PGRN induces impaired insulin sensitivity and defective autophagy in hepatic insulin resistance.

ORIGINAL

RESEARCH

PGRN Induces Impaired Insulin Sensitivity and Defective Autophagy in Hepatic Insulin Resistance Jiali Liu1#, Huixia Li1#, Bo Zhou1, Lin Xu1, Xiaomin Kang1, Wei Yang1, Shufang Wu1*, Hongzhi Sun1* 1 From the First Affiliated Hospital of Medical School of Xi’an Jiaotong University, Key Laboratory of Environment and Genes Related to Diseases, Ministry of Education, Medical School of Xi’an Jiaotong University, Xi’an, Shaanxi, 710061, China

Progranulin (PGRN) has recently emerged as an important regulator for glucose metabolism and insulin sensitivity. However, the underlying mechanisms of PGRN in regulation of insulin sensitivity and autophagy remain elusive. In this study, we aimed to address the direct effects of PGRN in vivo and to evaluate the potential interaction of impaired insulin sensitivity and autophagic disorders in hepatic insulin resistance. We found that mice treated with PGRN for 21 days exhibited the impaired glucose tolerance and insulin tolerance, and hepatic autophagy imbalance as well as defective insulin signaling. Furthermore, treatment of mice with TNFR1BP-Fc, a TNFR1 blocking peptide-Fc fusion protein to competitively block the interaction of PGRN and TNFR1, resulted in restoration of systemic insulin sensitivity, and recovery of autophagy and insulin signaling in liver. Consistent with these findings in vivo, we also observed that PGRN treatment induced defective autophagy, impaired insulin signaling in hepatocytes, with such effects being drastically nullified by the addition of TNFR1BP-Fc or TNFR1-siRNA via the TNFR1-NF-␬B-dependent manner, indicating the causative role of PGRN in hepatic insulin resistance. In conclusion, our findings supported the notion that PGRN is a key regulator of hepatic insulin resistance and that PGRN may mediate its effects, at least in part, by inducing defective autophagy via TNFR1/NF-␬B.

epatic insulin resistance is highly integrated with chronic inflammation, which contributes to a systemic, low-grade inflammatory state now known as “metabolic inflammation” and thereby promotes the development of systemic insulin resistance (1). However, the link between inflammation and hepatic insulin resistance is not fully understood. Progranulin (PGRN), a secreted protein that plays an important role in several processes including immune response (2), has recently emerged as an important regulator between inflammatory action and insulin resistance (3). PGRN, also known as proepithelin, granulin/ epithelin precursor (GEP) or PC cell-derived growth factor (PCDGF), has been shown to be a pluripotent growth factor that mediates cell growth, wound healing, tumor genesis and neurodegenerative disease such as frontotem-

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poral dementia (4 –7). However, recent studies supported the novel function of PGRN in regulating the energy metabolism (3, 8 –11). For instance, diet-induced obesitymice with PGRN deficiency exhibited lower body weight and ameliorated insulin sensitivity, whereas administration of recombinant PGRN induced glucose intolerance and insulin resistance in wild-type mice (3). Several clinical investigations also demonstrated that serum PGRN was associated with the parameters of adiposity, glucose tolerance, insulin resistance and inflammatory factors (12–17). Clinically, circulating PGRN is significantly higher in subjects with T2DM (type 2 diabetes mellitus) and positively associated with hsCRP (high-sensitivity Creaction protein), IL-6, and macrophage infiltration in omental adipose tissue (12–16). In particular, PGRN is more highly expressed in visceral fat area of the insulin-

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2015 by the Endocrine Society Received August 20, 2014. Accepted February 4, 2015.

Abbreviations: PGRN, progranulin; TNF-␣, Tumor necrosis factor-␣; GTT, Glucose tolerance testing; ITT, Insulin tolerance testing; TNFR, Tumor necrosis factor receptor; T2DM, Type 2 diabetes mellitus; siRNA, small interfering RNA; hsCRP, high-sensitivity C-reaction protein; IRS-1, insulin receptor substrate 1; IKK, multisubunit I␬B Kinase; ER, endoplasmic reticulum.

doi: 10.1210/me.2014-1266

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Progranulin Induces Impaired Insulin Sensitivity

resistant patients with morbid obesity than in their age-, sex- and BMI-matched insulin-sensitive counterparts (17). Although the role of PGRN in energy homeostasis has just recently been identified, the functional basis for PGRN-mediated insulin resistance remains elusive. However, the relevance of PGRN to autophagic response involving a variety of adipocytokines including IL-6 and TNF-␣ has recently drawn considerable attention (18 – 22). PGRN has been shown to impair insulin signaling in 3T3-L1 adipocytes through IL-6, an inducer of autophagy (3). Consistently, PGRN-/- mice reveals the autophagic disturbance with alterations in lysosomal homeostasis as evidenced by an abnormal accumulation of lipofuscin granules and p62 proteins (23) and inhibition of autophagy with chloroquine exhibit elevated endogenous PGRN levels (24, 25), indicating the causative link between PGRN and autophagic activity in insulin resistance. Collectively, these evidences support a newly regulatory role of PGRN on energy homeostasis and chronic inflammation, which raises the possibility that PGRN may contribute to the progression of hepatic insulin resistance and metabolic dysfunction. As the PGRN membrane receptor has not yet been identified, it is important to define the early stages of PGRN-mediated signaling from the plasma membrane. In this study, we aimed to address the direct effects of PGRN in vivo and to evaluate the potential interaction of impaired insulin sensitivity and autophagic disturbance during this process. Our results support the therapeutic potential of this new player in the regulation of hepatic insulin resistance and metabolic disorders.

