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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Protein tyrosine phosphatase 1B (PTP1B) inhibits hepatic insulin signaling by dephosphorylating tyrosine residues in insulin receptor (IR) and insulin receptor substrate (IRS). MicroRNAs may modulate metabolic functions. In view of the lack of understanding of the regulatory mechanism of PTP1B and its chemical inhibitors, this study investigated whether dysregulation of specific microRNA causes PTP1B-mediated hepatic insulin resistance, and if so, what the underlying basis is. In high-fat-diet-fed mice or hepatocyte models with insulin resistance, the expression of microRNA-122 (miR-122), the most abundant microRNA in the liver, was substantially down-regulated among those predicted to interact with the 3′-untranslated region of PTP1B messenger RNA (mRNA). Experiments using miR-122 mimic and its inhibitor indicated that miR-122 repression caused PTP1B induction. Overexpression of c-Jun N-terminal kinase 1 (JNK1) resulted in miR-122 down-regulation with the induction of PTP1B. A dominant-negative mutant of JNK1 had the opposite effect. JNK1 facilitated inactivating phosphorylation of hepatocyte nuclear factor 4α (HNF4α) responsible for miR-122 expression, as verified by the lack of HNF4α binding to the gene promoter. The regulatory role of JNK1 in PTP1B induction by a decrease in miR-122 level was strengthened by cell-based assays using isoliquiritigenin and liquiritigenin (components in Glycyrrhizae radix) as functional JNK inhibitors; JNK inhibition enabled cells to restore IR and IRS1/2 tyrosine phosphorylation and insulin signaling against tumor necrosis factor alpha, and prevented PTP1B induction. Moreover, treatment with each of the agents increased miR-122 levels and abrogated hepatic insulin resistance in mice fed a high-fat diet, causing a glucose-lowering effect. Conclusion: Decreased levels of miR-122 as a consequence of HNF4α phosphorylation by JNK1 lead to hepatic insulin resistance through PTP1B induction, which may be overcome by chemical inhibition of JNK. (HEPATOLOGY 2012;56:2209–2220)

The liver is the principal organ that regulates glucose homeostasis because of its capacity to consume and produce glucose. The prevalence of metabolic syndrome, as characterized by insulin resistance, dyslipidemia, and obesity, has dramatically increased and often causes liver disease; insulin resistance dampens insulin sensitivity in the liver as well as in other peripheral organs, being closely linked to liver steatosis and its progression to steatohepatitis.1 The abnormality in the signaling pathway, including insulin receptor (IR), insulin receptor substrate (IRS), and their downstream molecules,2 marks the main pathophysiological characteristics of insulin resistance. However, the molecular mechanisms underlying insulin resistance and impairment of insulin signaling network in the liver are not completely understood yet.

Protein tyrosine phosphatase (PTP) constitutes a family of phosphatases including PTP1B, SHP1, SHP2, and LAR.3, 4 As a negative regulator of insulin signaling cascade, PTP1B functions as a key phosphatase and reverses tyrosine kinase action.5 A deficiency of PTP1B improved glycemic control by enhancing sensitivity to insulin and made animals more resistant to diet-induced obesity.6 In addition, antisense oligonucleotides of PTP1B decreased blood glucose content, insulin level, and fat mass in diabetic animals.7 However, the upstream regulators or mediators that control transcription and translation of the PTP1B gene are not well defined. Moreover, pharmacological agents that intervene with PTP1B activity are not available, despite recognition of PTP1B inhibition as an effective way to improve insulin sensitivity.8

The microRNAs (miRNAs) have diverse functions in normal or pathological states.9 The consequences of changes in miRNA levels can affect not only lipid metabolism, but insulin signaling, potentially influencing glucose homeostasis and the development of diabetes. In fact, dysregulation of miRNA is implicated in defective insulin secretion, diabetic kidney, and heart diseases.10 Hepatocyte-specific deletion of Dicer, an enzyme essential for miRNA processing, resulted in the depletion of glycogen storage and led to mild hyperglycemia in the fed state and severe hypoglycemia in the fasting state.11 However, the genes targeted by miRNAs and their individual biological functions largely remain to be established. In particular, more information is necessary to understand the involvement of miRNAs in insulin resistance and find the pharmacological methods to modulate their levels.

