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

ENERGY

BALANCE-OBESITY

Circulating PGRN Is Significantly Associated With Systemic Insulin Sensitivity and Autophagic Activity in Metabolic Syndrome Huixia Li,* Bo Zhou,* Lin Xu, Jiali Liu, Weijin Zang, Shufang Wu, and Hongzhi Sun 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) is a secreted protein that has recently emerged as an important regulatory adipokine of glucose metabolism and insulin sensitivity. We report here that serum PGRN concentrations were significantly higher in patients with metabolic syndrome (MS) than in subjects without MS and correlated positively with body mass index, waist circumference, fasting insulin, fasting plasma glucose, glycated hemoglobin A1c, triglyceride, and homeostasis model assessment of insulin resistance, and were inversely related to high-density lipoprotein cholesterol and homeostasis model assessment of ␤ cell function. Subgroup analysis in 32 subjects showed that elevated expression levels of PGRN were positively correlated with increased autophagy markers LC3 and Atg7 proteins in omental adipose tissue of subjects with MS. Consistent with these findings, the enhanced PGRN levels were also observed in multiple insulin-resistant cellular models, whereas PGRN-deficient adipocytes were more susceptible to insulin action and refractory to tunicamycininduced autophagic disorders. PGRN remarkably attenuated insulin sensitivity, increased autophagic activity, and triggered endoplasmic reticulum (ER) stress in cultured human adipocytes, whereas these effects were nullified by reduction of ER stress with phenylbutyric acid chemical chaperone treatment. In addition, PGRN-induced ER stress and impaired insulin sensitivity were improved in TNFR1⫺/⫺ cells, indicating a causative role of TNF receptor in the action of PGRN. Collectively, our findings suggest that circulating PGRN is significantly associated with systemic insulin sensitivity and autophagic activity in adipose tissue and support the notion that PGRN functions as a potential link between chronic inflammation and insulin resistance. (Endocrinology 155: 3493–3507, 2014)

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rogranulin (PGRN), also known as proepithelin, granulin/epithelin precursor, or PC cell– derived growth factor, has recently emerged as an important regulatory adipokine of glucose metabolism and insulin sensitivity (1– 4). PGRN has been shown to be a pluripotent growth factor that mediates cell growth, wound healing, tumorigenesis, and neu-

rodegenerative disease such as frontotemporal dementia (5– 8). However, recent studies also supported the novel function of PGRN as an adipokine in regulating energy metabolism. For instance, diet-induced obesity mice with PGRN deficiency exhibited lower body weight and ameliorated insulin sensitivity, whereas administration of recombinant PGRN

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received January 22, 2014. Accepted June 21, 2014. First Published Online June 27, 2014

* H.L. and B.Z. contributed equally to the study.

doi: 10.1210/en.2014-1058

Abbreviations: AT, adipose tissue; BMI, body mass index; BP, blood pressure; FBS, fetal bovine serum; FINs, fasting insulin; FPG, fasting plasma glucose; GAPDH, glyceraldehyde3-phosphate dehydrogenase; HbA1c, glycated hemoglobin A1c; HDL-C, high-density lipoprotein-cholesterol; HOMA-␤, homeostasis model assessment for ␤-cell function; HOMAIR, homeostasis model assessment of insulin resistance; IR, insulin resistance; IRS-1, insulin receptor substrate 1; LDL-C, low-density lipoprotein cholesterol; MS, metabolic syndrome; mTOR, mammalian target of rapamycin; NF-␬B, nuclear factor-␬B; PBA, phenylbutyric acid; PDTC, pyrrolidine dithiocarbamate; PGRN, progranulin; PPAR␥, peroxisome proliferatoractivated receptor-␥; siRNA, small interfering RNA; T2DM, type 2 diabetes; TNFR, TNF receptor; WC, waist circumference.

Endocrinology, September 2014, 155(9):3493–3507

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Effects of PGRN on IR and Autophagy

induced glucose intolerance and insulin resistance (IR) in wild-type mice (3). Consistently, PGRN mediates TNF-␣induced IR through IL-6 expression in 3T3-L1 adipocytes (3). Several clinical investigations also demonstrated that serum PGRN was associated with the parameters of adiposity, glucose tolerance, IR, and inflammatory factors (9 –11). Clinically, circulating PGRN is significantly higher in subjects with type 2 diabetes (T2DM) and positively correlated with high-sensitivity C-reactive protein, IL-6, and macrophage infiltration in omental adipose tissue (AT) (12). In particular, PGRN is more highly expressed in visceral AT of insulin-resistant patients with morbid obesity than in their age-, sex- and body mass index (BMI)-matched insulin-sensitive counterparts (13–15). Collectively, these pieces of evidence support a new regulatory role for PGRN in glucose and energy homeostasis, which raises the possibility that PGRN may contribute to the progression of metabolic syndrome (MS) based on the critical role of insulin resistance (IR) and adiposity in the pathogenesis of metabolic disorders. Although the role of PGRN in energy homeostasis has just recently been identified, the functional basis for PGRN-mediated IR or inflammation remains elusive. Of note, the relationship between the autophagic process and PGRN has recently drawn considerable attention because of the close interaction of autophagy and a variety of adipocytokines, including adiponectin, IL-6, and TNF-␣ (16 –18). Recombinant PGRN has also been shown to impair insulin signaling in 3T3L1 adipocytes through IL-6, an inducer of autophagy (3). Consistently, PGRN⫺/⫺ mice reveal abnormal accumulation of lipofuscin granules and p62 proteins in brain regions, suggesting a disturbance of the autophagy-lysosomal system with alterations in lysosomal homeostasis (19, 20). Moreover, blockage of the mammalian target of rapamycin (mTOR) kinase with rapamycin effectively blunts PGRN-induced myotube hypertrophy and inhibition of autophagy with chloroquine generates elevated endogenous PGRN levels, indicating that the relationship between autophagic activity and PGRN is not straightforward (21, 22). In addition to the important function of autophagy in clearance of altered and damaged intracellular proteins and organelles, autophagy has been shown to regulate fat accumulation within a mass of AT, based on the fact that mice with targeted disruption of autophagy-related gene Atg5 or Atg7 are characterized by a robust reduction in white AT mass compared with that of wild-type animals (23–25). Moreover, a growing body of evidence suggests a relationship between autophagic disturbance and IR in AT (26 –28). It was shown that inhibition, rather than the stimulation, of autophagy in adipocytes gives them a brown fat cell–like appearance that favors fatty acid oxidation and increases insulin sensitivity, whereas the sup-


pression of hepatic Atg7 using short hairpin RNA interference in lean control mice resulted in IR and ER stress (29). Additional studies using AT explants of human origin demonstrated that the inhibition of autophagy led to the up-regulated production of IL-1␤, IL-6, and IL-8 gene expression (18). Thus, the pathophysiological functions of autophagy remain to be further determined, and it is not clear whether autophagic activity contributes to PGRNinduced IR. In the present study, we aimed to clarify the clinical significance of PGRN and autophagic imbalance in subjects with or without MS. We also sought to evaluate the correlations of PGRN, ER stress, and autophagic response in AT of MS and to characterize the potential interaction of ER stress and autophagic dysfunction involved in PGRN-impaired insulin sensitivity in adipocytes. Our results support the clinical significance of PGRN in the context of MS, indicating that PGRN functions as a causal link between ER stress, autophagy, and impaired insulin signaling in the pathophysiology of IR and AT dysfunction.

