4-HNE activates lipolysis in adipocytes.

<p>A, Fully-differentiated 3T3-L1 adipocytes were treated with 4-HNE (0, 10, 20, and 40 microM) for 4 hours, intracellular 4-HNE contents were determined by Western blot. B, Fully-differentiated 3T3-L1 adipocytes were stimulated with isoproterenol in the presence or absence of 4-HNE pretreatment for 5 hours. Glycerol and fatty acids releases were measured. All values are denoted as Means ± SD from three or more independent batches of cells. *p<0.05 and **p<0.01 vs. untreated cells. Bars with different characters differ significantly (p<0.05). C, Primary mouse adipocytes were freshly isolated from epididymal fat pad and stimulated with isoproterenol in the presence or absence of 4-HNE pretreatment for 5 hours. Glycerol releases were measured. All values are denoted as Means ± SD from three or more independent batches of cells. *p<0.05 and **p<0.01 vs. untreated cells. Bars with different characters differ significantly (p<0.05). D, Fully-differentiated 3T3-L1 adipocytes were treated with 40 microM 4-HNE for 5 hours. LDH activities in the media were measured. All values are denoted as Means ± SD from three or more independent batches of cells. *p<0.05 vs. untreated cells. E, Minced human visceral adipose tissue was treated with/without 40 microM 4-HNE for 4 hours and glycerol levels in the media were measured. All results are mean ± SD from at least three independent experiments (n ≥3), *p<0.05 vs. untreated adipose tissues.</p

4-HNE induces lipolysis via activating the cAMP/PKA signaling.

<p>A, Fully-differentiated 3T3-L1 cells were incubated with 4-HNE (20 and 40 microM) for 1 hour. Intracellular cAMP levels were measured by ELISA assay. All values are denoted as Means ± SD from three or more independent batches of cells. Bars with different characters differ significantly (p<0.05). B, Fully-differentiated 3T3-L1 cells were incubated with 4-HNE at 20 µM for indicated time points. Western blot was conducted to determine phosphorylated PKA and HSL protein abundance. The degree of changes (fold) was calculated as the optimal density ratio of the phosphorylated target protein to the corresponding total protein, normalized to the control cells. C, Fully-differentiated 3T3-L1 cells were pre-incubated with H89 (5 microM) for 2 hours before 4-HNE exposure (20 microM). Glycerol releases were measured 16 hours later. All values are denoted as Means ± SD from three or more independent batches of cells. Bars with different characters differ significantly (p<0.05).</p

Inhibition of AMPK contributes 4-HNE-induced lipolytic response.

<p>A, Fully-differentiated 3T3-L1 cells were treated with exogenous 4-HNE (20 microM) in the presence or absence of AICAR inclusion in the media for 2 hours. AMPK phosphorylation and HSL phosphorylation at serine 565 were examined. The degree of changes (fold) was calculated as the optimal density ratio of the phosphorylated target protein to either actin or the corresponding total protein, normalized to the control cells. B, Fully-differentiated 3T3-L1 cells were treated with exogenous 4-HNE (20 microM) in the presence or absence of AICAR inclusion in the media for 2 hours. AMPK activities were determined by ELISA kit. All values are denoted as Means ± SD from three or more independent batches of cells. Bars with different characters differ significantly (p<0.05). C, Fully-differentiated 3T3-L1 adipocytes were first treated with AICAR, an AMPK inducer, for 2 hours before 4-HNE exposure. Glycerol release with/without isoproterenol stimulation was determined 5 hours later. All values are denoted as Means ± SD from three or more independent batches of cells. Bars with different characters differ significantly (p<0.05).</p

Effects of mangiferin on the proteins expression of FFA metabolism including AMPK, CD36, CPT1, ACC and DGAT2 in liver of hyperlipidemic rats.

<p>Wistar rats were divided randomly into five groups (n = 10 per group): control group (fed an AIN-93G diet); hyperlipidemia group (fed a high-fat diet); mangiferin-supplemented groups, fed the high-fat diet and different doses of mangiferin (50, 100, 150 mg/kg BW/d). The experiment lasted for 6 weeks, and the liver was taken for western blot analysis. (A) AMPK phosphorylation level. (B) CD36 expression on cell membrane. (C) CPT1 expression in mitochondrion. (D) ACC level and activity. (E) DGAT2 expression. * P<0.05 compared with hyperlipidemic group.</p

Effects of mangiferin on the ratio of AMP to ATP and LKB1 protein expression in HepG2 cells.

