Leptin-induced mitochondrial fusion mediates hepatic lipid accumulation.

Accepted Article Preview: Published ahead of advance online publication Leptin-induced mitochondrial fusion mediates hepatic lipid accumulation W-H Hsu, B-H Lee, T-M Pan

Cite this article as: W-H Hsu, B-H Lee, T-M Pan, Leptin-induced mitochondrial fusion mediates hepatic lipid accumulation, International Journal of Obesity accepted article preview 29 June 2015; doi: 10.1038/ijo.2015.120. This is a PDF ﬁle of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its ﬁnal form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.

Received 4 April 2015; revised 2 June 2015; accepted 22 June 2015; Accepted article preview online 29 June 2015

©

2015 Macmillan Publishers Limited. All rights reserved.

Leptin-induced mitochondrial fusion mediates hepatic lipid accumulation

Wei-Hsuan Hsu†1,2

Bao-Hong Lee†1,2

Tzu-Ming Pan1*

Author address: 1

Department of Biochemical Science and Technology, College of Life Science,

National Taiwan University, Taipei, Taiwan.

2

Department of Basic Medical Sciences, College of Veterinary Medicine, and Center

for Cancer Research, Purdue University, West Lafayette, Indiana, USA.

†

These authors contributed equally to this work.

*Corresponding author: Department of Biochemical Science & Technology, College of Life Science, National Taiwan University, Taipei, Taiwan, No. 1, Sec. 4, Roosevelt Road, Taipei, 10617, Taiwan

Tel: +886-2-33664519 ext 10, Fax: +886-2-33663838, E-mail: [email protected]

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ABSTRACT BACKGROUND: Leptin alleviates metabolic conditions such as insulin resistance and obesity, although the precise mechanism of action is unclear. Mitochondrial fusion/fission states affect energy balance, but the association between mitochondrial fusion and lipid metabolism is also unknown. The aim of this study was to determine whether mitochondrial fusion/fission state regulates lipid accumulation and to understand the role of leptin in mitochondrial function and its mechanism of action in metabolic regulation. METHODS: Primary mouse hepatocytes were isolated from C57BL/6J mice and treated with leptin (25 ng ml-1) for 3 days prior to determinations of mitochondrial morphology and fatty acid accumulation. Hyperglycemia in C57BL/6J mice was induced by providing a 30% fructose-rich diet (FRD) for 6 months, followed by intraperitoneal (i.p.) injections of leptin (1 mg kg-1 bw-1) for 6 weeks (twice per week). RESULTS:

Leptin

triggered

mitochondrial

fusion

and

alleviated

high

glucose-induced fatty acid accumulation in primary hepatocytes by promoting mitochondrial

fusion-associated

transcription

factor

peroxisome

proliferative

activated receptor (PPAR)-α and co-activator peroxisome proliferative activated receptor-gamma coactivator (PGC)-1α. In turn, these activate the fusion protein

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mitofusin 1 (Mfn-1). RNA silencing of Mfn-1 or PGC-1 blocked the inhibitory effect of leptin. Leptin treatment also elevated liver Mfn-1 and PGC-1α and improved lipid profiles in FRD mice. CONCLUSIONS: Mitochondrial fusion plays a critical role in alleviating hepatic fatty acid accumulation. Leptin switches mitochondrial morphology via a PGC-1α-dependent pathway to improve hyperlipidemia.

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INTRODUCTION

Obesity is associated with chronic inflammatory responses, which are characterized by the abnormal production of cytokines such as adiponectin, leptin, and resistin in mature white adipose tissue (WAT).1 Leptin acts in the brain, liver, pancreas, mature adipose tissue, and the immune system. Overexpression of leptin results in an increase in suppressor of cytokine signaling-3 (SOCS3) expression, thereby blocking insulin receptor substrate-1/2 (IRS-1/2) activation and leading to insulin resistance.2,3 Recent evidence suggests leptin is a more potent regulator of blood glucose levels than an appetite suppressant.4-6 The action of leptin on glucose homeostasis is dependent on signal transduction via phosphatidylinositol-3-kinase (PI3K) activity,7 a pathway also used by the insulin receptor. Insulin stimulates PI3K activity and downstream mediator Akt.8 Unlike insulin, however, leptin does not induce Akt phosphorylation, but could improve diabetes.5 Leptin regulates hepatic insulin sensitivity and glucose homeostasis, as well as food intake and energy expenditures by activating the leptin receptor. The administration of leptin increases glucose turn-over and glucose uptake in peripheral tissues through the sympathetic nervous system.5 The transcription factor signal transducer and activator of transcription 3 (STAT3) is a downstream target of the leptin signaling pathway. STAT3 independently regulates hepatic gluconeogenesis