In view of the lack of understanding of the PTP1B-regulatory mechanism and limited information on its therapeutic manipulation, this study investigated whether dysregulation of a specific miRNA leads to hepatic insulin resistance by PTP1B, and if so, what the underlying basis is. Our findings reveal that dysregulation of miRNA-122 (miR-122) contributes to hepatic insulin resistance through PTP1B induction. Flavonoids are being actively studied as potential treatments for components of the metabolic syndrome. In our previous study, treatment with licorice flavonoid ameliorated liver steatosis.12 In the present study, we additionally discovered the effect of c-Jun N-terminal kinase 1 (JNK1) inhibition by isoliquiritigenin (IsoLQ) or liquiritigenin (LQ) on miR-122 dysregulation using in vivo models and cell-based assays. Here, we report that they have the ability to abolish hepatic insulin resistance by recovering the constitutive expression of miR-122 responsible for PTP1B down-regulation.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Materials.

Information on the materials used in this study is described in the Supporting Information.

Animal Treatments.

Animal studies were conducted in accordance with the guidelines of the Institutional Animal Use and Care Committee. Male C57BL/6 mice at 6 weeks of age were started on a high-fat diet (HFD) for 11 weeks. Detailed information is provided in the Supporting Information.

Cell Culture.

HepG2, H4IIE, C2C12, and 3T3-L1 cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD). The isolation of primary rat hepatocytes is described in the Supporting Information.

Transient Transfection and Reporter Gene Assays.

The plasmid containing Luc-PTP1B-3′UTR (3′-untranslated region; Product ID: HmiT015828-MT01) was specifically synthesized (GeneCopoeia, Rockville, MD) and was used in luciferase reporter assay. The plasmid contains firefly luciferase fused to the 3′UTR of human PTP1B, and Renilla luciferase that functions as a tracking gene. pMiR-122a luciferase reporter vector containing the firefly luciferase gene and miR-122 target site at 3′UTR was purchased from Signosis (Sunnyvale, CA). When miR-122 is expressed, it binds to the sequence and results in repression of the luciferase gene. The sources of other vectors and procedures used in this study for transient transfections and reporter gene assays are provided in the Supporting Information.

Real-Time Polymerase Chain Reaction (PCR) Assays.

Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA) and was reverse-transcribed. Quantitative real-time PCR (qRT-PCR) was performed with the Light Cycler 1.5 (Roche, Mannheim, Germany).

Chromatin Immunoprecipitation Assays.

Chromatin immunoprecipitation assay was done using the EZ ChIP kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer's protocol.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

MicroRNA Candidates Targeting the 3′UTR of PTP1B Messenger RNA (mRNA).

HFD feeding increased the mRNA and protein levels of PTP1B (Fig. 1A); the change in the level of PTP1B protein was greater than that in its mRNA, suggesting that a posttranscriptional mechanism might be involved in this event. RNA22 and TargetScan programs enabled us to select miRNAs that potentially bind to the 3′-untranslated region (3′UTR) of PTP1B (PTPN1) mRNA (Fig. 1B, upper); there existed one or two putative binding sites for eight miRNAs: miR-122, miR-203, miR-135, miR-29, miR-124, miR-506, miR-206, and miR-1. To find their expression profile, we tested whether hepatic insulin resistance elicited by HFD feeding affects the miRNA levels. In particular, the levels of miR-122 were significantly decreased in mice fed an HFD, implying that miR-122 dysregulation may be associated with the induction of PTP1B (Fig. 1B, lower). The levels of other miRNAs were not changed (Fig. 1B, lower). miR-1 (a muscle-specific miRNA) was not detected in the liver.

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Figure 1. Identification of miRNA that targets PTP1B mRNA. (A) qRT-PCR assays for PTP1B mRNA and immunoblottings for PTP1B in the liver of HFD-fed mice. Male C57BL/6 mice were fed on either a normal diet (ND) or a high-fat diet (HFD) (N = 3, each) for 11 weeks. (B) The locations of the predicted miRNA binding sites within the 3′UTR of PTP1B mRNA (upper) and qRT-PCR assays for the miRNAs (lower) (N = 5-7, each). For A and B, data represent the mean ± SE (significantly different as compared to ND, *P < 0.05, **P < 0.01). N.S., not significant; N.D., not detected). (C) PTP1B induction by TNF-α. HepG2 cells were treated with 20 ng/mL TNF-α for 3 hours. (D) qRT-PCR assays for the miRNAs. For C and D, data represent the mean ± SE of three separate experiments (significantly different as compared to control, *P < 0.05, **P < 0.01; N.D., not detected).