Materials and Methods Subjects Informed consent was obtained from all study participants in advance, and all procedures were performed in accordance with the guidelines in the Declaration of Helsinki and were approved by the ethics committee of Xi’an Jiao Tong University. MS was defined according to the definition of the Chinese Joint Committee for Developing Chinese Guidelines on Prevention and Treatment of Dyslipidaemia in Adults (30) as having ⱖ3 of the following metabolic risk factors: (1) central obesity (waist circumference [WC] of ⬎90 cm for men), (2) fasting triglycerides (TGs) of ⱖ1.70 mmol/L, (3) fasting high-density lipoprotein cholesterol (HDL-C) of ⬍1.04 mmol/L, (4) hypertension (sitting blood pressure [BP] of ⱖ130/85 mmHg or taking regular antihypertensive medications), or (5) hyperglycemia defined as fasting plasma glucose (FPG) of ⱖ6.1 mmol/L and/or 2-hour postchallenge glycemia of ⱖ7.8 mmol/L or receiving hypoglycemic therapy for treatment of diabetes. Cohort 1 consisted of 158 individuals with MS (n ⫽ 67) and without MS (n ⫽ 91) from whom blood samples were obtained to investigate the PGRN concentration. Individuals fulfilled the following inclusion criteria: (1) absence of any acute or chronic inflammatory disease as determined by a leukocyte count of ⬎7000 giga-particles/L, C-reactive protein of ⬎5.0 mg/dL, or clinical signs of infection; (2) undetectable antibodies against glutamic acid decarboxylase; (3) no thyroid dysfunction; (4) no alcohol or drug abuse; and (5) no pregnancy. Cohort 2 consisted of 15 subjects without MS and 17 with MS from whom omental AT was obtained during open abdominal surgery for cholecystectomy, abdominal injuries, or explorative laparotomy. In addition to the above-mentioned inclusion criteria, subjects with MS in cohort 2 were also characterized by homeostasis model assessment of insulin resistance (HOMA-IR) value of ⬎7.


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Anthropometric and biochemical measurements

Mitochondria histomorphometry

All baseline blood samples were collected between 8:00 and 10:00 AM after an overnight fast. Peripheral serum samples were subjected to ELISA using standard kits (R&D Systems, Inc) for PGRN. A complete physical examination including measurements of height, weight, WC, and BP was performed on each subject. Plasma glucose was determined using the glucose oxidase method. Serum insulin was assayed via radioimmunoassay (Linco Research). Glycated hemoglobin A1c (HbA1c) values were measured by HPLC (Bio-Rad Laboratories). Total cholesterol (TC), TGs, HDL-C, and low-density lipoprotein cholesterol (LDL-C) were determined by enzymatic procedures on an autoanalyzer (Hitachi 7600 – 020; Hitachi). HOMA-IR and homeostasis model assessment for ␤ cell function (HOMA-␤) were used to estimate insulin sensitivity and insulin secretion.

Human adipocytes were fixed in 4% paraformaldehyde-2% glutaraldehyde-0.1 M sodium cacodylate (pH 7.3), postfixed in 1% osmium tetroxide, and embedded in epoxy resin (Epon). Ultrathin sections (80 nm) were stained with aqueous uranyl acetate and lead citrate and examined with a 2000FX transmission electron microscope (JEOL). Sixteen electron micrographs per group of cells were digitized, and the area and number of clearly distinguishable mitochondria were analyzed using OsteoMeasure software (OsteoMetrics).

mRNA isolation and analysis by real-time PCR Quantitative real-time PCR analysis was performed as described previously (31). Primers were as follows (name, sense and antisense primer): Atg7, 5⬘-CACTGTGAGTCGTCCAGGAC-3⬘ and 5⬘-CGCTCATGTCCCAGATCTCA-3⬘; LC3B, 5⬘-AAAGCTGTGGATGATCCACG-3⬘ and 5⬘-AGCAGGTGACAGGAACTCCT-3⬘; LC3A, 5⬘-CCAGCAAAATCCCGGTGAT-3⬘ and 5⬘-TGGTCCGGGACCAAAAACT-3⬘; PGRN, 5⬘-GAAGGCTCGATCCTGCGAGA-3⬘ and 5⬘-CTCAAGGCTGGGTCCCTCAA-3⬘; ATF6, 5⬘CTCCGAGATCAGCAGAGGAA-3⬘ and 5⬘-AATGACTCAGGGATGGTGCT-3⬘; XBP-1s, 5⬘-GCTGAGTCCGCAGCAGGTGCAG-3⬘ and 5⬘-CGACTCAGGCGTCGTCCACGTC-3⬘; IL-6, 5⬘GGTACATCCTCGACGGCATCT-3⬘ and 5⬘-GTGCCTCTTTGCTGCTTTCAC-3⬘; IL-1␤, 5⬘-GCCCTAAACAGATGAAGTGCTC-3⬘ and 5⬘-GAACCAGCATCTTCCTCAG-3⬘; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5⬘-CCACCCATGGCAAATTCCATGGCA-3⬘and5⬘-TCTAGACGGCAGGTCAGGTCCACC-3⬘. The gene expression levels were normalized by the average levels of the mean-centered housekeeping gene GAPDH.

Cell differentiation and gene silencing Human preadipocytes-visceral cells (ATCC) were induced to differentiate mature fat cells with the induction medium (0.5 mM isobutylmethylxanthine, 1 ␮M dexamethasone, 10 ␮g/mL insulin, and DMEM with 10% fetal bovine serum [FBS]) and insulin medium (10 ␮g/ml insulin and DMEM with 10% FBS) in turn every 2 days. Four days later, the medium was changed to 10% FBS/DMEM. Cells were then fed with 10% FBS/DMEM every 2 days. Full differentiation is usually achieved on the 12th day. Gene silencing was performed according to the manufacturer’s instructions. Cells were transfected with a silencing RNA (small interfering RNA [siRNA]; Santa Cruz Technology) targeted for human PGRN (sc-39261), NF-␬B-p65 (sc-29410), and XBP-1 (sc-38627) using Lipofectamine 2000 (Invitrogen). siRNA consisting of a scrambled sequence of similar length was used as a control.

Glucose uptake After transfer of cells to medium without glucose, human adipocytes were incubated with 10 nmol/L insulin for 10 minutes, when glucose transport was determined as uptake of 50 ␮mol/L (10 ␮Ci/mL) 2-deoxy-D-[1-3H] glucose and then incubated for 30 min. Uptake was linear for at least 30 min.

