The herbal composition GGEx18 from Laminaria japonica, Rheum palmatum, and Ephedra sinica inhibits visceral obesity and insulin resistance by upregulating visceral adipose genes involved in fatty acid oxidation

Figures & data

Table 1. PCR primers and conditions.

Gene	Gene Bark	Primer sequence	cDNA size (bp)	Tm/cycle
ACC	NM_133360	Forward: 5′-GGGCTACCTCTAATGGTCTT-3′	439	52/40
		Reverse: 5′-CTACCTGATGGTAAATGGGA-3′
Adiponectin	NM_009605	Forward: 5′-GAAGATGACGTTACTACAAC-3′	704	58/40
		Reverse: 5′-GGTAGTTGCAGTCAGTTGGT-3′
AMPKα1	NM_001013367	Forward: 5′-AGAGGGCCGCAATAAAAGAT-3′	177	58/38
		Reverse: 5′-TGTTGTACAGGCAGCTGAGG-3′
AMPKα2	NM_178143	Forward: 5′-CGCCTCTAGTCCTCCATCAG-3′	219	58/38
		Reverse: 5′-ATGTCACACGCTTTGCTCTG-3′
aP2	K02109	Forward: 5′-CAAAATGTGTGATGCCTTTGTG-3′	416	58/34
		Reverse: 5′-CTCTTCDTTGGCTCATGCC-3′
β-actin	BC138614	Forward: 5′-TGGAATCCTGTGGCATCCATGAAAC-3′	349	58/30
		Reverse: 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′
CPT-1	L07736	Forward: 5′-TATGTGAGGATGCTGCTTCC-3′	629	58/34
		Reverse: 5′-CTCGGAGAGCTAAGCTTGTC-3′
Leptin	NM_008493	Forward: 5′-CCTGCTCCAGCAGCTGCAAG-3′	343	56/40
		Reverse: 5′-CTATCTGCAGCACATTTTGGGA-3′
MCAD	NM_007382	Forward: 5′-GACATTTGGAAAGCTGCTAGTG-3′	321	58/34
		Reverse: 5′-TCACGAGCTATGATCAGCCTCTG-3′
PPARα	NM_001113418	Forward: 5′-GCAGDCGTACAGGTCATCA-3′	202	58/34
		Reverse: 5′-CTCTTCATCCCCAAGCGTAG-3′
PPARγ	NM_013124	Forward: 5′-ATTCTGGCCCACCAACTTCGG-3′	339	58/34
		Reverse: 5′-TGGAAGCCTGATGDTTATCCCCA-3′
TNFα	NM_013693	Forward: 5′-GGCAGGTCTACTTTGGAGTCATTGC-3′	300	55/30
		Reverse: 5′-ACATTCGAGGRTCCAGTGAATTCGG-3′
VLCAD	AF017176	Forward: 5′-CGTCAGAGGTGTACTTTGATGG-3′	269	58/34
		Reverse: 5′-CATGGACTCAGTCACATACTGC-3′

Figure 1. Visceral adipose tissue weight and histology of visceral adipocytes. Adult male C57BL/6J mice were fed a low-fat diet (normal), a high-fat diet (control), or the high-fat diet supplemented with 125, 250, or 500 mg/kg/d GGEx18 for 8 weeks. (A) At the end of study, visceral adipose tissue weights were measured. (B) 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 with the normal group, *p < 0.05 compared with the obese control group. (C) Representative hematoxylin and eosin-stained sections (5 µm thick) of mesenteric adipose tissue.

Figure 2. Lipid accumulation in 3T3-L1 cells. (A) 3T3-L1 preadipocytes (ND) were differentiated into mature adipocytes (control). Differentiated control cells were treated with GGEx18, fenofibrate (FF) or Wy14,643 (Wy). Shown are representative Oil red O-stained cells. (B) Quantitative analysis of triglyceride contents. All values are expressed as the mean ± SD. #p < 0.05 compared with the ND group, *p < 0.05 compared with the control group.

Figure 3. The mRNA expression levels of genes involved in fatty acid oxidation in visceral adipose tissue of obese mice. (A) Adult male C57BL/6J mice were fed a low-fat diet (normal), a high-fat diet (control), or the high-fat diet supplemented with 250 mg/kg/d GGEx18 for 8 weeks. Total cellular RNA was extracted from visceral adipose tissue and mRNA levels were measured using RT-PCR. All values are expressed as the mean ± SD of relative density units using β-actin as a reference. #p < 0.05 compared with the normal group, *p < 0.05 compared with the obese control group. (B) Representative RT-PCR bands from one of the three independent experiments are shown.

Figure 4. The mRNA expression levels of genes involved in fatty acid oxidation in 3T3-L1 cells. (A) 3T3-L1 preadipocytes (ND) were differentiated into mature adipocytes (control). Differentiated control cells were treated with GGEx18, fenofibrate (FF) or Wy14,643 (Wy). Total cellular RNA was extracted from cells and mRNA levels were measured using RT-PCR. All values are expressed as the mean ± SD of relative density units using β-actin as a reference. #p < 0.05 compared with the ND group, *p < 0.05 compared with the control group. (B) Representative RT-PCR bands from one of the three independent experiments are shown.

Figure 5. The mRNA expression levels of adipocyte marker genes in visceral adipose tissue of obese mice. (A) Adult male C57BL/6J mice were fed a low-fat diet (normal), a high-fat diet (control), or the high-fat diet supplemented with 250 mg/kg/d GGEx18 for 8 weeks. Total cellular RNA was extracted from visceral adipose tissue and mRNA levels were measured using RT-PCR. All values are expressed as the mean ± SD of relative density units using β-actin as a reference. #p < 0.05 compared with the normal group, *p < 0.05 compared with the obese control group. (B) Representative RT-PCR bands from one of the three independent experiments are shown.

Figure 6. The mRNA expression levels of adipogenic genes in 3T3-L1 cells. (A) 3T3-L1 preadipocytes (ND) were differentiated into mature adipocytes (control). Differentiated control cells were treated with GGEx18, fenofibrate (FF) or Wy14,643 (Wy). Total cellular RNA was extracted from cells and mRNA levels were measured using RT-PCR. All values are expressed as the mean ± SD of relative density units using β-actin as a reference. #p < 0.05 compared with the ND group, *p < 0.05 compared with the control group. (B) Representative RT-PCR bands from one of the three independent experiments are shown.

Figure 7. Serum (A) glucose levels, (B) insulin levels, and (C) insulin-to-glucose ratios. Adult male C57BL/6J mice were fed a low-fat diet (normal), a high-fat diet (control), or the high-fat diet supplemented with 125, 250, and 500 mg/kg/d for 8 weeks. All values are expressed as the mean ± SD. #p < 0.05 compared with the normal group, *p < 0.05 compared with the control group.

Figure 8. Changes in blood glucose levels during the oral glucose tolerance test. Adult male C57BL/6J mice were fed a low-fat diet (normal), a high-fat diet (control), or the high-fat diet supplemented with 125, 250, and 500 mg/kg/d for 8 weeks. After a 12-h fast, mice orally received glucose (2 g/kg body weight). All values are expressed as the mean ± SD. #p < 0.05 compared with the normal group, *p < 0.05 compared with the control group.