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and

carbohydrate

metabolism.9

The

transcriptional

coactivator

peroxisome

proliferative activated receptor-gamma coactivator-1α (PGC-1α) regulates hepatic gluconeogenesis.10 Leptin administration increases PGC-1α expression.11 Suppression of PGC-1α by receptor-interacting protein 1 knockdown impairs mitochondrial oxidative phosphorylation and acceleration of glycolysis in cancer cell.12 Given that mitochondrial morphology affects energy imbalance and is continuously changed through fusion and fission events, a tight coordination between mitochondrial dynamics and interorganelle interactions is crucial. The fusion protein mitofusin (Mfn) 2 has been extensively implicated in mitochondrial fusion. Deficiency of Mfn 2 causes leptin resistance in diet-induced obesity mice.13 PGC-1α regulates mitochondrial morphology by activating the estrogen-related receptor (ERR) and nuclear respiratory factor (NRF) transcription factors to elevate fusion protein expression.14

Furthermore,

mitochondrial

fission

results

in

an

impaired

insulin-dependent glucose uptake.15 These findings suggest that leptin lowers blood glucose levels and may alter mitochondrial function by regulating PGC-1α. However, the mechanism by which leptin improves glucose uptake is unknown. We hypothesized that leptin exerts its glucose-lowering effects by regulating mitochondrial function via PGC-1α. We used in vitro and in vivo models to understand the effects of leptin on carbohydrate metabolism and lipid synthesis in

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hepatocytes. Our results provide new insight into the mechanism underlying leptin-mediated improvements in diabetes, hyperlipidemia, and hepatic steatosis.

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MATERIALS AND METHODS Reagents Leptin antagonist (mutant mouse recombinant) and recombinant murine leptin were purchased

from

MyBioSource

(Abingdon,

Oxon,

UK).

2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG) and medium-199, trypsin–EDTA, lipofectamine RNAiMAX reagent, and fetal bovine serum (FBS) were from Invitrogen (Carlsbad, CA, USA). F-12K medium was purchased from Gibco BRL Life Technologies, Inc. (Gaithersburg, MD, USA). Bovine serum albumin (BSA), and sodium dodecyl sulfate were purchased from Sigma Chem. Co. (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO), formaldehyde, and KH2PO4 were purchased from Wako Pure Chem. (Saitama, Japan). High-capacity cDNA reverse transcription kit was purchased from Applied Biosystems (Foster City, CA, USA). Mfn2, STAT3, and PGC-1, siRNA were purchased from Santa Cruz Biotechnology Inc. (Burlingame, CA, USA). Anti-PGC-1alpha antibody was purchased from Novus (Littleton, CO, USA). Anti-PPARalpha antibody and anti-NRF1 antibody were purchased from Abcam (Cambridge, MA, USA). Anti-ERRalpha antibody was purchased from GeneTex Inc. (San Antonio, TX, USA). Anti-p-STAT3 (Tyr705) antibody, anti-insulin antibody, anti-GAPDH antibody, anti-Mfn1 antibody, anti-Mfn2 antibody, and anti-STAT3 antibody were purchased

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from Santa Cruz (Santa Cruz, CA, USA). Anti-ACC antibody was purchased from Cell Signaling Technology (Beverly, MA, USA).

