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. 2015 Nov 23;10(11):e0141612.
doi: 10.1371/journal.pone.0141612. eCollection 2015.

Reduction of Adipose Tissue Mass by the Angiogenesis Inhibitor ALS-L1023 from Melissa officinalis

Byung Young Park  1   2 , Hyunghee Lee  3 , Sangee Woo  3 , Miso Yoon  3 , Jeongjun Kim  3 , Yeonhee Hong  3 , Hee Suk Lee  2 , Eun Kyu Park  2 , Jong Cheon Hahm  2 , Jin Woo Kim  1 , Soon Shik Shin  4 , Min-Young Kim  2 , Michung Yoon  3
Affiliations

Reduction of Adipose Tissue Mass by the Angiogenesis Inhibitor ALS-L1023 from Melissa officinalis

Byung Young Park et al. PLoS One. .

Abstract

It has been suggested that angiogenesis modulates adipogenesis and obesity. This study was undertaken to determine whether ALS-L1023 (ALS) prepared by a two-step organic solvent fractionation from Melissa leaves, which exhibits antiangiogenic activity, can regulate adipose tissue growth. The effects of ALS on angiogenesis and extracellular matrix remodeling were measured using in vitro assays. The effects of ALS on adipose tissue growth were investigated in high fat diet-induced obese mice. ALS inhibited VEGF- and bFGF-induced endothelial cell proliferation and suppressed matrix metalloproteinase (MMP) activity in vitro. Compared to obese control mice, administration of ALS to obese mice reduced body weight gain, adipose tissue mass and adipocyte size without affecting appetite. ALS treatment decreased blood vessel density and MMP activity in adipose tissues. ALS reduced the mRNA levels of angiogenic factors (VEGF-A and FGF-2) and MMPs (MMP-2 and MMP-9), whereas ALS increased the mRNA levels of angiogenic inhibitors (TSP-1, TIMP-1, and TIMP-2) in adipose tissues. The protein levels of VEGF, MMP-2 and MMP-9 were also decreased by ALS in adipose tissue. Metabolic changes in plasma lipids, liver triglycerides, and hepatic expression of fatty acid oxidation genes occurred during ALS-induced weight loss. These results suggest that ALS, which has antiangiogenic and MMP inhibitory activities, reduces adipose tissue mass in nutritionally obese mice, demonstrating that adipose tissue growth can be regulated by angiogenesis inhibitors.

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Conflict of interest statement

