Hematopoietic-Derived Galectin-3 Causes Cellular and Systemic Insulin Resistance

Abstract

In obesity, macrophages and other immune cells accumulate in insulin target tissues, promoting a chronic inflammatory state and insulin resistance. Galectin-3 (Gal3), a lectin mainly secreted by macrophages, is elevated in both obese subjects and mice. Administration of Gal3 to mice causes insulin resistance and glucose intolerance, whereas inhibition of Gal3, through either genetic or pharmacologic loss of function, improved insulin sensitivity in obese mice. In vitro treatment with Gal3 directly enhanced macrophage chemotaxis, reduced insulin-stimulated glucose uptake in myocytes and 3T3-L1 adipocytes and impaired insulin-mediated suppression of glucose output in primary mouse hepatocytes. Importantly, we found that Gal3 can bind directly to the insulin receptor (IR) and inhibit downstream IR signaling. These observations elucidate a novel role for Gal3 in hepatocyte, adipocyte, and myocyte insulin resistance, suggesting that Gal3 can link inflammation to decreased insulin sensitivity. Inhibition of Gal3 could be a new approach to treat insulin resistance.

Keywords: galectin-3; inflammation; insulin resistance.

Figure 1. Metabolic Studies in WT and Gal3 KO or Gal3 HET Mice

(A) Body weight, (B) Glucose tolerance tests, (C) basal insulin levels, and (D) Insulin tolerance tests in 6-hr fasted WT and Gal3 KO mice on HFD. (E) Glucose infusion rate (GIR) during hyperinsulinemic, euglycemic glucose clamp studies, (F) Glucose disposal rate (GDR), (G) Insulin-stimulated glucose disposal rate (IS-GDR), (H) Hepatic glucose production (HGP), and (I) HGP suppression during hyperinsulinemic euglycemic clamp studies in WT and Gal3 KO mice on HFD. (J) Blood Gal3 levels in WT, Gal3 HET and Gal3 KO mice on HFD. (K) Body weight of WT and Gal3 HET mice on 10 weeks HFD, (L) Insulin tolerance tests (0.45 U/kg), (M) Glucose tolerance tests, and (N) Insulin levels during glucose tolerance tests in WT and Gal3 HET mice on HFD. Values are expressed as mean ± SEM. n=8-10 in each group, * P<0.05, ** P<0.01 for KO versus WT (B-J), or HET versus WT (J-N). See also Figures S1 & S2.

Figure 2. Obesity Increases Circulating Gal3 Levels and Gal3 Induces Chemotaxis

(A) Blood Gal3 levels in chow and HFD fed mice. (B) Gal3 levels in clodronate or vehicle treated HFD mice. (C) Blood Gal3 levels in lean and obese human subjects. (D) Correlation analysis between Gal3 and BMI in human subjects. (E) Correlation analysis between Gal3 and HOMA-IR in human subjects. (F) In vitro macrophage chemotaxis stimulated by Gal3. (G) In vivo macrophage tracking experiments using normal WT donor monocytes and WT or Gal3 KO recipient mice. FACS analysis of labeled PKH26+, F4/80+, CD11b+ macrophages accumulating in recipient Epi-WAT (left panel) and liver (right panel). (H) FACS analysis of total F4/80+, Cd11b+ and (I) total F4/80+, CD11b+, CD11c+ adipose tissue macrophages in Epi-WAT of WT and KO mice on HFD. (J) Inflammatory gene expression in Epi-WAT. Values are expressed as mean ± SEM. n=8-10 in A, B and J, n=4-5 in F-I. * P<0.05, ** P<0.01 for KO versus WT. See also Figures S3 & S4 and Table S1.

