Deep Sea Water Inhibited Pancreatic β-Cell Apoptosis and Regulated Glucose Homeostasis by Affecting Lipid Metabolism in Db/Db Mice

Figures & data

Table 1 Primer Sequences

Gene	Primers
GAPDH	5’-CTGGAGAAACCTGCCAAGTA-3’;
	3’-AAGAGTGGGAGTTGCTGTTG-5’
Adiponectin	5’-CTCCACCCAAGGGAACTTGT-3’;
	3’-TAGGACCAAGAAGACCTGCATC-5’
ACC1	5’-TGTTGAGACGCTGGTTTGTAGAA-3’;
	3’-GGTCCTTATTATTGTCCCAGACGTA-5’
Acox1	5’-CTATGGGATCAGCCAGAAAGG-3’;
	3’-AGTCAAAGGCATCCACCAAAG-5’
CPT1a	5’-AGACCGTGAGGAACTCAAACCTAT-3’;
	3’-TGAAGAGTCGCTCCCACT-5’
FAS	5’-TTCCAAGACGAAAATGATGC-3’;
	3’-AATTGTGGGATCAGGAGAGC-5’
HSL	5’-CCAGGGAGGGCCTCAGC-3’;
	3’-TGTCTTCTGCGAGTGTCACC-5’
MACD	5’-TGACAAAAGCGGGGAGTACC-3’;
	3’-GCACCCCTGTACACCCATAC-5’
PPARα	5’-CAACGGCGTCGAAGACAAA-3’;
	3’-TGACGGTCTCCACGGACAT-5’

Table 2 Basic Information on Experimental Mice

	db/m	db/db	db/db+DOMC
Initial Weight (g)	24.61 ± 2.93	40.26 ± 1.80^#	39.94 ± 1.80^#
Final Weight (g)	27.74 ± 1.49	43.09 ± 3.95^#	40.96 ± 3.75^#
Liver (mg/g mice)	36.13 ± 1.63	64.75 ± 8.38^#	59.36 ± 4.30^#
Spleen (mg/g mice)	3.38 ± 0.71	1.68 ± 0.32^#	1.65 ± 0.10^#
Left Kidney (mg/g mice)	6.61 ± 0.75	4.72 ± 0.61^#	4.97 ± 0.49^#
Right Kidney (mg/g mice)	7.30 ± 0.37	4.98 ± 0.56^#	5.06 ± 0.65^#

Notes: Final weight and viscera weights were measured after 4 weeks of the experiment. All values were expressed as means ± SD (n=6-10/group). ^#Means p<0.0001 when compared to the db/m group.

Figure 1 DOMC reduced plasma lipids by regulation of lipolysis and β-oxidation. (A) Plasma TG, TC, LDL, and HDL measurements after a 4-week experiment. (B) RT-qPCR analysis for detection of lipolysis and fatty acid β-oxidation associated gene PPARα, CPT1a, MACD, and Acox1 on epididymal adipose tissue. (C) Plasma adiponectin and gene expression of adiponectin on epididymal adipose tissue. The data were presented as means ± SD; n=6-10/group. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001.

Abbreviation: ns, no significant difference.

Figure 2 DOMC ameliorated fatty liver by reduction of lipogenesis and β-oxidation. (A) HE and Oil red O staining of the liver to evaluate liver steatosis. (B) a. RT-qPCR analysis of fatty acid β-oxidation in the liver. b. Western blot to test PPAPα at the protein level. (C) lipogenesis-associated genes in the liver. The data were presented as means ± SD; n=6-10/group. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. Scale bar: 50 μm.

Abbreviation: ns, no significant difference.

Figure 3 DOMC regulated glucose homeostasis in db/db mice. (A) The test of fasting blood glucose levels and GSP. (B) IPGTT after a 4-week experiment. (C) HE staining of the pancreas: green arrows implied the vacuolar degeneration of pancreatic islet cells. (D) Immunofluorescence co-staining of the pancreas. Each section was stained for insulin, glucagon (green) and DAPI (blue). Insulin and glucagon-positive cells were counted per visual field. The data were presented as means ± SD; n=6-10/group. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. Scale bar: 50 μm.

Abbreviation: ns, no significant difference.

Figure 4 DOMC protected pancreatic β-cell by a long-term application. (A) The measurement of fasting blood glucose levels and AUC of IPGTT after a 12-week experiment. (B) HE staining of the pancreas. (C) Immunofluorescence of insulin and glucagon co-staining. The data were presented as means ± SD; n=8-14/group. *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001. Scale bar: 50 μm.

Figure 5 DOMC alleviated pancreatic β-cell apoptosis. (A) TUNEL staining of the pancreas slices after a 4-week experiment. (B) TUNEL staining of the pancreas slices after a 12-week experiment. White dotted boxes were depicted as pancreas islets, the number of TUNEL-positive cells was counted as green fluorescence cells in the boxes. The data were presented as means ± SD; n=6-14/group. *p<0.05, **p<0.005, ****p<0.0001. Scale bar: 50 μm.

Abbreviation: ns, no significant difference.