Competing Interests: HL, SW, MY, and JK received support in the form of salaries from AngioLab Inc. This does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Inhibitory effects of ALS on angiogenesis and MMP activity.
(A) Inhibition of VEGF-induced HUVEC proliferation by ALS. (B) Inhibition of bFGF-induced HUVEC proliferation by ALS. #p<0.05 compared to vehicle control, *p<0.05 compared to VEGF- or bFGF-treated control group. (C) The effect of ALS on HUVEC cell viability by MTT assay. * p<0.05 compared to the vehicle control. ALS-mediated inhibition of (D) MMP-2 and (E) MMP-9 activity measured by spectrofluorometry and the IC50 values were determined. * p<0.05 compared to the vehicle control.
Fig 2
Fig 2. Regulation of body weight gain, adipose tissue mass and food consumption in high fat diet-fed obese mice.
Adult male mice were fed a standard chow diet, a high fat diet, or high fat diet supplemented with 0.4 or 0.8% ALS for 8 weeks. (A) Modulation of body weight gain by ALS. Body weight gains at the end of the treatment period are significantly different between the chow diet group and the high fat diet group (#p<0.05) and between the high fat diet group and the groups fed a high fat diet supplemented with 0.4 or 0.8% ALS (*p<0.05). Regulation of VSC (B) and SC (C) fat mass by ALS. (D) Effects of ALS on food intake. (E) Appetite of ALS-treated mice. We fed the amount consumed per day by each treated mouse to a paired mouse and measured body weights daily. (F) Organ weights for liver, kidney, heart and pancreas. All values are expressed as the mean ± SD. #p<0.05 compared to the chow group, *p<0.05 compared to the high fat group.
Fig 3
Fig 3. Light microscopy analysis of adipocyte size in adipose tissue.
Adult male mice were fed a standard chow diet, a high fat diet, or the same high fat diet supplemented with 0.4 or 0.8% ALS for 8 weeks. Representative H&E-stained sections (5-μm thick) of (A) epididymal VSC and (B) inguinal SC adipose tissues are shown (original magnification ×100). Adipocyte size in the high fat diet plus ALS groups was smaller compared to the high fat diet groups. The size of adipocytes in a fixed area (1,000,000 μm2) was measured. All values are expressed as the mean ± SD. #p<0.05 compared to the chow group, *p<0.05 compared to the high fat group.
Fig 4
Fig 4. Histological analysis of blood vessels in adipose tissue stained with an antibody against vWF.
The blood vessels of (A) epididymal VSC and (B) inguinal SC adipose tissues derived from mice fed a high fat diet or a high fat diet supplemented with 0.8% ALS for 8 weeks were stained and analyzed (original magnification ×100). Higher magnitude of bracket area is shown (original magnification ×200). All values are expressed as the mean ± SD. *p<0.05 compared to the high fat group.
Fig 5
Fig 5. Zymographic analysis of adipose tissue.
Extracts from (A) epididymal VSC and (B) inguinal SC adipose tissues obtained from mice fed a high fat diet or a high fat diet supplemented with 0.4 and 0.8% ALS for 8 weeks were applied to a gelatin-containing gel. Gelatinolytic activity was measured by zymography. The HT1080 cell culture medium was used for the molecular weight markers for MMP-2 and MMP-9. All values are expressed as the mean ± SD. #p<0.05 compared to the chow group, *p<0.05 compared to the high fat group. M, molecular weight marker.
Fig 6
Fig 6. Effects of ALS on the expression of angiogenic factors, MMPs and their inhibitors in epididymal VSC and inguinal SC adipose tissues of diet-induced obese mice.
Adult male mice were fed a high fat diet or a high fat diet supplemented with 0.8% ALS for 8 weeks. Analysis of VEGF-A, FGF-2, MMP-2, MMP-9, TIMP-1, TIMP-2 and TSP-1 mRNA levels by RT-PCR in epididymal VSC (A) and inguinal SC (B) adipose tissues. Representative PCR bands from one of three independent experiments are shown. All values are expressed as the mean ± SD. *p<0.05 compared to the high fat group.
Fig 7
Fig 7. Effects of ALS on the protein expression of VEGF, MMP-2 and MMP-9 in epididymal VSC and inguinal SC adipose tissues of diet-induced obese mice.
The protein levels of epididymal VSC and inguinal SC adipose tissues derived from mice fed a high fat diet or a high fat diet supplemented with 0.8% ALS for 8 weeks were analyzed by Western blotting. Representative bands from one of three independent experiments are shown.
Fig 8
Fig 8. Effects of ALS on plasma lipid levels, hepatic lipid accumulation and liver PPARα target gene expression in diet-induced obese mice.
Adult male mice were fed a high fat diet or a high fat diet supplemented with 0.8% ALS for 8 weeks. Plasma levels of (A) triglycerides and (B) free fatty acids. (C) Histological analyses of hepatic lipid accumulation. Pathological scores of hepatic lipid accumulation are as follows: 0, no lesion; 1, mild; 2, moderate; 4, very severe. (D) Representative H&E-stained liver sections are shown (original magnification ×100). (E) Modulation of liver PPARα target gene expression. All values are expressed as the mean ± SD. *p<0.05 compared to the high fat group.
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Grants and funding

This work was supported by the National Research Foundation of Korea [http://www.nrf.re.kr/] Grants funded by the Korea Government (MEST) (2012R1A1A3002100, 2012R1A2A2A01004508, and 2015R1A1A3A04001016), Korea. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funder AngioLab Inc provided support in the form of salaries for authors [HL SW MY JK] and reagent materials, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.