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Next, a cell model was used to assess the expression profile of the miRNAs. Treatment of HepG2 cells with tumor necrosis factor-α (TNF-α) weakly, but significantly, increased PTP1B mRNA levels (Fig. 1C). TNF-α treatment induced PTP1B to a much greater extent (Fig. 1C), and decreased levels of the miRNAs predicted to bind the 3′UTR of the mRNA (Fig. 1D). Our in vivo and in vitro findings in conjunction with the database analyses suggested that miR-122 dysregulation might contribute to the posttranscriptional regulation of PTP1B.

PTP1B as a Direct Target of MiR-122.

Next, we explored the functional role of miR-122 in the repression of PTP1B. First, in vitro assays were performed using miR-122 inhibitor or its mimic. Transfection of HepG2 cells with miR-122 inhibitor induced PTP1B (Fig. 2A), whereas miR-122 mimic transfection diminished its expression (Fig. 2B). Consistently, transfection with miR-122 inhibitor increased the level of PTP1B mRNA, whereas miR-122 mimic transfection decreased it (Fig. 2C,D), indicating that miR-122 may facilitate the degradation of PTP1B mRNA. To further prove a direct interaction between miR-122 and its binding site within the mRNA, luciferase activities from the PTP1B 3′UTR reporter construct were measured. As expected, miR-122 inhibitor transfection significantly increased luciferase expression from pEZX-PTP1B luciferase construct, whereas its mimic transfection decreased it (Fig. 2E,F), which verifies PTP1B as a direct target of miR-122.

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Figure 2. The regulatory effect of miR-122 on PTP1B expression. (A) PTP1B induction by the miR-122 inhibitor. Immunoblottings were done on the lysates of HepG2 cells transfected with control or miR-122 inhibitor (left). qRT-PCR assays verified the transfection of miR-122 inhibitor (right). (B) PTP1B repression by miR-122. (C) The effect of miR-122 inhibitor on PTP1B mRNA levels. (D) The effect of miR-122 mimic on PTP1B mRNA levels. (E) The effect of miR-122 inhibitor on pEZX-PTP1B 3′UTR luciferase activity. Luciferase assays were done on HepG2 cells transfected with control or miR-122 inhibitor in combination with a construct comprising a luciferase cDNA fused to either pEZX-Control or the 3′UTR of PTP1B (pEZX-PTP1B 3′UTR). (F) The effect of miR-122 mimic on pEZX-PTP1B 3′UTR luciferase activity. For A-F, data represent the mean ± SE of three separate experiments (significantly different as compared to each control, *P < 0.05, **P < 0.01).

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JNK1 Repression of MiR-122 for the Induction of PTP1B.

JNK1 dampens the normal insulin response by inhibiting IR signaling through serine phosphorylation of IRS1/2, playing a role in the development of obesity and insulin resistance.13 Because JNK1 is closely linked to IR in mice fed an HFD or cell models exposed to TNF-α,12 we measured the levels of miR-122 and PTP1B in HepG2 cells transfected with the construct encoding for HA-tagged JNK1 (HA-JNK1) or a dominant-negative form of JNK1 (DN-JNK1). Overexpression of JNK1 decreased miR-122 levels, as verified by an increase in the miR-122 3′UTR luciferase activity, whereas that of DN-JNK1 had the opposite effect (Fig. 3A,B). In addition, enforced expression of JNK1 increased the pEZX-PTP1B luciferase activity and induced PTP1B protein levels (Fig. 3C). DN-JNK1 transfection exerted the opposite effect (Fig. 3D). JNK2 overexpression had no effect on miR-122 or PTP1B levels (Fig. 3E). These results showed that JNK1, but not JNK2, represses miR-122 levels, which may lead to the induction of PTP1B.

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Figure 3. MiR-122 down-regulation by JNK1 for the induction of PTP1B. (A) The effect of JNK1 overexpression on miR-122 levels. MiR-122 levels and miR-122 3′UTR luciferase activities were measured in HepG2 cells transfected with HA-JNK1. (B) The effect of DN-JNK1 transfection on miR-122 levels. (C) The effect of JNK1 overexpression on PTP1B expression. (D) The effect of DN-JNK1 transfection on PTP1B expression. (E) The effects of V5-JNK2 transfection on the expression of miR-122 and PTP1B. For A-E, data represent the mean ± SE of three separate experiments (significantly different from MOCK-transfected control, *P < 0.05, **P < 0.01; N.S., not significant).

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JNK1 Phosphorylation of HNF4α for Transcriptional Inhibition of MiR-122.