Culture of FL83B hepatocytes and primary hepatocytes isolation Mouse liver cell FL83B (Bioresource Collection and Research Center, Hsinchu, Taiwan) is a hepatocyte cell line isolated from a normal liver taken from a 15 to 17 day old fetal mouse. FL83B cells were cultured in a humidified atmosphere of 95% air and 5% CO2 at 37 oC in F-12K medium containing 10% FBS. The medium was renewed every 2–3 days and subcultured every 4 days. Primary hepatocytes were isolated from 8- to 12-week-old male C57BL/6J mice as described previously16 and were incubated overnight in Medium 199.

Animal induction Male C57BL/6J mice (6 weeks old) (n = 6) were kept and used for the experiment, which were purchased from BioLASCO Co., Ltd. (Taipei, Taiwan). The mice were housed in a temperature-controlled room (25 ± 1 oC) and kept on a 12-h light/dark cycle (light on at 08:00). (Protocol complied with guidelines described in the ‘‘Animal Protection Law’’, amended on June 29, 2011 Hua-Zong-(1)-Yi-Tzi10000136211, Council of Agriculture, Executive Yuan, Taiwan, ROC). The experiments were carried out in a qualified animal breeding room in the Animal 8

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Center at our institute. There is no randomization and blinding was used. The mice were fed with chow diet (Rodent Laboratory Chow 5001, Ralston Purina, St. Louis, MO, USA) for 1 week in order to accommodate them to the environment. Animals were randomly assigned to (A) control, (B) fructose-rich diet (FRD), (C) FRD + leptin (1 mg kg-1 bw-1). All animals were provided ad libitum access to the diets for 6 months. After FRD induction, mice were intraperitoneal (ip) injection with leptin (1 mg kg-1 bw-1) for subsequently 6 weeks (twice/week).

Western blot analysis Cell lysates were centrifuged (10,000xg for 10 min) to recover the supernatant. The supernatant was taken as the cell extract. The protein concentration in the cell extract was determined using a Bio-Rad protein assay kit. The samples were subjected to 10% SDS–polyacrylamide gel electrophoresis (PAGE). The protein spots were electrotransferred to a polyvinyldiene difluoride (PVDF) membrane. The membrane was incubated with block buffer and then probed with primary antibody overnight at 4 o

C. The membrane was washed, shaken in a solution of HRP-linked anti-rabbit IgG

secondary antibody. The expressions of proteins were detected by enhanced chemiluminescent (ECL) reagent (Millipore, Billerica, MA, USA).

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Real-time polymerase chain reaction (PCR) analysis Total RNA from liver tissue and hepatocytes was obtained using the Trizol reagent (Gibco BRL Life Technologies, Inc., Gaithersburg, MD, USA) according to the manufacturer’s instructions. Primers were synthesized by MD-Bio, Inc. (Taipei, Taiwan). The gene expression level was determined by relative quantitative real-time PCR (CFX Cycler System, Bio Rad Laboratories, Inc., Hercules, CA, USA).

Immunohistochemistry (IHC) stain The tissue sections were incubated with 3% H2O2 for 20 min to quench endogenous peroxidase activity. After being rinsed twice with PBS, the sections were incubated with skimmed milk (5%) for 1 h and the primary monoclonal antibody for 12 h at 4 oC. After being washed in PBS, the sections were incubated with the secondary antibody in PBS for 1 h. After the sections were rinsed twice with PBS, immunoreactions were visualized by incubation with 3,3’-diaminobenzidine tetrahydrochloride for 10 min. The sections were counterstained with hematoxylin.

Immunocytochemistry (ICC) stain Cells were stained with hoestest 33342 for 30 min. After being rinsed twice with PBS, the cells were fixed with formaldehyde (3.7%) for 10 min, and the primary

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monoclonal antibody for 12 h at 4 oC. After being washed in PBS, the sections were incubated with the secondary antibody (labeling FITC or rhodamine) in PBS for 1 h. After the sections were rinsed twice with PBS, cells were observed by confocal microscope.

Mitochondria stain Cells were treated by 250 nM of Mitotracker Deep-Red FM (Invitrogen, Carlsbad, CA, USA) for 30 min in serum-free culture medium. After wash with PBC twice, nuclei were stained by Hochest33342 for 10 min. The mitochondrial morphology was observed by confocal microscope.

