One-week exposure to a free-choice high-fat high-sugar diet does not disrupt blood–brain barrier permeability in fed or overnight fasted rats

Figures & data

Table 1 Antibody characteristics for western blots

Target	Antibody	µg protein	Antibody dilution	%gel	Expected size	Homogenization buffer
ZO-1	617300	30	1:1000	5	225 kD	RIPA
Claudin-5	LS-B5971	30	1:500	12.5	23 kD	Suspension
Occludin	711500	15	1:1000	10	53 kD	RIPA
GLUT-1	MA5–11315	20	1:1000	12.5	45/55 kD	RIPA

Table 2 Animal characteristics experiment 1

Diet group	Evans blue		Protein expression
	Chow	fcHFHS	Chow	fcHFHS
Intake (kcal)	82.6 ± 1.7	121.2 ± 5.7***	89.2 ± 3.9	115.1 ± 4.3***
Absolute BW (g)	353.8 ± 6.0	354.4 ± 7.1	339.6 ± 3.6	337.5 ± 5.5
BW gain (g)	28.5 ± 2.5	27.2 ± 4.9	29.0 ± 1.1	25.7 ± 2.0
eWAT (%)	0.42 ± 0.02	0.53 ± 0.04^#	0.43 ± 0.00	0.48 ± 0.02**

Kcal intake, mean daily caloric intake in diet week; absolute BW, BW (g) after diet week or week on chow; BW gain, weight gain (g) after diet week; eWAT, epididymal fat weight, % of BW. ^# trend; *P < 0.05; **P < 0.01, ***P < 0.001.

Figure 1 IOD of Evans blue dye in the ME after chow (n = 5, white bar) and 1-week fcHFHS diet (n = 4, black bar) (A) and representative images of Evans blue dye in the ME and Arc of the hypothalamus of a chow and fcHFHS diet animal. White asterix depicts third ventricle (B). Data are expressed as mean ± SEM.

Table 3 Animal characteristics experiment 2

Diet group	Evans blue			Protein expression
	Chow-fed	Chow	fcHFHS	Chow	fcHFHS
Intake (kcal)	78.3 ± 2.8	83.84 ± 2.6	117.4 ± 7.1***	85.6 ± 1.8	122.3 ± 5.7****
Absolute BW (g)	367.6 ± 7.4	363.8 ± 2.6	359.5 ± 10.6	315.1 ± 3.8	324 ± 4.6
BW gain (g)	27.8 ± 4.2	28.0 ± 1.9	26.33 ± 4.2	31.63 ± 3.19	38.75 ± 5.15
eWAT (%)	0.49 ± 0.04	0.49 ± 0.02	0.59 ± 0.05	0.41 ± 0.01	0.52 ± 0.02**

Kcal intake, mean daily caloric intake in diet week; absolute BW, BW (g) after diet week or week on chow; BW gain, weight gain (g) after diet week; eWAT, epididymal fat weight, % of BW. ^# trend; *P < 0.05; **P < 0.01, ***P < 0.001.

Figure 2 Representative western blots on hypothalamic tissue lysates of two fcHFHS diet-fed (left bands) and two chow-fed animals (right bands) for ZO-1, occludin, and GLUT-1 protein, with β-actin protein as control for amount of protein loaded. Claudin-5 bands are shown of lysates of two fcHFHS diet animals on the left and right, and three chow-fed animals in the middle. Molecular weight is depicted next to blots.

Figure 3 Effects of 1 week fcHFHS (n = 6, black bars) compared to chow (n = 8, white bars), on ME-Arc area protein expression of ZO-1 (A), claudin-5 (B), occludin (C), and GLUT-1 (D). Data are expressed as mean ± SEM. Right: ZO-1 immunoreactivity in coronal sections of the ME and third ventricle of a representative chow-fed and fcHFHS-fed rat. Arrowheads indicate immunopositive staining for ZO-1 (scale bar 100 µm). Lower two images are a magnification of the border of the ME with the honeycomb pattern of ZO-1 (green, scale bar 10 µm) and. Cells where counterstained with Hoechst 33342 (blue).

Figure 4 IOD of Evans blue dye in the ME in chow-fed (n = 4, white bar) and fasted chow (n = 5, gray bar) and fcHFHS animals (n = 5, black bar) (A) and representative images of Evans blue dye in the ME and Arc of the hypothalamus of a chow-fed, fasted chow and fcHFHS diet animal. White asterix depicts third ventricle (B). Data are expressed as mean ± SEM.

Figure 5 Effects of 1 week fcHFHS (black bars) diet compared to chow (white bars) (n = 8, per group), after overnight fast, on ME-Arc area protein expression of ZO-1 (A), claudin-5 (B), occludin (C), and GLUT-1 (D). Data are expressed as mean ± SEM. Right: ZO-1 immunoreactivity in coronal sections of the ME and third ventricle of a representative fasted chow and fcHFHS diet rat. Arrowheads indicate immunopositive staining for ZO-1 (scale bar 100 µm). Lower two images are a magnification of the border of the ME with the honeycomb pattern of ZO-1 (green, scale bar 10 µm) and. Cells where counterstained with Hoechst 33342 (blue).