Pathogenesis of hypothyroidism-induced NAFLD is driven by intra- and extrahepatic mechanisms - PubMed (original) (raw)

Pathogenesis of hypothyroidism-induced NAFLD is driven by intra- and extrahepatic mechanisms

Giuseppe Ferrandino et al. Proc Natl Acad Sci U S A. 2017.

Abstract

Hypothyroidism, a metabolic disease characterized by low thyroid hormone (TH) and high thyroid-stimulating hormone (TSH) levels in the serum, is strongly associated with nonalcoholic fatty liver disease (NAFLD). Hypothyroidism-induced NAFLD has generally been attributed to reduced TH signaling in the liver with a consequent decrease in lipid utilization. Here, we found that mildly hypothyroid mice develop NAFLD without down-regulation of hepatic TH signaling or decreased hepatic lipid utilization. NAFLD was induced by impaired suppression of adipose tissue lipolysis due to decreased insulin secretion and to a reduced response of adipose tissue itself to insulin. This condition leads to increased shuttling of fatty acids (FAs) to the liver, where they are esterified and accumulated as triglycerides. Lipid accumulation in the liver induces hepatic insulin resistance, which leads to impaired suppression of endogenous glucose production after feeding. Hepatic insulin resistance, synergistically with lowered insulin secretion, increases serum glucose levels, which stimulates de novo lipogenesis (DNL) in the liver. Up-regulation of DNL also contributes to NAFLD. In contrast, severely hypothyroid mice show down-regulation of TH signaling in their livers and profound suppression of adipose tissue lipolysis, which decreases delivery of FAs to the liver. The resulting lack of substrates for triglyceride esterification protects severely hypothyroid mice against NAFLD. Our findings demonstrate that NAFLD occurs when TH levels are mildly reduced, but, paradoxically, not when they are severely reduced. Our results show that the pathogenesis of hypothyroidism-induced NAFLD is both intra- and extrahepatic; they also reveal key metabolic differences between mild and severe hypothyroidism.

Keywords: NAFLD; de novo lipogenesis; hypothyroidism; insulin resistance; sodium/iodide symporter.

Published under the PNAS license.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Iodide restriction leads to mild hypothyroidism, increased fat gain, and NAFLD. Eight-week-old male C57BL/6J mice were fed a CD or a LID. (A_–_C) Serum T4, T3, and TSH levels of CD and LID mice after 12 wk on their respective diets; n = 8. (D) Percentage of body made up by VAT after 12 wk on a CD or LID; n = 9. (E) Percentage of body weight made up by fat and (F) percentage made up by lean mass; n = 5. (G) Body weight of CD and LID mice at the time points indicated. (H) Oil Red O staining of frozen liver sections from CD and LID mice; n = 8. (Scale bar: 10 μm.) (I) Liver triglyceride content; n = 5. Data shown as mean ± SEM; *P < 0.05.

Fig. S1.

Fig. S1.

Mice metabolic parameters measured after 8 wk on a CD or a LID. (A) Activity. (B) Energy expenditure (EE). (C) RER. (D) VO2 and (E) VCO2; n = 5 for all experiments. (F) Glycerol tolerance test. CD and LID mice were i.p. injected with glycerol (1 g/kg of body weight), and glucose was measured in the tail blood at the time points indicated; n = 10. (G) Pyruvate tolerance test. Mice were treated as described for the glycerol tolerance test; n = 10. Data are shown as mean ± SEM; *P < 0.05.

Fig. S2.

Fig. S2.

(A) GIR necessary to maintain euglycemia during insulin infusion; n = 6–7. (B) Total insulin content of isolated islets; n = 4. (C) Serum insulin measured during fasting (F.I., fasting insulin) or 20 min after i.p. injection of 1.5 g/kg of glucose (G.C., glucose challenge); n = 10. (D) Plasma glucose measured over the course of the clamp studies; n = 6–7. (E) Body weight of CD and LID mice used for the clamp studies; n = 6–7. (F) Steady-state GIR; n = 6–7. (G) Whole-body glucose uptake; n = 6–7. (H) Plasma insulin measured over the course of the clamp studies; n = 6–7. (I) Serum FA levels of nonfasted CD and LID mice; n = 10. (J) Glycerol levels of CD and LID mice fasted overnight; n = 10. (K) Food intake of CD and LID mice after 10 wk on a LID; n = 5. (L) Food intake of WT and _Slc5a5_−/− mice after 10 wk on a LID; n = 7. Data are shown as mean ± SEM; *P < 0.05; **P < 0.01.

Fig. 2.

Fig. 2.

Mild hypothyroidism induces NAFLD without reducing lipid oxidation in the liver. (A) Expression levels of genes involved in the β-oxidation pathway. Data are presented as ratios between expression levels in the livers of LID and CD mice; n = 8. Levels in CD mice are set to 1. (B) Expression levels of genes regulated by THs in liver; n = 7. Data are presented as ratios between expression levels in LID and CD mice; n = 8. Levels in CD mice are set to 1. (C and D) Conversion of 14C-palmitate to CO2 and acid-soluble metabolites mediated by liver mitochondria extracted from livers of CD and LID mice; n = 4. (E) Expression levels of genes involved in de novo lipogenesis and lipid uptake in liver; n = 8. Data are presented as ratios between expression levels in LID and CD mice; n = 8. Levels in CD mice are set to 1. (F) Western blot of liver extracts showing ACC, FAS, and β-actin; n = 5. (G) Quantification of the intensity of the bands in F; n = 5. Data shown as mean ± SEM; *P < 0.05; **P < 0.01.

