Diacerhein Improves Glucose Tolerance and Insulin Sensitivity in Mice

DIABETES-INSULIN-GLUCAGON-GASTROINTESTINAL

Diacerhein Improves Glucose Tolerance and Insulin Sensitivity in Mice on a High-Fat Diet Nata´lia Tobar, Alexandre G. Oliveira, Dioze Guadagnini, Renata A. Bagarolli, Guilherme Z. Rocha, Tiago G. Arau´jo, Junia C. Santos-Silva, Ricardo L. Zollner, Luiz H. B. Boechat, Jose´ B. C. Carvalheira, Patrícia O. Prada, and Mario J. A. Saad Department of Internal Medicine, State University of Campinas, 13081-970, Campinas, Saõ Paulo, Brazil

Obesity and type 2 diabetes are characterized by insulin resistance, and the common basis of these events is a chronic and systemic inflammatory process marked by the activation of the c-Jun Nterminal kinase (JNK) and inhibitor-␬B kinase (IKK␤)/nuclear factor-␬B (NF␬B) pathways, up-regulated cytokine synthesis, and endoplasmic reticulum dysfunction. The aim of this study was to evaluate the effects of diacerhein administration, an antiinflammatory drug that reduces the levels of inflammatory cytokines, on insulin sensitivity and signaling in diet-induced obese (DIO) mice. Swiss mice were fed with conventional chow (control group) or a high-fat diet (DIO group). Later, DIO mice were randomly subdivided into a new subgroup (DAR) that received 20 mg/kg diacerhein for 10 d. Western blotting was used to quantify the expression and phosphorylation of insulin receptor, insulin receptor substrate 1, and Akt and of inflammatory mediators that modulate insulin signaling in a negative manner (IKK␤, JNK, and inducible nitric oxide synthase). We show here, for the first time, that the administration of diacerhein in DIO mice improved endoplasmic reticulum stress, reduced JNK and IKK␤ phosphorylation, and resulted in a marked improvement in fasting glucose, a decrease in macrophage infiltration in adipose tissue, and a reduced expression and activity of proinflammatory mediators accompanied by an improvement in the insulin signaling mainly in the liver and adipose tissue. Taken together, these results indicate that diacerhein treatment improves insulin sensitivity in obesity, mediated by the reversal of subclinical inflammation, and that this drug may be an alternative therapy for insulin resistance. (Endocrinology 152: 4080 – 4093, 2011)

besity, type 2 diabetes (T2D), and insulin resistance are metabolic disturbances that are closely interrelated. The intersection of these conditions is linked to a systemic and subclinical inflammatory process that is characterized by the abnormal production of cytokines and NO and increased levels of acute-phase proteins (1, 2). In this interdependent context, it has become evident that many inflammatory mediators act as negative modulators of insulin action, playing an important role in insulin resistance and T2D pathogenesis (3). Cytokine-mediated molecular mechanisms that promote insulin resistance have been further clarified recently and particularly demonstrate the activation of serine kinases proteins, such as

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c-Jun N-terminal kinase (JNK) and the inhibitor-␬B kinase (IKK␤)/nuclear factor-␬B (NF␬B) complex. JNK and IKK␤ pathways cause serine residue phosphorylation on the insulin receptor (IR) and IR substrate 1 (IRS-1), reducing the IR binding ability and contributing to the deregulation of the insulin signaling (4 – 6). Thus, negative modulators of the insulin intracellular cascade such as JNK and IKK␤ are partly responsible for the establishment of insulin resistance and represent potential therapeutic targets for T2D treatment. Endoplasmic reticulum (ER) stress has been thought to be a core molecular mechanism involved in triggering and integrating inflammation and insulin action in T2D and

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/en.2011-0249 Received March 3, 2011. Accepted August 2, 2011. First Published Online September 6, 2011

Abbreviations: 2-DG, 2-[14C]Deoxy-D-glucose; DIO, diet-induced obesity; ER, endoplasmic reticulum; I␬B␣, inhibitor of NF␬B; IKK␤, inhibitor-␬B kinase; iNOS, inducible nitric oxide synthase; IR, insulin receptor; IRS-1, IR substrate 1; JNK, c-Jun N-terminal kinase; NF␬B, nuclear factor-␬B; PERK, PKR-like ER kinase; PTP1B, protein-tyrosine phosphatase 1B; SVF, stromal vascular fraction; T2D, type 2 diabetes; TG, triglyceride.