<p>HepG2 cells were incubated to 0.2 mmol/L oleic acid only or with different concentrations of mangiferin (12.5, 25, 50, 100 µmol/L) simultaneously for 24 h. The ratio of AMP to ATP was detected by HPLC (A). The LKB1 protein expression was carried out by western blot analysis (B). The experiments were repeated 3 times. Data are presented as means ± SD (n = 3). ^*<i>P</i><0.05 compared with only OA stimulation group.</p

HepG2 cells were incubated with 0.2 mmol/L OA only or with different concentrations of mangiferin (12.5, 25, 50, 100 µmol/L) simultaneously for 24 h.

<p>Proteins were isolated from the cell lysates and analyzed by western blot analysis for AMPK (A), CD36 (B), CPT1 (C), ACC (D) and DGAT (E) expressions. The experiments were repeated 3 times. Data are presented as means ± SD (n = 3). ^*<i>P</i><0.05 compared with only OA stimulation group.</p

MAP kinases activation is not involved in 4-HNE-elicited lipolytic response.

<p>A, Fully-differentiated 3T3-L1 cells were treated with exogenous 4-HNE (20 microM) for indicated time points. The activations of the MAP kinases, including ERK1/2, p38, and JNK, were detected by immunoblotting. B, Fully-differentiated 3T3-L1 cells were pretreated with specific kinase inhibitors for ERK1/2 (U0126, 10 microM), JNK (SP600125, 10 microM), and p38 (SB203580, 10 microM) for 1 hours, followed by exogenous 4-HNE (20 microM) exposure. Glycerol releases were measured 6 hours later. All values are denoted as Means ± SD from three or more independent batches of cells. Bars with different characters differ significantly (p<0.05).</p

Effect of mangiferin on fasting metabolic variables at 6 weeks in hyperlipemic rats.

<p>Data are means ± SD (n = 10),</p>#<p><i>P</i><0.05</p>##<p><i>P</i><0.01 indicate statistically significant differences when compared with control group.</p><p>*<i>P</i><0.05 and</p><p>**<i>P</i><0.01 indicate statistically significant differences when compared with hyperlipidemia group.</p

Schematic representation of the proposed mechanism underlying 4-HNE-induced enhancement of lipolytic response in adipocytes.

<p>4-HNE-triggered lipolysis in adipocytes involves the activation of the cAMP/PKA/HSL pathway as well as the suppression of the AMPK pathway. Both AC activation and PDE3B inhibition potentially contribute to 4-HNE-induced increase of intracellular cAMP levels. PKA, protein kinase A; HSL, hormone-sensitive lipase; AMPK, AMP-activated protein kinase; P, phosphorylation; TG, triglyceride; FFA, free fatty acids; AC, adenylyl cyclase; PDE3B, phosphodiesterase 3B; ? hypothetic mechanism.</p

Increased triglyceride accumulation in adipocyte is associated with increased intracellular 4-HNE contents.

<p>A, Adipogenesis in 3T3-L1 fibroblasts was induced by standard adipogenesis-inducing mix. Intracellular 4-HNE-protein adducts formation were detected by Western blot on day 3, 5, 7, and 13 during the process of adipogenesis. Significant elevations in intracellular 4-HNE contents were observed on day 7 (D7) in comparison to these at day 3 and 5, and the elevations continued to day 13 (D13). B, Adipogenesis in 3T3-L1 fibroblasts was induced by standard adipogenesis-inducing mix. Time-course changes of intracellular triglyceride (TG) levels were determined on day 0, 3, 5, 7, and 13 during the process of adipogenesis. All values are denoted as Means ± SD from three or more independent batches of cells. *p<0.05 vs. D0. C & D, Oleate, at the concentration of 0.2 mM was next added into the culture medium on day 6 after the induction of adipogenesis and TG contents were determined 24 hours later (day 7) by Oil Red O Staining and biochemical assay in adipocytes as described in the materials and method. UT, untreated 3T3-L1 cells (day6); OA, Oleate. All values are denoted as Means ± SD from three or more independent batches of cells. *p<0.05 vs. D6. D, Oleate, at the concentration of 0.2 mM was next added into the culture medium on day 6 after the induction of adipogenesis and intracellular 4-HNE adducts formation was determined by Western blot on day 7. UT, untreated 3T3-L1 cells (day6); OA, Oleate.</p