Materials and Methods Materials. All chemicals used were of analytical grade and were purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA) unless stated otherwise. The following antibodies were used: anti-Atg7 (1:400), anti-p62 (1:300), and anti-LC3 (1:500) (Cell Signaling Technology Inc. Danvers, MA, USA); anti-PGRN (1: 400), anti-p-IRS-1 (1:300), anti-pY20 (1:300), anti-p-AKT (1: 400), anti-TNFR1 (1:400), anti-IKK␤ (1:400), anti-NF-␬B (1: 1000), anti-p-I␬B␣ (1:500), anti-GAPDH (1:5000) and peroxidase goat antirabbit IgG (1:3000) and peroxidase goat antimouse IgG (1:3000) (Santa Cruz Biotechnology Inc., CA, USA). Preparation of recombinant mouse PGRN and human IgG1 Fc fused TNFR1 blocking peptide (TNFR1BP-Fc). The methods were followed by the literature (3) except for the source of CHO-K1 cells (ATCC), and the venders of CD OptiCHO medium (Life Technologies Grand Island, NY, USA). The pFLAG-

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CMV1 vector was purchased from Addgene (Cambridge MA, USA). pCAGIPuro-FLAG was constructed by subcloning the insert encoding the preprotrypsin signal peptide and FLAG epitope of pFLAG-CMV1 into pCAGIPuro. pCAGIPuro-FLAG was constructed by Sangon Biotech (Shanghai, CN). Purified PGRN was made endotoxin-free using the Detoxi-Gel endotoxin removing column (Thermo Scientific, Rockford, IL USA) as recommended by the manufacturer. Preparation of human IgG1 Fc fused TNFR1 blocking peptide (TNFR1BP-Fc) was described as previously (26). The characteristics of TNFR1BPFc: A TNFR1 blocking peptide originated from screening a phage 12-mer peptide library (New England Biolabs, Ipswich, MA) using soluble TNFR1 as bait (26). DNA sequence encoding the TNFR1 blocking peptide was directly synthesized by PTI (Protein technologies Inc. Tucsan, AZ). Animals Treatment and Cell Culture. Four-week-old C57BL/6J female mice were obtained from Medical Experimental Animal Center, Xi’an Jiaotong University. All studies were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of Xi’an Jiaotong University. Mice were maintained in a temperature- (22°C), humidity-, and light- (12 hours of light, 12 hours of darkness, darkness from 7:30 pm to 7:30 am) - controlled environment. Sixty mice fed regular diet and allowed ad libitum access to chow and water were distributed in six groups: 1) vehicle (saline solution intraperitoneally i.p.); 2) PGRN (recombinant mouse PGRN, i.p. 1 ␮g/g body weight/d, once a day); 3) IgG1Fc (injection via tail vein, 1 ␮g/g body weight, every three days); 4) TNFR1BP-Fc (injection via tail vein, 1 ␮g/g body weight, every three days); 5) PGRN (i.p. 1 ␮g/g body weight/d, once a day) ⫹ IgG1Fc (injection via tail vein, 1 ␮g/g body weight, every three days); 6) PGRN (i.p. 1 ␮g/g body weight/d, once a day) ⫹ TNFR1BP-Fc (injection via tail vein, 1 ␮g/g body weight, every three days). At the end of the 21-days study period, mice received vehicle or an intraperitoneal injection of insulin at a dosage of 2 IU/kg for the insulin signaling test; 15 minutes after the injection, all animals were euthanized and their liver tissues and blood samples were obtained and stored at – 80°C for subsequent analysis. BNL cl.2 cell were purchased from American Tissue Culture Collection (ATCC, Manassas, Virginia, USA) and maintained at 37°C in a humidified atmosphere of 5% CO2 in DMEM (Gibco, Grand Island, New York, USA) with 10% fetal bovine serum (Gibco). In some experiments, cells were treated with 500 ␮M of palmitate (FFA) (Sigma-Aldrich, Cat. No. P5585) for 16 hours. For the effect of recombinant mouse PGRN, cells were treated with 100ng/ml of PGRN for 10 hours. For insulin signaling, cells were stimulated with 10 nM insulin for 10 minutes. Immunohistochemistry of liver tissues and metabolic tests. The immunohistomorphometry was performed as previously described (27). Glucose tolerance testing (GTT) was performed after the mice were fasted overnight. Insulin tolerance testing (ITT) was performed after the animals were fasted for 4 hours. Metabolic tests were performed using a standard protocol as described previously (28). The in vivo fatty acid oxidation was performed based on previous report (29, 30). Briefly, [1-14C] oleic acid [specific activity, 56.3 mCi/mmol (2083MBq/mmol)] was purchased from PerkinElmer Inc (Boston, MA, USA). Oleic acid was dissolved in 10 ml chloroform to prepare 50 mg/ml stock solution and stored in glass container at –20°C. The work-