Fig. 3.

Fig. 3.

Mild hypothyroidism induces glucose intolerance due to hepatic insulin resistance and impaired pancreatic β-cell insulin secretion. (A) Glucose tolerance test; mice were i.p. injected with 1.5 g/kg of glucose, and blood glucose was measured at the time points indicated; n = 10. (B) Quantification of the area under the curves shown in A. (C) EGP before and after insulin perfusion during a euglycemic-hyperinsulinemic clamp; n = 6–7. (D) Pancreatic islets were isolated from CD and LID mice after 12 wk on their respective diets. Isolated islets were perfused with 2 mM or 16 mM glucose, and insulin was measured at the time points indicated; n = 4. (E) Phases of insulin secretion obtained by pooling the values shown in D. Basal: 5–14 min; first phase: 14–24 min; second phase: 30–71 min: KCl: 72–79 min. Data are shown as mean ± SEM unless specified otherwise; *P < 0.05; **P < 0.01.

Fig. 4.

Fig. 4.

Mild hypothyroidism leads to impaired insulin-mediated lipolysis suppression and macrophage-dependent sterile inflammation in VAT. (A) Serum glycerol levels of fed CD and LID mice after 12 wk on their respective diets; n = 10. (B) HSL phosphorylation at activating Ser-563, activating Ser-660, and inactivating Ser-565 in VAT excised from fed CD and LID mice. (C) Quantification of the intensity of the bands in B; n = 5. (D) Glycerol and (E) palmitate turnover in the basal condition and after insulin infusion during a euglycemic-hyperinsulinemic clamp; n = 6–7. (F) FACS analysis of VAT CD11b+ macrophage and (G) CD11c+ proinflammatory macrophage infiltration of VAT; n = 10. (H) Expression levels of macrophage-specific genes in VAT; n = 7–8. (I) Glycerol release from VAT (∼20 mg) after 2 h incubation with 1 µM isoproterenol (Isop) or Isop plus the PKA inhibitor H89; n = 4. Data shown as mean ± SEM; N.S., nonsignificant; *P < 0.05; **P < 0.01.

Fig. S3.

Fig. S3.

FACS analysis of VAT stromal vascular fraction in (A) B cells, (B) T cells, and (C) dendritic cells; n = 10.

Fig. S4.

Fig. S4.

The FlowJo gating strategy used to isolate the different types of immune cells shown in Fig. 2 and Fig. S3.

Fig. 5.

Fig. 5.

Severely reduced serum thyroid hormone levels alter cholesterol levels and impair fat and body weight gain. Eight-week-old male WT and _Slc5a5_−/− mice harboring C57BL/6J/_N_-A mixed genetic backgrounds were fed a LID. (A) Serum T4 and (B) serum T3 measured on CD and after 3 wk on a LID; n = 7. (C) Serum TSH measured after 12 wk on a LID; n = 7. (D) Serum levels of total cholesterol, cholesterol in HDL, and cholesterol in LDL measured after 17 wk on a CD or a LID; n = 4. (E) Percentage of body weight made up by fat and (F) percentage made up by lean mass at the time points indicated; n = 7. (G) Body weight of WT and _Slc5a5_−/− mice at the time points indicated; n = 7. Data are shown as mean ± SEM; N.D., nondetectable; *P < 0.05; **P < 0.01.

Fig. 6.

Fig. 6.

Severe hypothyroidism impairs adipose tissue lipolysis stimulation and protects mice against NAFLD. Eight-week-old male WT and _Slc5a5_−/− mice harboring C57BL/6J/_N_-A mixed genetic backgrounds were fed a LID. (A) Serum glycerol and (B) serum FA levels after 12 h fasting before switching from a CD to a LID and after 3 wk on a LID; n = 7. (C) Western blot of VAT extracts showing total HSL and HSL-activating phosphorylation (Ser-563 and Ser-660) from WT and _Slc5a5_−/− mice after 12 wk on a LID and 12 h fasting. (D) Quantification of the intensity of the bands in C; n = 5. (E) Glycerol release from VAT (∼20 mg) after 2 h incubation with 1 µM isoproterenol (Isop); n = 4. (F and G) Conversion of 14C-palmitate to CO2 and acid-soluble metabolites mediated by liver mitochondria extracted from livers of WT and _Slc5a5_−/− mice; n = 3–4. (H) Expression levels of genes regulated by THs in liver; n = 7. (I) Oil Red O staining of frozen liver sections from WT and _Slc5a5_−/− mice; n = 7. (Scale bars: 10 μm.) (J) Liver triglyceride content; n = 7. Data are shown as mean ± SEM; N.S., nonsignificant; *P < 0.05; **P < 0.01.

Fig. 7.

Fig. 7.

Summary of the mechanisms that contribute to NAFLD in mild hypothyroidism and those that protect against NAFLD in severe hypothyroidism. Under euthyroid conditions (Left), insulin secretion is intact and inhibits adrenergic stimulation of lipolysis in adipose tissue, thereby preventing NAFLD. In mild hypothyroidism (Center), reduced thyroid hormone levels impair insulin secretion, whereas adrenergic stimulation of lipolysis is still functional. Insulin fails to suppress adipose tissue lipolysis with a consequent increased delivery of FAs to the liver, where they are esterified and accumulated as triglycerides. In severe hypothyroidism (Right), adipose tissue lipolysis is constitutively suppressed. The decreased release of FAs protects against NAFLD.

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