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. 2014 Jan;34(1):43-51.
doi: 10.1038/jcbfm.2013.166. Epub 2013 Sep 25.

Diet-induced obesity suppresses expression of many proteins at the blood-brain barrier

Suidong Ouyang  1 , Hung Hsuchou  1 , Abba J Kastin  1 , Yuping Wang  1 , Chuanhui Yu  1 , Weihong Pan  1
Affiliations

Diet-induced obesity suppresses expression of many proteins at the blood-brain barrier

Suidong Ouyang et al. J Cereb Blood Flow Metab. 2014 Jan.

Abstract

The blood-brain barrier (BBB) is a regulatory interface between the central nervous system and the rest of the body. However, BBB changes in obesity and metabolic syndrome have not been fully elucidated. We hypothesized that obesity reduces energy metabolism in the cerebral microvessels composing the BBB, reflected by downregulation of protein expression and function. We performed comparative proteomic analyses in enriched microvessels from the cerebral cortex of mice 2 months after ingestion of a high-fat diet or regular rodent chow. In mice with diet-induced obesity (DIO), there was downregulation of 47 proteins in the cerebral microvessels, including cytoskeletal proteins, chaperons, enzymes, transport-related proteins, and regulators for transcriptional and translational activities. Only two proteins, involved in messenger RNA (mRNA) transport and processing, were upregulated. The changes of these proteins were further validated by quantitative polymerase chain reaction (qPCR), western blotting, and immunofluorescent staining of freshly isolated microvessels, in samples obtained from different batches of mice. The predominant downregulation suggests that DIO suppresses metabolic activity of BBB microvessels. The finding of a hypometabolic state of the BBB in mice at the chronic stage of DIO is unexpected and unprecedented; it may provide novel mechanistic insight into how obesity influences CNS function via regulatory changes of the BBB.

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Figures

Figure 1
Figure 1
Changes of body weight and fat composition after diet-induced obesity (DIO). (A) The DIO group started 45% high-fat diet (HFD) at age 2 months and continued for 8 weeks; the control group was maintained on regular rodent chow (n=6/group). Significantly higher weight gain was seen at 3 weeks of HFD and persisted to the end of the study. (B) Diet-induced obesity mice had a higher % fat than the control mice starting at 3 weeks of HFD feeding, till the end of 8 weeks of feeding. *P<0.05, **P<0.01, ***P<0.001 when the DIO group was compared with the control group. BW, body weight.
Figure 2
Figure 2
Processing of samples for comparative proteomics. (A) Isolated cerebral microvessels from control and diet-induced obesity (DIO) groups were similar in morphology. (B) The master gel image of two-dimensional difference gel electrophoresis of proteins extracted from the cerebral microvessels. (C) Representative two-dimensional gel images from control mice (C) and DIO mice (D). Spots 1 to 4: unchanged proteins; 5 to 8: downregulated proteins; 9 to 10: upregulated proteins. HFD, high-fat diet; MW, molecular weight.
Figure 3
Figure 3
Gene ontology classification of differentially expressed protein in blood–brain barrier microvessels from control and diet-induced obesity mice according to their subcellular location and biologic function. (A) Functional classification of differentially expressed proteins. (B) Subcellular location of differentially expressed proteins.
Figure 4
Figure 4
Messenger RNA mRNA of blood –brain barrier BBB microvessels from control and diet-induced obesity (DIO) mice (n=5/group). (A) Eight genes representing different classes of proteins showed significant downregulation in DIO mice in comparison with lean control mice. (B) Two proteins, among the 49 total proteins identified to have differential expression, showed upregulation. *P<0.05, **P<0.01, ***P<0.001. DRP-1 dihydropyrimidinase-related protein 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GRP78, glucose-regulated protein precursor; HFD, high-fat diet; hnRNP, heterogeneous nuclear ribonucleoprotein; HSP70, heat shock 70 kDa protein; mRNA, messenger RNA; UCH-L1, ubiquitin carboxyl-terminal hydrolase isoenzyme L1.
Figure 5
Figure 5
Western blot validation of downregulated proteins and transporters in blood–brain barrier microvessels from control and diet-induced obesity (DIO) mice (n=2 mice/group). (A) The expression of vimentin and tubulin was decreased by DIO, whereas P-glycoprotein (Pgp), glucose transporter-1 (Glut1), transferrin receptor (Trf), and housekeeping genes β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) did not show apparent change. (B) Densitometric analysis confirmed the difference of the proteins shown in the gel image. *P<0.05. HFD, high-fat diet.
Figure 6
Figure 6
Immunofluorescent staining of isolated microvessels from control and diet-induced obesity (DIO) mice. (A) In cerebral microvessels from DIO mice (right panel), the levels of vimentin, tubulin, and zona occludin (ZO-1) were all lower than those of the respective controls (left panel). (B) Densitometric analysis confirmed the significant reduction for these three proteins in the DIO group. *P<0.05. HFD, high-fat diet.
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