Glucose uptake analysis Glucose uptake of cells was assessed using the fluorescent glucose analog, 2-NBDG. Briefly, hepatocytes were treated with leptin (25 ng ml-1) in serum-free medium. After incubation, the medium was replaced with Krebs–Ringer-Bicarbonate (KRB) buffer containing 2-NBDG (160 μM; final concentration) for 30 min incubation at 37 oC. Free 2-NBDG was washed out from cultures after treatment and measured 2-NBDG positive cells with a FACS flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed using Cell-Quest software.

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Oil-red O stain Oil red O working solution was added to each well and stained for 15 min. The staining photos were taken under the microscope.

Statistical analysis Results were analyzed in triplicates and expressed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) and Duncan’s multiple range tests were carried out. Differences were considered significant when P ≤ 0.05.

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RESULTS Leptin and mitochondrial fusion As shown in Figure 1A, 3-day leptin treatment (25 ng ml-1) of primary mouse hepatocytes isolated from C57BL/6J mice resulted in mitochondrial fusion; however, this effect was not observed in the leptin(mut) (25 ng ml-1) treatment group. Both transcription factors ERRα and NRF1 induce mitochondrial fusion and activation of transcriptional coactivator PGC-1α. Leptin treatment clearly promoted PPARα and PGC-1α expression but not ERRα and NRF1 in primary hepatocytes isolated from C57BL/6J mice (Figures 1B and 1C), suggesting leptin may induce a PGC-1α-dependent change in mitochondrial morphology. Mfn-1 and Mfn-2 participate in mitochondrial fusion regulated by ERRα and NRF1; we found that leptin treatment for 24 h increased transcript levels of Mfn-1 in primary hepatocytes (Supplemental Figure 1). Moreover, STAT3 phosphorylation was elevated in primary hepatocytes after 12-h leptin treatment (25 ng ml-1). In addition, 3-day leptin treatment (25 ng ml-1) resulted in increases in acetyl-CoA carboxylase (ACC), Mfn-1, and Mfn-2 levels in primary hepatocytes (Figure 1D).

Association between mitochondrial fusion and lipid accumulation regulated by leptin Obese and/or diabetic individuals develop cellular resistance to the action of

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insulin, characterized by an impaired ability of insulin to inhibit glucose output from the liver and to promote glucose uptake in fat and muscle. Hence, elevating glucose uptake ability is benefit from obesity and/or diabetes. Primary hepatocytes from C57BL/6J mice were treated with leptin or leptin(mut) (25 ng ml-1) for 3 days, and the results indicated that 2-NBDG uptake was greater in the leptin-treated group than in the leptin(mut)-treated group (Supplemental Figure 2), suggesting leptin treatment may improve insulin resistance, consistent with the findings of a recent report.7 We treated FL83B hepatocytes with mannitol-balanced glucose (33 mM) for 7 days to induce hepatic fatty acid accumulation, and then treated the cells with leptin (25 ng ml-1) for 3 days. Leptin attenuated fatty acid accumulation in hepatic cells versus the mock controls, but the effect was abolished by knockdown of Mfn-1, PGC-1, or STAT3 (Figure 2). Furthermore, leptin (25 ng ml-1) resulted in mitochondrial fusion in FL83B hepatocytes—this was also reversed by 3-day knockdown of Mfn-1, PGC-1, or STAT3 (Figure 3A), and this effect was attributed to the leptin-mediated elevation of Mfn-1 expression after 3 days (Figure 3B). STAT3 phosphorylation was also markedly increased in leptin-treated FL83B hepatocytes (Figure 3C). These findings indicated that mitochondrial fusion was essential for the activity of leptin in the reduction of hepatic fatty acid accumulation and hepatic steatosis.