Hepatocyte nuclear factor 4α (HNF4α), a transcription factor abundantly expressed in the liver, may directly bind to the promoter region of the miR-122 gene.14 The phosphorylation status of HNF4α at serine/threonine residues governs its activity.15 Because JNK1 negatively phosphorylates HNF4α,16 we assessed the effect of JNK1 overexpression on miR-122 expression and HNF4α phosphorylation. JNK1 overexpression decreased the expression of primary precursor of miR-122 in HepG2 cells (Fig. 4A) and facilitated the serine/threonine phosphorylation of HNF4α (Fig. 4B). Chromatin-immunoprecipitation assays revealed that overexpressed JNK1 prohibited HNF4α binding to the promoter region of the miR-122 gene (Fig. 4C). Consistently, enforced expression of HNF4α increased miR-122 levels, which was reversed by JNK1 (Fig. 4D). The miR-122 3′UTR reporter assays confirmed the ability of JNK1 to inhibit HNF4α-mediated miR-122 expression (Fig. 4E). Our results demonstrate that JNK1 inhibits miR-122 expression through HNF4α phosphorylation, which results in the induction of PTP1B.

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Figure 4. HNF4α phosphorylation by JNK1 for miR-122 down-regulation. (A) The effect of JNK1 overexpression on pri-miR-122 levels. Data represent the mean ± SE of three separate experiments (significantly different from MOCK-transfected control, **P < 0.01). (B) The effect of JNK1 overexpression on HNF4α phosphorylation. HNF4α immunoprecipitates were immunoblotted with anti-phosphorylated serine (p-Ser) or anti-phosphorylated threonine (p-Thr) antibodies (at least three separate experiments were performed). (C) Chromatin immunoprecipitation assays for HNF4α. Cross-linked protein-DNA complexes were immunoprecipitated using anti-RNA polymerase II antibody (Pol II, positive control), preimmune-IgG (negative control), or anti-HNF4α antibody in cells transfected with MOCK or HA-JNK1. The DNA samples from immunoprecipitates were PCR-amplified using primers specific for the 160-bp conserved region of miR-122 gene promoter. One-tenth of the total input was used as a loading control. Results were confirmed by repeated experiments. (D) The effect of JNK1 overexpression on miR-122 induction by HNF4α. (E) The effect of JNK1 overexpression on miR-122 3′UTR luciferase activity. For D,E, data represent the mean ± SE of three separate experiments (significantly different from MOCK-transfected control, **P < 0.01; significantly different from HNF4α transfection, #P < 0.05, ##P < 0.01).

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PTP1B Repression by JNK Inhibition.

In our previous studies, IsoLQ isolated from Glycyrrhizae radix inhibited the activity of JNK1.12 In HepG2 cells, the inhibition of TNF-α-induced JNK1/2 phosphorylation by ILQ (or LQ) was confirmed (Fig. 5A). Mice fed an HFD for 11 weeks showed a significant increase in PTP1B level in the liver, which was abolished by treatment with either IsoLQ or LQ (30 mg/kg, 5 times per week, for the last 5 weeks) (Fig. 5B). Consistently, treatment with each agent prevented PTP1B induction by TNF-α (Fig. 5C). IsoLQ treatment enabled the cells to restore tyrosine phosphorylations in IRβ and IRS1/2 against TNF-α (Fig. 5D). Tyrosine phosphorylations of IRβ and IRS1/2 transmit an insulin signal to PI3-kinase/Akt.2 As expected, IsoLQ was capable of increasing the phosphorylation of Akt or glycogen synthase kinase 3β in the cells treated with insulin and/or TNF-α (Fig. 5E). In this model, PTP1B overexpression virtually eliminated the ability of IsoLQ to increase the tyrosine phosphorylation of IRβ or IRS1 (Fig. 5F). These results demonstrate that IsoLQ and LQ as functional JNK1 inhibitors sensitize IR signaling against TNF-α by repressing PTP1B levels.

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Figure 5. The role of PTP1B repression in IR sensitization by IsoLQ or LQ. (A) The effect of IsoLQ or LQ on the phosphorylation of JNK by TNF-α. Phosphorylated JNK or total JNK was immunoblotted on the lysates of HepG2 cells treated with IsoLQ or LQ for 1 hour, followed by continuous incubation with vehicle or TNF-α (20 ng/mL) for indicated times. Data represent the mean ± SE of three to five separate experiments. The statistical significance of differences between each treatment group and the control (**P < 0.01) or TNF-α alone (#P < 0.05, ##P < 0.01) was determined. (B) PTP1B repression in the liver. PTP1B was immunoblotted on the liver homogenates from mice fed either ND or HFD for 11 weeks with or without IsoLQ or LQ. Mice were treated with IsoLQ or LQ (10 or 30 mg/kg/day, orally, 5 times per week) during the last 5 weeks of the diet feeding. Data represent the mean ± SE (N = 6, each). The statistical significance of differences between each treatment group and ND (**P < 0.01) or HFD alone (##P < 0.01) was determined. (C) PTP1B repression. PTP1B was immunoblotted on the lysates of HepG2 cells treated with IsoLQ (5, 10, or 20 μM) or LQ (10, 30, or 100 μM) for 1 hour and continuously exposed to 20 ng/mL TNF-α for 3 hours. (D) Phosphorylation of IRβ and IRS1/2. HepG2 cells were treated with 20 μM IsoLQ for 1 hour and exposed to TNF-α for 6 hours followed by 10 nM insulin treatment for 10 minutes. (E) Akt and GSK3β phosphorylation. (F) The effect of PTP1B overexpression on IR sensitization by IsoLQ. For C-F, at least three separate experiments were performed.