doi: 10.1210/me.2014-1266

ing solution was prepared by adding 500␮l of cold oleic acid solution (50 mg/ml) into 0.33 ml of fatty acid-free BSA (0.1 mg/ml) to prepare the working solution (30 ␮g/␮l). 10 ␮l of the working solution was injected intraperineally into the mice with [1-14C] oleic acid [30 ␮l, 0.1 mCi/ml (1 Ci ⫽ 37GBq)] at 12 AM. Mice were put in metabolic chambers connected with 1N NaOH trap to capture died 14CO2. 14C radioactivity from NaOH trap was counted at 30-minute interval over the next 4 hours, and the slope for the initial 2 hours was plotted because captured radioactivity is saturated after 2 hours. Hepatic triglyceride content were measured using Triglyceride Detection Kit (Sigma-Aldrich, TRI19 –1KT) according to the manufacturers’ instructions. Autophagy flux analysis. Chloroquine experiment was described as previously (31, 32). Chloroquine (CQ; Sigma-Aldrich, St. Louis, MO, USA) dissolved in phosphate-buffered saline (PBS) was injected subcutaneously at a dose of 50 mg/Kg body weight and mice sacrificed 24 hours following the injection. Vehicle-treated mice were injected subcutaneously with equal volume of PBS. The injection dose of CQ was determined based on previous reports and our preliminary study, which had no detectable effect on liver function. After the dissection, the liver tissues were immediately homogenized in lysis buffer for western blotting analysis. Electronic microscopy (EM) analysis. Cell samples were fixed in 4% paraformaldehyde/ 2% glutaraldehyde/ 0.1M sodium cacodylate pH7.3, postfixed in 1% osmium tetraoxide and embedded in epoxy resin (Epon). Ultrathin section (80 nm) were stained with aqueous uranyl acetate and lead citrate and examined with a JEOL 2000EX transmission electron microscope (EM) (JEOL, Peabody, MA, USA). For quantification of autophagolysosome-like vacuoles, the numbers of autophagolysosomal-like vacuoles were counted in each field and normalized by the surface area. Extraction of mRNA, real-time PCR and Gene silencing. Total RNA was prepared from isolated liver tissues and cells by Trizol extraction (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Small interference RNA (siRNA) oligos targeted for mouse PGRN (Santa Cruz, catalog number sc39 262), TNFR1 (Santa Cruz, catalog number sc-36 688) and Atg7 (Santa Cruz, catalog number sc-41 448) were transfected using lipofectamine 2000 (Invitrogen, Carlsbad, California, USA). A pool of siRNAs consisting of scrambled sequences of similar length was transfected similarly as control siRNA. Western Blot Analysis. Tissues and cells under various treatments were lysed in lysis buffer containing 25 mM Tris HCl (pH 6.8), 2% SDS, 6% glycerol, 1% 2-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride, 0.2% bromphenol blue, and a protease inhibitor cocktail for 20 minutes. Western blotting was performed using a standard protocol as described previously (33). Statistical Analysis. Statistical analyses were performed using IBM SPSS 20.0 software. The data are expressed as means ⫾ S.D. Statistical analysis between the 2 groups was performed using an unpaired, two-tailed Student t test or ANOVA fol-


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lowed by post hoc tests. P value of ⬍ 0.05 was considered statistically significant.

Results Effects of PGRN on glucose metabolism and insulin sensitivity in mice To address the effects of PGRN on glucose metabolism and insulin sensitivity in vivo, mice were administered recombinant PGRN intraperitoneally daily at the dosage of 1ug/g body weight/d under standard diet condition for 3 weeks. We found that serum PGRN levels were elevated to 1.4 ␮g/ml, about 1.6-fold of control, after treatment with PGRN for 21 days (Figure 1B), while no difference was found between control and PGRN treated mice with respect to other indicators, such as food intake, blood glucose, serum insulin levels under these conditions (Figure 1A, C, and D). We also noticed that the TNF-␣ level had no significant change either in serum or in the liver tissues (Figure S1A-B). Administration of PGRN decreased glucose tolerance and insulin sensitivity as measured by glucose tolerance testing (GTT) and insulin tolerance testing (ITT), with no difference observed in body weight (Figure 1E-1F), indicating the causative role of PGRN involved in insulin resistance in vivo. Meanwhile, there is no significant difference observed either in liver weight or in serum triglyceride among the groups (Figure 2A and 2B), and mice injected with recombinant PGRN or/and TNFR1BP-Fc did not accumulate lipids in the liver (Figure 2C). Moreover, injection of PGRN and TNFR1BP-Fc was associated with improved glucose tolerance and insulin tolerance compared with mice injected with PGRN and IgG1Fc (Figure 1E-1F), indicating that TNFR1BP-Fc, at least partially, blocks the negative effect of PGRN on glucose metabolism in vivo. Systemic effects of PGRN on hepatic autophagic response and insulin signaling To test whether PGRN is required to affect hepatic insulin signaling and autophagy in vivo, we first examined the expression patterns of several autophagic indicators by real-time PCR in hepatic tissues. Of note, we found that mice treated with PGRN displayed significant decrease in mRNA level of Atg7 and dramatic increase in P62 expression compared to mice received vehicle, with such effects being reversed by treatment of TNFR1BP-Fc concomitantly (Figure 2D). Consistent with a decrease in mRNA levels of Atg7, immunohistochemical detection of hepatic tissues revealed that Atg7 expression was decreased in PGRN treated mice compared with that of mice received vehicle, supporting the biochemical alterations


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of Atg7 expression in mice hepatic tissues. The level of Atg7 expression had a significant increase in mice cotreated with PGRN and TNFR1BP-Fc (Figure 2E). Then we detected the protein expression of indicators of autophagy by western blot. As expected, remarkable inhibition of autophagy as evidenced by down-regulation of LC3II, in particular, Atg7 protein levels. Meanwhile, p62 was elevated in the liver tissue of mice treated with PGRN compared to that of mice received vehicle. As a result of PGRN and TNFR1BP-Fc cotreatment, the mice had an increased Atg7 and LC3II expression compared with the mice cotreated by PGRN and IgGFc (Figure 3A). A number of studies have previously indicated that mRNA levels of autophagy genes were correlated with decreased autophagic flux in a variety of experiment systems (34 –36). Likewise, significant changes were evident in the expression of Atg7, P62 and LC3 II mRNAs in PGRN or/ and TNFR1BP-Fc treatment subjects (Figure 2D). We next employed the lysosomal protease inhibitor chloroquine to evaluate autophagic flux. Although an increase of LC3II level was readily observable with the treatment of chloroquine, the addition of PGRN caused a significant difference in LC3II expression in hepatic tissue with chloroquine (Figure 3B). Consistently, the insulin sensitivity was also markedly decreased in hepatic tissues of mice treated

