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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. References

Objective:

Hepatic iron overload (HIO) and iron-induced oxidative stress have recently emerged as an important factor for the development and progression of insulin resistance. The aim of this study was to evaluate the effect of tamibarotene, a selective retinoic acid receptor α/β agonist, on hepatic iron metabolism, based on our previous findings that retinoids suppress hepatic iron accumulation by increasing hepatic iron efflux through the regulation of hemojuvelin and ferroportin expression.

Design and Methods:

We quantitated the non-heme iron content and iron metabolism-related gene expression in the liver, and serum lipid and blood glucose levels in KK-Ay mice after dietary administration of tamibarotene.

Results:

It was demonstrated that tamibarotene significantly reduced blood glucose and hepatic iron, but not serum lipids, and that hemojuvelin expression significantly decreased while ferroportin increased, as observed previously.

Conclusions:

These results suggest that tamibarotene is a promising alternative for the treatment of insulin resistance associated with HIO.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. References

Increased oxidative stress is widely regarded as one of the key factors for the development and progression of insulin resistance (1). It is well established that chronic liver diseases, including non-alcoholic fatty liver disease (NAFLD) and chronic hepatitis C (CH-C), are commonly associated with insulin resistance. Because excess iron accumulation causes severe cellular dysfunction by facilitating the production of reactive oxygen species, the observation that a high frequency of hepatic iron overload (HIO) exists in patients with chronic liver diseases suggests its possible link with insulin resistance (1).

The liver expresses several iron metabolism-related genes to maintain appropriate cellular and tissue iron levels. Iron response element (IRE) is a conserved sequence found in 5′- or 3′-untranslated regions (UTRs) of the mRNA of several genes related to iron metabolism (2). An abundance of cellular iron induces the release of iron response proteins from IRE, which stimulates the translation of mRNA with IRE in a 5′-UTR (ferroportin, ferritin heavy chain, and ferritin light chain) and degradation of mRNA with IRE in a 3′-UTR (transferrin receptor 1) (2). Hepcidin plays an indispensable role in systemic iron homeostasis by suppressing ferroportin-mediated cellular iron export, whereas hemojuvelin is one of the major inducers of hepcidin expression (3, 4). However, the exact mechanism that causes the disturbance of iron metabolism in the liver of patients with insulin resistance remains unclear.

We previously demonstrated that all-trans-retinoic acid (ATRA) suppresses hepatic iron accumulation by increasing the hepatic iron efflux through the regulation of hemojuvelin and ferroportin expression, while hepatic retinoid signaling is severely impaired in patients with chronic liver diseases (5, 6). Moreover, we recently clarified that ATRA improves insulin sensitivity in a leptin-dependent manner (7). Tamibarotene is a selective retinoic acid receptor (RAR) α/β agonist and is used as a molecular targeting drug for acute promyelocytic leukemia with more potent efficiency than ATRA (8). On the basis of our previous findings, this study investigated the effect of tamibarotene on hepatic iron metabolism.

Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. References

Mouse experiments

Five-week-old male KK-Ay mice were purchased from CLEA Japan (Tokyo, Japan). The mice were kept under pathogen-free conditions and were maintained in a temperature-controlled room with a 12-h light/dark illumination cycle. Animals received humane care in accordance with study guidelines, and this animal study was approved by the animal ethics committee at Kyoto Pharmaceutical University. Normal (MF diet) and 20 mg/kg tamibarotene-supplemented normal diets were purchased from Oriental Yeast (Tokyo, Japan). Following acclimation for 1 week, KK-Ay mice were fed the normal and tamibarotene-supplemented normal diets for 4 weeks (seven mice in each group). The tamibarotene diet did not change the daily dietary consumption and body weight of the mice (data not shown). The collection of blood and liver samples was conducted as described previously (9).

Biochemical study

Determination of serum lipid content, blood glucose levels, and hepatic non-heme iron content was performed according to previous reports (9).

Gene expression analysis

Liver protein and total RNA samples were prepared as described previously (9). The mRNA levels of iron metabolism-related genes were determined by quantitative reverse transcriptase-polymerase chain reaction as previously reported (5) using primers described therein. Anti-mouse ferroportin antibody was purchased from Alpha Diagnostics (San Antonio, TX). The mRNA and protein expression of β-actin was used as an internal control.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. References

Blood glucose levels in KK-Ay mice on the normal diet gradually increased, whereas tamibarotene significantly suppressed the increase (Figure 1a). Moreover, decreases in fasting blood glucose and serum insulin levels have also been observed (7), suggesting that tamibarotene improved insulin sensitivity in KK-Ay mice. In agreement with our previous findings (5), the hepatic non-heme iron content significantly lowered in mice on the tamibarotene diet (Figure 1b). However, serum lipid levels including free fatty acid, triglyceride, and total cholesterol remained unchanged (data not shown).

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Figure 1. Effect of tamibarotene on hyperglycemia and hepatic iron metabolism. Blood glucose levels (a) and hepatic non-heme iron content (b) levels in KK-Ay mice on the Am80-supplemented normal diet for 4 weeks. The expression levels of the retinoic acid receptor β mRNA (Rarβ; c), ferritin heavy-chain mRNA (Fth; d), ferritin light-chain mRNA (Ftl; e), transferrin receptor 1 mRNA (Tfr1; f), ferroportin mRNA (Fpn; g) and protein (h), hemojuvelin mRNA (Hjv; i), and hepcidin mRNA (Hepc; j) in the livers of KK-Ay mice on the Am80-supplemented normal diet for 4 weeks. β-Actin was used as an internal control. *P < 0.05; **P < 0.01 vs. the control diet.