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Leptin improved dyslipidemia in FRD-induced mice We investigated the effect of leptin on FRD-induced hyperglycemia in C57BL/6J mice. C57BL/6J mice received FRD (30%) for 6 months to induce hyperglycemia and were then treated with leptin (1 mg kg-1 bw-1) by biweekly i.p. injection for 6 weeks. Mean daily food intake in each group was shown in Supplemental Figure 3A. FRD induction significantly increased fasting blood glucose and serum insulin by the 30th week, suggesting that insulin resistance was induced by FRD. However, leptin suppressed these effects after 6 weeks (Figures 4A and 4B). The levels of adipokines, including leptin and adiponectin, were measured: leptin administration markedly increased serum leptin (Figure 4C) but did not affect serum adiponectin (Supplemental Figure 3B). Our data indicated that serum triacylglycerol (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and free fatty acid (FFA) were elevated in the FRD-treated group but were alleviated in the leptin-treated group (Figures 4D-4H). Moreover, fat accumulation in the liver occurred in FRD-induced mice, while leptin administration suppressed hepatic steatosis induced by FRD (Figure 5). However, leptin treatment did not affect pancreatic islets in mice (Figure 5). Hepatic PPARα (Supplemental Figure 4) as well as PGC-1α and STAT3

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phosphorylation levels increased in FRD-induced mice treated with leptin (Figure 6A), suggesting that leptin promoted PGC-1α in a leptin signal-dependent pathway. In addition, PGC-1α downstream factors associated with mitochondrial fusion such as ERRα, NRF1, Mfn-1, and Mfn-2 were significantly elevated in FRD + leptin-treated mice versus FRD-treated mice (Figure 6B).

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DISCUSSION Mitochondria play critical roles in energy production and metabolism.17 Mitochondrial fusion/fission state is thought to be crucial for mitochondria division. Proteins such as Mfn 1 and 2 (Mfn-1/2) and GTPases optic atrophy-1 (Opa-1) governing mitochondrial membrane fusion between two individual mitochondria. In contrast, proteins like dynamin-related protein-1 (Drp1) and fission protein-1 (Fis1) controlling mitochondrial fission.18 In type 2 diabetic patients, smaller mitochondria has been found, and mitochondrial fusion protein levels were decreased.

19,20

In

skeletal muscle, mitochondrial fusion protein modulates insulin signal pathway and glucose homeostasis via regulating mitochondrial function also have been reported.21 Insulin stimulates mitochondrial fragmentation and Ca2+ uptake, and promotes Akt activation and mitochondrial glucose uptake to produces energy.15 Mitochondrial fusion proteins are potent modulators of mitochondrial carbohydrate metabolism with an impact on energy metabolism in liver and skeletal muscle. However, the association between mitochondrial fusion and lipid metabolism is unclear. A murine model showed that inhibiton of mitochondrial fission abates hepatic steatosis in nonalcoholic fatty liver disease, revealing that mitochondrial function may regulates hepatic steatosis.22 There are numerous clinical evidence have described various pathways and mechanisms of leptin-improved metabolic syndrome. Also,

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leptin ameliorates dyslipidemia and fatty acid liver was recently reported.23 However, the precise mechanism of leptin on regulating fatty acid oxidation is still unclear, and the association between mitochondrial fusion and lipid metabolism is also unknown. Hence, how leptin mediates fatty acid oxidation in liver and whether mitochondria fusion/fission state involved in this regulation need to be clarified. Our present data reveal that mitochondrial fusion/fission state regulates lipid accumulation and we point out the role of leptin on mitochondrial function and its mechanism of action in metabolic regulation. In this study of hyperglycemic C57BL/6J mice, we explored the effect of leptin on mitochondrial fusion and lipid accumulation in both liver tissue of mice and primary hepatocytes and found that leptin functions through a PGC-1α-dependent pathway to improve hyperlipidemia. This is the first study to elucidate that leptin regulates mitochondrial function, and we point out the inhibitory effect of leptin on hepatic lipid accumulation is via leptin-mediated mitochondrial fusion. Both ERR and NRF elevate the expression of mitochondrial fusion-associated protein.14 Here, we found that leptin promoted mitochondrial fusion in both primary mouse hepatocytes and FL83B hepatic cells (Figures 1 and 4), as well as increased mRNA expression of mitochondrial fusion-associated factors in hepatocytes of HGD-induced mice, including PGC-1α, NRF1, NRF2, Mfn1, and Mfn2 (Figure 6B).