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obesity. Although the exact mechanisms of this link are unclear, it involves multiple characteristic signals of these metabolic diseases, including excess lipid accumulation, abnormalities in intracellular energy fluxes, and nutrient availability (7–9). Much in vitro and in vivo genetic evidence demonstrates a strong and causal relation between the functional capacity of the ER and insulin action (7). Notably, JNK and IKK complex are activated by ER stress situations, and inflammatory mediators can trigger ER dysfunction, leading to propagation of general cellular stress responses (10 –12). In this context, it has been postulated that the ER might be a site for the sensing of metabolic stress and the translation of that stress into inflammatory signaling that impairs the insulin intracellular signal. Therefore, pharmacological manipulation of the ER stress pathway seems to offer novel opportunities for treating metabolic diseases like insulin resistance and T2D. Diacerhein (1,8-diacetoxy-9,10-dioxo-dihydroanthracene-3-carboxylic acid) is an anthraquinone found in Cassia gender plants that presents antiinflammatory properties in addition to moderate analgesic and antipyretic characteristics (13). Based on in vitro and in vivo experiments in animals and humans, rhein, the active metabolic of diacerhein, has been demonstrated to inhibit the synthesis and activity of proinflammatory cytokines such as TNF-␣, IL-6, and specially IL-1␤ (14 –19). Besides this, the compound acts directly on inflammatory cells inhibiting superoxide anion production by human neutrophils, release of lysosomal enzymes, chemotaxis, and phagocytic activity of neutrophils and macrophages (13, 14, 18, 20 – 23). Moreover, diacerhein negatively modulates the synthesis and activity of inducible nitric oxide synthase (iNOS) by decreasing its mRNA levels (18, 24) and may also inhibit IKK␤ activity (19). In this regard, this drug has a potential to improve insulin resistance in obesity and T2D and has an established safety profile after many years of use for osteoarthritis; nevertheless, there are few studies that involve it in the treatment of nonrheumatic diseases. For this reason, this study aimed to investigate the effects of diacerhein (10 d treatment) on insulin sensitivity and signaling in the liver, skeletal muscle, and adipose tissue of diet-induced obese Swiss mice as well as its role on ER stress and JNK and IKK␤ activation. Taken together, our findings demonstrate that diacerhein treatment improves glucose tolerance and insulin sensitivity and signaling in all tissues studied by reducing ER stress, expression of proinflammatory mediators, and activation of insulin negative modulators, highlighting it as a potential alternative treatment of insulin resistance and type 2 diabetes.


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Materials and Methods Materials Male Swiss mice were provided by the State University of Campinas Central Breeding Center (Campinas, Brazil). All antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), except anti-Akt, anti-phospho-Akt, anti-phospho-IKK␤, and anti-␣-tubulin, which were obtained from Cell Signaling Technology (Beverly, MA), and anti-protein-tyrosine phosphatase 1B (PTP1B), which was from Abcam (Cambridge, MA). Human recombinant insulin was from Eli Lilly and Co. (Indianapolis, IN). Diacerhein was kindly provided by TRB-Pharma (Campinas, Brazil). Routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless specified otherwise.

Animal characterization All experiments were approved by the Ethics Committee of the State University of Campinas. Eight-week-old male Swiss mice were maintained under specific pathogen-free conditions in a regimen of 12-h dark, 12-h light cycles and room temperature of 21 C. The animals were randomly divided into two groups with similar body weights (30.22 ⫾ 3.95 g), according to the diet that they were assigned to receive for 12 consecutive weeks; a standard rodent chow (control group) or a high-fat diet [dietinduced obesity (DIO) group], as previously used (25). Food and water were ad libitum. Food intake was determined by measuring the difference between the weights of the high-fat diet given and their weights at the end of a 24-h period. After 12 wk of feeding, the animals underwent insulin, glucose, and pyruvate tolerance tests, as previously described (26, 27).

Diacerhein administration protocol Dried diacerhein was diluted in 0.01 M PBS to a final concentration of 1.5 mg/ml. Some of the DIO animals were randomly distributed into a new subgroup that started to receive diacerhein solution at a dose of 20 mg/kg䡠d (DAR group). In previous experiments, we performed dose-response experiments with 1, 5, 10, and 20 mg/kg diacerhein, and although the results showed an improvement in insulin signaling with the lowest dose, the maximal response was observed with 20 mg/kg. We then decided to use the dose of 20 mg/kg. The drug was administered in one gavage per day for 10 consecutive days. The other mice of the DIO group and those of the control group received only a vehicle solution (0.01 M PBS).

Hyperinsulinemic-euglycemic clamp procedures After 12 h fasting, animals were anesthetized ip with ketamine and diazepam (70:30) (50 mg/kg), and catheters were then inserted into the left jugular vein (for tracer infusions) and femoral artery (for blood sampling), as previously described (28). A 120-min hyperinsulinemic-euglycemic clamp procedure was conducted in the anesthetized catheterized mice, as shown previously (29, 30), with a prime continuous infusion of human insulin at a rate of 30 mU/kg䡠min. Blood samples were collected at 5-min intervals for the immediate measurement of plasma glucose concentration, and 5% of unlabeled glucose was infused at variable rates to maintain plasma glucose at fasting levels. To estimate insulin-stimulated glucose-transport activity and metabolism in skeletal muscle, 2-[14C]deoxy-D-glucose (2-DG) was administered as a bolus (10 ␮Ci) 45 min before the end of the

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clamp procedure. All infusions were performed using Harvard infusion pumps. At the end of the clamp procedure, animals were killed by a ketamine and diazepam iv injection. Within 2 min, gastrocnemius muscles from both hind limbs were taken. The tissue was dissected out within 2 sec, weighed, frozen with liquid N2, and stored at ⫺80 C for later analysis. Glucose transport activity in skeletal muscle was calculated from the plasma 2-DG profile, as described before (31, 32).


Real-time PCR The mRNA was determined in the muscle, liver, and adipose tissue using RT-PCR, as previously reported (37). Primer sequences are shown in Supplemental Table 1 (published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). Results are expressed as relative expression values, as published previously (38).

Morphometry After the treatment with diacerhein, blood samples were taken for the determination of serum concentration of basal insulin (Millipore, St. Charles, MO) and IL-6 and TNF-␣ (Thermo Fischer Scientific Inc., Rockford, IL) by ELISA. Glucose values were measured from the tail venous blood of all animals with a glucose monitor (Glucometer; Bayer, Tarrytown, NY).