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with PGRN compared with that of control as assessed by phosphorylation of IRS-1 and AKT. While the insulin sensitivity was restored in the mice cotreated with PGRN and TNFR1BP-Fc (Figure 3C). These results support a clear tendency towards the relevance of PGRN to hepatic insulin signaling and autophagic actions. Defective hepatic autophagy in PGRN treated mice resulted in decreased glycogen and unusual lipolytic and lipogenic genes expression As the main detoxifying organ of the body, liver is a major regulator of glucose and lipid metabolism in the body and it plays a central role in the synthesis and degradation (oxidation) of fatty acids (1). To address the role of PGRN in hepatic glucose homeostasis, we examined the hepatic glycogen content and lipid metabolism of the mice. As shown in the results, treatment of PGRN reduced the levels of glycogen in hepatic tissues, while the hepatic glycogen content had a significant increase in mice cotreated with PGRN and TNFR1BP-Fc (Figure 4A). Moreover, we found there was no significant change in the expression of gluconeogenic genes such as G6pc and Pck1 among the groups (Figure 4B). We next evaluated ␤-oxidation by measuring release of 14CO2 after administration of [1-14C] oleic acid. No significant differ-

Figure 1. Effects of recombinant mouse PGRN on glucose metabolism and insulin sensitivity in vivo. Mice were injected daily with saline solution as vehicle, 1 ␮g/g body weight/d recombinant PGRN, 1 ␮g/g body weight every three days IgG1Fc and 1 ␮g/g body weight every three days TNFR1BP-Fc for 21 days. A, Food intake. B, Serum PGRN. C, Blood glucose. D, Serum insulin. E, GTT. F, ITT. Data are expressed as means ⫾ SD in each bar graph from 12 to 15 mice per group. * P ⬍ .05.


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ence was observed in ␤-oxidation rate (Figure 4C). To gain further insights into the unchanged ␤-oxidation rate, we performed mRNA expression using real-time PCR. We observed markedly down-regulated expression of the ␤-oxidation-related genes such as Ppara and Cptla in hepatic tissues of mice treated with PGRN. Meanwhile, TNFR1BP-Fc treatment partially restored the expression levels of Ppara and Cptla in mice treated with PGRN (Figure 4D). In contrast, lipogenic genes expression in the hepatic tissues was up-regulated in mice injected with PGRN. Mice coinjected with PGRN and TNFR1BP-Fc showed a significant down-regulation of lipogenic genes in hepatic tissues (Figure 4E). An increase trend was also observed in the hepatic triglyceride content in mice treated with PGRN and/or TNFR1BP-Fc (Figure 4F), though these changes did not seem to be remarkable compared with the controls (P ⫽ .064 and 0.057 respectively), implicating that multiple mechanisms may contribute to PGRN-induced effects.


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Ablation of PGRN increased autophagy and improved insulin signaling on FFA treated hepatocytes To address the effects of PGRN on autophagy and insulin sensitivity, BNL cl.2 hepatocytes and primary mouse hepatocytes were treated with 100ng/ml PGRN at different time points, respectively. As shown in Figure 5A and Figure S2A, PGRN caused a time dependent decrease in protein expression of Atg7 and LC3-II, as well as an increase in protein expression of p62. Electron microscopic examination in BNL cl.2 hepatocytes demonstrated a significant reduction in autophagosome/autolysosome formation in cells treated with PGRN compared with control, with this effect of PGRN being reversed by addition of TNFR1BP-Fc (Figure 5B). Since abnormal autophagy has been shown to be implicated in the Akt signaling pathway, we postulated that PGRN might affect insulin signaling in hepatocytes. As expected, decreased insulin sensitivity was observed by treatment of PGRN both in BNL cl.2 hepatocytes and in primary mouse hepatocytes, as assessed by phosphorylation of IRS-1 and Akt

Figure 2. Effects of PGRN and TNFR1BP-Fc on liver steatosis and hepatic autophagy. Mice were injected daily with 0.9% saline solution as vehicle, 1 ␮g/g body weight/d recombinant PGRN, 1 ␮g/g body weight every three days IgG1Fc and 1 ␮g/g body weight every three days TNFR1BP-Fc for 21 days. A, Liver weight. B, Serum triglycerides. C, Oil Red O staining of liver sections (magnification, X100). Scale bar, 20 ␮m. D, Relative expression of Atg7, p62 and LC3A/B in the liver normalized to ␤-actin (real-time PCR). E, Immunohistochemical staining (10⫻) of the Atg7 protein in tissue sections. The pictures shown below are high magnification (40⫻) of the field marked with a red rectangle above. Data expressed as means ⫾ SD in each bar graph from 12 to 15 mice per group. Scale bar, 20 ␮m. * P ⬍ .05.