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Leptin treatment has reported to increase PGC-1α levels in mice.11 PGC-1α regulates gluconeogenesis9, glycolysis12, lipid metabolism24, and mitochondrial function,25 suggesting that leptin may act an important role on hepatic steatosis inhibition through PGC-1α regulation. Our results indicated that leptin treatment significantly increased hepatic PGC-1α levels in hepatocytes (Figure 1C) and FRD-induced mice (Figure 6B). In addition, Mfn-2 or PGC-1α inhibition has been linked with mitochondrial fusion and causes lipid accumulation in the liver, heart, and muscle of rats, these results were demonstrated that Mfn-2 and PGC-1α play a role in organs against lipid deposit.25 Moreover, induction of mitochondrial fusion by silencing of fission protein (Drp1) is able to decrease accumulation of cellular TG; in contrast, induction of mitochondrial fission by silencing the fusion proteins Mfn-2 or OPA-1 causes an increase in TG accumulation.26 One study suggested hepatic lipid accumulation in ob/ob mice is primarily targeted for export by leptin repletion.27,28 Hence, leptin may promote fatty acid oxidation via regulating mitochondrial fusion through elevating PGC-1α. Our results indicated that leptin treatment attenuated lipid accumulation in hepatocytes (Figure 3), suppression of hyperlipidemia (Figures 4D-4H) and attenuation of hyperglycemia (Figure 4). Our findings showed that leptin improved dyslipidemia and dysglycemia by up-regulating PGC-1α and mitochondrial fusion,

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thereby protecting hepatocytes from liver steatosis (Figure 5). PPARα is a major transcription factor for fatty acid oxidation. This transcription factor

may

participate

in

leptin-improved

hepatic

steatosis.

Despite

the

leptin-mediated improvement of hepatic steatosis in ob/ob mice, neither mitochondrial function nor the abundance of PPARα increased.27 We demonstrated that leptin also increased PGC-1α, STAT3 phosphorylation (Figure 6A), and PPARα (Supplemental Figure 4) levels in the liver of FRD-induced mice. However, the association between fatty acid oxidation mediated by mitochondrial fusion and activation of PPARα mediated by leptin treatment should be further investigated. The mechanism by which STAT3 regulates hepatic gluconeogenic gene expression and carbohydrate metabolism has been thoroughly investigated.10 ERR target promoters are enriched for NRF-1, CREB, and STAT3 binding sites.29 STAT3 also regulates mitochondrial respiration.30 Here, we reported that the effect of leptin on mitochondria may occur partly through STAT3. The leptin-PGC-1α pathway promoted mitochondrial fusion and STAT3 phosphorylation, and these effects were blocked by STAT3 knockdown (Figures 4 and 5). In addition, STAT3 siRNA treatment reversed the leptin-mediated suppression of lipid accumulation in FL83B hepatocytes (Figure 3). In conclusion, we found that leptin improved fatty liver and hepatic steatosis in a

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PGC-1α-dependent

pathway,

thereby

stimulating

mitochondrial

fusion.

Leptin-mediated mitochondrial fusion plays an important role on hepatic lipid accumulation, and reagents or drugs involving in the regulation of mitochondrial fusion/fission proteins may have novel implication for fatty acid liver and hyperlipidemia.

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CONFLICT OF INTEREST The authors have declared no conflict of interest.

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Supplementary information is available at IJO's website.

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Figure legends Figure 1. The effect of leptin on mitochondrial fusion. (A) Three-day leptin (25 ng ml-1) treatment altered mitochondrial morphology in primary hepatocytes isolated from C57BL/6J mice. (B) The effect of leptin (25 ng ml-1; 3 days) on the expression of PPARα and PGC-1α in primary hepatocytes isolated from C57BL/6J mice, as demonstrated by fluorescent stain and confocal microscopy. (C) Leptin (25 ng ml-1; 3 days) regulated mitochondrial fusion-associated transcription factors (ERRα, NRF-1, and PPARα) and co-activator PGC-1α in primary hepatocytes isolated from C57BL/6J mice. (D) Western blot analysis of STAT3 phosphorylation in primary hepatocytes isolated from C57BL/6J mice after treatment with leptin (25 ng ml-1) for 12 h. The levels of ACC, Mfn-1, and Mfn-2 were measured in primary hepatocytes isolated from C57BL/6J mice treated with leptin (25 ng ml-1) for 3 days. Leptin(mut) (25 ng ml-1) was used as the negative control. Results are expressed as mean ± SD (n = 3).

a.b.c

values with one different letter superscript are significantly different from

each other (p < 0.05).