Five-micrometer sections from epididymal adipose tissue and liver were observed with a Zeiss Axiophot light microscope using ⫻10 and ⫻40 objectives, respectively, and digital images were captured with a Canon PowerShot G5. To mark macrophages in the adipose tissue, specific antibodies were used according to previous methodologies (34). Measurements of adipocyte were determined using the image analysis system Image J (http://rsbweb.nih.gov/ij/).

Tissue extraction

Statistical analysis

After a 12- to 14-h fasting period, the mice were anesthetized by ip injection of sodium thiopental and opened 10 –15 min later, i.e. as soon as anesthesia was assured by the loss of pedal and corneal reflexes. The abdominal cavity was opened, the portal vein was exposed, and 0.2 ml normal saline was injected with or without insulin (10⫺6 mol/liter). At 30 sec after the insulin injection, the liver was removed, and 90 sec later, gastrocnemius muscle and epididymal adipose tissue were extracted, minced coarsely, and homogenized immediately in extraction buffer, as described elsewhere (33). Extracts were then centrifuged at 15,000 rpm at 4 C for 40 min to remove insoluble material. The whole-tissue extracts were subjected to SDS-PAGE and immunoblotting, as previously described (25, 27). NF␬B p65 activation was also determined by an immunoblotting method using nuclear extracts from the same tissues (34).

Data are expressed as means ⫾ SEM of the number of independent experiments indicated. The results of blots are presented as direct comparisons of bands or spots in autoradiographs and quantified by optical densitometry (Scion Image). For statistical analysis, the groups were compared using a two-way ANOVA with the Bonferroni test for post hoc comparisons. The level of significance adopted was P ⬍ 0.05 unless specified otherwise.

Assays

Isolation of the stromal vascular fraction (SVF) and adipocyte fraction of the adipose tissue Epididymal fat pads were excised, and isolation of the SVF and adipocyte fraction of the adipose tissue was performed, as previously described (35).

Protein analysis by immunoblotting The whole-tissue and nuclear extracts were treated with Laemmli sample buffer containing 100 mM dithiothreitol and heated in a boiling water bath for 5 min, after which they were subjected to SDS-PAGE in a Bio-Rad (Hercules, CA) miniature slab gel apparatus (Mini-Protean). Electrotransfer of proteins from the gel to nitrocellulose membranes was performed for 120 min at 120 V in a Bio-Rad Mini-Protean transfer apparatus (36). Nonspecific protein binding to the nitrocellulose was reduced by preincubating the filter for 2 h in blocking buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaCl, and 0.02% Tween 20). The nitrocellulose blot was incubated overnight at 4 C with specific antibodies. Results were visualized by autoradiography with preflashed Kodak XAR film, and band intensities were quantified by optical densitometry (Hoefer Scientific Instruments, San Francisco, CA; model GS300).

Results Physiological and metabolic parameters Figure 1 shows comparative data regarding the controls (C), DIO mice, and DIO mice treated with diacerhein for 10 d (DAR). All animals from the DIO group, treated or not, presented similar body weights and epididymal fat pad weights, which were higher when compared with the control group (Fig. 1, A and B). Diacerhein did not change food ingestion in DIO mice (Fig. 1, C and D). After the 10th day of treatment, the fasting serum glucose concentrations were significantly lower in the treated group than in the DIO and very similar to the control group (Fig. 1E). Fasting serum insulin levels were also reduced in the DAR animals and controls, compared with the DIO group (Fig. 1F). During the insulin tolerance test, the reduction in the glucose disappearance rate (KITT), induced by the highfat diet, was seen to be restored after treatment with diacerhein (Fig. 1G). To investigate glucose tolerance, we performed an ip glucose tolerance test. Throughout the test, glucose levels of DIO animals were higher at all time points. Conversely, DAR mice demonstrated improved glucose profiles that were closer to those of the control group at some points (Fig. 1H). A hyperinsulinemic-euglycemic clamp with tracer infusions was also performed to examine the effects of di-



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FIG. 1. Physiological and metabolic parameters in control mice, obese mice, and obese mice submitted to the administration of diacerhein for 10 d. Panel A, Body weight; panel B, epididymal fat pad weight; panel C, total food intake; panel D, consumption of calories; panel E, fasting serum glucose; panel F, fasting serum insulin; panel G, KITT (calculated using the formula 0.693/t1/2; the plasma glucose t1/2 was calculated from the slope of the least-square analysis of the plasma glucose concentrations during the linear decay phase); panel H, glucose response curve and the area under curve (AUC) during the glucose tolerance test; panel I, steady-state glucose infusion rates obtained from averaged rates of 90 –120 min of 5% unlabeled glucose infusion during hyperinsulinemic-euglycemic clamp; panel J, glucose transport in gastrocnemius muscle evaluated by 2-deoxy-D-glucose (2-DG) uptake during the last 45 min of the hyperinsulinemic-euglycemic clamp; panel K, glucose response curve and the area under curve (AUC) during the pyruvate test; panel L, levels of TG in the gastrocnemius muscle; panel M, levels of TG in the liver. Data are presented as means ⫾ SEM of 10 mice per group. #, P ⬍ 0.05 vs. control; *, P ⬍ 0.05 vs. DIO; †, P ⬎ 0.05 vs. DIO. C, Control.

acerhein treatment on glucose metabolism in the skeletal muscle. The glucose infusion rate was lower in the DIO group than in the control group and increased in DAR mice (Fig. 1I). As shown in Fig. 1J, DIO mice presented an impressive reduction in glucose uptake by skeletal muscle when compared with the control group. The diacerhein treatment improved this parameter only mildly (Fig. 1J).

