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Nonthyrotoxic prevention of diet-induced insulin resistance by 3,5-diiodo-L-thyronine in rats.

de Lange P, Cioffi F, Senese R, Moreno M, Lombardi A, Silvestri E, De Matteis R, Lionetti L, Mollica MP, Goglia F, Lanni A - Diabetes (2011)

Bottom Line: T2 did so by rapidly stimulating hepatic fatty acid oxidation, decreasing hepatic triglyceride levels, and improving the serum lipid profile, while at the same time sparing skeletal muscle from fat accumulation.At the mechanistic level, 1) transfection studies show that T2 does not act via thyroid hormone receptor β; 2) AMP-activated protein kinase is not involved in triggering the effects of T2; 3) in HFD rats, T2 rapidly increases hepatic nuclear sirtuin 1 (SIRT1) activity; 4) in an in vitro assay, T2 directly activates SIRT1; and 5) the SIRT1 targets peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC-1α) and sterol regulatory element-binding protein (SREBP)-1c are deacetylated with concomitant upregulation of genes involved in mitochondrial biogenesis and downregulation of lipogenic genes, and PPARα/δ-induced genes are upregulated, whereas genes involved in hepatic gluconeogenesis are downregulated.Proteomic analysis of the hepatic protein profile supported these changes.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, Caserta, Italy.

ABSTRACT

Objective: High-fat diets (HFDs) are known to induce insulin resistance. Previously, we showed that 3,5-diiodothyronine (T2), concomitantly administered to rats on a 4-week HFD, prevented gain in body weight and adipose mass. Here we investigated whether and how T2 prevented HFD-induced insulin resistance.

Research design and methods: We investigated the biochemical targets of T2 related to lipid and glucose homeostasis over time using various techniques, including genomic and proteomic profiling, immunoblotting, transient transfection, and enzyme activity analysis.

Results: Here we show that, in rats, HFD feeding induced insulin resistance (as expected), whereas T2 administration prevented its onset. T2 did so by rapidly stimulating hepatic fatty acid oxidation, decreasing hepatic triglyceride levels, and improving the serum lipid profile, while at the same time sparing skeletal muscle from fat accumulation. At the mechanistic level, 1) transfection studies show that T2 does not act via thyroid hormone receptor β; 2) AMP-activated protein kinase is not involved in triggering the effects of T2; 3) in HFD rats, T2 rapidly increases hepatic nuclear sirtuin 1 (SIRT1) activity; 4) in an in vitro assay, T2 directly activates SIRT1; and 5) the SIRT1 targets peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC-1α) and sterol regulatory element-binding protein (SREBP)-1c are deacetylated with concomitant upregulation of genes involved in mitochondrial biogenesis and downregulation of lipogenic genes, and PPARα/δ-induced genes are upregulated, whereas genes involved in hepatic gluconeogenesis are downregulated. Proteomic analysis of the hepatic protein profile supported these changes.

Conclusions: T2, by activating SIRT1, triggers a cascade of events resulting in improvement of the serum lipid profile, prevention of fat accumulation, and, finally, prevention of diet-induced insulin resistance.