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We further investigated the expression of iron metabolism-related genes in the liver. The expression of a retinoid signaling target gene Rarβ (10) was significantly upregulated in the tamibarotene group, suggesting that the retinoid signaling pathway was activated in the liver (Figure 1c). The genes for ferritin heavy chain, ferritin light chain, and transferrin receptor 1, whose expression is regulated by IRE, significantly changed, consistent with the reduced hepatic iron content in the tamibarotene group (Figure 1d-f) (2). The expression of ferroportin mRNA and protein was not changed significantly (Figure 1g,h). Because ferroportin expression is also under the regulation of IRE, this result suggests that there is an additional mechanism counteracting the IRE-mediated translational inhibition of ferroportin in response to the decreased hepatic iron content.

It has been reported that hepcidin facilitates the degradation of ferroportin protein, leading to a decrease in ferroportin-mediated cellular iron export (4). In agreement with the finding of a constant ferroportin protein level despite the decreased hepatic iron content, hemojuvelin and its target, hepcidin, were significantly downregulated by the tamibarotene diet (Figure 1i,j). These results were almost consistent with the finding that ATRA suppresses hepatic iron accumulation by increasing the hepatic iron efflux through the regulation of hemojuvelin and ferroportin expression (5).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. References

The cumulative evidence strongly indicates that chronic liver diseases, including NAFLD and CH-C, commonly develop and progress in association with insulin resistance. Moreover, HIO is frequently observed, and has been a potential therapeutic target for the treatment of NAFLD and CH-C (1). The fact that the liver is a major target organ for insulin and plays an important role in iron storage as well as the control of iron homeostasis suggests that the liver is the primary site where HIO and insulin resistance are linked. However, the precise relationship between both pathologies is currently under investigation.

ATRA, an active form of vitamin A, exhibits broad physiological properties. In the liver, decreased vitamin A storage in hepatic stellate cells during pathological conditions such as liver cirrhosis has been observed (11). We recently demonstrated that the hepatic retinoid signaling is severely impaired in patients with NAFLD and CH-C (6). Taking into account that retinoids including ATRA and tamibarotene not only improve insulin sensitivity in mouse models (7) but also suppress HIO, it is reasonable to postulate that retinoids and hepatic retinoid signaling are key to understanding the mechanism for the development of NAFLD and CH-C associated with insulin resistance and HIO.

The decreased hepatic iron content inhibits ferroportin translation by an IRE-mediated mechanism (2). However, ferroportin protein levels between the control and tamibarotene groups were similar. Hemojuvelin is involved in the regulation of hepcidin expression cooperatively with bone morphogenic proteins (12). Hepcidin induction results in the inhibition of cellular export of iron by the degradation of ferroportin in enterocytes (4). Ramey et al. also recently demonstrated that ferroportin expressed in hepatocytes is a target of hepcidin, which induces the lysosomal degradation of ferroportin (13). Therefore, our observation may be attributable to these reciprocal regulations for ferroportin expression in the liver. This notion suggests that tamibarotene may have an effect on maintaining ferroportin-mediated iron export despite the decreased hepatic iron content. In contrast to this study, ATRA induced a marked upregulation of ferroportin expression in the liver of mice developing HIO by administration of iron dextran (5). However, in this study, KK-Ay mice did not exhibit HIO because their hepatic non-heme iron content was in the normal range [0.5-1.5 μmol/g liver; from our previous observations (5, 9)]. Although the effect of tamibarotene on mice with HIO must be investigated, retinoids may accelerate mobilization of iron from the liver to other organs. We previously observed that a hemojuvelin-overexpressing hepatoma cell line exhibits a significant increase in the cellular iron content without changes in ferroportin expression levels compared with its parent cell line (5). This result suggests that hemojuvelin itself may have an additional function to increase intracellular iron contents independent of ferroportin and that the retinoid-induced decrease in the hepatic iron content could be explained by the effects of hemojuvelin.

As described above, although KK-Ay mice did not develop HIO, tamibarotene significantly improved their insulin sensitivity, accompanied by decreased hepatic iron contents. It is not clear if the decrease in hepatic iron contents contributed to the tamibarotene-induced insulin sensitization. However, phlebotomy normalizes insulin resistance in patients without HIO (14). Although a large-scale randomized clinical trial is required to establish the definitive clinical benefit of iron reduction therapy in insulin resistance, iron-induced oxidative stress may have a role in insulin resistance despite normal hepatic iron contents. Therefore, retinoids may provide dual benefits for patients with insulin resistance.

Tamibarotene exerted similar efficacy to that seen with ATRA (5). However, tamibarotene did not affect dyslipidemia in contrast to ATRA. It has been reported that ATRA enhances lipolysis in mature adipocytes in a RAR-independent manner (15). Thus, ligand-dependent RARα/β activation offers a more potent improvement on glucose and iron metabolisms than lipid metabolism.

In conclusion, this study demonstrated that tamibarotene, like other retinoids, holds great potential for the therapy and/or prevention of insulin resistance associated with HIO.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods and Procedures
  5. Results
  6. Discussion
  7. References