To investigate hepatic glucose output after a pyruvate load, we injected ip pyruvate and measured blood glucose for 3 h. The results demonstrated a higher increase in blood glucose during the pyruvate test in DIO mice compared with the control group, whereas DAR mice showed a lower increase in blood glucose (Fig. 1K), suggesting a more marked suppression of glucose production.

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Effects of diacerhein on intracellular lipid levels To investigate whether diacerhein treatment induces changes in the intracellular lipid levels, we measured the triglyceride (TG) content in the gastrocnemius muscle and liver of the groups studied with a colorimetric kit (Trig/ GB; Roche Diagnostics, Indianapolis, IN), as previously described (39). The DIO mice exhibited increased TG in the muscle and liver when compared with the control group, and after 10 d diacerhein administration, the TG content decreased only in the liver (Fig. 1, L and M). Histological characterization of liver and adipose tissue after treatment with diacerhein We assessed whether the metabolic improvements induced by diacerhein were accompanied by alterations in hepatic and adipose morphologies. Histological sections


from the liver of the DIO group showed the presence of innumerous fat vesicles, compared with the control animals, characterizing the first group as presenting hepatic steatosis. Nevertheless, the treatment with diacerhein prevented fat accumulation in the liver parenchyma of DIO mice (Fig. 2, A–C). Morphological analysis of the epididymal fat pad revealed large adipocytes and a significant infiltration of inflammatory cells in DIO animals, as demonstrated by the presence of numerous crown-like structures and confirmed by the detection of the specific macrophage marker F4/80 (Fig. 2, D–G). Mice from the DAR group presented fewer and smaller adipocytes than the DIO group, and apart from a discrete presence of macrophages, a very similar profile was found in control animals (Fig. 2, H–K).

FIG. 2. Morphological characterization of liver and adipose tissue samples. A–C, Hematoxylin and eosin staining of 5-␮m histological sections of the liver parenchyma of control mice (panel A), DIO mice (panel B), and DAR mice (panel C) (original magnification, ⫻400); panels D–F, representative immunohistochemical staining of 5-␮m histological sections of epididymal fat pad using the specific macrophage marker F4/80⫹ in control mice (panel D), DIO mice (panel E), and DAR mice (panel F); panel G, quantification of crown-like structures (mean) (original magnification, ⫻100), with red arrows indicating crown-like structures through F4/80⫹ detection; panels H–J, hematoxylin and eosin staining of 5-␮m histological sections of the epididymal fat pad of control mice (panel H), DIO mice (panel I), and DAR mice (panel J); panel K, quantification of adipocyte area (square micrometers) (original magnification, ⫻100) with red arrows indicating crown-like structures. Scale bar, 50 ␮m for all pictures. Data are presented as means ⫾ SEM of 10 mice per group. #, P ⬍ 0.05 vs. control; *, P ⬍ 0.05 vs. DIO.



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not significant (Fig. 3, A–I). The relative amount of TNF-␣, IL-6, and IL-1␤ transcripts were significantly reduced in all tissues of DAR animals compared with the DIO group (Fig. 3, A–I). To investigate deeply the reduction of proinflammatory cytokine mRNA levels in the adipose tissue, we separated SVF and parenchymal cells from adipose tissue of control, DIO, and DAR groups. A significant reduction in the expression of IL-1␤, IL-6, and TNF-␣ mRNA was observed in adipocytes and of IL-1␤ and TNF-␣ in SVF (Supplemental Fig. 1A) of animals that received diacerhein. In addition, we analyzed serum levels of TNF-␣ and IL-6, which were lower after the diacerhein treatment (Fig. 3, J and K).

FIG. 3. Effects of diacerhein on TNF-␣, IL-1␤, and IL-6 mRNA tissue expression and serum TNF-␣ and IL-6 levels in control mice, obese mice, and obese mice submitted to the administration of diacerhein for 10 d. Panels A–C, Determination of TNF-␣ mRNA expression by real-time PCR in the liver (panel A), gastrocnemius muscle (panel B), and epididymal adipose tissue (panel C); panels D–F, determination of IL-6 mRNA expression by real-time PCR in the liver (panel D), gastrocnemius muscle (panel E), and epididymal adipose tissue (panel F); panels G–I, determination of IL-1␤ mRNA expression by real-time PCR in the liver (panel G), gastrocnemius muscle (panel H), and epididymal adipose tissue (panel I). Data are presented as means ⫾ SEM of 10 mice per group. #, P ⬍ 0.0001 vs. control (C) group; *, P ⬍ 0.0001 vs. DIO. Panels J and K, Determination of serum TNF-␣ (panel J) and serum IL-6 (panel K) by ELISA. Data are presented as means ⫾ SEM of six mice per group. #, P ⬍ 0.05 vs. control group; *, P ⬍ 0.05 vs. DIO.