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(Figure 5C and Figure S2B). These results indicated that PGRN expression is strongly associated with autophagy dysfunction and insulin resistance at cell level. To further confirm the involvement of PGRN in autophagy and insulin signaling of BNL cl.2 hepatocytes and primary mouse hepatocytes, we utilized siRNA against PGRN to determine whether ablation of PGRN could increase autophagy and improve insulin signaling on different cellular models of insulin resistance induced by palmitate (FFA) or dexamethasone. PGRN siRNA was validated by measurement of reduced PGRN protein expression in PGRN siRNA transfected BNL cl.2 hepatocytes (Figure 6A). After transfection with PGRN siRNA, we found that the autophagic imbalance was corrected as evidenced by the enhanced expression of Atg7 and LC3II and the reduced expression of p62 in the presence of FFA (Figure 6B). The insulin stimulated phosphorylation of

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both IRS-1 and Akt was increased in the presence of FFA in PGRN-knockdown BNL cl.2 hepatocytes (Figure 6C). In addition, PGRN protein expression was increased in hepatocyte interfered with Atg7 siRNA (Figure 6D). Furthermore, dexamethasone induced autophagy dysfunction and decrement of insulin-stimulated phosphorylation of IRS-1 and Akt were restored in primary mouse hepatocytes (Figure S2C and S2D) by transfection of PGRN siRNA. Similarly, insulin signaling and autophagy were also improved in dexamethasone-treated BNL cl.2 hepatocytes after PGRN ablation (Figure S4A and S4B). PGRN induced autophagy imbalance and impaired insulin signaling via the TNFR/NF-␬B dependent manner Recent finding suggested that PGRN bind to TNFR1 and mediate its anti-inflammatory effects in collagen an-

Figure 3. Effects of PGRN on hepatic autophagy and insulin sensitivity via the TNFR1-dependent manner in vivo. Mice were injected daily with 0.9% saline solution as vehicle, 1 ␮g/g body weight/d recombinant PGRN, 1 ␮g/g body weight every three days IgG1Fc and 1 ␮g/g body weight every three days TNFR1BP-Fc for 21 days. For insulin signaling, they were injected with 2 IU/kg for insulin. A, Atg7, P62 and LC3II in liver samples. B. Determination of autophagic flux in mice receiving PGRN and/or TNFR1BP-Fc in the absence or presence of chloroquine. Liver tissue lysates were analyzed by western blot using anti-LC3 antibody. Results are given as fold increase normalized to internal control GAPDH. C, IRS-1 phosphorylation and Akt phosphorylation in liver samples. Quantification was performed on 3 different fields per animals. Data are expressed as means ⫾ SD in each bar graph from 12 to 15 mice per group. IB, immunoblotting; IP, immunoprecipitation. *P ⬍ .05.


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tibody-induced arthritis and collagen-induced arthritis (37). Therefore, we next sought to explore the molecular mechanism of PGRN/TNFR1 interaction on metabolic regulation in hepatocytes. We employed different strategies (TNFR1BP-Fc or TNFR1 siRNA) to interfere with TNFR1 in cultured hepatocytes. We examined autophagic response and insulin action in the presence of 100ng/ml TNFR1BP-Fc or/and 100ng/ml PGRN in BNL cl.2 hepatocytes. We found the decreased expression of Atg7 and LC3II and elevated p62 expression by PGRN were abolished with treatment of TNFR1BP-Fc (Figure 7A). Moreover, blockage of TNFR1 by TNFR1BP-Fc could also enhance the IRS-1 and Akt phosphorylation after PGRN treatment (Figure 7C). As expected, TNFR1 siRNA treatment alone had no effect on autophagic expression, while addition of TNFR1 siRNA could restore the defective autophagy and impaired insulin sensitivity induced by PGRN (Figure 7B and 7D). Consistent with these results, there were expected results that knockdown of TNFR1 could reverse PGRN-induced autophagy suppression and impaired insulin signaling in primary mouse


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hepatocytes (Figure S3A and S3B). Taken together, these observations confirmed that PGRN induced autophagy defection and impaired insulin signaling, at least in part, through the TNFR1 dependent manner. The classical NF-␬B pathway is stimulated by a wide variety of cellular stressors and members of the Toll-like and the TNF receptor families particularly recognized by TNFR1 (38). Recent studies suggested that NF-␬B activation contributes to hepatic insulin resistance via inhibitory crosstalk within the hepatocytes, resulting in blockade of specific steps in downstream insulin signaling pathways (39, 40). To investigate whether PGRN impaired insulin signaling and induced autophagy defection via the TNFR1/NF-␬B dependent manner, we first observed an induction of I␬B␣ phosphorylation following PGRN treatment in hepatocytes, indicating an activation of the canonical NF-␬B pathway (Figure 8A).As stimulation of the multisubunit I␬B Kinase (IKK) complex triggers phosphorylation-dependent proteasomal degradation of I␬B␣, we then demonstrated that IKK␤ inhibitor VI, a selective inhibitor of the canonical pathway-specific

Figure 4. Effects of PGRN and TNFR1BP-Fc on hepatic glucose and lipid metabolism in vivo. Mice were injected daily with 0.9% saline solution as vehicle, 1 ␮g/g body weight/d recombinant PGRN, 1 ␮g/g body weight every three days IgG1Fc and 1 ␮g/g body weight every three days TNFR1BP-Fc for 21 days. A, glycogen levels. B, Relative mRNA level of gluconeogenesis-associated genes in the liver. C, In vivo ␤ oxidation of infused [1-14C] oleic acid. D, Relative mRNA levels of genes associated with ␤ – oxidation in hepatic tissues. E, Relative mRNA levels of genes associated with fatty acid and triacylglycerol (TG) synthesis in the liver. F, Hepatic triglyceride content, results are expressed per gram of liver tissue. Data are expressed as means ⫾ SD in each bar graph from 12 to 15 mice per group. * P ⬍ .05.


