Processed Aloe vera gel (PAG) (20), Aloesin (ALS), Aloe QDM, and Aloe QDM complex (21) were provided by Univera, Inc. (Seoul, Republic of Korea). Pioglitazone (Actos®) was purchased from Eli Lilly (Toronto, Canada); chromium (Cr) was purchased from Lallemand Inc. (Montreal, Canada); and leupeptin, aprotinin, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Antibodies of Hypoxia induced factor 1 alpha (HIF-1α) and NF-κB p65 were purchased from Novus Biologicals (Littleton, CO, USA) and Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA), respectively. Anti-mouse AMP-activated protein kinase alpha (AMPKα) and phospho-AMPKα (Thr172) were purchased from Cell Signaling Technology (MA, USA), and all other chemicals and reagents used in this study were reagent grade.

Male C57BL/6NCrjBgi mice were purchased from the Charles River Laboratory of Animal Science (Orient Co., Seoul, Republic of Korea) at four weeks old and fed a normal diet for one week. Animals were housed in individual cages with free access to water and food in a temperature-controlled animal facility under a 12 h light-dark cycle at 22±2℃ and 55±5% humidity. Mice were fed either a high-fat diet (HFD) (Open Source diets #D12492; Research Diets Inc., New Brunswick, NJ) to induce obesity, or a regular diet (RD; Open Source diets #D12450B; Research Diets Inc.). The nutritional contents of the HFD were similar to those on the regular diet except for low carbohydrate content and a high level of fat.

At 26 weeks of age, mice exhibiting blood glucose levels >160 mg/dl were selected as non-insulin-dependent diabetes mellitus (NIDDM) animals and were divided into five groups of 20 animals per group. One group was administrated PBS only and served as diabetic controls; four groups received daily 100 mg/kg of PAG, 2 mg/kg of Aloesin (ALS), 100 mg/kg of PAG containing 2% ALS (Aloe QDM), and Aloe QDM plus 500 mg/kg of Cr-enriched yeast containing 0.2% Cr (Aloe QDM complex) respectively, and the fifth group was administered pioglitazone (PGZ, 2.5 mg/kg), an anti-diabetic drug currently in clinical use. Mice were weighed and blood samples were collected weekly by tail bleeding into heparin-coated tubes after 4 h fasts.

At the end of the experimental period, mice were sacrificed and WAT were immediately removed from periepididymal and perirenal fat for morphological examination. Mice were treated in accordance with the guidelines issued by Sahmyook University for the care and use of laboratory animals.

Adipose tissue was isolated from mice, fixed in 10% formalin, and embedded in paraffin. Four-micrometer thin sections were obtained, mounted on two glass-slides, and stained with hematoxylin-eosin. Sections were viewed with an Olympus microscope (Olympus, Tokyo, Japan) at 400X magnification.

Twenty mice were selected and used for this analysis. WAT and muscles were immediately frozen in liquid nitrogen and stored at -70℃ for the determination of gene transcripts. Total RNA was extracted from tissues using the RNeasy Mini kit (QIAGEN, Valencia, USA) in an RNase-free environment. RNA was then quantified by reading the absorbance at 260 nm. The reverse transcription of 1µg RNA was carried out using M-MLV reverse transcriptase (Promega, USA), an oligo (dT) 16 primer, dNTP (0.5µM), and 1 U RNase inhibitor. After incubation at 65℃ for 5 min and 37℃ for 60 min, M-MLV reverse transcriptase was inactivated by heating at 70℃ for 15 min. The polymerase chain reaction (PCR) was performed in 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, and 2.5 mM dNTPs with 5 units of Taq DNA polymerase, and 10 pM of each primer set for CD36, scavenger receptor A (SR-A), interleukin (IL)-1β, and interleukin (IL)-6. The cDNA was amplified by 35 cycles of denaturing at 94℃ for 45 s, annealing at 62℃ for 45 s, and extension at 72℃ for 1 min. Final extension was performed at 72℃ for 5 min. The PCR products were then electrophoresed on 1.5% agarose gels and stained with ethidium bromide. The primers selected were 5' AGT AGG CGT GGG TCT GAA GG 3' (forward) and 5' CTT GCT TGC CCA GTC ACA GG 3' (reverse) for CD36, 5' GGA GAC AGA GGG CTT ACT GG 3' (forward) and 5' GTT GAT CCG CCT ACA CTC CC 3' (reverse) for SR-A, 5' CAG GAT GAG GAC ATG ACA CC' (forward) and 5' CTC TGC AGA CTC AAA CTC CAC3' (reverse) for IL-1β, 5' GTA CTC CAG AAG ACC AGA GG 3' (forward) and 5' TGC TGG TGA CAA CCA CGG CC 3' (reverse) for IL-6, and 5' CAA CTT TGG CAT TGT GGA AGG 3' (forward) and 5' ATG GAA ATT GTG AGG GAG ATG C 3' (reverse) for GAPDH, which was used as an internal control.