Cytokine analyses TNF-␣, IL-6, and IL-1␤ mRNA expression was examined in the liver, gastrocnemius muscle, and epididymal adipose tissue of the groups studied. As expected, cytokine mRNA expressions in the DIO mice tissues were higher than in the control group, with the exception of the expression of TNF-␣ in the muscle, where this increase was

Effects of diacerhein treatment on insulin signaling in liver, muscle, and adipose tissue We then examined the effects of diacerhein administration on the insulin-signaling pathway in its main target tissues. As expected, in the liver, muscle, and adipose tissue of mice fed on a high-fat diet, insulin-stimulated tyrosine phosphorylation levels of IR␤ and IRS-1 and Akt serine phosphorylation were significantly reduced compared with those of the control animals (Fig. 4, A–I). Moreover, the group treated with diacerhein exhibited a higher phosphorylation of these proteins in all tissues studied when compared with the nontreated group, with the exception of Akt phosphorylation in the muscle, which was lower in the DAR animals (Fig. 4F). There were no differences in IR␤, IRS-1, and Akt protein expression among the control, DIO, and DAR groups.

Effects of diacerhein on the JNK pathway, IRS-1 serine 307 phosphorylation, and PTP1B expression JNK activation was determined by monitoring phosphorylation of JNK1 and of its substrate, c-Jun. The DIO group exhibited a higher expression and phosphorylation of JNK in the liver, muscle, and adipose tissue in relation

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FIG. 4. Effects of diacerhein administration on insulin signaling in high-fat diet-fed mice. Panels A–C, Representative blots of liver tyrosine phosphorylation of IR␤ (panel A), IRS-1 (panel B), and serine phosphorylation of Akt (panel C) of control mice, DIO mice, and DAR mice (top panels) and total protein expression (bottom panels); panels D–F, representative blots of muscle tyrosine phosphorylation of IR␤ (panel D), IRS-1 (panel E), and serine phosphorylation of Akt (panel F) of control mice, DIO mice, and DAR mice (top panels) and total protein expression (bottom panels); panels G–I, representative blots of adipose tissue tyrosine phosphorylation of IR␤ (panel G), IRS-1 (panel H), and serine phosphorylation of Akt (panel I) of control mice, DIO mice, and DAR mice (top panels) and total protein expression (bottom panels). Data are presented as means ⫾ SEM of six mice per group. *, P ⬍ 0.05 vs. control group; #, P ⬍ 0.05 vs. DIO. C, Control; IB, immunoblot; p, phosphorylated.

to the control animals (Fig. 5, A–C). Accordingly, the phosphorylation levels of c-Jun were also higher in the obese nontreated group (Fig. 5, D–F). Conversely, the expression and phosphorylation of JNK, as well as the phosphorylation of c-Jun, in these tissues of the DAR group were significantly lower when compared with the DIO group. Next, we analyzed the IRS-1 serine 307 phosphorylation levels in the three tissues studied. The results showed that diacerhein was able to reduce markedly serine 307

phosphorylation levels in the liver and adipose tissue. Regarding muscle, we found only a mild improvement in this parameter (Fig. 5, G–I). Finally, we investigated the effect of diacerhein on the expression of PTP1B. The results showed an increase in PTP1B protein expression in tissues of DIO mice, and there was a notable reduction in the expression of PTP1B in the liver, muscle, and adipose tissue of DAR group compared with the nontreated group (Fig. 5, J–L).



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Effects of diacerhein on the IKK␤/ I␬B␣/NF␬B pathway and on iNOS expression IKK␤ activity was monitored through IKK␤ and inhibitor of NF␬B (I␬B␣) I␬B␣ phosphorylation, as previously described (40), and complemented by the analysis of the nuclear expression of NF␬B p65. In the liver and adipose tissue of the DIO group, the phosphorylation levels of IKK␤ (Fig. 6, A and D) and I␬B␣ (Fig. 6, B and E) were higher when compared with those of the control group. Regarding the DIO animals, the treated mice presented a lower phosphorylation of IKK␤ and I␬B␣ in both tissues studied. For this reason, we assessed the translocation of NF␬B p65 to the nuclei of hepatocytes and adipocytes in animals from the DAR group. As expected, in nuclear tissue extracts from these mice, we detected a lower expression of NF␬B p65 compared with the nontreated group, which was similar to that found in the control animals (Fig. 6, C and F). DIO animals treated with diacerhein presented an impressive reduction of iNOS expression in the liver, muscle, and adipose tissue compared with DIO mice (Fig. 7, A–C).

FIG. 5. Effects of diacerhein administration on the JNK pathway, IRS-1 serine 307 phosphorylation, and PTP1B expression in DIO mice. Panels A–C, JNK phosphorylation in liver (panel A), muscle (panel B), and adipose tissue (panel C) of control mice, DIO mice, and DAR mice (upper panels) and total protein expression of JNK (lower panels); panels D–F, c-Jun phosphorylation in the liver (panel D), muscle (panel E), and adipose tissue (panel F) of control mice, DIO mice, and DAR mice and total protein expression of pc-Jun (lower panels); panels G–I, IRS-1 serine 307 phosphorylation in liver (panel G), muscle (panel H), and adipose tissue (panel I) of control mice, DIO mice, and DAR mice (upper panels) and total protein expression of IRS-1 (lower panels); panels J–L, PTP1B expression in liver (panel J), muscle (panel K), and adipose tissue (panel L) of control mice, DIO mice, and DAR mice (upper panels) and total protein expression of PTP1B (lower panels). Data are presented as means ⫾ SEM of six mice per group. *, P ⬍ 0.05 vs. control group; #, P ⬍ 0.05 vs. DIO. C, Control; IB, immunoblot; p, phosphorylated.