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IKK␤ subunit, blocks I␬B␣ phosphorylation, as does siRNA-mediated knockdown of IKK␤ protein (Figure 8B and 8C). Treatment of PGRN suppressed autophagy and impaired insulin signaling, whereas blockade of NF-␬B by the addition of SD50, a known NF-␬B inhibitor in the culture medium nullified the effect of PGRN (Figure 8D and 8E), suggesting that effects of PGRN upon autophagy and insulin signal pathway are in IKK␤-NF-␬B dependent

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mechanisms. Taken together, these results indicated that NF-␬B activation is required for PGRN to suppress autophagy and to inhibit insulin signaling and implicated NF-␬B as a factor in promoting hepatocellular insulin resistance.

Figure 5. Effects of PGRN in autophagy and insulin receptor signaling in hepatocyte. BNL cl.2 cells were treated with or without recombination PGRN (100ng/ml). For insulin signaling, cells were stimulated with 10 nM insulin for 10 minutes. Indicators of autophagy and insulin receptor signaling were measured at protein levels. The relative quantity of proteins was analyzed with Quantity One software. A, Time course for autophagy in hepatocyte following PGRN treatment. B, Representative electron micrographs (10,000⫻) of hepatocyte. Quantification of autophagolysosome-like vacuoles per field in the EM images, Scale bars, 1 ␮m. C, Phosphorylation of IRS-1 and Akt in hepatocyte. Data expressed as means ⫾ SD in each bar graph represent the average of 3 independent experiments. IB, immunoblotting; IP, immunoprecipitation. *P ⬍ .05 or **P ⬍ .01.


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Discussion Recent studies have implicated PGRN in diet-induced obesity and insulin resistance (3, 13–16). However, much remains to be elucidated regarding the mechanism of PGRN action before its putative potential as therapeutic target can be realized. In the present study, mice developed signs of insulin resistance and defective hepatic autophagy upon long-term treatment with PGRN and these effects were ameliorated when TNFR1 blockade was administered simultaneously. Consistent with these findings in vivo, PGRN also plays a pivotal role in insulin sensitivity involving autophagic mechanism in cultured hepatocytes. Collectively, these findings support the notion that PGRN functions as a potential link between chronic inflammation and hepatic insulin resistance at least partially through TNFR1 via NF-␬B signaling. Clinically, several evidences also indicate that PGRN is an important link between insulin resistance and aggressive inflammatory condition (12, 14). By these data, circulating PGRN levels correlate with BMI, macrophage infiltration into adipose tissue, chemotactic activity, suggesting that PGRN reflects the chronic inflammation in obesity, which is in line with that PGRN may be a novel


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biomarker of the chronic inflammatory response in insulin resistance and associated disturbances. PGRN is composed of 7.5 repeats of a cysteine-rich motif. Proteolytic cleavage of this precursor protein by extracellular proteases gives rise to smaller peptide fragments termed granulins (GRNs) or epithelins (5, 41, 42). In contrast to the anti-inflammatory properties of intact PGRN, GRNs has been shown to promote inflammatory activity (43, 44). It is unclear at this point whether GRNs derived by proteolysis of PGRN is implicated in the inhibition of hepatic insulin sensitivity and autophagy. Since GRNs range in size from 6 to 25 kDa, so far it is difficult to measure serum GRNs or to investigate the function of GRNs in vivo (45). However, our preliminary results showed that elastase-digested PGRN had little effects on insulin signaling and autophagy in cultured hepatocytes, whereas cells exposed to PGRN showed considerable attenuation in insulin signaling and autophagic activity (Supplemental Figure S6). Although PGRN plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been hampered by the inability to identify

Figure 6. Ablation of PGRN increased autophagy and improved insulin signaling on FFA-treat treated hepatocyte. Cells were cultured with or without 100 nM PGRN siRNA. For insulin signaling, cells were stimulated with 10 nM of insulin for 10 minutes. Indicators of autophagy and insulin receptor signaling were measured at protein levels. The relative quantity of proteins was analyzed with Quantity One software. A, PGRN siRNA was validated by reduction of PGRN protein level. B, Autophagy indicators in hepatocyte. C, Phosphorylation of IRS-1and Akt in hepatocyte. D, Effect of Atg7 siRNA on PGRN expression in hepatocyte. Data expressed as means ⫾ SD in each bar graph represent the average of 3 independent experiments. IB, immunoblotting; IP, immunoprecipitation. *P ⬍ .05.


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its binding receptor(s), and it is still hard to clearly define the early stages of PGRN-mediated signaling from the plasma membrane. Some report that PGRN action is not mediated through TNFR, while more studies suggest that PGRN is TNFR antagonist or a cofactor for TNF␣ action (46). Recently, it has been shown that PGRN binds to tumor necrosis factor (TNF)-receptors (TNFR), interfering with the interaction between TNF␣ and TNFR (37). Further evidence also demonstrated that mice deficient in PGRN are susceptible to collagen-induced arthritis, an experimental model of rheumatoid arthritis (RA), and that administration of PGRN reversed the arthritic process (37). Several other groups also independently reproduced the binding of PGRN to TNFR1 and TNFR2, and inhibitory effect of this binding on TNF-␣-induced effects (47–50). TNFR1 and TNFR2 do not share homology in the cytoplasmic domains but exhibit a low degree of similarity in the ligand-binding region located in the extracellular domains, which suggests that they are capable of inducing distinct cellular responses (26). Some studies imply PGRN elicits its action more through TNFR2 than TNFR1, because disturbed the interaction of PGRN with