Western blot analysis and immunoprecipitation Western blotting and immunoprecipitation were performed as described for the previous study (31). A standard sample was run in each blot to compare density values between blots, and the data were quantified by expressing each band density value as fold of this standard sample.

Statistical analyses Data were analyzed by SPSS Statistics 19.0. Characteristics of subjects between the 2 groups were compared by an unpaired Student t test for normally distributed variables and a MannWhitney U test for skewed variables. Multiple comparisons of quantitative variables among groups were made using one-way ANOVA with the least significant difference post hoc test. Interrelationships between variables were analyzed by Spearman correlation analysis. A value of P ⬍ .05 was considered to be significant.

Results Characteristics of the study participants and association of serum PGRN levels with metabolic parameters The demographic and clinical characteristics of the subjects are summarized in Table 1. Compared with individuals without MS of each sex, subjects with MS had remarkably higher BMI, IL-1␤, IL-6, fasting insulin (FINs), HbA1c, and HOMA-IR besides the MS-associated parameters (WC, BP, TG, low HDL-C, and FPG; P ⬍ .001). The PGRN serum concentration was dramatically higher in the MS group than in the non-MS group (203.1 mg/mL [122–305.5 ng/mL] vs 173.7 ng/mL [90 –282 ng/ mL], P ⬍ .001) (Figure 1A), whereas no significant difference in PGRN concentrations could be demonstrated between men and women without and with MS (data not shown). When subjects were further divided into different groups on the basis of the components of metabolic disorders, circulating PGRN increased remarkably with the increment of the components of metabolic disorders, with the highest levels seen in subjects with 5 components (P ⬍ .001) (Figure 1B). Significantly higher serum PGRN concentrations were also found in subjects with 3, 4, and 5 components than in those in the non-MS group (P ⬍ .05)


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Table 1.



Demographic and Clinical Characteristics of the Cohort 1 Subjects Non-MS Group

No. of subjects Age, y BMI, kg/m2 Systolic BP, mmHg Diastolic BP, mmHg WC, cm FINs, mU/L FPG, mmol/L HbA1c, % (mmol/mol) TC, mmol/L TG, mmol/L HDL-C, mmol/L LDL-C, mmol/L IL-6, pg/mL IL-1␤, pg/mL HOMA-IR HOMA-␤

MS Group

Men

Women

Men

Women

35 53.5 ⫾ 8.74 22.8 ⫾ 1.9 122.2 ⫾ 7.98 82.2 ⫾ 4.97 85.7 ⫾ 5.34 10.13 ⫾ 2.15 5.12 ⫾ 0.7 5.75 ⫾ 0.79 (39.4 ⫾ 8.83) 4.83 ⫾ 1.1 1.23 ⫾ 0.34 1.4 ⫾ 0.2 2.51 ⫾ 0.42 1.13 ⫾ 0.76 0.46 ⫾ 0.41 2.32 ⫾ 0.67 155.3 ⫾ 95.28

32 52.2 ⫾ 9.28 23.1 ⫾ 2.13 121.3 ⫾ 9.45 81.8 ⫾ 8.43 82.4 ⫾ 6.21 10.79 ⫾ 2.28 5.02 ⫾ 0.42 5.68 ⫾ 0.78 (38.91 ⫾ 8.62) 4.87 ⫾ 0.95 1.27 ⫾ 0.32 1.43 ⫾ 0.16 2.49 ⫾ 0.48 1.19 ⫾ 0.80 0.53 ⫾ 0.45 2.43 ⫾ 0.63 149.7 ⫾ 40.99

48 53.6 ⫾ 10.2 26.7 ⫾ 2.39a 136.1 ⫾ 9.21a 87.5 ⫾ 9.25b 94.8 ⫾ 5.79a,c 16.59 ⫾ 2.74a 6.14 ⫾ 0.7a 6.52 ⫾ 0.53a (47.77 ⫾ 5.88)a 4.91 ⫾ 0.52 1.72 ⫾ 0.36a 1.17 ⫾ 0.2b 2.55 ⫾ 0.43 2.91 ⫾ 0.95a 0.75 ⫾ 0.53b 4.56 ⫾ 1.08 a 141.6 ⫾ 84.74

43 51.6 ⫾ 12.15 26.7 ⫾ 2.67a 132.5 ⫾ 10.28a 84.7 ⫾ 9.78b 92.1 ⫾ 5.98a,c 16.92 ⫾ 2.53a 6.16 ⫾ 0.76a 6.69 ⫾ 0.65a (49.51 ⫾ 7.03)a 5.02 ⫾ 0.39 1.79 ⫾ 0.45a 1.15 ⫾ 0.23b 2.53 ⫾ 0.49 3.05 ⫾ 1.06a 0.76 ⫾ 0.42b 4.69 ⫾ 1.11a 137.4 ⫾ 42.67

Data are means ⫾ SD. a P ⬍ .001 for men and women in the MS vs non-MS group. b P ⬍ .05. c P ⬍ .05 for difference between men and women in the MS vs non-MS group.

(Figure 1B). We next examined the relationship between PGRN levels and IR without the confounding effects of BMI. Paired subjects who were matched for BMI (⫾3 kg/ m2), age (⫾10 years), and sex but who were discordant for the diagnosis of MS were identified. As shown in Supplemental Table 1, these subjects were well matched for age and BMI, and there were significant differences in serum PGRN and other metabolic parameters. Bivariate correlation analyses revealed prominent and positive correlations between serum PGRN and BMI, WC, and TG (r ⫽ 0.275, 0.319, and 0.256, respectively, P ⬍ .05) (Table 2). The PGRN concentrations also correlated positively and significantly with IL-1␤, IL-6, FPG, FINs, HbA1c, and HOMA-IR (r ⫽ 0.174, 0.298, 0.405, 0.254, 0.341, and 0.34, respectively, P ⬍ .01) (Table 2), but negatively with HOMA-␤ and HDL-C (r ⫽ ⫺0.305 and ⫺0.184, respectively, P ⬍ .05). Furthermore, even after further adjustment for BMI, correlations remained distinct between PGRN and WC, FPG, HbA1c, HOMA-IR, and HOMA-␤ (Table 2). Circulating PGRN levels correlate with ER stress and autophagic activity in omental AT of subjects with MS To gain further insight into the resultant elevation of serum PGRN, we first assessed the expression levels of the PGRN gene in omental AT samples obtained during elective abdominal surgery. The demographic and clinical characteristics of the cohort 2 subjects are summarized in Supple-

mental Table 2. As expected, PGRN mRNA and protein expression was profoundly higher in AT of MS subjects compared with those without MS (Figure 1, C and D). In this cohort, subjects with MS also have remarkably higher serum PGRN levels than those without MS (Figure 1E). In addition, correlation analyses revealed highly remarkable correlations between mRNA levels of PGRN and WC, BMI, FPG, HbA1c, and HDL-C, suggesting that PGRN gene expression in AT was strongly associated with MS-related phenotypes (Supplemental Table 3). In particular, the marked positive relationship between omental PGRN mRNA expression and circulating PGRN indicates a potential contribution of AT alterations to the elevated serum PGRN. We next sought to detect whether the autophagic response in AT was related to circulating PGRN. Consistent with the previous studies, the mRNA levels of Atg7, LC3A, and LC3B were significantly higher in AT of the subjects with MS (Figure 1F), which in consequence were paralleled by higher protein levels of Atg7 and the lipidated/cleaved form of LC3 (LC3-II), compared with those in subjects without MS (Figure 1G). These observations demonstrated an elevation of autophagic activity and a correlative involvement for abnormal autophagy in the processes of MS. In this cohort, we also found a strong positive correlation between circulating PGRN and mRNA levels of Atg7, LC3A, and LC3B, respectively (Figure 1H). Meanwhile, we also examined the ER stress response in human ATs. Real-time PCR analysis revealed that the


