diabetes. Prior to the onset of diabetes, β-Tfb1 m(-/-) mice exhibited retarded glucose clearance due to impaired insulin secretion. β-Tfb1 m(-/-) islets released less insulin in response to fuels, contained less insulin and secretory granules, and displayed reduced β-cell mass. Moreover, mitochondria in Tfb1 m-deficient β-cells were more abundant with disrupted architecture. TFB1M is known to control mitochondrial protein translation by adenine-dimethylation of 12S ribosomal RNA (rRNA). Here, we found that levels of TFB1M and mitochondrial encoded proteins, mitochondrial 12S rRNA methylation, ATP production and oxygen consumption were reduced in β-Tfb1 m(-/-) islets. Furthermore, levels of reactive oxygen species in response to cellular stress were increased while induction of defense mechanisms was attenuated. We also show increased apoptosis and necrosis as well as infiltration of macrophages and CD4(+)-cells in the islets. Taken together, our findings demonstrate that Tfb1 m-deficiency in β-cells caused mitochondrial dysfunction and subsequently diabetes due to combined loss of β-cell function and mass. These observations reflect pathogenetic processes in human islets: using RNA sequencing, we found that the TFB1M risk variant exhibited a negative gene-dosage effect on islet TFB1M mRNA levels, as well as insulin secretion. Our findings highlight the role of mitochondrial dysfunction in impairments of β-cell function and mass, the hallmarks of T2D.
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- Stamenkovic, Jelena
(författare)
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Control of metabolism and hormone secretion from pancreatic alpha and beta cells
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Type 2 Diabetes (T2D) is a multifactorial disease, which has made it difficult to resolve its pathophysiology. We investigated processes that could contribute to the development of T2D. In addition to well-known hallmarks of the disease, such as defective insulin secretion, insulin resistance and perturbed glucagon secretion, we aimed to find a possible role for CLOCK genes in the pathogenesis of T2D. In paper 1, we showed that there is a link between the expression levels of core CLOCK genes in human islets and functional parameters important for T2D. This link to T2D was particularly strong for the core CLOCK components, PER2, PER3 and CRY2. We further investigated this possibility in paper 2, where we found that silencing of Per3 in a beta cell line disrupted insulin secretion in response to glucose and other secretagogues. Our observations suggest that exocytosis may be an underlying cause. In support of this assumption, we found down-regulation of crucial genes involved in the exocytotic machinery upon silencing of Per3. Together, our observations suggest that there is a link between the circadian clock machinery and beta cell function. In paper, 3 we employed an alpha and a beta cell line, which were challenged with the same stimuli. We compared their responses in hormone release and metabolism in order to understand metabolic control of glucagon secretion. We found that two cell lines responded similarly to glucose: alpha cells increased glucagon secretion upon glucose stimulation while beta cells increased secretion of insulin. Differences, however, were primarily found in the coupling of glycolytic and mitochondrial metabolism. Moreover, inhibiting the malate-aspartate shuttle completely abolished glucagon secretion, while insulin secretion was largely preserved. This was likely due to a compensatory activity in the glycerolphosphate shuttle in beta cells. So far, most observations seemed to involve mitochondrial metabolism in some way. Therefore we studied how mitochondria become molecularly equipped for metabolic coupling in beta cells. Characterization of a beta cell-specific knockout of Tfb1m (beta Tfb1m-/-), which encodes a protein controlling translation of mitochondrial proteins, showed mitochondrial dysfunction. This confirmed previous findings where TFB1M was identified as T2D risk gene in human islets. Islets from β-Tfb1m-/- mice showed impaired insulin secretion, contained less insulin in secretory granules and exhibited reduced beta cell mass. Mitochondria exhibited disrupted architecture. All measured metabolic parameters in mitochondria were impaired. Reactive oxygen species were increased, and signs of apoptosis and necrosis with accompanying inflammation were observed. These studies have illustrated the complexity of the mechanisms involved in the pathogenesis of T2D. Thus, investigating different metabolic aspects of its pathogenesis, supported the multifactorial nature of the underlying mechanism of T2D development.
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- Stamenkovic, Jelena, et al.