Effects of diacerhein on ER stress in obesity The phosphorylation status of PKRlike ER kinase (PERK) is one of the key indicators of the presence of ER stress. As expected, PERK phosphorylation in DIO mice was higher in the liver and adipose tissue if compared with the control group. Moreover, animals treated with diacerhein exhibited significantly lower activity of PERK in both tissues if compared with the mice that received no treatment (Fig. 7, D and F). Regarding the muscle samples, there was no impressive increase in PERK activation in the DIO group compared with the control one, but there was evidence of a significant reduction in phospho-PERK levels in DAR animals compared with the DIO mice (Fig. 7E).

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gering inflammatory signaling through the activation of both JNK and IKK pathways (8, 11, 12, 42). The ER is an attractive potential therapeutic target in part because of proper ER function may be able to prevent chronic metabolic disease (43). We show here, for the first time, that the administration of the antiinflammatory diacerhein for 10 d in DIO mice reduced tissue expression and serum levels of IL-6 and TNF-␣, improved ER stress, and resulted in an improvement in glucose tolerance, a decrease in macrophage infiltration in adipocytes, and a reduced expression and activity of proinflammatory mediators accompanied by an improvement in insulin signaling in the liver, muscle, and adipose tissue. Taken together, these results indicate that diacerhein treatment FIG. 6. Effects of diacerhein administration on IKK␤/I␬B␣/NF␬B pathway in DIO mice. Panels A–C, Representative blots of IKK␤ phosphorylation (panel A), I␬B␣ phosphorylation (panel B), improves insulin sensitivity in obesity, and NF␬B p65 expression in nuclear extracts (panel C) from the liver of control mice, DIO mediated by the reversal of subclinical mice, and DAR mice; panels D–F, representative blots of IKK␤ phosphorylation (panel D), I␬B␣ chronic inflammation and that this drug phosphorylation (panel E), and NF␬B p65 expression in nuclear extracts (panel F) from the adipose tissue of control mice, DIO mice, and DAR mice. Total protein expression of IKK␤, may be an alternative therapy for insulin I␬B␣, and NF␬B p65 in both tissues are represented in the lower panels in all graphics. Data resistance. are presented as means ⫾ SEM of six mice per group. *, P ⬍ 0.05 vs. control group; #, P ⬍ As demonstrated in previous study 0.05 vs. DIO. C, Control; IB, immunoblot; p, phosphorylated. (44), the ER stress in DIO mice was evident in liver and adipose tissue but not One day of diacerhein treatment was able to in muscle. In both the adipose tissue and liver of obese reduce IL-6 mRNA in the adipose tissue and IL-6 mice, the treatment with diacerhein was able to reduce serum levels in DIO mice To investigate a possible mechanism by which diacer- significantly the PERK phosphorylation. The ER stress hein suppress inflammation, we analyzed the effects of 1 d described in obesity induces a complex signaling pathway of diacerhein treatment on IL-1␤, IL-6, and TNF-␣ mRNA that activates IKK␤ and JNK (41). Interestingly, it is also in both SVF and adipocytes and also in the liver and skel- known that interventions that inhibit their expression or etal muscle of all studied groups. This treatment induced activity significantly improve peripheral insulin sensitivity a reduction in mRNA levels of IL-6 only in the adipocyte (45, 46). Accordingly, our data demonstrate that reducfraction, and no significant decreases were found regard- tions in IKK␤ and JNK activation after diacerhein admining the IL-1␤ and TNF-␣ mRNA levels in both adipose istration in obese animals increase the phosphorylation fractions (Supplemental Fig. 1B) or in the liver and muscle levels of important proteins of the insulin signaling cas(data not shown). Interestingly, we observed a reduction cade (IR, IRS-1, and Akt), improving the insulin resistance in IL-6 circulating levels in DAR group compared with state as a whole. These findings are in accordance with DIO animals, but no difference was observed in TNF-␣ previous data showing that these serine kinases are possibly key modulators in the cross talk between inflammaserum levels (Supplemental Fig. 1B). tory and metabolic pathways (41, 47). The phosphorylation of IRS-1 at serine 307 is an important mechanism by which the activation of JNK and Discussion IKK␤ can inhibit insulin signaling in DIO animals (48, 49). It is generally agreed that inflammation is a key feature of Previous study showed that IRS-1 serine phosphorylation obesity and type 2 diabetes (9, 41). It has been recently is able to mediate inhibition of insulin receptor tyrosine shown that obesity is a chronic stimulus for ER stress in kinase activity (4). Diacerhein treatment improved insuperipheral tissues, and this, in turn, plays a central role in lin-induced tyrosine phosphorylation levels of IRS-1 and the development of insulin resistance and diabetes by trig- IR by suppressing JNK and IKK pathways and TNF-␣