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Figure 1. Serum PGRN concentrations and autophagic activity of omental AT in subjects with and without MS. A, Serum PGRN levels in cohort 1 subjects with (n ⫽ 91) and without (n ⫽ 67) MS. B, Serum PGRN levels stratified by the number of the components of MS (n ⫽ 36, 33, and 22, respectively, in 3-, 4-, and 5-component groups). mRNA and protein expressions of PGRN or autophagy-related genes were measured in omental AT samples from the subjects of cohort 2 without (n ⫽ 15) and with MS (n ⫽ 17). C, Relative expression of PGRN mRNA examined by quantitative real-time PCR analysis in all groups normalized to the average levels of the mean-centered housekeeping gene GAPDH; the non-MS samples were set as 1. D, Representative Western blots of PGRN in subjects without and with MS. E, Serum PGRN levels in cohort 2 subjects. F, Relative expression of autophagy-related genes Atg7, LC3A, or LC3B normalized to GAPDH. G, Representative Western blots of Atg7, LC3-I/II, and p62 in AT. H, Univariate correlations between circulating PGRN and mRNA levels of autophagy genes in AT. I, ER stress markers XBP-1s and ATF6 were examined by quantitative real-time PCR analysis in omental AT samples obtained from the cohort 2 subjects. Results are presented as gene expression levels in all groups normalized to the average levels of the mean-centered housekeeping gene GAPDH. J, mRNA levels of IL-1␤ and IL-6. Data are means ⫾ SEM in each bar graph. *, P ⬍ .05 vs the non-MS group; **, P ⬍ .001 vs the non-MS group. #, P ⬍ .05 vs. the 3-component group.

mRNA levels of ER stress markers (XBP-1s and ATF6) were significantly elevated in AT of subjects with MS (Figure 1I), indicating the presence of ER stress. Considering the previous evidence on the relationship between IL-6, ER stress, and PGRN (3, 32), we also measured the expression of 2 key inflammatory genes encoding IL-1␤ and IL-6. Consistently, IL-1␤ and IL-6 mRNA were also significantly up-regulated in the AT of subjects with MS (Figure 1J). Expression of endogenous PGRN in multiple cellular models of insulin-resistant adipocytes Given the elevated expression of PGRN in AT and the remarkable correlations of PGRN with metabolic parameters or autophagy-related genes in subjects, we hypoth-

esized that PGRN might be involved in IR and autophagy in insulin-resistant cellular models. As expected, ER stress induced by tunicamycin significantly triggered autophagic dysfunction and impaired insulin signaling, as confirmed by the increased protein levels of Atg7 and the conversion of LC3-I into LC3-II, the reduced p62 levels, and the suppressed insulin-stimulated tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) as well as Akt phosphorylation (Figure 2, A and B). Both PGRN protein and mRNA expressions increased with tunicamycin treatment (Figure 2, A–C). The secretion of PGRN in the medium was also measured in cultured adipocytes not treated or treated with tunicamycin (5 ␮g/mL). After 24 hours of treatment with tunicamycin, PGRN was detected at a


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Table 2. Correlation of Serum PGRN Levels With Anthropometric Parameters and Biochemical Indexes of the Cohort 1 Subjects PGRN (Adjusted for BMI)

PGRN

BMI WC FINs FPG HbA1c TC TG HDL-C LDL-C IL-1␤ IL-6 HOMA-IR HOMA-␤

r

P

r

P

0.275 0.319 0.254 0.405 0.341 0.081 0.256 ⫺0.184 0.106 0.174 0.298 0.34 ⫺0.305

⬍.001 ⬍.001 .001 ⬍.001 ⬍.001 .311 .001 .021 .186 .028 ⬍.001 ⬍.001 ⬍.001

0.196 0.087 0.284 0.199 0.086 0.081 ⫺0.004 0.022 0.137 0.178 0.165 ⫺0.17

.014 .28 ⬍.001 .012 .285 .311 .965 .784 .086 .026 .039 .034

trace level in the culture medium (Figure 2D). To further confirm this involvement of PGRN in different cellular models of IR, we analyzed PGRN expression and secretion as well as autophagic activity by treatment with dexamethasone or TNF-␣, respectively. As shown in Figure 2E, dexamethasone or TNF-␣ induced a marked reduction in insulin-stimulated phosphorylation of Akt with an imbalance of autophagy as evidenced by an increase in Atg7, the elevated conversion of LC-I to LC-II, and a reduction in p62. Meanwhile, dexamethasone or TNF-␣ also resulted in a considerable increase in both PGRN expression and PGRN secretion (Figure 2, E and F). PGRN exposure results in impaired insulin signaling and abnormal autophagy in adipocytes We next determined whether PGRN has a direct effect on insulin signaling and autophagy in adipocytes. Mature adipocytes were pretreated with 100 ng/mL PGRN at different time points and then stimulated with insulin for 10 minutes. As shown in Figure 3A, insulin-stimulated phosphorylation of both IRS-1 and Akt was diminished slightly in adipocytes upon exposure to PGRN for 8 hours, and a further reduction was observed after treatment with PGRN for 16 hours, indicating that insulin receptor signaling was time dependently inhibited. Consistent with these results, a time-dependent reduction in insulin-stimulated glucose uptake was also observed in cells pretreated with PGRN (Figure 3B). Concurrently, an increase in Atg7 and LC3-II and a decrease in p62 were also evident in adipocytes exposed to PGRN for 8 hours, and occurred in a time-dependent manner (Figure 3C). Simultaneously, electron microscopic examination demonstrated a significant elevation in au-

