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Deletion of the alpha-arrestin protein Txnip in mice promotes adiposity and adipogenesis while preserving insulin sensitivity.

Chutkow WA, Birkenfeld AL, Brown JD, Lee HY, Frederick DW, Yoshioka J, Patwari P, Kursawe R, Cushman SW, Plutzky J, Shulman GI, Samuel VT, Lee RT - Diabetes (2010)

Bottom Line: Thioredoxin interacting protein (Txnip), a regulator of cellular oxidative stress, is induced by hyperglycemia and inhibits glucose uptake into fat and muscle, suggesting a role for Txnip in type 2 diabetes pathogenesis.RNA interference gene-silenced preadipocytes and Txnip(-/-) MEFs were markedly adipogenic, whereas Txnip overexpression impaired adipocyte differentiation.As increased adipogenesis and insulin sensitivity suggested aspects of augmented peroxisome proliferator-activated receptor-gamma (PPARgamma) response, we investigated Txnip's regulation of PPARgamma function; manipulation of Txnip expression directly regulated PPARgamma expression and activity.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Cambridge, Massachusetts, USA.

ABSTRACT

Objective: Thioredoxin interacting protein (Txnip), a regulator of cellular oxidative stress, is induced by hyperglycemia and inhibits glucose uptake into fat and muscle, suggesting a role for Txnip in type 2 diabetes pathogenesis. Here, we tested the hypothesis that Txnip- (knockout) mice are protected from insulin resistance induced by a high-fat diet.

Research design and methods: Txnip gene-deleted (knockout) mice and age-matched wild-type littermate control mice were maintained on a standard chow diet or subjected to 4 weeks of high-fat feeding. Mice were assessed for body composition, fat development, energy balance, and insulin responsiveness. Adipogenesis was measured from ex vivo fat preparations, and in mouse embryonic fibroblasts (MEFs) and 3T3-L1 preadipocytes after forced manipulation of Txnip expression.

Results: Txnip knockout mice gained significantly more adipose mass than controls due to a primary increase in both calorie consumption and adipogenesis. Despite increased fat mass, Txnip knockout mice were markedly more insulin sensitive than controls, and augmented glucose transport was identified in both adipose and skeletal muscle. RNA interference gene-silenced preadipocytes and Txnip(-/-) MEFs were markedly adipogenic, whereas Txnip overexpression impaired adipocyte differentiation. As increased adipogenesis and insulin sensitivity suggested aspects of augmented peroxisome proliferator-activated receptor-gamma (PPARgamma) response, we investigated Txnip's regulation of PPARgamma function; manipulation of Txnip expression directly regulated PPARgamma expression and activity.

Conclusions: Txnip deletion promotes adiposity in the face of high-fat caloric excess; however, loss of this alpha-arrestin protein simultaneously enhances insulin responsiveness in fat and skeletal muscle, revealing Txnip as a novel mediator of insulin resistance and a regulator of adipogenesis.

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Txnip deletion augments PPARγ-stimulated adipogenesis and PPARγ activity. A: Wild-type and Txnip knockout adipocyte differentiation at day 9 after DMI induction + increasing rosiglitazone concentrations. Total lipid levels are quantified after ORO extraction at OD520 staining. γ-antagonist = 1 μmol/l GW9229. *P < 0.001 for wild-type vs. Txnip knockout at each rosiglitazone concentration; n = 6 replicates per group. The representative color images were nonlinearly color-contrast enhanced using equivalent settings, and were not used for quantitative analysis. White bars = wild type, black bars = Txnip knockout. B: Endogenous PPARγ activation with increasing rosiglitazone dosing in 3T3-L1 cells stably transduced with Txnip or control lentivirus. PPARγ activity was determined by a transfected PPAR response element luciferase reporter stimulated by 18 h of rosiglitazone treatment (48 h after transfection). Transfection efficiency was normalized to β-galactosidase (β-gal) activity from a cotransfected β-gal reporter. n = 4 replicates per group. C and D: PPARγ LBD activation assay. PPARγ-LBD::GAL4 DNA-BD fusion protein was cotransfected with a GAL4 promoter-luciferase reporter into 3T3-L1 preadipocytes expressing Txnip lentivirus (C) or Txnip shRNA lentivirus (D) compared with relevant controls. Luciferase activity was determined after 18-h rosiglitazone stimulation. Transfection efficiency was normalized to β-gal activity from a cotransfected β-gal reporter. *P < 0.05, n = 4 replicates per condition. Graphs depict single representative experiments with error bars reflecting replicates. antag, antagonist. (A high-quality digital color representation of this figure is available in the online issue.)
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Figure 5: Txnip deletion augments PPARγ-stimulated adipogenesis and PPARγ activity. A: Wild-type and Txnip knockout adipocyte differentiation at day 9 after DMI induction + increasing rosiglitazone concentrations. Total lipid levels are quantified after ORO extraction at OD520 staining. γ-antagonist = 1 μmol/l GW9229. *P < 0.001 for wild-type vs. Txnip knockout at each rosiglitazone concentration; n = 6 replicates per group. The representative color images were nonlinearly color-contrast enhanced using equivalent settings, and were not used for quantitative analysis. White bars = wild type, black bars = Txnip knockout. B: Endogenous PPARγ activation with increasing rosiglitazone dosing in 3T3-L1 cells stably transduced with Txnip or control lentivirus. PPARγ activity was determined by a transfected PPAR response element luciferase reporter stimulated by 18 h of rosiglitazone treatment (48 h after transfection). Transfection efficiency was normalized to β-galactosidase (β-gal) activity from a cotransfected β-gal reporter. n = 4 replicates per group. C and D: PPARγ LBD activation assay. PPARγ-LBD::GAL4 DNA-BD fusion protein was cotransfected with a GAL4 promoter-luciferase reporter into 3T3-L1 preadipocytes expressing Txnip lentivirus (C) or Txnip shRNA lentivirus (D) compared with relevant controls. Luciferase activity was determined after 18-h rosiglitazone stimulation. Transfection efficiency was normalized to β-gal activity from a cotransfected β-gal reporter. *P < 0.05, n = 4 replicates per condition. Graphs depict single representative experiments with error bars reflecting replicates. antag, antagonist. (A high-quality digital color representation of this figure is available in the online issue.)