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Having identified miR-122 repression by HFD feeding or TNF-α treatment, we determined the effects of IsoLQ and LQ on miR-122 expression. HFD feeding for 11 weeks decreased miR-122 levels, whereas treatment with either IsoLQ or LQ reversed it (Fig. 6A). Similarly, IsoLQ or LQ treatment enabled HepG2 cells to restore miR-122 levels against TNF-α (20 ng/mL, for 3 hours) (Fig. 6B). As expected, JNK1 overexpression abolished the ability of IsoLQ to inhibit TNF-α-induced luciferase activity from pEZX-PTP1B reporter construct (Fig. 6C), confirming the effect of JNK1 inhibition on the repression of PTP1B. Immunoblottings for PTP1B also supported this effect (Fig. 6D).

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Figure 6. The effect of JNK1 inhibition on the expression of miR-122 and PTP1B. (A) Increase in hepatic miR-122 levels. miR-122 levels were measured using qRT-PCR assays on the liver of mice treated with vehicle or IsoLQ or LQ (30 mg/kg/day) as described in the legend to Fig. 5B. The statistical significance of differences between each treatment group and ND (*P < 0.05) or HFD alone (#P < 0.05) was determined (at least 4 animals/group). (B) Increase in miR-122 levels in vitro. HepG2 cells were treated with 20 μM IsoLQ or 100 μM LQ for 1 hour and continuously exposed to 20 ng/mL TNF-α for 3 hours. (C) The effect of JNK1 overexpression on PTP1B 3′UTR reporter activity. HepG2 cells were transfected with MOCK or HA-JNK1 and were treated with TNF-α or TNF-α+IsoLQ for 12 hours. (D) JNK1 reversal of PTP1B repression by IsoLQ. Immunoblottings for PTP1B were done on the lysates of HepG2 cells treated as described in Fig. 6C except for the condition of 3 hours TNF-α treatment. For B-D, data represent the mean ± SE of three determinations. The statistical significance of differences between each treatment group and the control (**P < 0.01) or TNF-α alone (#P < 0.05, ##P < 0.01) was determined.

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Inhibition of IRS1 Serine Phosphorylation.

The phosphorylation of IRS1/2 at Ser312 in human, corresponding to Ser307 in rodents, is a marker of insulin resistance.17 Treatment of HFD-fed mice with IsoLQ or LQ caused a decrease in IRS1 phosphorylation at Ser307 (Fig. 7A), verifying the improvement of insulin signaling. Consistently, treatment of H4IIE cells with IsoLQ (5-20 μM) or LQ (10-100 μM) effectively prevented the serine phosphorylation of IRS1 (Fig. 7B). The inhibition of IRS1 serine phosphorylation by IsoLQ or LQ was also confirmed in other cell models such as HepG2 cells, C2C12 myotubes, 3T3-L1 adipocytes, and primary rat hepatocytes (Fig. 7C).

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Figure 7. Inhibition of IRS1 serine phosphorylation by IsoLQ or LQ. (A) The effect of IsoLQ or LQ on IRS1 serine phosphorylation. Immunoblottings for phosphorylated IRS1 were done on the liver homogenates of mice treated as described in the legend to Fig. 5B. Data represent the mean ± SE (N = 3, each). The statistical significance of differences between each treatment group and ND (**P < 0.01) or HFD alone (##P < 0.01) was determined. (B) Inhibition of IRS1 serine phosphorylation by IsoLQ or LQ in H4IIE cells. The cells were treated as described in the legend to Fig. 5C. (C) Inhibition of IRS1 serine phosphorylation in other cell models. Immunoblottings were done on the lysates of cells treated with 20 μM IsoLQ or 100 μM LQ for 1 hour and subsequently exposed to 20 ng/mL TNF-α for 3 hours. For B and C, immunoblottings were performed in at least three separate experiments.

