Global knockout of VEGFB improves lipoprotein lipase activity leading to an improved lipid profile during diabetes

Figures

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  FIGURE 1. Characterization of the global VegfbKO rat model (A) PCR amplification of gDNA extracted from heterozygous, WT and VegfbKO rat ear notch samples, confirming VegfbKO via beta-galactosidase knock-in (Vegfb at 610 bp, LacZ at 771 bp). (B,C) mRNA expression of Vegfb in WT and VegfbKO rat heart ventricles (n = 7–25) and skeletal muscle (n = 5–6) as determined using quantitative RT-PCR, confirming Vegfb gene deletion. (D) Spearman’s correlation heatmap of transcriptomic analysis of WT and VegfbKO hearts (n = 5). Data are presented as mean ± SEM. Unpaired t-tests were used to compare the two groups, ****p < 0.0001.

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  FIGURE 2. The severity of diabetes is similar in WT and VegfbKO rats injected with STZ (A) Blood glucose changes over the 4 days of STZ diabetes (n = 4–9). (B) Change in body weight in WT and VegfbKO rats after 4 days of STZ diabetes (n = 6–14). (C) Following acid-ethanol extraction, pancreatic insulin levels in the different groups were measured with an insulin ELISA (n = 4–8). (D) Plasma insulin in the different groups as measured (n = 8–15). (E) Key differentially expressed gene related to classic glucose metabolism genes as described using heat map (n = 5). Data are presented as mean ± SEM. Two-way ANOVA was used to analyze the data, ****p < 0.0001.

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  FIGURE 3. VegfbKO increases coronary vascular LPL activity following diabetes (A) Vascular LPL activity was determined by perfusing the isolated Langendorff heart with the highly negatively charged heparin and collecting the coronary perfusate over time for the determination of LPL activity. (B) Representative example of heparin-releasable LPL activity from a WT control heart following 10 min of heparin (5 U/mL; arrow) perfusion. Fractions of perfusate (over 15 s) were collected at the indicated times and analyzed for LPL activity. (C) Peak heparin-releasable LPL activity from hearts from the different groups was normalized to the peak LPL from WT control rats. Only the fold change is presented (n = 4–5). (D) Western blot of LPL protein from intact hearts isolated from different groups of rats (n = 5–14). The inset is a representative example of WT and VegfbKO heart samples following diabetes. (E) Potential mechanism by which Vegfb knockout affects coronary vascular LPL. In VegfbKO hearts, LPL is prevented from translocating to the vascular lumen and possibly accumulates in the cardiomyocytes (left panel); this effect is reversed with diabetes (right panel). Two-way ANOVA was used to…

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  FIGURE 4. Plasma lipid profile in WT and VegfbKO control and diabetic animals (A) Plasma triglycerides during diabetes (n = 13–17). (B) Total plasma FA levels following diabetes (n = 7–12). (C) Percentage of palmitic acid in the plasma (n = 5–6). (D) Percentage of linoleic acid in the plasma (n = 5–6). (E) Percentage of saturated FA (SFA) in the plasma (n = 5–6). (F) Expression of the FA transporter, Cd36 in the heart (n = 5–10). (G) Mechanism by which NEFA and LPL-derived FA contribute towards TG storage or oxidation. VegfbKO hearts use more LPL-derived FA as opposed NEFA, which in WT diabetes saturates mitochondria oxidation, leading to lipotoxicity. Data are presented as mean ± SEM. Two-way ANOVA was used to analyze the data, *p < 0.05,**p < 0.005, ****p < 0.0001.

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  FIGURE 5. Dotplots of the ventricular transcriptome of rats with diabetes clustered based on biological process. Dot plots display the top 20 most enriched GO biological process terms by gene ratio (the number of genes related to GO term/total number of significant genes). The adjusted p value is represented by color, and the size of the circle represents the counts involved in each biological process. Left panel is WT control versus WT diabetic rats. Right panel is VegfbKO control versus VegfbKO diabetic rats. The insets describe the total number of genes whose expression was either up- or downregulated in each comparison (n = 5).

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  FIGURE 6. Ventricular transcriptome with emphasis on FA metabolism and angiogenesis in rats with diabetes (A) The volcano plots describe the profile of differentially expressed genes associated with FA metabolism in WT and VegfbKO rats following diabetes (n = 5). (B) The heat map was used to represent the key differentially expressed gene related to beta oxidation (n = 5). (C) Volcano plot expresses differentially expressed genes linked with angiogenesis in WT and VegfbKO rats following diabetes (n = 5). The x-axis represents Log2FC expression of genes versus Log2CPM on the y-axis. CPM indicates counts per million.

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  FIGURE 7. Ventricular transcriptome and protein expression related to cell death in rats with diabetes (A) The volcano plots describe the profile of differentially expressed genes associated with cell death in WT and VegfbKO following diabetes (n = 5). The x-axis represents Log2FC expression of genes versus Log2CPM on the y-axis. CPM indicates counts per million. (B) The expression of cleaved PARP/full length PARP in hearts from the different groups (n = 5–13). The inset is a Western blot representing WT and VegfbKO hearts following diabetes. Two-way ANOVA was used to analyze the data. Data are presented as mean ± SEM, *p < 0.05.