A new antidiabetic compound attenuates inflammation and insulin resistance in Zucker diabetic fatty rats

Abstract

Tissue macrophage inflammatory pathways contribute to obesity-associated insulin resistance. Here, we have examined the efficacy and mechanisms of action of a novel anti-inflammatory compound (HE3286) in vitro and in vivo. In primary murine macrophages, HE3286 attenuates LPS- and TNFalpha-stimulated inflammation. In Zucker diabetic fatty rats, inflammatory cytokine/chemokine expression was downregulated in liver and adipose tissue by HE3286 treatment, as was macrophage infiltration into adipose tissue. In line with reduced inflammation, HE3286 treatment normalized fasting and fed glucose levels, improved glucose tolerance, and enhanced skeletal muscle and liver insulin sensitivity, as assessed by hyperinsulinemic euglycemic clamp studies. In phase 2 clinical trials, HE3286 treatment led to an enhancement in insulin sensitivity in humans. Gluconeogenic capacity was also reduced by HE3286 treatment, as evidenced by a reduced glycemic response during pyruvate tolerance tests and decreased basal hepatic glucose production (HGP) rates. Since serum levels of gluconeogenic substrates were decreased by HE3286, it indicates that the reduction of both intrinsic gluconeogenic capacity and substrate availability contributes to the decrease in HGP. Lipidomic analysis revealed that HE3286 treatment reduced liver cholesterol and triglyceride content, leading to a feedback elevation of LDL receptor and HMG-CoA reductase expression. Accordingly, HE3286 treatment markedly decreased total serum cholesterol. In conclusion, HE3286 is a novel anti-inflammatory compound, which displays both glucose-lowering and cholesterol-lowering effects.

Fig. 1.

HE3286 attenuates LPS-induced inflammation in murine macrophages. A: murine intraperitoneal macrophages (IP Mac) pretreated with HE3286 (100 nM) or vehicle (DMSO) were treated with 100 ng/ml LPS for the times indicated. Relative phosphoproteins levels normalized to respective kinase or β-tubulin are shown as means ± SD. B: phosphoproteins and total proteins were detected by immunoblotting. C: IP Mac pretreated with HE3286 (100 nM) or DMSO for overnight were stimulated with 50 ng/ml LPS for 6 h. Total RNA extracted from cells was subjected to quantitative nuclease protection assay (qNPA) analysis. Data shown are the fold induction of gene expression normalized with housekeeping genes (means ± SD) from 3 experiments. *Statistical significance (P < 0.05) between conditions connected by bars.

Fig. 2.

HE3286 ameliorate TNFα-induced inflammation in murine macrophages A: murine IP Mac pretreated with HE3286 (100 nM) or vehicle (DMSO) were treated with 100 ng/ml LPS for the times indicated. Relative phosphoprotein levels normalized to respective kinase or β-tubulin are shown as means ± SD. B: phosphoproteins and total proteins were detected by immunoblotting. C: IP Mac pretreated with HE3286 (100 nM) or DMSO were stimulated with 10 ng/ml TNFα for 3 h. Total RNA extracted from cells was subjected to qNPA analysis. Data shown are the fold induction of gene expression normalized with housekeeping genes (means ± SD) from 3 experiments. *Statistical significance (P < 0.05) between conditions connected by bars.

Fig. 3.

HE3286 impairs adipocyte conditioned media-induced macrophage chemotaxis. A: 3T3-L1 adipocytes were treated with vehicle and TNFα (10 ng/ml) in the absence or presence of HE3286 (100 nM). Conditioned media were then collected to induce chemotaxis of suspended RAW 264.7 macrophages. Migrated RAW 264.7 cells were quantified, and data are shown as means ± SE. B and C: 3T3-L1 adipocytes were treated with vehicle and TNFα (10 ng/ml) in the absence or presence of HE3286 (100 nM). Conditioned media were collected to induce cytokine release from RAW 264.7 macrophages. Concentrations of monocyte chemoattractant protein-1 (MCP-1; B) and regulated upon activation, normal T cell expressed and secreted (RANTES; C) in adipocyte-conditioned media were measured by ELISA. Data are shown as means ± SD. *Statistical significance (P < 0.05) between conditions connected by bars.

Fig. 4.