tophagosome/autolysosome formation in adipocytes treated with PGRN at the indicated time points (Figure 3D). Considering the possibility that the increased levels of autophagy markers such as LC3-II may either signify increased autophagosome production or, conversely, impaired autophagosome-lysosome fusion that in turn stabilizes the autophagy-related proteins, we pretreated adipocytes in serum-free medium with chloroquine, a lysosomotropic alkalinizing agent. As expected, increased p62 and LC3-II levels were readily observable, and adipocytes predisposed with PGRN and chloroquine exhibited more remarkable increases in LC3-II and p62 than the controls (Figure 3E), suggesting that autophagy flux was increased. In parallel, excessive autophagy was also supported by mitochondrial loss in PGRN-treated cells, as evident by the reduced amount of mitochondria and smaller mitochondria volume fraction (Supplemental Figure 1). Reduction of ER stress via a chemical chaperone abrogates the effect of PGRN on insulin signaling and autophagy in adipocytes To investigate the potential involvement of ER stress in PGRN action, we determined the expression patterns of several molecular indicators of ER stress and confirmed that the phosphorylation status of PERK and eIF2␣ was significantly increased in PGRN-treated cells at 2 hours compared with that in the controls, also in a time-dependent manner (Figure 4A). In contrast, we observed no significant changes in insulin-promoted phosphorylation of IRS-1 and Akt and the expression of Atg7 and LC3-II in adipocytes exposed to PGRN until 8 hours (Figure 3). We then determined that phenylbutyric acid (PBA), a small chemical chaperone to reduce ER stress, reversed PGRNinduced phosphorylation of PERK and eIF2␣ (Figure 4B). PBA also significantly improved IRS-1 and Akt phosphorylation and inhibited PGRN-induced elevation of the autophagy indicators such as Atg7 and LC3-II upon exposure to PGRN, indicating that the ameliorated ER stress via PBA could abrogate the effect of PGRN on insulin signaling and autophagy in vitro. It has previously been reported that XBP-1⫺/⫺ mouse embryonic fibroblasts are hypersensitive to ER stress because of decreased ER folding capacity (33). We therefore reasoned that modulation of XBP-1 levels in cells should alter PGRN action via its potential impact on the magnitude of the ER stress responses. To test this possibility, XBP-1 siRNA was transfected in cells, and its validation was measured by the reduction of XBP-1 protein expression by Western blot. As shown in Figure 4C, treatment of wild-type cells with low-dose PGRN caused no alterna-


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Figure 2. Expression of endogenous PGRN in various cellular models of insulin-resistant adipocytes. A and B, Tunicamycin (Tun, 5 ␮g/mL) for 3 hours, was used to induce ER stress and insulin resistance. For insulin signaling, cells were stimulated with 10 nM insulin (Ins) for 10 minutes. All indicators were measured at the protein level. The relative quantity of proteins was analyzed using Quantity One software. A, ER stress markers including eIF2␣ phosphorylation (p-eIF2␣) and PERK phosphorylation (p-PERK), autophagy-related proteins Atg7, p62, and LC3, and PGRN in human adipocytes. B, IRS-1 tyrosine phosphorylation (pY), Akt serine 473 phosphorylation (p-Akt), and their total protein levels were examined in human adipocytes either with immunoprecipitation (IP) followed by immunoblotting (IB) or by direct immunoblotting. C, mRNA levels of PGRN in response to tunicamycin treatment. D, Secretion of PGRN by adipocytes treated with 5 ␮g/mL tunicamycin for 24 hours. E, Effects of TNF-␣ or dexamethasone (Dex) on PGRN and autophagy-related gene expression in human adipocytes. F, Secretion of PGRN by adipocytes treated with TNF-␣ or dexamethasone. Quantitative data are presented as means ⫾ SEM from at least 3 independent experiments. *P ⬍ .05 vs. the control (Ctrl).

tion in ER stress, insulin action, or autophagy, but the effects were more pronounced in XBP-1⫺/⫺cells. Ablation of PGRN partially reverses tunicamycinor TNF-␣-induced insulin insensitivity and autophagic disorders in adipocytes To determine the effect of endogenous PGRN, we used siRNA against PGRN in cultured human adipocytes, and the efficiency of PGRN siRNA was validated by the reduction in PGRN protein expression in PGRN⫺/⫺ adipocytes (Figure 5A). Tunicamycin significantly induced impaired insulin sensitivity as evident by a decrease in phosphorylation of IRS-1 and Akt with the stimulation of insulin, whereas knockdown of PGRN in adipocytes resulted in a mild increase of the phosphorylations of IRS-1 and Akt by insulin treatment (P ⬍ .05) and partially reversed the impaired insulin signaling induced by tunica-

mycin (Figure 5B). In addition, we also found that the autophagic activity decreased about 40% at the basal state in PGRN⫺/⫺ adipocytes, as evidenced by the elevation of p62 and down-regulation of Atg7 levels. Concomitantly, PGRN siRNA also partially recovered the tunicamycininduced autophagic imbalance (Figure 5C). Because previous studies have shown that PGRN activates ERK and ERK activity has also been associated with autophagy and ER stress-induced IR in vitro (34, 35), we hypothesized that ERK might be the potential mediator in the reversal mechanism of PGRN knockdown. Based on the preceding results, we found that ERK phosphorylation is significantly down-regulated in PGRN⫺/⫺ cells treated with tunicamycin as depicted in Supplemental Figure 2A. Accordingly, blockade of ERK by U0126 remarkably nullified the inhibitory effects of PGRN in adipocytes, as observed


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Figure 3. Inhibition of insulin signaling and up-regulation of autophagic activity by PGRN in mature adipocytes. Mature adipocytes were treated with 100 ng/mL recombinant human (h) PGRN at the indicated times. For insulin signaling, cells were subsequently stimulated with 10 nM insulin for 10 minutes. All indicators were measured at protein levels. The relative quantity of proteins was analyzed using Quantity One software. A, IRS1 tyrosine phosphorylation (pY)and Akt serine 473 phosphorylation (p-Akt) at the indicated times after PGRN treatment in adipocytes. The ratio of phosphorylation to total protein levels is presented in the graph. IB, immunoblotting; IP, immunoprecipitation. B, Effects of PGRN on glucose uptake. C, Time-dependent effects of PGRN on indicators of autophagy, Atg7, p62, and LC3-I/II, in mature adipocytes. D, Representative electron micrographs of adipocytes treated with PGRN for 0, 4, 8, and 16 hours. E, Protein expressions of p62 and LC3-I/II in human adipocytes in the presence or absence of PGRN with or without chloroquine. Results are given as fold increase in chloroquine exposure normalized to the absence of inhibitors. A representative blot is shown, and all the data expressed as means ⫾ SEM in each bar graph represent the average of at least 3 independent experiments. *, P ⬍ .05 vs the control (Ctrl); #, P ⬍ .01 vs the control; †, P ⬍ .05 vs chloroquine.

by the improvement of IRS-1 tyrosine phosphorylation and the restoration of Atg7 and LC3-II (Supplemental Figure 2B), reflecting a regulatory role of ERK in PGRN-mediated effects. In addition, we also found that knockdown of PGRN resulted in an increase in mTOR phosphorylation along with improved insulin signaling and restored autophagy in the presence of tunicamycin (Supplemental Figure 2A). Meanwhile, blockade of ERK activity by U0126 could induce a considerable elevation in mTOR phosphorylation (Supplemental Figure 2B), indicating that mTOR might serve as a potential link for PGRN action and ERK signaling. These results are in line with previous evidence that inhibition of mTOR by rapamycin impeded insulin action or triggered an autophagic disorder in adipocytes (36 –39). It is therefore reasonable to speculate that mTOR activation followed by PGRN depletion might at least partially neutralize the inhibitory effects of tunicamycin-induced ER stress and support an involvement of mTOR in the PGRN-mediated effects.