Mentions: Increased insulin sensitivity, increased adiposity, and augmented adipogenesis are similar to the clinical response of PPARγ agonism, as seen in thiazolidinedione treatment of patients with type 2 diabetes (5,28). To test the hypothesis that Txnip- cells show increased PPARγ activity, we repeated MEF adipogenesis in the presence of the PPARγ-selective agonist rosiglitazone, and demonstrated PPARγ specificity with the coadministration of the PPARγ-selective irreversible antagonist GW9662 (29). Txnip- MEFs had approximately fivefold greater adipogenic response than wild-type controls at each dose of rosiglitazone (Fig. 5A, P < 0.0001), which was attenuated with the coapplication of the antagonist GW9662. Net adipogenesis was uniformly greater at each dose of rosiglitazone, suggesting that Txnip did not affect ligand responsiveness.


Deletion of the alpha-arrestin protein Txnip in mice promotes adiposity and adipogenesis while preserving insulin sensitivity.

Chutkow WA, Birkenfeld AL, Brown JD, Lee HY, Frederick DW, Yoshioka J, Patwari P, Kursawe R, Cushman SW, Plutzky J, Shulman GI, Samuel VT, Lee RT - Diabetes (2010)

Txnip deletion augments PPARγ-stimulated adipogenesis and PPARγ activity. A: Wild-type and Txnip knockout adipocyte differentiation at day 9 after DMI induction + increasing rosiglitazone concentrations. Total lipid levels are quantified after ORO extraction at OD520 staining. γ-antagonist = 1 μmol/l GW9229. *P < 0.001 for wild-type vs. Txnip knockout at each rosiglitazone concentration; n = 6 replicates per group. The representative color images were nonlinearly color-contrast enhanced using equivalent settings, and were not used for quantitative analysis. White bars = wild type, black bars = Txnip knockout. B: Endogenous PPARγ activation with increasing rosiglitazone dosing in 3T3-L1 cells stably transduced with Txnip or control lentivirus. PPARγ activity was determined by a transfected PPAR response element luciferase reporter stimulated by 18 h of rosiglitazone treatment (48 h after transfection). Transfection efficiency was normalized to β-galactosidase (β-gal) activity from a cotransfected β-gal reporter. n = 4 replicates per group. C and D: PPARγ LBD activation assay. PPARγ-LBD::GAL4 DNA-BD fusion protein was cotransfected with a GAL4 promoter-luciferase reporter into 3T3-L1 preadipocytes expressing Txnip lentivirus (C) or Txnip shRNA lentivirus (D) compared with relevant controls. Luciferase activity was determined after 18-h rosiglitazone stimulation. Transfection efficiency was normalized to β-gal activity from a cotransfected β-gal reporter. *P < 0.05, n = 4 replicates per condition. Graphs depict single representative experiments with error bars reflecting replicates. antag, antagonist. (A high-quality digital color representation of this figure is available in the online issue.)
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Related In: Results  -  Collection