Figure 3. Gal3 Directly Induces Cellular Insulin Resistance

(A) Dose response effect of Gal3 on insulin-stimulated glucose uptake in L6 myocytes. (B) Dose response effect of Gal3 on insulin stimulated glucose uptake in 3T3-L1 differentiated adipocytes. (C) Effect of Gal3 on gluconeogenesis in primary mouse hepatocytes. (D) Effect of CRD-Gal3 on insulin stimulated glucose uptake in L6 myocytes. (E) Circulating Gal3 levels after injection of 0.2 mg/kg recombinant Gal3. (F) Effect of exogenous Gal3 treatment (0.2 mg/kg) on glucose tolerance, (G) glucose-induced insulin secretion, (H) and insulin tolerance in chow fed insulin sensitive mice. Effect of Ad-Gal3 on glucose tolerance, (I) insulin tolerance, (J) and insulin levels (K) in chow fed mice. (L) Effect of CM from IPMacs on insulin stimulated glucose uptake in L6 myocytes. Values are expressed as mean ± SEM. n=8-10 in each group, * P<0.05, ** P<0.01. See also Figure S5.

Figure 4. In Vitro Gal3 Treatment Impairs Insulin Signaling

(A) Gal3 impairs insulin-stimulated IR tyrosine phosphorylation as shown by IP and IB for IRβ in L6 myocytes and (B) 3T3-L1 adipocytes. (C) Gal3 impairs insulin-stimulated IRS1 tyrosine phosphorylation as shown by ELISA and (D) WB in L6 myocytes. (E) Gal3 impairs insulin-stimulated IRS1 tyrosine phosphorylation as shown by ELISA and (F) Western blot (WB) in 3T3-L1 adipocytes. (G) Gal3 impairs insulin-stimulated p-PDK1 and (H) p-Akt in 3T3-L1 adipocytes. (I) Gal3 decreases insulin-stimulated Glut4 translocation as measured by the by sheet assay and (J) immunofluorescence Glut4 staining ring assay in 3T3-L1 adipocytes. Values are expressed as mean ± SEM. * P<0.05, ** P<0.01. See also Figure S6.

Figure 5. Gal3 Interacts With the Insulin Receptor and does not Interfere with Insulin Binding

(A) Co-immunoprecipitation of Bio-Gal3 with the IR, but not IRS1, at 37°C in L6 myocytes. (B) Co-immunoprecipitation of Bio-Gal3 with the IR at 16°C using 3T3-L1 adipocytes. (C) Immunoblotting of IR after bead capture of Bio-Gal3 from L6 myocytes or 3T3-L1 adipocytes treated with Bio-Gal3. (D) Comparable effect of Bio-Gal3 and recombinant Gal3 to inhibit insulin-stimulated glucose uptake in L6 myocytes. (E) Insulin binding to 3T3-L1 adipocytes in the presence of 200 ng/ml Gal3. (F) Insulin binding to primary hepatocytes in presence of 200mg/ml Gal3. Values are expressed as mean ± SEM.

Figure 6. Metabolic Studies in Chimeric BMT Gal3−/− mice

(A) Blood Gal3 levels. (B) Body weight curves. (C) Glucose tolerance tests. (D) Insulin tolerance tests. (E) Basal insulin levels in WT and Gal3 KO BMT mice on HFD. Hyperinsulinemic, euglycemic glucose clamp studies performed in WT and KO BMT mice on HFD for measurements of (F) GIR, (G) IS-GDR, (H) HGP, and (I) HGP suppression during the clamp study. Values are expressed as mean ± SEM. and n=8-10 in each group, *P < 0.05, **P < 0.01.

Figure 7. Gal3 Inhibitor Improves Insulin Sensitivity

(A) The Gal3 inhibitor (compound 47) mitigates the Gal3 and (B) CRD Gal3-induced decrease in insulin stimulated glucose transport in L6 myocytes. (C) The Gal3 inhibitor blocks Gal3 and (D) CRD Gal3-stimulated HGO in primary hepatocytes. (E) Single dose treatment with the Gal3 inhibitor (50 mg/kg, intraperitoneal injection) improves glucose tolerance in HFD mice. (F) Two week treatment with the Gal3 inhibitor (6.4 mg/kg) via an osmotic mini pump improves glucose tolerance in HFD mice. Values are expressed as mean ± SEM, n=8-10 in each group, *P<0.05, **P<0.01.