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Related in: MedlinePlus

Hepatic induction of fatty acid oxidation by T2 in HFD animals does not involve AMPK but is associated with SIRT1 activation. A: Compound C effectively inhibits AMPK Thr172 and ACC Ser79 phosphorylation within 1 and 6 h, respectively, of its simultaneous administration with T2. Positions of ACC isoforms 1 and 2 are indicated at the right. The increase of fatty acid oxidation persisted at 6 h after injection of T2 plus Compound C. SIRT1 nuclear protein activity increased at 6 h after injection of T2 and was not inhibited by Compound C. B: T2 is a specific activator of SIRT1. The effects of T2 and RSV (positive control) were compared using a fluorescence-based deacetylation assay (x-axis: logarithmic scale). Inlay: Other tested TH metabolites either inhibit SIRT1 activity (T3) or are ineffective (T1, T0). C: T2 induces prolonged activation of SIRT1. Upper: Nuclear SIRT1 protein. Middle: mRNA levels in livers of animals treated as indicated underneath the bars. Lower: Hepatic nuclear SIRT1 activity. Ratios are shown for values from HFD-T2 animals over those from HFD animals at the indicated time points. D: T2 treatment causes deacetylation of SIRT1 target proteins, consistent with increased fatty acid oxidation. Left panel: Mitochondrial fatty acid oxidation in HFD or HFD-T2 animals treated for 1 day (left panel) in the presence or absence of the specific SIRT1 inhibitor EX-527 (EX). Left and right panel: In the same animals, as well as in animals treated for 4 weeks, hepatic nuclear extracts (2 mg) were immunoprecipitated with an anti–PGC-1α (upper) or anti–SREBP-1c antibody (lower) and analyzed with anti-acetyllysine antibody vs. PGC-1α or SREBP-1c. Numbers indicate the ratio acetylated over total protein. Upper right panel: No variation in total PGC-1α, SREBP-1c, and tubulin (control) protein levels in nuclear extracts between N, HFD, and HFD-T2 animals (input). A, C, and D: Representative blots are shown. D: Dividing lines indicate omissions/rearrangements of lanes from the same gel. Error bars represent SEM. *P < 0.05 vs. untreated controls; **P < 0.05 vs. both untreated controls and HFD-fed groups. □, N; ■, HFD; ▨, HFD-T2; ▧, HFD-T2–Compound C; dotted bars, HFD-T2-EX-527; light dotted bars, HFD-EX-527. B: ◇, RSV; □, T2. FFA, free fatty acid; FU, fluorescence units; IP, immunoprecipitated; IB, immunoblotted.
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Figure 3: Hepatic induction of fatty acid oxidation by T2 in HFD animals does not involve AMPK but is associated with SIRT1 activation. A: Compound C effectively inhibits AMPK Thr172 and ACC Ser79 phosphorylation within 1 and 6 h, respectively, of its simultaneous administration with T2. Positions of ACC isoforms 1 and 2 are indicated at the right. The increase of fatty acid oxidation persisted at 6 h after injection of T2 plus Compound C. SIRT1 nuclear protein activity increased at 6 h after injection of T2 and was not inhibited by Compound C. B: T2 is a specific activator of SIRT1. The effects of T2 and RSV (positive control) were compared using a fluorescence-based deacetylation assay (x-axis: logarithmic scale). Inlay: Other tested TH metabolites either inhibit SIRT1 activity (T3) or are ineffective (T1, T0). C: T2 induces prolonged activation of SIRT1. Upper: Nuclear SIRT1 protein. Middle: mRNA levels in livers of animals treated as indicated underneath the bars. Lower: Hepatic nuclear SIRT1 activity. Ratios are shown for values from HFD-T2 animals over those from HFD animals at the indicated time points. D: T2 treatment causes deacetylation of SIRT1 target proteins, consistent with increased fatty acid oxidation. Left panel: Mitochondrial fatty acid oxidation in HFD or HFD-T2 animals treated for 1 day (left panel) in the presence or absence of the specific SIRT1 inhibitor EX-527 (EX). Left and right panel: In the same animals, as well as in animals treated for 4 weeks, hepatic nuclear extracts (2 mg) were immunoprecipitated with an anti–PGC-1α (upper) or anti–SREBP-1c antibody (lower) and analyzed with anti-acetyllysine antibody vs. PGC-1α or SREBP-1c. Numbers indicate the ratio acetylated over total protein. Upper right panel: No variation in total PGC-1α, SREBP-1c, and tubulin (control) protein levels in nuclear extracts between N, HFD, and HFD-T2 animals (input). A, C, and D: Representative blots are shown. D: Dividing lines indicate omissions/rearrangements of lanes from the same gel. Error bars represent SEM. *P < 0.05 vs. untreated controls; **P < 0.05 vs. both untreated controls and HFD-fed groups. □, N; ■, HFD; ▨, HFD-T2; ▧, HFD-T2–Compound C; dotted bars, HFD-T2-EX-527; light dotted bars, HFD-EX-527. B: ◇, RSV; □, T2. FFA, free fatty acid; FU, fluorescence units; IP, immunoprecipitated; IB, immunoblotted.