To further confirm these findings, we also used another cellular model of IR induced by TNF-␣. Similarly, TNF-␣ resulted in defective insulin sensitivity and autophagic imbalance, whereas PGRN knockdown could restore TNF-␣– induced impaired insulin signaling upon insulin stimulation as exhibited by a recovery in the phosphorylation of Akt (Figure 5D). Meanwhile, the autophagic imbalance was also partially recovered in TNF-␣–treated PGRN⫺/⫺ adipocytes, as evident by the reduction of Atg7 and the increase in p62 levels (Figure 5D). Collectively, the effects of PGRN knockdown might be at least partially mediated by ERK via mTOR pathways and, in turn, contribute to the improvement in insulin signaling and restoration of autophagic balance. Effect of PGRN on ER stress, IR, and autophagy is mediated through TNF receptor (TNFR)/nuclear factor ␬B (NF-␬B) pathway in adipocytes Recent findings suggested that PGRN bound to the TNFR in chondrocytes and that the administration of PGRN could


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Figure 4. Identification of the involvement of ER stress in PGRN-induced impaired insulin signaling and autophagy imbalance. A, Time-dependent effects of PGRN on ER stress markers and phosphorylation of PERK (p-PERK) and eIF2␣ (p-eIF2␣). B, Adipocytes were treated with 20 mM PBA in the presence of 100 ng/mL PGRN and then stimulated with insulin (10 nM) for 10 minutes. Phosphorylation of PERK, IRS-1 (pY), and Akt (p-Akt) and expression of Atg7 and LC3 were measured in adipocytes. C, Phosphorylation of PERK, IRS-1, and Akt and expression of Atg7 and LC3 with or without treatment with low-dose PGRN (20 ng/mL) for 16 hours in XBP-1⫺/⫺ adipocytes. The graph next to the blots shows the quantification of the protein levels under experimental conditions. A representative blot is shown, and all the data expressed as means ⫾ SEM in each bar graph represent the average of at least 3 independent experiments. *, P ⬍ .05 and **, P ⬍ .01 vs the control (Ctrl); #, P ⬍ .05 vs PGRN. IB, immunoblotting; IP, immunoprecipitation; siXBP-1, XBP-1 siRNA.

prevent TNF-␣–induced inflammatory arthritis (40, 41). To clarify whether the effects of PGRN on ER stress, IR, and autophagic imbalance were dependent on the TNFR pathway, an siRNA pool specific to TNFR1 (p55⫺/⫺) and TNFR2 (p75⫺/⫺) was used to reduce the endogenous levels of TNFR1 and TNFR2, and the efficiency was validated by Western blotting (data not shown). As observed in Figure 6A, PGRN induced impaired insulin sensitivity, autophagic dis-

orders, and ER stress, whereas p55⫺/⫺ cells displayed an improved response to insulin, as evident by the phosphorylation of IRS-1, and less effect was observed in p75⫺/⫺ cells than in their PGRN-treated controls. Likewise, PGRN-induced ER stress and autophagic disorders were partially alleviated in p55⫺/⫺ cell, and knockdown of p75 resulted in a similar trend in the alleviation of ER stress and the restoration of autophagy although these changes did not reach sta-


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Figure 5. Effect of PGRN ablation on insulin signaling and autophagy in mature adipocytes. A, Total protein was isolated from siPGRN adipocytes and immunoblotted with antibodies for PGRN. B, IRS-1 tyrosine phosphorylation (pY) and Akt serine 473 phosphorylation (p-Akt) in PGRN⫺/⫺ adipocytes with or without treatment with tunicamycin (Tun, 5 ␮g/ml) for 3 hours. The graph next to the blots shows the quantification of IRS-1 and Akt phosphorylation. Ins, insulin; IB, immunoblotting; IP, immunoprecipitation. C, Effect of PGRN knockdown on autophagic activity in the presence of tunicamycin. D, PGRN⫺/⫺ adipocytes and their controls were treated with 10 ng/mL TNF-␣ for 8 hours and then were stimulated with insulin (10 nM) for 10 minutes, followed by immunoblotting using antibodies against Atg7, p62, p-Akt, or Akt. A representative blot is shown, and all graphs show means ⫾ SEM from at least 3 independent experiments. *, P ⬍ .05 and **, P ⬍ .001 vs Ctrli; †, P ⬍ .05 vs Ctrli ⫹ Tun; #, P ⬍ .05 vs Ctrli ⫹ TNF-␣. Ctrli, control siRNA. PGRNi, PGRN siRNA.

tistical significance. Double knockdown of TNFR p55 and p75 produced a greater effect than single knockdown of either in the presence of PGRN, indicating a complementary effect of both subunits and a potential role of TNFR in mediating PGRN function (Figure 6A). In addition, we also investigated the potential role of IL-6 in PGRN-mediated effects. As shown in Supplemental Figure 3, we found that IL-6 knockdown by administration of IL-6 siRNA partially alleviated ER stress although this recovery did not seem to be remarkable, as evident by the induction of eIF2␣ and PERK phosphorylation in response to PGRN treatment. This pattern is reminiscent of the previous evidence in some respects and indicates a complementary role of IL-6 in PGRN function (3). We next ascertained whether the downstream pathway of TNF-␣, such as NF-␬B, could be involved in the effects of

PGRN on autophagy and insulin action. We used two different strategies to suppress the NF-␬B pathway in adipocytes, either by pyrrolidine dithiocarbamate (PDTC), an NF-␬B inhibitor, or by p65 siRNA. As shown in Figure 6B, the effects of PGRN on autophagy and insulin signaling were partially nullified by the blockade of the NF-␬B pathway through either the addition of PDTC or the transfection with siRNA, indicating that the regulation of PGRN on autophagy and insulin sensitivity occurs at least partially through TNFR via the NF-␬B-p65 pathway in vitro. Inhibitory effect of PGRN on human adipocytes differentiation To clarify the agonistic potential of PGRN in adipocyte differentiation, human preadipocytes were induced to un-


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Figure 6. Effect of PGRN on ER stress, IR, and autophagy is mediated through the TNFR/NF-␬B pathway in adipocytes. For the effects of PGRN, adipocytes were treated with 100 ng/mL PGRN for 16 hours. For insulin signaling, cells were stimulated with 10 nM insulin for 10 minutes. Indicators of ER stress, insulin signaling, and autophagy were measured at protein levels. The relative quantity of proteins was analyzed with Quantity One software. A, Phosphorylation of PERK (p-PERK) and IRS-1 and expression of Atg7 in p55⫺/⫺, p75⫺/⫺, and p55⫺/⫺p75⫺/⫺ adipocytes. B, Human adipocytes were cultured in the presence or absence of 100 ng/mL PGRN with or without 1 ␮M PDTC (an NF-␬B inhibitor) and 100 nM NF-␬B-p65 siRNA. Quantification of protein levels under the experimental conditions is displayed next to the blots. A representative blot is shown, and the data expressed as means ⫾ SEM in each bar graph represent the average of 3 independent experiments. *, P ⬍ .05 vs Ctrli; #, P ⬍ .05 and ##, P ⬍ .01 vs PGRN. IB, immunoblotting; IP, immunoprecipitation; pY, IRS-1 tyrosine phosphorylation; p-Akt, Akt serine 473 phosphorylation; Ctrls, controls; Veh, vehicle; Ctrli, control siRNA; NF-␬B p65i, NF-␬B p65 siRNA.