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In Vivo and In Vitro Inhibition of Insulin Resistance.

To further assess the effect of IsoLQ or LQ on glucose homeostasis and insulin sensitivity, each agent was administered to mice fed an HFD: treatment of mice with the agents at the dose of 10 or 30 mg/kg/day for 5 days during the last 5 weeks of total 11 weeks of HFD feeding displayed a significantly improved glucose tolerance (2 g glucose/kg) compared to vehicle-treated control (Fig. 8A; normal diet [ND] and HFD controls were shared to simultaneously compare the compound effects), showing their effects on systemic insulin sensitivity. In mice fed an HFD for 9 weeks, IsoLQ treatment almost completely reduced fasting glucose, fasting serum insulin levels, and HOMA-IR values (Fig. 8B, upper). Similar results were obtained using Lepob/ob mice (Fig. 8B, lower). Our results indicate that licorice flavonoids have the ability to reduce obesity-induced insulin resistance.

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Figure 8. In vivo and in vitro effects of IsoLQ or LQ on IR sensitization. (A) Improvement of glucose tolerance in mice fed an HFD. Glucose tolerance tests were conducted in mice administered IsoLQ or LQ during the last 5 weeks of HFD feeding for 11 weeks (at least 6 animals/group, diet controls were shared). (B) The effect of IsoLQ treatment on blood glucose, serum insulin, and HOMA-IR in HFD-fed mice or Lepob/ob mice. For A and B, the statistical significance of differences between each treatment group and ND (**P < 0.01) or HFD alone (#P < 0.05, ##P < 0.01) was determined. Lepob/ob mice were orally administered vehicle or IsoLQ (30 mg/kg/day, 5 times per week) for 20 days and were fasted for 6 hours (N = 4, each). Data represent the mean ± SE. The statistical significance of differences compared with vehicle (*P < 0.05, **P < 0.01) was determined. (C) Glucose production and glucose uptake assays. Basal glucose output was assessed by measuring glucose contents in collected culture media of HepG2 cells incubated in serum-free Dulbecco's modified Eagle's medium (DMEM) with or without 20 μM IsoLQ, 100 μM LQ, or 10 nM insulin for 6 hours, and continuously incubated in glucose-free DMEM for 3 hours (left). Glucose uptake was measured in C2C12 myotubes or 3T3-L1 adipocytes treated with 100 nM insulin for 10 minutes after treatment with TNF-α, TNF-α+IsoLQ, or TNF-α+LQ for 6 hours (right). Data represent the mean ± SE of three determinations. The statistical significance of differences between each treatment group and control (**P < 0.01) or insulin (#P < 0.05, ##P < 0.01) was determined. (D) qRT-PCR assays for G6Pase mRNA. Hepatic mRNA levels were measured in mice treated as described in panel A (upper). The statistical significance of differences between each treatment group and HFD alone (*P < 0.05, **P < 0.01) was determined. Primary rat hepatocytes were treated with 20 μM IsoLQ, 100 μM LQ, or 10 nM insulin for 3 hours and continuously incubated with 1 μM cAMP and 100 nM dexamethasone for 6 hours (lower). Data represent the mean ± SE of three determinations. The statistical significance of differences between each treatment group and the control (**P < 0.01) or cAMP/dexamethasone (##P < 0.01) was determined. (E) A schematic diagram illustrating the proposed mechanism by which JNK1 regulates PTP1B levels for insulin resistance.

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As a continuing effort to assess the effect of IsoLQ or LQ on insulin action, we measured glucose production and uptake in representative cell models. Incubation of HepG2 cells with each agent resulted in a significant decrease in glucose production, which was comparable to that caused by insulin (Fig. 8C, left). TNF-α inhibited an increase in glucose uptake by insulin in C2C12 myotubes or differentiated 3T3-L1 adipocytes, which was also abrogated by IsoLQ treatment (Fig. 8C, middle and right). Our results indicate that IsoLQ (or LQ) treatment prevents glucose production from hepatocytes and stimulates glucose uptake into muscle cells or adipocytes.