Mentions: To examine whether a transient phosphorylation of AMPK between 0 and 6 h might have triggered the observed T2-induced increase in liver fatty acid oxidation, we concomitantly administered T2 and Compound C to a subgroup of HFD animals. There was an almost complete inhibition of AMPK phosphorylation by Compound C at both 1 and 6 h after T2 injection (Fig. 3A, left and center panels). However, the T2-induced increase in mitochondrial fatty acid oxidation in the liver was not different between the HFD-T2–Compound C and HFD-T2 groups (Fig. 3A, far right upper panel), although CPT activity was significantly lower in the former group than in all other conditions examined (Fig. 3A, far right center panel). Besides AMPK, SIRT1 has emerged as an interesting target in the amelioration of diet-induced metabolic disorders. To examine the involvement of SIRT1 in the effects brought about by T2 in vivo, we isolated hepatic nuclei and immunoprecipitated nuclear SIRT1 protein under native conditions. In agreement with the rapid preventive effect of T2 on fat accumulation, in the HFD-T2 group, hepatic SIRT1 activity was already elevated at the 6-h time point by around twofold versus the HFD group (Fig. 3A, far right lower panel). A quantitatively similar result was obtained for the HFD-T2–Compound C group, showing that the activating effect of T2 on SIRT1 is AMPK independent (Fig. 3A, far right lower panel). We then examined whether T2 could directly activate purified SIRT1. For this, we used a fluorescence-based deacetylation assay, with the naturally occurring SIRT1 activator RSV as a positive control. The half-maximal inhibitory concentration (IC50) values were 8.5 and 17 μmol/L for RSV and T2, respectively, with both activities being inhibitable by nicotinamide (Fig. 3B). Other iodothyronines (T1 and T0) did not stimulate SIRT1 activity at concentrations of 1 mmol/L, whereas T3 inhibited it (see inlay, Fig. 3B). With time, The SIRT1-activity ratio between the HFD-T2 and HFD groups remained elevated (at around two- to threefold vs. control) (up to 4 weeks) (Fig. 3C, lower). In the HFD-T2 group, SIRT1 nuclear protein levels did not alter significantly with time (Fig. 3C, upper), nor did SIRT1mRNA levels (Fig. 3C, middle). HFD treatment caused acetylation of SIRT1 targets SREBP-1c and PGC-1α, which was effectively prevented by T2 coadministration (Fig. 3D). Overnight inhibition of SIRT1 activity with EX-527 abolished T2-induced mitochondrial fatty acid oxidation (Fig. 3D, left upper panel) as well as deacetylation of SREBP-1c and PGC-1α (Fig. 3D, left lower panel).


Nonthyrotoxic prevention of diet-induced insulin resistance by 3,5-diiodo-L-thyronine in rats.

de Lange P, Cioffi F, Senese R, Moreno M, Lombardi A, Silvestri E, De Matteis R, Lionetti L, Mollica MP, Goglia F, Lanni A - Diabetes (2011)

