http / / cjbmb. bjmu. edu. cn Chinese Journal of Biochemistry and Molecular Biology Aβ42 Alzheimer's disease AD insulin degrading enzyme IDE.

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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 75 PPARγ IDE TNF-α Aβ metformin MET MET β- β-amyloid Aβ Alzheimer's disease AD AD MET Aβ OB MET 4 W MET Aβ42 α TNF-α γ peroxisome proliferator-activated receptor γ PPARγ insulin degrading enzyme IDE OB CTL P < 0 01 OB TNF-α Aβ42 CTL PPARγ IDE P < 0 05 MET OB P < 0 05 TNF-α Aβ42 P < 0 01 PPARγ IDE OB P < 0 01 TNF-α PPARγ IDE IDE Aβ42 AD α γ β- 42 R96 Metformin Down-regulates PPARγ and IDE and Up-regulates TNF-α and Aβ42 in the Hippocampus of Obese Rats YANG Si-Si JIANG Teng ZHANG Mu-Xun Department of Endocrinology TongJi Hospital Huazhong University of Science and Technology Wuhan China Abstract Metformin was commonly used in patients with obesity and insulin resistance Several studies indicated that metformin increased the risk of Alzheimer's disease but the molecular mechanism remained unclear Extracellular deposits of the amyloid β-peptide as the originating pathophysiology of Alzheimer's disease may be the key point This study was designed to determine the effects of metformin on β- amyloid Aβ and related factors in hippocampus of diet-induced obese rat model The SD rats were fed with high glucose high fat and high protein for 12 weeks to establish the OB models Metformin was administered intragastrically for 4 weeks and the MET group was made The expression of TNF-α peroxisome proliferator-activated receptor γ PPARγ and insulin degrading enzyme IDE gene in hippocampus were analyzed by real time-pcr and the protein level of TNF-α Aβ42 PPARγ IDE were determined by Western blotting and immunohistochemistry The plasma insulin and insulin resistance calculated by HOMA-IR were significantly higher in OB group P < 0 01 than that in CTL group P < 0 05 The level of TNF-α and Aβ42 was up-regulation in OB group P < 0 05 while PPARγ and Tel zhangmuxun@ sina com Received July Accepted September Corresponding author Tel zhangmuxun@ sina com

2 1 PPARγ IDE TNF-α Aβ42 71 IDE were descent P < 0 05 In MET group the level of TNF-α and Aβ42 was highest while PPARγ and IDE were lowest P < 0 01 These findings suggest that metformin could reduce the level of plasma insulin and IR in obese rat models At the same time the drug could increase the degree of TNF-α in hippocampus then down-regulate the level of PPARγ and IDE which induces the deposition of Aβ42 Key words obesity metformin TNF-α PPARγ insulin degrading enzyme β-amyloid42 obesity Alzheimer s 4 w MET CTL OB disease AD 1 3 d 2 AD 2 EP RT-PCR Western 4% IHC β 42 β-amyloid 42 Aβ Tau 1 Aβ AD 2 3 ~ Tau 5 1 ml - 20 TNF-α 3 Aβ 6 ~ 8 insulin degrading enzyme Aβ IDE HOMA-IR = fasting plasma insulin Aβ miu / L fasting plasma glucose FPG IDE mmol / L /22 5 γ peroxisome proliferator-activated receptor γ PPARγ IDE metformin 2 - ΔΔCT ΔCT = CT Aβ42 MET 4 W Aβ ΔΔCT Table 1 TNF-α PPARγ IDE 1 4 Aβ SD min 12 μg 180 ~ 220 g 10 ~ 12 10% SDS-PAGE PVDF CTL OB CTL 26 0% 15 2% 58 8% 12 OB 1 h Table 2 4 IgG Santa Cruz 20% OB 1 h TBST 3 10 min OB mg / kg / d 1 3 RT-PCR Tnf-α Pparγ Ide mrna 10 PPARγ Trizol RNA MET 260 nm 280 nm A RNA - cdna 20 μl Real time-pcr 11 Aβ 95 3 min s s s AD MET Aβ 40 PCR CT - CT ΔΔCT = ΔCT - ΔCT cdna g 10 min 10 μl Bradford % TBST 37 TBST 3 10 min ECL ECL