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TNFR2, and in turn abolished PGRN-mediated activation of Erk1/2 and Akt signaling and protection against apoptosis in response to ER-stress (47). In parallel, our recent study indicated a complimentary effect of both TNFR1 and TNFR2 in mediating PGRN function in cultured adipocytes (51). These discrepancies might be possibly because there is a different distribution between TNFR1 and TNFR2 in different cell types, and the function of PGRN might be diverse in different tissues, which should warrant further investigations. When compared with TNF-␣, PGRN exhibited a higher affinity to both TNFR1 and TNFR2, and PGRN has approximately 600-fold higher binding affinity than TNF-␣ (44, 52–54). Similar to PGRN, Atsttrin, an engineered protein made of three PGRN fragments, inhibited the interaction between TNF and TNFR, and in turn, the downstream events of TNF/TNFR signaling. In contrast to TNF-␣, Atsttrin exhibited a higher binding affinity for TNFR2, but a lower affinity for TNFR1 (37). In addition, it was also observed that TNF family ligands bind to the extracellular regions of TNFR1 and TNFR2 in which each receptor subunit contacts two adjacent ligand sub-

Figure 7. Blockade of TNFR1 or knockdown TNFR1 on autophagy and insulin signaling in hepatocyte. Cells were cultured with TNFR1BP-Fc or 100 nM TNFR1 siRNA. For insulin signaling, cells were stimulated with 10 nM of insulin for 10 minutes. Indicators of autophagy and insulin receptor signaling were measured at protein levels. The relative quantity of proteins was analyzed with Quantity One software. A and B, Expression of Atg7, P62 and LC3II in hepatocyte. C and D, Phosphorylation of IRS-1 and Akt in hepatocytes. Data expressed as means ⫾ SD in each bar graph represent the average of 3 independent experiments. IB, immunoblotting; IP, immunoprecipitation. *P ⬍ .05.


doi: 10.1210/me.2014-1266

units typically via CRD2 and CRD3( cysteine-rich repeat domains, CRDs) (46, 55). Deletion mutants of TNFR1 and TNFR2 used to map the binding of PGRN revealed that CRD2 and CRD3 of TNFR are essential for the interaction with PGRN, similar to the binding to TNF-␣ (49, 50, 56, 57). However, as PGRN is administered systemically in this study, the observed findings cannot exclusively be attributed to PGRN itself and a systemic state of inflammation could be the reason for the observations, since PGRN can induce the release of cytokines such as IL-6 and IL-6 is a key player of hepatic insulin resistance (3). In our supplementary study, IL-6 ablation partially ameliorated autophagic defects though this recovery did not seem to be most remarkable. In another aspect, recent evidence indicates that PGRN directly binds to TNF receptors (TNFR) (37). In our study, knockdown of TNFR1 resulted in restoration of hepatic insulin signaling and autophagic balance, indicating a potential role of TNFR1 in mediating PGRN function. On the other hand, Several lines of evi-


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dences suggested that TNF-␣ up-regulates mTOR activity in a NF-␬B-dependent manner, since this activity is induced in TNF-␣-treated NF-␬B-competent cells and, inversely impaired in TNF-␣-treated cells lacking NF-␬B activity (58). NF-␬B activation might be necessary to PGRN-induced hepatic insulin resistance and autophagic imbalance, thus our findings are reminiscent of previous evidence that NF-␬B activation represses TNF-␣-induced autophagy (38). These supported the conclusion that the NF-␬B dependent inhibition of autophagy may be a general cellular response. Thus, our results provide a more complementary insight into the regulation of PGRN, which suggest that the induction of impaired insulin sensitivity by PGRN is not only secondary to IL-6, but more likely to be mediated through TNFR1 via NF-␬B signaling during hepatic insulin resistance. Though defective autophagy induced by PGRN appeared causal to hepatic insulin resistance, a definitive link could be established if the restoration of autophagy could rescue the impaired insulin sensitivity. Our study

Figure 8. PGRN-dependent NF-␬B activation in hepatocytes. Cells were cultured with or without 100ng/ml PGRN. For blocking IKK␤, cells were treated with or without inhibitor VI (10 mM) and 100 nM IKK␤ siRNA. For blocking NF-␬B, cells were treated with or without 10 nM SD50 (an NF-␬B inhibitor). For insulin signaling, cells were stimulated with 10 nM of insulin for 10 minutes. Indicators of autophagy and insulin receptor signaling were measured at protein levels. The relative quantity of proteins was analyzed with Quantity One software. A, Phosphorylation of I␬B␣ and NF-␬B in liver cells. B, Effect of inhibitor VI (10 mM) on PGRN dependent NF-␬B activation. C, Effect of IKK␤ siRNA on PGRN dependent NF-␬B activation. D, Expression of Atg7, P62 and LC3II in hepatocytes. E, Phosphorylation of IRS-1 and Akt. Data expressed as means ⫾ SD in each bar graph represents the average of 3 independent experiments. IB, immunoblotting; IP, immunoprecipitation. * P ⬍ .05.