Hepatic induction of fatty acid oxidation by T2 in HFD animals does not involve AMPK but is associated with SIRT1 activation. A: Compound C effectively inhibits AMPK Thr172 and ACC Ser79 phosphorylation within 1 and 6 h, respectively, of its simultaneous administration with T2. Positions of ACC isoforms 1 and 2 are indicated at the right. The increase of fatty acid oxidation persisted at 6 h after injection of T2 plus Compound C. SIRT1 nuclear protein activity increased at 6 h after injection of T2 and was not inhibited by Compound C. B: T2 is a specific activator of SIRT1. The effects of T2 and RSV (positive control) were compared using a fluorescence-based deacetylation assay (x-axis: logarithmic scale). Inlay: Other tested TH metabolites either inhibit SIRT1 activity (T3) or are ineffective (T1, T0). C: T2 induces prolonged activation of SIRT1. Upper: Nuclear SIRT1 protein. Middle: mRNA levels in livers of animals treated as indicated underneath the bars. Lower: Hepatic nuclear SIRT1 activity. Ratios are shown for values from HFD-T2 animals over those from HFD animals at the indicated time points. D: T2 treatment causes deacetylation of SIRT1 target proteins, consistent with increased fatty acid oxidation. Left panel: Mitochondrial fatty acid oxidation in HFD or HFD-T2 animals treated for 1 day (left panel) in the presence or absence of the specific SIRT1 inhibitor EX-527 (EX). Left and right panel: In the same animals, as well as in animals treated for 4 weeks, hepatic nuclear extracts (2 mg) were immunoprecipitated with an anti–PGC-1α (upper) or anti–SREBP-1c antibody (lower) and analyzed with anti-acetyllysine antibody vs. PGC-1α or SREBP-1c. Numbers indicate the ratio acetylated over total protein. Upper right panel: No variation in total PGC-1α, SREBP-1c, and tubulin (control) protein levels in nuclear extracts between N, HFD, and HFD-T2 animals (input). A, C, and D: Representative blots are shown. D: Dividing lines indicate omissions/rearrangements of lanes from the same gel. Error bars represent SEM. *P < 0.05 vs. untreated controls; **P < 0.05 vs. both untreated controls and HFD-fed groups. □, N; ■, HFD; ▨, HFD-T2; ▧, HFD-T2–Compound C; dotted bars, HFD-T2-EX-527; light dotted bars, HFD-EX-527. B: ◇, RSV; □, T2. FFA, free fatty acid; FU, fluorescence units; IP, immunoprecipitated; IB, immunoblotted.
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Figure 3: Hepatic induction of fatty acid oxidation by T2 in HFD animals does not involve AMPK but is associated with SIRT1 activation. A: Compound C effectively inhibits AMPK Thr172 and ACC Ser79 phosphorylation within 1 and 6 h, respectively, of its simultaneous administration with T2. Positions of ACC isoforms 1 and 2 are indicated at the right. The increase of fatty acid oxidation persisted at 6 h after injection of T2 plus Compound C. SIRT1 nuclear protein activity increased at 6 h after injection of T2 and was not inhibited by Compound C. B: T2 is a specific activator of SIRT1. The effects of T2 and RSV (positive control) were compared using a fluorescence-based deacetylation assay (x-axis: logarithmic scale). Inlay: Other tested TH metabolites either inhibit SIRT1 activity (T3) or are ineffective (T1, T0). C: T2 induces prolonged activation of SIRT1. Upper: Nuclear SIRT1 protein. Middle: mRNA levels in livers of animals treated as indicated underneath the bars. Lower: Hepatic nuclear SIRT1 activity. Ratios are shown for values from HFD-T2 animals over those from HFD animals at the indicated time points. D: T2 treatment causes deacetylation of SIRT1 target proteins, consistent with increased fatty acid oxidation. Left panel: Mitochondrial fatty acid oxidation in HFD or HFD-T2 animals treated for 1 day (left panel) in the presence or absence of the specific SIRT1 inhibitor EX-527 (EX). Left and right panel: In the same animals, as well as in animals treated for 4 weeks, hepatic nuclear extracts (2 mg) were immunoprecipitated with an anti–PGC-1α (upper) or anti–SREBP-1c antibody (lower) and analyzed with anti-acetyllysine antibody vs. PGC-1α or SREBP-1c. Numbers indicate the ratio acetylated over total protein. Upper right panel: No variation in total PGC-1α, SREBP-1c, and tubulin (control) protein levels in nuclear extracts between N, HFD, and HFD-T2 animals (input). A, C, and D: Representative blots are shown. D: Dividing lines indicate omissions/rearrangements of lanes from the same gel. Error bars represent SEM. *P < 0.05 vs. untreated controls; **P < 0.05 vs. both untreated controls and HFD-fed groups. □, N; ■, HFD; ▨, HFD-T2; ▧, HFD-T2–Compound C; dotted bars, HFD-T2-EX-527; light dotted bars, HFD-EX-527. B: ◇, RSV; □, T2. FFA, free fatty acid; FU, fluorescence units; IP, immunoprecipitated; IB, immunoblotted.
Mentions: To examine whether a transient phosphorylation of AMPK between 0 and 6 h might have triggered the observed T2-induced increase in liver fatty acid oxidation, we concomitantly administered T2 and Compound C to a subgroup of HFD animals. There was an almost complete inhibition of AMPK phosphorylation by Compound C at both 1 and 6 h after T2 injection (Fig. 3A, left and center panels). However, the T2-induced increase in mitochondrial fatty acid oxidation in the liver was not different between the HFD-T2–Compound C and HFD-T2 groups (Fig. 3A, far right upper panel), although CPT activity was significantly lower in the former group than in all other conditions examined (Fig. 3A, far right center panel). Besides AMPK, SIRT1 has emerged as an interesting target in the amelioration of diet-induced metabolic disorders. To examine the involvement of SIRT1 in the effects brought about by T2 in vivo, we isolated hepatic nuclei and immunoprecipitated nuclear SIRT1 protein under native conditions. In agreement with the rapid preventive effect of T2 on fat accumulation, in the HFD-T2 group, hepatic SIRT1 activity was already elevated at the 6-h time point by around twofold versus the HFD group (Fig. 3A, far right lower panel). A quantitatively similar result was obtained for the HFD-T2–Compound C group, showing that the activating effect of T2 on SIRT1 is AMPK independent (Fig. 3A, far right lower panel). We then examined whether T2 could directly activate purified SIRT1. For this, we used a fluorescence-based deacetylation assay, with the naturally occurring SIRT1 activator RSV as a positive control. The half-maximal inhibitory concentration (IC50) values were 8.5 and 17 μmol/L for RSV and T2, respectively, with both activities being inhibitable by nicotinamide (Fig. 3B). Other iodothyronines (T1 and T0) did not stimulate SIRT1 activity at concentrations of 1 mmol/L, whereas T3 inhibited it (see inlay, Fig. 3B). With time, The SIRT1-activity ratio between the HFD-T2 and HFD groups remained elevated (at around two- to threefold vs. control) (up to 4 weeks) (Fig. 3C, lower). In the HFD-T2 group, SIRT1 nuclear protein levels did not alter significantly with time (Fig. 3C, upper), nor did SIRT1mRNA levels (Fig. 3C, middle). HFD treatment caused acetylation of SIRT1 targets SREBP-1c and PGC-1α, which was effectively prevented by T2 coadministration (Fig. 3D). Overnight inhibition of SIRT1 activity with EX-527 abolished T2-induced mitochondrial fatty acid oxidation (Fig. 3D, left upper panel) as well as deacetylation of SREBP-1c and PGC-1α (Fig. 3D, left lower panel).