(författare)
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Inhibition of the malate-aspartate shuttle in mouse pancreatic islets abolishes glucagon secretion without affecting insulin secretion
- 2015
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Ingår i: Biochemical Journal. - 0264-6021. ; 468, s. 49-63
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Tidskriftsartikel (refereegranskat)abstract
- Altered secretion of insulin as well as glucagon has been implicated in the pathogenesis of Type 2 diabetes (T2D), but the mechanisms controlling glucagon secretion from alpha-cells largely remain unresolved. Therefore, we studied the regulation of glucagon secretion from alpha TC1-6 (alpha TC1 clone 6) cells and compared it with insulin release from INS-1 832/13 cells. We found that INS-1 832/13 and alpha TC1-6 cells respectively secreted insulin and glucagon concentration-dependently in response to glucose. In contrast, tight coupling of glycolytic and mitochondrial metabolism was observed only in INS-1 832/13 cells. Although glycolytic metabolism was similar in the two cell lines, TCA (tricarboxylic acid) cycle metabolism, respiration and ATP levels were less glucose-responsive in alpha TC1-6 cells. Inhibition of the malate-aspartate shuttle, using phenyl succinate (PhS), abolished glucose-provoked ATP production and hormone secretion from alpha TC1-6 but not INS-1 832/13 cells. Blocking the malate-aspartate shuttle increased levels of glycerol 3-phosphate only in INS-1 832/13 cells. Accordingly, relative expression of constituents in the glycerol phosphate shuttle compared with malate-aspartate shuttle was lower in alpha TC1-6 cells. Our data suggest that the glycerol phosphate shuttle augments the malate-aspartate shuttle in INS-1 832/13 but not alpha TC1-6 cells. These results were confirmed in mouse islets, where PhS abrogated secretion of glucagon but not insulin. Furthermore, expression of the rate-limiting enzyme of the glycerol phosphate shuttle was higher in sorted primary beta-than in alpha-cells. Thus, suppressed glycerol phosphate shuttle activity in the alpha-cell may prevent a high rate of glycolysis and consequently glucagon secretion in response to glucose. Accordingly, pyruvate-and lactate-elicited glucagon secretion remains unaffected since their signalling is independent of mitochondrial shuttles.
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- Stamenkovic, Jelena, et al.
(författare)
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Regulation of core clock genes in human islets.
- 2012
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Ingår i: Metabolism, Clinical and Experimental. - : Elsevier BV. - 1532-8600. ; 61:7, s. 978-985
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Tidskriftsartikel (refereegranskat)abstract
- Nearly all mammalian cells express a set of genes known as clock genes. These regulate the circadian rhythm of cellular processes by means of negative and positive autoregulatory feedback loops of transcription and translation. Recent genomewide association studies have demonstrated an association between a polymorphism near the circadian clock gene CRY2 and elevated fasting glucose. To determine whether clock genes could play a pathogenetic role in the disease, we examined messenger RNA (mRNA) expression of core clock genes in human islets from donors with or without type 2 diabetes mellitus. Microarray and quantitative real-time polymerase chain reaction analyses were used to assess expression of the core clock genes CLOCK, BMAL-1, PER1 to 3, and CRY1 and 2 in human islets. Insulin secretion and insulin content in human islets were measured by radioimmunoassay. The mRNA levels of PER2, PER3, and CRY2 were significantly lower in islets from donors with type 2 diabetes mellitus. To investigate the functional relevance of these clock genes, we correlated their expression to insulin content and glycated hemoglobin levels: mRNA levels of PER2 (ρ = 0.33, P = .012), PER3 (ρ = 0.30, P = .023), and CRY2 (ρ = 0.37, P = .0047) correlated positively with insulin content. Of these genes, expression of PER3 and CRY2 correlated negatively with glycated hemoglobin levels (ρ = -0.44, P = .0012; ρ = -0.28, P = .042). Furthermore, in an in vitro model mimicking pathogenetic conditions, the PER3 mRNA level was reduced in human islets exposed to 16.7 mmol/L glucose per 1 mmol/L palmitate for 48 hours (P = .003). Core clock genes are regulated in human islets. The data suggest that perturbations of circadian clock components may contribute to islet pathophysiology in human type 2 diabetes mellitus.
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