The L-4F mimetic peptide prevents insulin resistance through increased levels of HO-1, pAMPK, and pAKT in obese mice

Abstract

We examined mechanisms by which L-4F reduces obesity and diabetes in obese (ob) diabetic mice. We hypothesized that L-4F reduces adiposity via increased pAMPK, pAKT, HO-1, and increased insulin receptor phosphorylation in ob mice. Obese and lean mice were divided into five groups: lean, lean-L-4F-treated, ob, ob-L-4F-treated, and ob-L-4F-LY294002. Food intake, insulin, glucose adipocyte stem cells, pAMPK, pAKT, CB1, and insulin receptor phosphorylation were determined. Subcutaneous (SAT) and visceral adipose tissue (VAT) were determined by MRI and hepatic lipid content by magnetic resonance spectroscopy. SAT and VAT volumes decreased in ob-L-4F-treated animals compared with control. L-4F treatment decreased hepatic lipid content and increased the numbers of small adipocytes (P < 0.05) and phosphorylation of insulin receptors. L-4F decreased CB1 in SAT and VAT and increased pAKT and pAMPK in endothelium. L-4F-mediated improvement in endothelium was prevented by LY294002. Inhibition of pAKT and pAMPK by LY294002 was associated with an increase in glucose levels. Upregulation of HO-1 by L-4F produced adipose remodeling and increased the number of small differentiated adipocytes. The anti-obesity effects of L-4F are manifested by a decrease in visceral fat content with reciprocal increases in adiponectin, pAMPK, pAKT, and phosphorylation of insulin receptors with improved insulin sensitivity.

Fig. 1.

Effect of L-4F on body weight and visceral fat content in lean and ob mice. A: Effect of L-4F administration on body appearance. Examples of subcutaneous and visceral fat (B) and dissected fat (C) from lean and ob mice. Representative photographs showing one mouse from each group after 6 weeks of treatment (n = 8).

Fig. 2.

Effect of L-4F treatment on Global SAT and VAT as measured. A: Effect of L-4F treatment on global SAT and VAT as measured by MRI. B: Global fat was increased in ob mice compared with ob-L-4F-treated mice (* P < 0.01, n = 3).

Fig. 3.

Effect of L-4F on gastrocnemius muscle fibril diameter and muscle weight. A: Hematoxylin eosin staining of gastrocnemius and Oil Red O staining within myofibril interstitial spaces in muscle cross section. Small arrow indicates extracellular fat deposits and large bar indicates intracellular fat deposits. Lean (A), ob fat (B), ob fat L-4F (C). A: The mean diameter of myofibril of the lean versus obese (P < 0.05). B: Muscle fibril diameter, arrow in lean, ob, and ob-L4F-treated mice. C: Muscle weight was measured as described in Materials and Methods. The results are expressed as mean ± SEM, n = 4. D: Oil Red O muscle fat staining showing increased staining in ob mice and not reduced by L-4F (*P < 0.005).

Fig. 4.

Effect of L-4F on Subcutaneous (A) and visceral (B) fat in ob mice. Haematoxylin-eosin staining of subcutaneous (A) and visceral (B) fat in ob and L-4F-treated ob mice. Bars = 50 μm. Quantitative analysis of adipocyte size in subcutaneous fat and visceral fat surrounding the aorta of ob or L-4F-treated ob mice is displayed. Data are expressed as means ± SD (* P < 0.05 vs. ob).

Fig. 5.

Expression of CB-1 expression in subcutaneous fat and visceral fat surrounding aorta of ob mice. A: IOD of CB-1 expression in subcutaneous fat and visceral fat surrounding aorta of ob or L-4F-treated ob mice is displayed. Data are expressed as mean ± SD (* P < 0.05 vs. obese mice). B: Effect of L-4F on CB1 levels in SAT and VAT.

Fig. 6.

Effect of L-4F on HO-1 expression in subcutaneous fat and visceral fat surrounding aorta of ob mice. A: Immunohistochemistry staining for HO-1 of subcutaneous fat and visceral fat surrounding aorta of ob or L-4F-treated ob mice. Arrows indicate HO-1 immunoreactivity. Bars = 50 μm. B: IOD of HO-1 expression in subcutaneous fat and visceral fat surrounding aorta of ob or L-4F-treated ob mice. Data are expressed as means ± SD (* P < 0.05 SAT and VAT).

Fig. 7.

Effect of L-4F on percentage of fat in the liver of ob mice measured by MRS. Analysis of lipid content percentage of fat was calculated as described in Materials and Methods (*P < 0.01 vs. obese, n = 3).

Fig. 8.

Effect of L-4F on insulin receptor phosphorylation. A: Western blot and densitometry analysis of insulin receptor phosphorylation and actin proteins in liver of lean, ob, and ob-treated with L-4F. B: Quantitative densitometry evaluation of P-Tyr 972 and Tyr-1146 and actin proteins ratio was determined. Representative immunoblots are shown (n = 5).

Fig. 9.

Effect of inhibition of pAKT by LY294002 on glucose tolerance and signaling. A: Effect of LY294002 [three times/week for 3 weeks, administered by intraperitoneal injection on glucose tolerance (IPGTT)] tests in obese, L-4F obese, was performed as described in Materials and Methods. The results are expressed mean ± SEM, n = 3. B: Western blot of HO-1, adiponectin, pAKT, pAMPK, and actin proteins in kidney of ob, ob-treated with L-4F, and ob-treated with L-4F and LY294002. Representative immunoblots are shown (n = 4).

Fig. 9.

Effect of inhibition of pAKT by LY294002 on glucose tolerance and signaling. A: Effect of LY294002 [three times/week for 3 weeks, administered by intraperitoneal injection on glucose tolerance (IPGTT)] tests in obese, L-4F obese, was performed as described in Materials and Methods. The results are expressed mean ± SEM, n = 3. B: Western blot of HO-1, adiponectin, pAKT, pAMPK, and actin proteins in kidney of ob, ob-treated with L-4F, and ob-treated with L-4F and LY294002. Representative immunoblots are shown (n = 4).

Fig. 10.

Effect of LY294002 on L-4F-mediated MSC-derived adipogenesis. A: Scan of lipid size of a representative experiment. B: Adipogenesis was measured as the relative absorbance of Oil Red O at day 10 after inducing adipogenesis as described in Materials and Methods. C: Measurement of number of lipid droplet size. D: Comparison of the effect of L-4F, LY294002, on number of lipid droplet sizes. Results are mean ± SEM, n = 4 (*P < 0.05 vs. control medium; **P < 0.02 vs. LY294002 or control culture).