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have contributed to this absence of activation of Akt in muscle after diacerhein treatment. The lack of effect of diacerhein on Akt in muscle had important consequences for glucose utilization in this tissue. It is important to mention that the glucose clamp, associated with tracer isotope, which quantifies insulin sensitivity and muscle glucose uptake, showed that despite the animals of DAR displayed a moderate increase in insulin sensitivity, their muscle glucose uptake improves only mildly. In addition, we also performed a pyruvate test that demonstrated that the hepatic glucose output was normalized by diacerhein treatment. By comparing the effects of diacerhein in the liver and muscle, these results suggest that the drug induces improvement in insulin sensitivity only in liver, reflected by a reduction in fasting plasma glucose, but only a mild FIG. 7. Effects of diacerhein on iNOS tissue expression and ER stress in DIO mice. Panels A–C, iNOS expression in the liver (panel A), muscle (panel B), and adipose tissue (panel C) of control improvement in glucose levels at 120 min mice, DIO mice, and DAR mice (upper panels) and total protein expression of iNOS (lower during the glucose tolerance test. panels); panels D–F, PERK activity in the liver (panel D), muscle (panel E), and adipose tissue As previously described, the overlap (panel F) of control mice, DIO mice, and DAR mice (upper panels) and total protein expression of PERK (lower panels). Data are presented as means ⫾ SEM of six mice per group. *, P ⬍ 0.05 between stimuli that activate IKK␤ and vs. control group; #, P ⬍ 0.05 vs. DIO. C, Control; IB, immunoblot. JNK and conditions that promote insulin resistance also includes proinflamtissue expression and serum levels. Another possible matory cytokines (41, 46). IKK␤ activation, through the mechanism to explain the improvement in insulin-induced phosphorylation and degradation of I␬B␣, initiates NF␬BIR tyrosine phosphorylation is related to the lower ex- mediated transcription, which in certain cells enhance the pression of PTP1B found in the tissues of animals treated production of TNF-␣, IL-6, and IL1-␤, which further acwith diacerhein. Although the adipose tissue inflamma- tivate JNK and IKK␤ pathways through a feed-forward tion and TNF␣ up-regulate PTP1B expression in insulin- mechanism (41, 46). This positive feedback loop could target tissues of several obese and/or diabetic animal mod- perpetuate a vicious cycle of low-grade inflammatory sigels (50), diacerhein administration was able to reduce naling, contributing to enhanced insulin resistance. It is PTP1B expression, contributing to improve the insulin- also important to mention that these cytokines, possibly induced IR and IRS-1 tyrosine phosphorylation in the through deregulation of the TNF-␣-converting enzyme/ liver, muscle, and adipose tissue. tissue inhibitor of metalloproteinase 3 proteolytic system, The results showed that diacerhein was able to reduce have been shown to play an important role in subclinical markedly serine 307 phosphorylation levels in the liver inflammation in obesity/type 2 diabetes insulin resistance and adipose tissue, but regarding muscle, we found only a (51–53). Our findings indicate that IKK␤ inhibition mild improvement in this parameter. There was only a breaks that vicious cycle. The treatment with diacerhein mild improvement in insulin-induced IRS-1 tyrosine phos- was able to reduce IKK␤ phosphorylation, then I␬B␣ phorylation, which was not accompanied by an improve- phosphorylation, and to prevent the translocation of the ment in insulin-induced Akt phosphorylation. It is possi- NF␬B subunit p65 to the cellular nucleus, confirming that ble that the mild improvement in IRS-1 tyrosine the inactivation of IKK/NF␬B axis represents an alternaphosphorylation was not sufficient to modulate Akt phos- tive target for the therapy of insulin resistance as indicated phorylation, or some other factors downstream of IRS-1, by other studies (54 –56). In this line, the inhibition of NF␬B such as phosphatidylinositol-3-kinase (PI3K) activity or nuclear translocation may, at least in part, explain the rephosphatase that dephosphorylates Akt (PP2A) activity, duced levels of TNF-␣, IL-6, and IL-1␤ detected, in general, or even the absence of reduction in TG content may also in the liver and adipose tissue of diacerhein-treated animals

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and, consequently, the improvement in the systemic inflammatory process. In addition to IKK␤ pathway down-regulation, diacerhein also inhibited the phosphorylation of JNK as well its substrate c-Jun. In obesity, JNK activity is increased in liver, muscle, and fat tissues (9). A number of JNK inhibitors, or JNK1 deficiency, have been demonstrated to prevent the development of insulin resistance, T2D, and fatty liver disease as well as presenting beneficial effects on insulin sensitivity in both genetic and dietary models of obesity (47, 57). The reduction in JNK phosphorylation found in this study agrees with previous study in which the inhibition of JNK activity in the liver improves insulin sensitivity and hepatic steatosis (47). Although it has been reported that muscle JNK does not contribute to insulin resistance (58), these data are not uniformly observed (59), and in the present study, we observed an increase in JNK phosphorylation in muscle of DIO mice, which was significantly reduced by diacerhein administration, suggesting that this reduction may have contributed to the improvement in the systemic insulin responsiveness.


Considering the reduced JNK total expression in diacerheintreated animals, we cannot exclude the possibility that diacerhein exerts a direct effect on JNK transcription and/or the degradation processes. Modest weight loss, achieved by diet and exercise, can enhance insulin sensitivity and even reverse insulin resistance (60 – 62). Many JNK inhibitors with greater potency and selectivity have been described (63, 64), and one of these has demonstrated beneficial effects on weight gain (63). However, 10 d treatment with diacerhein in mice did not cause any alteration in body weight, epididymal fat pad, or in the amount of food intake, which could explain the improvement in insulin sensitivity. Considering the differences in the caloric ingestion between all groups, we may hypothesize that diacerhein-treated animals may not have the same regulation of energy expenditure, although this point requires further investigation. In obesity, the analysis of macrophages isolated from adipose tissue demonstrates that they are responsible for most of the TNF-␣ tissue expression and significant

FIG. 8. Proposed mechanism by which diacerhein improves insulin sensitivity and glucose homeostasis. Diacerhein treatment for just 1 d induced a reduction in IL-6 mRNA levels in adipose tissue and also in serum IL-6 levels. Diacerhein administration for 10 d reduced serum levels of IL-6 and TNF-␣ and expression of TNF-␣, IL-6, and IL-1␤ in liver, muscle, and adipose tissue. In addition, it improved ER stress and reduced hepatic glucose production, fasting plasma glucose, and the expression and activity of proinflammatory mediators, accompanied by an improvement in insulin signaling mainly in the liver and adipose tissue. p, Phosphorylated.