3 72 29 Table 1 Sequence for primers Gene Primer sequence Product / bp Pparγ Forward 5'-CCCTGGCAAAGCATTTGTAT-3' 222 Reverse 5'-ACTGGCACCCTTGAAAAATG-3' Tnf-a Forward 5'-CTTCTCATTCCTGCTCGTGG-3' 202 Reverse 5'-CTCCGCTTGGTGGTTTGC-3' Ide Forward 5'-TTTCCTGAGCACCCTTTCCA-3' 150 Reverse 5-'CAGATGACCGAGATAATGACCG-3' Gapdh Forward 5'-CGCTAACATCAAATGGGGTG-3' 201 Reverse 5 '-TTGCTGACAATCTTGAGGGAG-3' Table 2 Primary antibodies used in this study Antibody Specificity Reference / source Aβ42 Rabbit Epitomics IDE Rabbit Epitomics PPARγ Mouse Santa TNF-α Rabbit Abcom 1 6 SPSS11 5 x 珋 ± s ANOVA P < Quantity One 2 1 Table 3 OB MET CTL 1 5 TNF-α Aβ42 PPARγ IDE OB 20% CTL P < 3 4% 48 h 0 01 P < 0 05 MET CTL OB P < 0 05 MET DAB CTL OB P < 0 05 Table 3 Design of experimental rats and laboratory data n = 10 珔 x ± s Groups CTL OB MET Wight / g ± ± ± 14 6 Length / cm 23 2 ± ± ± 0 48 Plasma glucose mmol / L 6 45 ± ± ± 0 26 Plasma insulin IU / L 8 89 ± ± ± 2 68 HOMA-IR 2 12 ± ± ± 0 48 Results are expressed as meansd for groups indicated as control CTL obesity OB obesity P < 0 05 P < 0 01 vs control + metformin treatment MET 2 2 TNF-α PPARγ IDE MET TNF-α Aβ42 IDE PPARγ MET Aβ AD TNF-α Aβ42 PPARγ IDE Aβ42 mrna Fig 1 OB TNF-α mrna CTL 2 3 PPARγ IDE mrna CTL 35% 67% MET Aβ42 WB Fig 2 TNF-α mrna OB TNF-α Aβ42 CTL 4 5 PPARγ IDE mrna CTL PPARγ IDE CTL 18% 22% CTL 74% 56% MET

4 1 PPARγ IDE TNF-α Aβ42 73 TNF-α Aβ42 CTL PPARγ IDE CTL 35% 40% MET TNFα Aβ42 IDE PPARγ Aβ42 Fig 1 Expression of TNF-α PPARγ and IDE mrna in hippocampus Graphics showed the result of RT-PCR experiments to measure relative mrna levels of TNF-α PPARγ and IDE and the date were T calculated by 2 - ΔΔCT Results showed that expression of TNF-α gene was upregulation in OB and MET groups while the levels of PPARγ and IDE mrna were down-regulation n = 10 P < 0 05 P < 0 01 VS control CTL control group OB obesity group MET metformin groups Fig 2 Western blot analysis of TNF-α Aβ42 PPARγ and IDE in rat hippocampus Western blotting for TNF-α Aβ42 PPARγ and IDE proteins were showed in panel A The panel B were showed scanning graphic of Western blotting quantified The results were normalized by the corresponding β-actin The results showed that expression of TNF-αand Aβ42 proteins were up-regulation in OB and MET groups while the levels of PPARγ and IDE proteins were down-regulation All results were presented as means ± SD n = 10 P < 0 05 P < 0 01 compared with CTL CTL control group OB obesity group Met OB + Metformin treatment group Fig 3 Immunohistochemical assay of TNF-α PPARγ and Aβ42 in hippocampus 400 A B C Paraffinembadded tissue sections of rat hippocampus were immunostained with antibody anti-tnfα Rat hippocampus were immunostained with antibody anti-pparγ for D E F Rat hippocampus were immunostained with antibody anti-aβ42 for G H I Left lane was control group CTL Middle lane was obesity group OB Right lane was metformin group MET