dergo adipocyte differentiation by treatment with insulin, dexamethasone, and isobutylmethylxanthine in the presence or absence of PGRN. The normal adipocyte differentiation was assessed by increased expression of adipocyte-specific genes including peroxisome proliferatoractivated receptor-␥ (PPAR␥) and fatty acid binding protein 4 and decreased the preadipocyte-specific gene Pref-1 (Supplemental Figure 4A). As differentiation progressed, autophagy was a dynamic process supported by the dramatically increased LC3-II and Atg7 and reduced p62 levels on day 6 and the tendency to be stable on days 9 and 12. Next, we investigated the levels of unfolded protein response markers during adipogenesis. Interestingly, the ratio of phosphorylated PERK to total PERK

was increased at day 6 but restored at day 12 (Supplemental Figure 4A). As predicted, concomitant treatment with PGRN halted those cells at the undifferentiated stage, as evident by the sustained expression of Pref-1, and the blunted expression of PPAR␥ and fatty acid binding protein 4 on days 6, 9, and 12, respectively (Supplemental Figure 4A). Consistent with the above observations, exposure of preadipocytes to a PGRN supply led to marked increases in Atg7 and LC3-II and the phosphorylation of PERK. To examine whether the induction of ER stress and autophagy by PGRN caused the blockade of adipocyte differentiation, concomitant treatment of preadipocytes with low-dose tunicamycin substantially induced phosphorylation of PERK and expression of Atg7 and LC3-II.


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Likewise, when ER stress was introduced during adipogenesis, adipocyte differentiation was significantly attenuated, with reduced expression of mature adipocyte marker genes (Supplemental Figure 4B). These results indicated a possibility that ER stress suppresses adipocyte differentiation and that PGRN has the potential for interfering with adipogenesis.

Discussion Recent studies have implicated PGRN in diet-induced obesity and IR (10 –14). However, much remains to be elucidated regarding the mechanism of PGRN action before its putative potential as a therapeutic target can be realized. In the present study, circulating PGRN was significantly higher in subjects with MS and increased correspondingly with the increasing number of components of MS. Correlation analysis indicated that serum PGRN was correlated with TG, FPG, HbA1c, HOMA-IR, and HOMA-␤, even after adjustment for BMI. Moreover, PGRN plays a pivotal role in insulin sensitivity and adipogenesis involving ER stress and autophagy mechanism. These observations suggest that circulating PGRN is significantly associated with systemic insulin sensitivity and autophagic activity in AT and support the notion that PGRN functions as a potential link between chronic inflammation and IR. A few clinical studies have demonstrated a positive correlation between circulating PGRN and the components of MS including IR, obesity, and dyslipidemia (14). In accordance with these studies, we provided evidence that serum PGRN was correlated positively with BMI, WC, and TGs and inversely with HDL-C and showed a remarkable correlation between PGRN and FPG, HbA1c, HOMA-IR, and HOMA-␤, even after adjustment for BMI. Our study is in line with the previous observation that PGRN is clinically associated with macrophage infiltration and is contributing to the inflammation profile of obese insulin-resistant patients compared with that in their age-, sex-, and BMI-matched insulin-sensitive counterparts (12, 13), indicating that both obesity and abnormal glucose tolerance are involved in the elevated circulating PGRN concentrations. Dyslipidemia, including increased serum levels of TGs and decreased HDL, is a characteristic feature of the metabolic syndrome and T2DM (42, 43). PGRN was correlated with TGs and HDL in the present study (r ⫽ 0.256 and ⫺0.184 in the overall population, respectively), consistent with reports that PGRN can affect lipid metabolism by inhibiting free fatty acid uptake and lipogenesis and/or by enhancing intracellular lipolysis (44). Of interest, our study also showed that


correlations of PGRN with WC were larger than those with BMI (r ⫽ 0.319 vs 0.275 for WC and BMI in the overall population), indicating that activation of the PGRN system is more closely related to visceral adiposity than to subcutaneous fat and that PGRN plays causal roles in obesity-mediated IR (13). Another key finding of our study was the identification of the significant association between PGRN and autophagic activity in omental AT. We provided evidence that expression of autophagy-related genes in omental AT was significantly higher in subjects with MS and positively correlated with serum PGRN levels. Consistently, tunicamycin- or TNF-␣–induced autophagic imbalance was accompanied by increased PGRN protein expression and mitochondria dysfunction in adipocytes, whereas the ablation of PGRN could at least partially restore the attenuated insulin signaling and autophagic dysfunction. Our previous study demonstrated that autophagic deficiency triggers ER stress and the impaired insulin signaling in experimental models of IR (45), indicating that disturbance of autophagy renders cells vulnerable to ER stress. It has been also proved that autophagic disorders and ER stress exhibit key roles as chronic stimuli in the development of T2DM in terms of the integrated deterioration of systemic glucose homeostasis (27, 29, 33). It is therefore possible that autophagic dysfunction represents an integral part of the ER stress and provides a protective response in PGRN-induced imbalance (46, 47). However, the complex network governing PGRN, autophagic disturbance, and ER stress is likely to involve multiple signals, including a chronically increased demand on synthetic machinery along with profound alterations in energy fluxes in metabolically active tissues. Our findings strongly suggest that PGRN was coupled to ER stress, disturbed autophagy, and also defective insulin action. Strikingly, exposure to PGRN did not lead to the expected disturbance of insulin sensitivity and autophagic response until 16 hours, despite a remarkable increase in ER stress markers at 2 hours. These results verified our hypothesis that initiation of ER stress by exposure to various detrimental factors may promote restrained insulin action and abnormal autophagy. Consistently, the reduction in ER stress via chemical chaperones, such as PBA, prevents further worsening of IR and the autophagy disturbance induced by PGRN (33). It is nevertheless possible that, during the early stages of obesity, ER stress may contribute to the dampening of the initiation of autophagic machinery and glucose homeostasis, which would further the organelle dysfunction, disrupt metabolic homeostasis, and promote the emergence of the associated disease, thus suggesting a critical role of ER stress in the induction of autophagy by PGRN. In our