After homogenized tissues were collected and washed twice with cold PBS, they were resuspended in ice lysis buffer (20 mM Trizma base, 50 mM NaCl, 250 mM sucrose, 50 mM NaF, 5 mM Na₄P₂O₇·10H₂O, 1% Triton-X100, 5µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 5µg/ml aprotinin). After 15 min incubation on ice, the cells were lysed by the addition of 0.1% NP-40 and vigorous vortexing for 1 min. The nuclei were pelleted by centrifugation at 12,000×g for 1 min at 4℃ and resuspended in high salt buffer (20 mM HEPES, pH 7.9, 25% glycerol, 400 mM KCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 1 mM NaF, 1 mM sodium orthovanadate). The supernatant fluid was stored in aliquots at -70℃.

Frozen epididymal adipose tissue and muscles were homogenized in 3 volumes of ice lysis buffer (20 mM Trizma base, 50 mM NaCl, 250 mM sucrose, 50 mM NaF, 5 mM Na₄P₂O₇·10H₂O, 1% Triton-X100, 5µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 5µg/ml aprotinin). Twenty micrograms of protein from cell lysates were applied to 8~12% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in TBST solution for 1 hr. They were then incubated with anti-HIF1α, anti-AMPK, anti-p-AMPK, and anti-p65 monoclonal antibodies for 2 hrs and washed 3 times with TBST. After incubation with alkaline phosphatase-labeled secondary antibody for 2 hrs, the bands were visualized using a Western Blot Kit with an alkaline phosphatase substrate (Vector, Burlingame, USA).

All data have been presented as mean±SEM values. Significant differences (p<0.05) between groups were evaluated using a one-way analysis of variance with SPSS (Chicago, IL, USA) for Windows and Duncan's Multiple Range Test where appropriate.

CD36 and SR-A are scavenger receptors for oxidized low-density lipoprotein (oxLDL) and cellular transporters of long-chain fatty acid (22). In the present study, we examined changes in the level of expression of scavenger receptors and their affect on the amount of adipose tissues in obese mice. We obtained WAT, stained with H&E, then counted the adipocytes. Numerous adipocytes completely disappeared after aloe formula supplementation, especially after Aloe QDM complex supplementation (Fig. 1). In agreement with this, levels of scavenger receptors such as SR-A and CD36 in the WAT were significantly lower in obese mice suppplemented with dietary aloe formula than in the HFD group (Fig. 2). These findings indicated that dietary aloe formula supplementation suppressed fat size through inhibition of scavenger receptors in ATM.

We demonstrated that aloe formulas increased AMPKα phosphorylation in both WAT and muscles. Since the AMPK cascade has been shown to activate insulin signaling (10), we hypothesized that AMPK might be involved in the effect of lipolysis on WAT. The Aloe QDM complex enhanced phosphorylation of AMPK in tissues (Fig. 3), a finding indicating that dietary aloe formula reduced fat levels through mitochondrial biogenesis.

To examine whether the dietary aloe formula modulated obesity-induced inflammation, we measured a proinflammatory cytokine gene and its transcription factor, NF-κB p65, and protein expression in WAT by RT-PCR and western blot. As shown in Figs. 4A and B, HIF1α protein and proinflammatory cytokines, IL-1β and IL-6 mRNA were lower in the obese mice supplemented with aloe formula than in the HFD group. The transcription factor, NF-κB p65 was also inhibited by aloe formulas, and in particular, the Aloe QDM complex (Fig. 5). These findings indicated that aloe formulas, and especially the Aloe QDM complex reduce WAT inflammatory responses through inactivation of NF-κB p65.