5 TNF-α Aβ42 PPARγ IDE PPAR γ IDE OB 4 W TNF- 3 α Aβ IDE PPAR γ TNF-α Fig 3 CTL PPAR γ IDE Aβ OB TNF-α Aβ42 PPARγ MET MET TNF-α Aβ42 OB Aβ PPARγ Aβ 3 MET TNF-α Aβ AD PPAR γ IDE Aβ42 MET AD Aβ42 MET Aβ 13 TNF-α PPAR γ Aβ AD Aβ 6 ~ Aβ 8 IDE References Aβ Aβ IDE PPAR γ 14 PPAR γ IDE PPAR γ IDE PPAR 2 Jiao P Ma J pathways J PPRE 13 PPARγ 15 PPARγ PPAR γ PPAR γ in Alzheimer's disease J Hum Mol Genet 2010 R12-20 TNF-α 6 ~ TNF-α Aβ 8 TNF-α PPAR γ IDE Aβ TNF-α Aβ IDE PPAR γ TNF-α PPAR γ PPAR γ IDE IDE Aβ TNF-α 1 Lee Y H Martin R L Maple W G et al Plasma amyloid peptide levels correlate with adipocyte amyloid precursor protein gene expression in obese individuals J Neuroendo-crinology 2009 Feng B et al FFA-induced adipocyte inflammation and insulin resistance involvement of ER stress and IKKβ Obesity Silver Spring Chouliaras L Rutten B P Kenis G et al Epigenetic regulation in the pathophysiology of Alzheimer's disease J Prog Neurobiol Lesné S Koh M T Kotilinek L et al A specific amyloid-beta protein assembly in the brain impairs memory J Nature Crews L Masliah E Molecular mechanisms of neurodegeneration 19 R1 6 Craft S Insulin resistance syndrome and Alzheimer s disease age-and obesity-related effects on memory amyloid and inflammation J Neurobiol Aging Suppl Malm T Koistinaho M Muona A et al The role and therapeutic potential of monocytic cells in Alzheimer s disease J Glia Azzu V Jastroch M Divakaruni A S et al The regulation and turnover of mitochondrial uncoupling proteins J Biochim Biophys Acta Revett T J Baker G B Jhamandas J et al Glutamate system amyloid β peptides and tau protein functional interrelationships and relevance to Alzheimer disease pathology J J Psychiatry Neurosci Espuny-Camacho I Dominguez D Merchiers P et al

6 1 PPARγ IDE TNF-α Aβ42 75 Peroxisome proliferator-activated receptor gamma enhances the activity of an insulin degrading enzyme-like metalloprotease for amyloid-β clearance J J Alzheimer's Dis Rice S Pellatt L J Bryan S J et al Action of metformin on the insulin-signaling pathway and on glucose transport in human granulosa cells J J Clin Endocrinol Metab E Matthews D R Hosker J P Rudenski A S et al Homeostasis model assessment insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man J Diabetologia Chen Y Zhou K Wang R et al Antidiabetic drug metformin GlucophageR increases biogenesis of Alzheimer 's amyloid peptides via up-regulating BACE1 transcription J Proc Natl Acad Sci U S A Tamboli I Y Barth E Christian L et al Statins promote the degradation of extracellular amyloid-peptide by microglia via stimulation of exosome-associated insulin-degrading enzyme IDE secretion J J Biol Chem Kershaw E E Schupp M Guan H P et al PPARgamma regulates adipose triglyceride lipase in adipocytes in vitro and in vivo J Am J Physiol Endocrinol Metab E