In HFD-fed mice, we measured the levels of glucose 6-phosphatase (G6Pase) mRNA as a marker of gluconeogenesis. IsoLQ or LQ treatment inhibited the G6Pase gene induction (Fig. 8D, upper). Consistently, either IsoLQ or LQ treatment antagonized the ability of cyclic adenosine monophosphate (cAMP) and dexamethasone to increase G6Pase mRNA levels in primary rat hepatocytes, as did insulin (Fig. 8D, lower). These results demonstrate that the inhibition of glucose production by IsoLQ or LQ may be mediated by the suppression of G6Pase.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

PTP1B negatively regulates insulin signaling by catalyzing the dephosphorylation of IR and IRS1/2.5 A decrease in PTP1B activity accompanies improved insulin sensitivity in obese subjects.18 In addition, evidence is accumulating that PTP1B polymorphisms in humans might be associated with insulin resistance.19 PTP1B is linked to the liver-specific aspect of metabolic syndrome; mice deficient in PTP1B displayed an increase in insulin sensitivity with increased or prolonged tyrosine phosphorylation of IRβ and IRS1.20 In hepatocytes of fructose-fed animals, PTP1B expression levels and activities were higher.21 Our results shown here confirmed an alteration in hepatic PTP1B level in mice fed an HFD, being consistent with the observation that the protein was up-regulated in patients with nonalcoholic steatohepatitis.22

Mature miRNAs work as posttranscriptional regulators by hybridizing to complementary binding sites in the 3′UTR of target mRNAs.23 This property allows a single miRNA sequence to have multiple binding sites on various mRNAs. The discovery of posttranscriptional gene silencing as an additional regulatory principle to control protein levels suggests that dysregulation of miRNAs may affect the development of hepatic insulin resistance.24 Dicer-deficient mice showed markedly increased apoptosis, proliferation, and lipid accumulation in hepatocytes, showing steatosis; a deficiency in dicer down-regulated the levels of miRNAs highly enriched in the liver,11 highlighting the role of miRNAs in regulating glucose and lipid metabolism.

MiR-122 is the most abundantly (accounting for 52%) expressed miRNA in the liver,25 and may be involved in lipid and cholesterol metabolism.26 Transfection of miR-122 inhibitor significantly increased the mRNA levels of lipogenic genes such as FAS, HMG-CoA reductase, SREBP-1c, and SREBP-2.9 Thus, miR-122 down-regulation may alter lipid metabolism, potentially facilitating the pathogenesis of nonalcoholic steatohepatitis. Our results provide evidence that miR-122 has an inhibitory effect on PTP1B levels. Luciferase assays using plasmids harboring the PTP1B 3′UTR confirmed this regulatory effect. Moreover, bioinformatic analyses of the miRNA array data obtained from human nonalcoholic steatohepatitis samples (Supporting Table S1)9 and our in vivo and in vitro results enabled us to identify miR-122 as an miRNA that critically controls PTP1B-associated insulin resistance in the liver. Moreover, miR-122 levels were consistently decreased in the liver of several different in vivo models with insulin resistance (i.e., HFD-fed rats, ob/ob mice, and streptozotocin-induced diabetic mice) (Supporting Table S2).27, 28

The conditions responsible for increased PTP1B gene transcription are not well defined except the finding that D-glucose enhanced transcription of the PTP1B gene in a human hepatocyte cell line by way of protein kinase C (PKC).29 Macrophage activation enhanced PTP1B induction by palmitate in myotubes.30 TNF-α induces PTP1B by nuclear factor kappa B (NF-κB) activation.31 So, TNF-α from macrophages increases insulin resistance. The target scan results shown in the present study raised the proposal that the miRNAs including miR-203, 135, 29, 124, 506, and 206 might also interact with the binding sites within the 3′UTR of human PTP1B mRNA. Although the levels of these miRNAs were also decreased in HepG2 cells exposed to TNF-α, they were not changed in the in vivo model. In our supplemental experiment, palmitate treatment decreased miR-122, miR-203, miR-506, and miR-206 in HepG2 cells (Supporting Fig. S1). So, the in vitro results did not perfectly match the in vivo outcome presumably because obesity-induced insulin resistance is complex and may be accompanied by alterations not restricted to the liver. Because the in vivo model reflects human situation better, it is highly likely that miR-122 plays a key role in regulating PTP1B expression.