Bottom Line: T2 did so by rapidly stimulating hepatic fatty acid oxidation, decreasing hepatic triglyceride levels, and improving the serum lipid profile, while at the same time sparing skeletal muscle from fat accumulation.At the mechanistic level, 1) transfection studies show that T2 does not act via thyroid hormone receptor β; 2) AMP-activated protein kinase is not involved in triggering the effects of T2; 3) in HFD rats, T2 rapidly increases hepatic nuclear sirtuin 1 (SIRT1) activity; 4) in an in vitro assay, T2 directly activates SIRT1; and 5) the SIRT1 targets peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC-1α) and sterol regulatory element-binding protein (SREBP)-1c are deacetylated with concomitant upregulation of genes involved in mitochondrial biogenesis and downregulation of lipogenic genes, and PPARα/δ-induced genes are upregulated, whereas genes involved in hepatic gluconeogenesis are downregulated.Proteomic analysis of the hepatic protein profile supported these changes.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Scienze della Vita, Seconda Università degli Studi di Napoli, Caserta, Italy.

ABSTRACT

Objective: High-fat diets (HFDs) are known to induce insulin resistance. Previously, we showed that 3,5-diiodothyronine (T2), concomitantly administered to rats on a 4-week HFD, prevented gain in body weight and adipose mass. Here we investigated whether and how T2 prevented HFD-induced insulin resistance.

Research design and methods: We investigated the biochemical targets of T2 related to lipid and glucose homeostasis over time using various techniques, including genomic and proteomic profiling, immunoblotting, transient transfection, and enzyme activity analysis.

Results: Here we show that, in rats, HFD feeding induced insulin resistance (as expected), whereas T2 administration prevented its onset. T2 did so by rapidly stimulating hepatic fatty acid oxidation, decreasing hepatic triglyceride levels, and improving the serum lipid profile, while at the same time sparing skeletal muscle from fat accumulation. At the mechanistic level, 1) transfection studies show that T2 does not act via thyroid hormone receptor β; 2) AMP-activated protein kinase is not involved in triggering the effects of T2; 3) in HFD rats, T2 rapidly increases hepatic nuclear sirtuin 1 (SIRT1) activity; 4) in an in vitro assay, T2 directly activates SIRT1; and 5) the SIRT1 targets peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC-1α) and sterol regulatory element-binding protein (SREBP)-1c are deacetylated with concomitant upregulation of genes involved in mitochondrial biogenesis and downregulation of lipogenic genes, and PPARα/δ-induced genes are upregulated, whereas genes involved in hepatic gluconeogenesis are downregulated. Proteomic analysis of the hepatic protein profile supported these changes.

Conclusions: T2, by activating SIRT1, triggers a cascade of events resulting in improvement of the serum lipid profile, prevention of fat accumulation, and, finally, prevention of diet-induced insulin resistance.

Show MeSH
Related in: MedlinePlus