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Figure 5: Txnip deletion augments PPARγ-stimulated adipogenesis and PPARγ activity. A: Wild-type and Txnip knockout adipocyte differentiation at day 9 after DMI induction + increasing rosiglitazone concentrations. Total lipid levels are quantified after ORO extraction at OD520 staining. γ-antagonist = 1 μmol/l GW9229. *P < 0.001 for wild-type vs. Txnip knockout at each rosiglitazone concentration; n = 6 replicates per group. The representative color images were nonlinearly color-contrast enhanced using equivalent settings, and were not used for quantitative analysis. White bars = wild type, black bars = Txnip knockout. B: Endogenous PPARγ activation with increasing rosiglitazone dosing in 3T3-L1 cells stably transduced with Txnip or control lentivirus. PPARγ activity was determined by a transfected PPAR response element luciferase reporter stimulated by 18 h of rosiglitazone treatment (48 h after transfection). Transfection efficiency was normalized to β-galactosidase (β-gal) activity from a cotransfected β-gal reporter. n = 4 replicates per group. C and D: PPARγ LBD activation assay. PPARγ-LBD::GAL4 DNA-BD fusion protein was cotransfected with a GAL4 promoter-luciferase reporter into 3T3-L1 preadipocytes expressing Txnip lentivirus (C) or Txnip shRNA lentivirus (D) compared with relevant controls. Luciferase activity was determined after 18-h rosiglitazone stimulation. Transfection efficiency was normalized to β-gal activity from a cotransfected β-gal reporter. *P < 0.05, n = 4 replicates per condition. Graphs depict single representative experiments with error bars reflecting replicates. antag, antagonist. (A high-quality digital color representation of this figure is available in the online issue.)
Mentions: Increased insulin sensitivity, increased adiposity, and augmented adipogenesis are similar to the clinical response of PPARγ agonism, as seen in thiazolidinedione treatment of patients with type 2 diabetes (5,28). To test the hypothesis that Txnip- cells show increased PPARγ activity, we repeated MEF adipogenesis in the presence of the PPARγ-selective agonist rosiglitazone, and demonstrated PPARγ specificity with the coadministration of the PPARγ-selective irreversible antagonist GW9662 (29). Txnip- MEFs had approximately fivefold greater adipogenic response than wild-type controls at each dose of rosiglitazone (Fig. 5A, P < 0.0001), which was attenuated with the coapplication of the antagonist GW9662. Net adipogenesis was uniformly greater at each dose of rosiglitazone, suggesting that Txnip did not affect ligand responsiveness.

Bottom Line: Thioredoxin interacting protein (Txnip), a regulator of cellular oxidative stress, is induced by hyperglycemia and inhibits glucose uptake into fat and muscle, suggesting a role for Txnip in type 2 diabetes pathogenesis.RNA interference gene-silenced preadipocytes and Txnip(-/-) MEFs were markedly adipogenic, whereas Txnip overexpression impaired adipocyte differentiation.As increased adipogenesis and insulin sensitivity suggested aspects of augmented peroxisome proliferator-activated receptor-gamma (PPARgamma) response, we investigated Txnip's regulation of PPARgamma function; manipulation of Txnip expression directly regulated PPARgamma expression and activity.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Cambridge, Massachusetts, USA.

ABSTRACT

Objective: Thioredoxin interacting protein (Txnip), a regulator of cellular oxidative stress, is induced by hyperglycemia and inhibits glucose uptake into fat and muscle, suggesting a role for Txnip in type 2 diabetes pathogenesis. Here, we tested the hypothesis that Txnip- (knockout) mice are protected from insulin resistance induced by a high-fat diet.

Research design and methods: Txnip gene-deleted (knockout) mice and age-matched wild-type littermate control mice were maintained on a standard chow diet or subjected to 4 weeks of high-fat feeding. Mice were assessed for body composition, fat development, energy balance, and insulin responsiveness. Adipogenesis was measured from ex vivo fat preparations, and in mouse embryonic fibroblasts (MEFs) and 3T3-L1 preadipocytes after forced manipulation of Txnip expression.

Results: Txnip knockout mice gained significantly more adipose mass than controls due to a primary increase in both calorie consumption and adipogenesis. Despite increased fat mass, Txnip knockout mice were markedly more insulin sensitive than controls, and augmented glucose transport was identified in both adipose and skeletal muscle. RNA interference gene-silenced preadipocytes and Txnip(-/-) MEFs were markedly adipogenic, whereas Txnip overexpression impaired adipocyte differentiation. As increased adipogenesis and insulin sensitivity suggested aspects of augmented peroxisome proliferator-activated receptor-gamma (PPARgamma) response, we investigated Txnip's regulation of PPARgamma function; manipulation of Txnip expression directly regulated PPARgamma expression and activity.

Conclusions: Txnip deletion promotes adiposity in the face of high-fat caloric excess; however, loss of this alpha-arrestin protein simultaneously enhances insulin responsiveness in fat and skeletal muscle, revealing Txnip as a novel mediator of insulin resistance and a regulator of adipogenesis.

Show MeSH
Related in: MedlinePlus