amounts of IL-6, IL-1␤, and iNOS expression (38). Moreover, adipose tissue macrophage numbers increase in obesity, and these cells participate significantly in inflammatory pathways that are activated in obese individuals (40). In addition, many other studies have shown that a reduction in macrophage infiltration can decrease local inflammation in adipose tissue (65). In accordance with these data, our findings reveal that in the adipose tissue of DIO mice treated with diacerhein, in addition to the reduction in macrophage infiltration and decreased adipocytes size, there was lower expression of TNF-␣, IL-6, IL-1␤, and iNOS, providing evidence that this drug decreases local inflammation in the adipose tissue of obese mice. Furthermore, our results demonstrated that 10 d diacerhein treatment can reduce the mRNA levels of IL-1␤, IL-6, and TNF-␣ in both fractions of adipose tissue, SVF and adipocytes, although best results were observed in adipocytes. We extended this finding by demonstrating that 1 d diacerhein treatment can reduce the IL-6 mRNA levels only in adipocytes, accompanied by a reduction in the circulating levels of this cytokine. Thus, we hypothesized that the antiinflammatory effect observed in liver and muscle is probably a secondary systemic effect induced by this adipose tissue amelioration (Fig. 8). Lipid accumulation in the liver is a hallmark of high-fat diet-induced insulin resistance (57). Nonalcoholic fatty liver disease often accompanies abdominal adiposity, and its pathological spectrum ranges from simple steatosis to steatohepatitis, advanced fibrosis, and cirrhosis (41). Inflammation has been demonstrated to play a pivotal role in the progression of this disease process. In addition, gene disruption studies in mice have proven that interference in insulin signaling in hepatocytes activates fat-synthesizing enzymes in these cells and results in steatosis (57). Although in hepatic sections of DIO mice there was a large number of fat vesicles that were amply distributed across the parenchyma, mice treated with diacerhein presented liver parenchyma very similar to that of the controls, with no evidence of hepatic steatosis, in parallel to a marked reduction in TG content, in inflammatory pathways and cytokine expression in this tissue. Proinflammatory cytokines increase NO production via an increased expression of iNOS in the rat skeletal muscle and cultured myocytes and adipocytes (27). Therefore, we may speculate that the significant reduction of iNOS expression in the liver, muscle, and adipose tissue of diacerhein-treated groups may be the consequence of the reduction of cytokine expression in the tissues of these animals. In addition, it is known that iNOS induction and NO may be involved in the pathogenesis of obesity-linked T2D, affecting the insulin pathway through the S-nitrosation of proteins of its signaling cascade (27). For this


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reason, the improvement in insulin sensitivity may be also related to the reduced expression of iNOS found in the tissues of treated animals. These findings are supported by a previous study, demonstrating that genetic disruption of iNOS protects against obesity-linked insulin resistance (66). Aspirin and salicylates, whose beneficial effects in the treatment of diabetic patients have been known for decades (41, 47), not only are cyclooxygenase blockers but also function as IKK␤ inhibitors, improving the insulin sensitivity of obese mice (56, 67). However, the therapeutic usefulness of high-dose aspirin is limited by the antithrombotic and anti-platelet-aggregation effects, coupled with gastrointestinal irritation and unacceptably high risks of bleeding (41). In this context, diacerhein presents a better risk to benefit ratio than aspirin because it has the advantage of a higher gastric tolerance and rare side effects (68). In summary, our data show that the administration of the antiinflammatory diacerhein to animals on a high-fat diet reduced tissue expression and serum levels of IL-6 and TNF-␣, improved ER stress, and resulted in an improvement in glucose tolerance, a decrease in macrophage infiltration in adipocytes, and a reduced expression and activity of proinflammatory mediators accompanied by an improvement in insulin signaling mainly in the liver and adipose tissue. These results indicate that diacerhein treatment improves insulin sensitivity in obesity, mediated by the reversal of subclinical chronic inflammation, and that this drug may be an alternative therapy for insulin resistance.

Acknowledgments We thank L. Janieri, J. Pinheiro, T. Zanotto, and L. Weissmann (Department of Internal Medicine, UNICAMP, Campinas, Saõ Paulo) for their technical assistance. Address all correspondence and requests for reprints to: Mario J. A. Saad, M.D., Departamento de Clínica Me´dica, FCM (Faculdade de Ciências Médicas)-UNICAMP (Universidade Estadual de Campinas), Cidade Universita´ria Zeferino Vaz, Campinas, Saõ Paulo, Brazil, 13081-970. E-mail: [email protected]. N.T. researched data, contributed to discussion, and wrote, reviewed, and edited the manuscript; A.G.O. researched data and reviewed and edited the manuscript; D.G., R.A.B., G.Z.R., T.A., J.C.R.M., R.L.Z., and L.H.B.B.: researched data; J.B.C.C. and P.O.P. contributed to discussion and reviewed and edited the manuscript; and M.J.A.S. contributed to discussion and wrote, reviewed, and edited the manuscript.

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This work was supported by FAPESP (Fundaçaõ de Amparo a Pesquisa do Estado de Saõ Paulo) and INCT-CNPq (Instituto Nacional de Cieˆncia e Tecnologia-Conselho Nacional de Desenvolvimento Científico e Tecnolo´gico). Disclosure Summary: The authors have nothing to disclose.


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