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provided the evidences that reconstitution of Atg7 results in enhanced autophagic activity and improved insulin signaling in cultured hepatocytes (Supplemental Figure S5). Atg7, which encodes an ubiquitin-activating enzyme (E1)-like enzyme, is central for autophagosome formation responsible for both Atg12-Atg5 conjugation and LC3 conversion (59, 60). It has been proposed that reduction of insulin level in obese mice fails to recover Atg7 deficiency, but suppression of Atg7 directly results in hepatic insulin resistance in lean mice (59, 60). It is nevertheless possible that PGRN may contribute to the dampening of the initiation of autophagic machinery, which would further the organelle dysfunction, disrupt metabolic homeostasis, and promote the emergence of metabolic disorders. The causative role of PGRN was implicated in the pathogenesis of the adipose insulin resistance of obese mice (3), however the mechanisms of this defect could be diverse. Since obesity is characterized with enhanced intracellular lipid accumulation, the role of PGRN with sustained lipogenesis might be much more complicated than expected. It is plausible that chronic lipid overloading, which impairs insulin signaling and insulin-stimulated glucose uptake, might be one of the triggers in metabolic disturbance. Thus, systematic investigation of the metabolic consequences of PGRN administration should be warranted in the absence of other chronic changes that accompany the obese state. In this study, although we did not investigate lipid metabolism in our model under prolonged conditions, it is possible that severe defects induced by PGRN disrupt major autophagy components and major homeostatic pathways. In fact, suppression of autophagy also results in alterations of glycogen accumulation in the liver of lean mice (59, 60). As autophagy is involved in gluconeogenesis and lysosome plays an important role in glycogen breakdown, hence defective autophagy and insulin resistance are highly integrated in mice and impose a major effect on systemic metabolism. However, the possibility remains that some part of Atg7 action on liver metabolism may still involve yet unknown and autophagy-independent mechanisms. Future studies are warranted to explore such possibilities to fully understand the metabolic impact of this adaptive response and how insulin action is influenced by different autophagy mediators. In our study, there was still a clear tendency towards increased hepatic triglycerides (TGs) level in mice injected with PGRN and/or TNFR1BP-Fc, though these changes did not reach statistical significance. Hepatic TG storage mainly originates from lipolysis of TGs released from white adipose tissue (60%) and the rest derive from dietary fatty acids (15%) and de novo lipogenesis (25%)

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(29, 61). The fatty acid supply from adipose tissues and diet remains relatively constant in our study, thus TG pool derived from these sources only accounts for a minor part of hepatic TG accumulation. Another consideration is from recent observation that PGRN remarkably triggers ER stress in cultured human adipocytes (51). The unfolded protein response (UPR) by an accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER) regulates lipogenesis (62), and the induction of chaperone molecules guiding unfolded proteins through the ER (such as GRP78/BiP) could suppress activation of the UPR and thus reduce liver lipid content (63). It seems reasonable that the dysfunctional endoplasmic reticulum (ER) might be able to interfere with triacylglycerol synthesis, and thereby contribute to liver triglycerides contents during PGRN exposure. Despite ER stress, several other possibilities can also been taken into consideration such as different stage or duration of PGRN intervention, and the controversial effects secondary to inflammatory disorders. Thus, the underlying mechanism of hepatic TG deposition is not fully understood and it remains to be conclusively shown the definite role of ER responses induced by PGRN in parallel with the development of hepatic TG metabolic profile. In conclusion, our study revealed that administration of PGRN attenuated hepatic insulin sensitivity, and suggested a causal link between PGRN, impairment of hepatic insulin signaling and attenuation of hepatic autophagy, which may implicate that promising therapeutic approach through the modulation of PGRN secretion/ action and consequent amelioration of insulin resistance in subjects with the metabolic disorders.

Acknowledgments We appreciate the technical support and materials from the EM center of Xi’an Jiaotong University. This work was supported by the programs from the National Natural Science Foundation of China (Program no. 81 370 899, no. 81 170 741, no.81472038), National Excellent Young Scientist Program (no.81222026) and the New Century Excellent Talents in University from the Ministry of Education, China (NCET). Received August 20, 2014. Accepted February 4, 2015. Address all correspondence and requests for reprints to: *Hongzhi Sun, PhD, MD, [email protected] or Shufang Wu, PhD, MD, [email protected] Medical School of Xi’an Jiaotong University, 76 Yanta West Road, Xi’an, Shaanxi 710 061, China. Tel.:01186029082655046. Fax: 01186029082655046. # These authors contributed equally to this work. Disclosure Summary: The authors have nothing to disclose. This work was supported by.


doi: 10.1210/me.2014-1266


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Impaired macrophage autophagy induces systemic insulin resistance in obesity.

Insulin resistance and impaired adipogenesis.

Insulin Secretory Defect and Insulin Resistance in Isolated Impaired Fasting Glucose and Isolated Impaired Glucose Tolerance.

resistance.

Exercising insulin sensitivity: AMPK turns on autophagy!

resistance.

Circulating PGRN is significantly associated with systemic insulin sensitivity and autophagic activity in metabolic syndrome.

Insulin receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance.

Higher insulin sensitivity and impaired insulin secretion in cachetic solid ehrlich tumour-bearing mice.

Administration of progranulin (PGRN) triggers ER stress and impairs insulin sensitivity via PERK-eIF2α-dependent manner.

Acute glucagon induces postprandial peripheral insulin resistance.

Insulin resistance and impaired mitochondrial function in obese adolescent girls.

Autophagy in diabetes: β-cell dysfunction, insulin resistance, and complications.

Caveolin-1 phosphorylation regulates vascular endothelial insulin uptake and is impaired by insulin resistance in rats.

Peripheral insulin resistance and impaired insulin signaling contribute to abnormal glucose metabolism in preterm baboons.

High-fat diet induces hepatic insulin resistance and impairment of synaptic plasticity.

Elevated hepatic 11β-hydroxysteroid dehydrogenase type 1 induces insulin resistance in uremia.

Diabetes, impaired glucose tolerance and insulin resistance with diuretics.

Autophagy downregulation contributes to insulin resistance mediated injury in insulin receptor knockout podocytes in vitro.

Insulin resistance, impaired glucose tolerance and alpha-thalassemia carrier state.

Heart Rate Variability, Insulin Resistance, and Insulin Sensitivity in Japanese Adults: The Toon Health Study.

The diabetes gene Zfp69 modulates hepatic insulin sensitivity in mice.

Hepatic circadian-clock system altered by insulin resistance, diabetes and insulin sensitizer in mice.

Restoration of autophagy alleviates hepatic ER stress and impaired insulin signalling transduction in high fructose-fed male mice.