Figure 2. The alleviation of fatty acid accumulation by leptin treatment. After treatment with glucose (33 mM) for 7 days, leptin (25 ng ml-1) was added to the FL83B hepatocytes with or without specific knockdown by siRNA (Mfn-1, PGC-1, or

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STAT3). Medium containing leptin was refreshed every day during the 3-day induction. Fatty acid accumulation was observed by oil-red O stain.

Figure 3. The effect of leptin on mitochondrial fusion was blocked by Mfn-1, PGC-1, or STAT3 knockdown in FL83B hepatocytes. (A) Confocal images of mitochondrial fusion in FL83B hepatocytes after 3-day treatment with leptin (25 ng ml-1) and siRNA (Mfn-1, PGC-1, or STAT3). (B) Transcript levels of Mfn-1 in FL83B hepatocytes after treatment with specific siRNA (Mfn-1, PGC-1, or STAT3) for 3 days. (C) Western blot analysis of STAT3 phosphorylation. After 3-day treatment with specific siRNA (Mfn-1, PGC-1, or STAT3), leptin (25 ng ml-1) was added to FL83B hepatocytes for 12 h before analysis. Results are expressed as mean ± SD (n = 3).

a.b.c

values with one different letter superscript are significantly different from each other (p < 0.05).

Figure 4. Lipid profiles in FRD-induced C57BL/6J mice. Hyperglycemia was induced in C57BL/6J mice by providing a 30% fructose-rich diet (FRD) for 6 months. The mice then received biweekly intraperitoneal leptin injections (1 mg kg-1 bw-1) for 6 weeks and improved hyperlipidemia. Levels of (A) fasting blood glucose, (B) serum insulin, (C) serum leptin, (D) Serum triacylglycerol (TG), (E) total cholesterol (TC),

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(F)

low-density

lipoprotein-cholesterol

(LDL)-C,

(G)

high-density

lipoprotein-cholesterol (HDL-C), and (H) free fatty acid (FFA) levels were measured. Results are expressed as mean ± SD (n = 6).

a.b.c

values with one different letter

superscript are significantly different from each other (p < 0.05).

Figure 5. Histopathology images of the liver of FRD-induced C57BL/6J mice (left). The specific stain for pancreatic insulin (right).

Figure 6. (A) Protein levels of hepatic PGC-1α (top) and p-STAT3 (bottom) in FRD-induced C57BL/6J mice. (B) Hepatic mRNA levels of factors associated with mitochondrial fusion in FRD-induced C57BL/6J mice. Results are expressed as mean ± SD (n = 6).

a.b

values with one different letter superscript are significantly different

from each other (p < 0.05).

31

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Supplemental Figure Legends Supplemental Figure 1. Mfn-1 transcript levels in primary hepatocytes isolated from C57BL/6J mice and treated with leptin (25 ng ml-1) for 24 h. Leptin(mut) was used as the negative control. Results are expressed as mean ± SD (n = 3).

Supplemental Figure 2. Primary hepatocytes from C57BL/6J mice after 3-day treatment with leptin or leptin(mut) (25 ng ml-1). Leptin(mut) was used as the negative control. 2-NBDG uptake was measured by flow cytometry. Results are expressed as mean ± SD (n = 3).

Supplemental Figure 3. (A) Mean daily food intake in each group of C57BL/6J mice. (B) Serum adiponectin levels in FRD-induced hyperglycemic C57BL/6J mice after 6 weeks of biweekly intraperitoneal leptin injections (1 mg kg-1 bw-1). Results are expressed as mean ± SD (n = 6).

Supplemental Figure 4. Hepatic PPARα expression in FRD-induced C57BL/6J mice. Results are expressed as mean ± SD (n = 6).

a.b

values with one different letter

superscript are significantly different from each other (p < 0.05).

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