JNK activation impairs insulin-induced tyrosine phosphorylation of IRS1/2 through serine phosphorylation, causing insulin resistance: the increase in IRS1 phosphorylation at Ser307 by JNK is closely associated with insulin resistance.13 In the current study, JNK1 was identified as a kinase that causes miR-122 repression. miR-122 expression may be transcriptionally regulated by HNF4α, C/EBPα, HNF1α, and HNF3β.14 Previously, it has been shown that JNK activated by TNF-α or IL-1β catalyzes phosphorylation of HNF4 for the inhibition of CYP7A1 and CYP8B1 genes.16 JNK1 and JNK2 are expressed in most types of cells including hepatocytes.13, 32 Each isoform has an overlapping or distinct role in liver pathophysiology; a deficiency of JNK1, but not JNK2, improved insulin sensitivity with decreased adiposity.13 JNK2 might negatively regulate JNK1 and its downstream c-Jun phosphorylation and stabilization.32, 33 In another study, JNK1 and JNK2 antisense oligonucleotides treatment improved HFD-induced insulin resistance.34 In the present study, JNK1 served as a novel inhibitory regulator of miR-122 expression, contributing to PTP1B induction, whereas JNK2 had no effect. So, JNK1 may play a key role in insulin resistance. This idea was supported by the finding that JNK1 transfection decreased miR-122 levels with an increase in miR-122 3′UTR reporter activity, as verified by the opposite changes in cells transfected with DN-JNK1. Our finding that JNK1 enhanced HNF4α phosphorylation at serine and threonine residues confirmed its role in HNF4α regulation. An important finding of our study is that the decrease in miR-122 levels by JNK1 results from the inactive phosphorylation of HNF4α (Fig. 8E), which parallels the ability of JNK1 to induce PTP1B.

Although Ertiprofatib was launched as an investigational drug that targets the activity of PTP1B (i.e., phase I trials in 2000), the clinical trial was discontinued after 2 years because of its poor efficacy and dose-limiting side effects. Hence, developing other approaches modulating PTP1B for the treatment of insulin resistance is anticipated.35 Licorice (Glycyrrhizae radix) is largely used as sweetening agent, and its extract is applied for analgesic and antitussive remedies.36 Among the constituents in licorice, IsoLQ and LQ are structurally related flavonoids; IsoLQ is the biosynthetic precursor and an isomer of LQ.37 IsoLQ and LQ have anticarcinogenic and antiinflammatory activities.38, 39 In addition, IsoLQ has an antisteatotic effect, which depends on MKK7-JNK1 inhibition.12 Another important finding of our study is the discovery of JNK inhibitors originated from licorice as the regulators of miR-122 for PTP1B repression; decreased levels of PTP1B allowed cells to maintain the phosphorylation of IRβ and IRS1 at tyrosine residues with the inhibition of IRS1/2 serine phosphorylation. IsoLQ or LQ treatment caused 50%-60% inhibition of JNK1 in a cell model exposed to TNF-α (15 minutes), being consistent with our previous report.12 More important, IsoLQ treatment at 10 and 30 mg/kg inhibited HFD-induced JNK1 activation by 50% and 100%, respectively,12 which matches the repression of PTP1B shown in the present study. IR sensitization was further supported by not only the inhibition of glucose production in hepatocytes, but the increase in glucose uptake by myotubes or adipocytes. The improved glucose homeostasis was strengthened by the glucose-lowering effect shown in an animal model. Our results that IsoLQ treatment decreased body weight and liver weight gains and the plasma triacylglycerol contents in HFD-fed mice also support their beneficial effects on metabolic syndrome.12

SIRT1 levels are decreased in insulin-resistant cells or tissues. The effect of SIRT1 on insulin resistance is also affected by the repression of PTP1B transcription at the chromatin level.40 The agents used in the present study had the capability to activate SIRT1, which may be associated with the regulation of glucose metabolism. However, PTP1B inhibition by IsoLQ seems to be independent of SIRT1, as supported in part by our finding that IsoLQ treatment inhibited PTP1B expression even after SIRT1 inhibition using sirtinol and nicotinamide (data not shown). Nrf2 contributes to the inhibition of LXRα-dependent lipogenesis,41 whereas AMPK activation of SIRT1 inhibits gluconeogenesis and increases energy expenditure.42 Although the enhanced energy metabolism by IsoLQ may involve Nrf2 and/or AMPK, PTP1B repression by IsoLQ seems to be irrelevant to the molecules. This possibility is supported by the finding that the agent decreased PTP1B levels in hepatocytes deficient in Nrf2 or those treated with compound C (an AMPK inhibitor) (data not shown).

In conclusion, the present study identified miR-122 dysregulation as a cause of hepatic insulin resistance, which may depend on the posttranscriptional induction of PTP1B, as mediated by JNK1-dependent inactive phosphorylation of HNF4α. Our finding that PTP1B induction can be overcome by the pharmacological inhibitors of JNK provides key information in understanding liver pathobiology and designing a therapeutic strategy for hepatic insulin resistance.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Dr. Young Woo Kim (Daegu Haany University) for the kind additional supply of IsoLQ.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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HEP_25912_sm_SuppInfo.doc120KSupporting Information

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