HE3286 improves glucose tolerance in Zucker diabetic fatty (ZDF) rats. Eight-week-old male ZDF rats were administered orally with vehicle (n = 12), HE3286 (100 mg·kg⁻¹·day⁻¹; n = 11), or rosiglitazone (Rosi; 10 mg·kg⁻¹·day⁻¹; n = 8) for 30 days. On day 7, glucose (A) and insulin levels (B) were measured at fed, 6-h fasting, and refed state (6-h refeeding after overnight fasting). C: on day 14, rats were subjected to oral glucose tolerance tests (OGTT). D: on day 21, rats were subjected to insulin tolerance tests (ITT). Data shown in D are the percentages compared with glucose levels at 0 min. E and F: on day 14, plasma glycerol (E) and free fatty acid (F) levels were measured by colorimetric assay kits. For A, B, E, and F: *statistical significance vs. vehicle-treated rats (P < 0.05); †P < 0.01; ‡P < 0.001. For C and D: *statistical significance (P < 0.05) between vehicle-treated and HE3286-treated rats; †statistical significance (P < 0.05) between vehicle-treated and Rosi-treated rats. All data are shown as means ± SE.

Fig. 5.

HE3286 enhances insulin sensitivity in vivo. A–D: ZDF rats treated with vehicle (n = 14), HE3286 (n = 11), or Rosi (n = 8) for 4 wk were subjected to euglycemic hyperinsulinemic clamps. Glucose infusion rate (GIR; A), insulin-stimulated glucose disposal rate (GDR; B), basal hepatic glucose production (HGP; C), and HGP suppression by insulin (D) are shown as means ± SE. Statistical significance vs. vehicle-treated rats is indicated by *P < 0.05, †P < 0.01, or ‡P < 0.001. E: protein extracted from the skeletal muscle of treated rats was subjected to SDS-PAGE. Phospho-Akt (Ser⁴⁷³) was detected by immunoblotting. Relative protein levels normalized to respective kinase are shown as means ± SE. *Statistical significance (P < 0.05) between conditions connected by bars.

Fig. 6.

HE3286 enhances insulin sensitivity in humans. A: clinical characteristics of obese, insulin-resistant subjects treated with HE3286 (5 mg BID, n = 5, or 10 mg BID, n = 5; total, n = 10) for 4 wk. B: whole body insulin sensitivity was determined by hyperinsulinemic euglycemic clamps, and the results are expressed in terms of M values. Statistical significance was calculated via a paired (2-tailed) t-test.

Fig. 7.

HE3286 regulates gene expression involved in inflammation and metabolism. A: tissue sections of epididymal white adipose tissue were subjected to Mac-2 staining (brown). B: total RNA extracted from epididymal white adipose tissue of treated ZDF rats was subjected to quantitative PCR analysis. Data shown are the fold induction of gene expression normalized with housekeeping genes and expressed as means ± SE. C: serum levels of TNFα and IL-1 were measured by ELISA. Data are shown as means ± SE. Statistical significance vs. vehicle-treated rats is indicated by *P < 0.05, †P < 0.01, or ‡P < 0.001.

Fig. 8.

HE3286 regulates lipid metabolism in vivo. A and B: lipid classes in the liver (A) and serum (B) were quantified by lipidomic analysis. Data are expressed as means ± SE. C: protein extracted from the livers of treated rats was subjected to SDS-PAGE. Sterol regulatory element-binding protein-2 (SREBP-2) precursor (P) and mature form (M) were detected by immunoblotting. Culture cells infected with adenovirus expressing SREBP-2 NH₂ terminus (Ad-2N) were used as a positive control for blotting. D: total RNA extracted from the liver of treated ZDF rats was subjected to quantitative PCR analysis. Data shown are the fold induction of gene expression normalized with housekeeping genes and expressed as means ± SE. Statistical significance vs. vehicle-treated rats is indicated by *P < 0.05, †P < 0.01, or ‡P < 0.001. TAG, triacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; CE, cholesteryl ester; FC, free chholesterol; TC, total cholesterol.

Fig. 9.

HE3286 modulates fatty acid composition in vivo. A and B: mole percentages (nmol%) of palmitate (16:0), palmitoleate (16:1n7), linoleic acid (18:2n6), α-linolenic acid (18:3n3), arachidonic acid (20:4n6), eicosapentaenoic acid (20:5n3), and docosahexaenoic acid (22:6n3) in the liver (A) and white adipose tissue (B) were measured. C and D: nmol% of palmitoleate in lipid classes (16:1n7 compared with total fatty acids in indicated lipid classes) were calculated in serum (C) and muscle (D). Data are expressed as means ± SE. Statistical significance vs. vehicle-treated rats is indicated by *P < 0.05, †P < 0.01, or ‡P < 0.001. CL, cardiolipin; DAG, diacylglycerol; FFA, free fatty acid; LYPC, lysophosphatidylcholine; PS, phosphatidylserine.