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study, blockade of ERK activity or PGRN knockdown remarkably rescued PGRN-induced inhibition of mTOR activity, which is consistent with previous reports that inhibition of mTORC1 by sustained MAPK kinase/ERK activation could trigger pronounced autophagic dysfunction, and treatment with the specific ERK inhibitor U0126 could rescue impaired insulin signaling induced by ER stress (34, 35). In parallel, several lines of evidence also suggest that inhibition of mTOR could impede insulin action or trigger an autophagic disorder in adipocytes (36 –39). Thus, the effects of PGRN depletion might be at least partially mediated by ERK via mTOR pathways and, in turn, lead to an improved autophagic response. Overall, our study supports a cause and effect relationship between ER stress, autophagy, and impaired insulin sensitivity involved in the PGRN action, which indicates that initiation of ER stress may promote restrained insulin action and abnormal autophagy. It had been shown that the expression of PPAR␥ and CCAAT/enhancer-binding protein ␣, as well as that of serum response element binding protein normally associated with adipocyte differentiation, was down-regulated in obesity and diabetes (48 –50). In this study, we also observed PGRN-mediated repression of adipocyte differentiation. Our previous finding revealed that preadipocyte adipogenesis is associated with increased autophagic activity (45), and inefficient adipogenesis in Atg7⫺/⫺cells is also evident by decreased induction of PPAR␥ and CCAAT/enhancer-binding protein ␣ compared with controls. Collectively, these results indicate that autophagy appears to be essential for completion of adipogenesis, acting as a potential survival mechanism for cells during the differentiation process (24). Thus, one may hypothesize that the deficient autophagy may also serve to augment ER stress and promote the further worsening of IR in adipocytes. This hypothesis is supported by our previous observation that inhibition of autophagy renders cells vulnerable to low-dose tunicamycin compared with tunicamycin-treated wild-type cells. In line with our results, previous studies demonstrated that inhibition of autophagy stimulates proinflammatory gene expression levels in both human and murine AT (18), whereas rapamycin markedly attenuate ER stress through stimulation of autophagy, suggesting that autophagy may actually function as a mechanism to prevent chronic inflammation and ER stress in AT. A recent study implicated PGRN in the pathogenesis of IR (3), and although IL-6 was implicated in that study (51–54), the mechanisms of PGRN action have not been fully elucidated. In our study, ablation of IL-6 partially ameliorated ER stress, but this recovery does not reach a physiological condition, indicating that some potential


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mechanisms other than IL-6 might also contribute to the initiation of these disorders. On the other hand, a recent unbiased approach was based on PGRN directly binding to TNFRs (41); however, the possibility that TNFR could be a specific receptor for PGRN-mediated effects in IR has never been tested through biochemical or genetic means, and it is still hard to clearly define the early stages of PGRN-mediated signaling from the plasma membrane. In our study, double knockdown of TNFR p55 and p75 caused a higher effect than single knockdown of either, indicating a complementary effect of both subunits and a potential role of TNFR mediating PGRN function. Thus, our results might provide a more complementary insight into the regulation of PGRN on IR (Supplemental Figure 5), which may indicate that the induction of ER stress and autophagy by PGRN is not only indirectly secondary to IL-6 but also more likely to be mediated through TNFR1 via NF-␬B signaling during IR (55). On the other hand, ERK-dependent inactivation of mTOR and the desensitized unfolded protein response caused by ER stress on PGRN stimuli were both sufficient to trigger the autophagic response, whereas disturbance of autophagy in turn leads to the development of IR. Taken together, the scheme depicted in Supplemental Figure 5 suggests that multiple mechanisms underlie the suppressive effect of PGRN on insulin signaling. Nevertheless, we cannot rule out other unidentified factors that may be involved in the complicated actions of PGRN on IR in vivo. In conclusion, our study revealed that serum levels of PGRN were increased in subjects with MS and correlated closely with parameters of IR and metabolic disorders. Notably, PGRN attenuated insulin signaling and autophagic balance in adipocytes, suggesting a causative role of PGRN in insulin sensitivity and indicating that decreasing PGRN levels by influencing its turnover or production is, consequently, a promising therapeutic approach for application to metabolic disorders.

Acknowledgments We appreciate the technical support and materials from the EM Center of Xi’an Jiaotong University. Address all correspondence and requests for reprints to: Hongzhi sun, PhD, MD, Xi’an Jiaotong University College of Medicine, 76 Yanta West Road, Xi’an 710061, China. E-mail: [email protected]; or Shufang Wu, Xi’an Jiaotong University College of Medicine, 76 Yanta West Road, Xi’an 710061, China. E-mail: [email protected]. This work was supported by the programs from the National Natural Science Foundation of China (General Program 30971392, 81170741, 81071440, and 81222026) and the the


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Ministry of Education, China (New Century Excellent Talents in University NCET-08-0435) H.L. wrote the manuscript and researched data, B.Z., J.L., and L.X. researched data and contributed to the discussion, W.Z. contributed to the discussion, and S.W. and H.S. contributed to the experimental designs and reviewed and edited the manuscript. H.S. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Disclosure Summary: The authors have nothing to disclose.


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Circulating docosahexaenoic acid levels are associated with fetal insulin sensitivity.

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

A fasting insulin-raising allele at IGF1 locus is associated with circulating levels of IGF-1 and insulin sensitivity.

Selective enhancement of insulin sensitivity in the mature adipocyte is sufficient for systemic metabolic improvements.

Adipose tissue μ-crystallin is a thyroid hormone-binding protein associated with systemic insulin sensitivity.

Epicardial adipose tissue is associated with visceral fat, metabolic syndrome, and insulin resistance in menopausal women.

Letter: irritable bowel syndrome is significantly associated with somatisation.

Circulating triacylglycerol signatures and insulin sensitivity in NAFLD associated with the E167K variant in TM6SF2.

Circulating adipokines and insulin resistance in subjects with combined cardiac and metabolic syndrome X.

Maternal insulin sensitivity is associated with oral glucose-induced changes in fetal brain activity.

Sense of coherence is significantly associated with both metabolic syndrome and lifestyle in Japanese computer software office workers.

Biochemical hyperandrogenism is associated with metabolic syndrome independently of adiposity and insulin resistance in Romanian polycystic ovary syndrome patients.

Peripheral neuropathy is associated with insulin resistance independent of metabolic syndrome.

Associations of objective physical activity with insulin sensitivity and circulating adipokine profile: the Framingham Heart Study.

Metabolic syndrome, but not insulin resistance, is associated with an increased risk of renal function decline.

Higher omega-3 index is associated with increased insulin sensitivity and more favourable metabolic profile in middle-aged overweight men.

Afamin serum concentrations are associated with insulin resistance and metabolic syndrome in polycystic ovary syndrome.

Effect of resveratrol administration on metabolic syndrome, insulin sensitivity, and insulin secretion.

Effects of cold sensitivity in the extremities on circulating adiponectin levels and metabolic syndrome in women.

Metabolic syndrome is associated with atrial electrical and mechanical dysfunction.

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

Headache associated with metabolic and systemic disorders.

Arterial norepinephrine concentration is inversely and independently associated with insulin clearance in obese individuals with metabolic syndrome.

Spontaneous periodontitis is associated with metabolic syndrome in rhesus monkeys.