1. Ketogenic diet induced weight loss and affected metabolite levels in blood and tissues
After feeding for 2 weeks, we measured the body weights, and found it to be significantly lower in the KD group than in the ND group (Fig. 1a). We also measured random blood glucose and blood ketone levels (represented by blood β-hydroxybutyrate) using glucose and ketone meters during 4 weeks of feeding. We did not find a significant change in random blood glucose levels in ND-fed mice; in KD-fed mice, however, blood glucose gradually decreased in the first 2 weeks then remained stable in the third and fourth weeks (Fig. 1b). Similarly, there was no significant change in blood ketone levels of ND-fed mice; conversely, in mice fed the KD, there was a remarkable increase in blood ketone levels in the first week of feeding, reaching 2.7 mmol/L, then gradually returning to approximately 2 mmol/L in the second week and mostly remaining stable in third and fourth weeks (Fig. 1c). Finally, we measured β-hydroxybutyrate content in different tissues including the muscle, heart, and liver using a β-hydroxybutyrate assay kit, and found that β-hydroxybutyrate levels increased in the heart and muscle tissues of mice in the KD group, with a greater increase in the heart tissue compared with mice in the ND group. There was no significant difference in β-hydroxybutyrate content in liver tissues between the two groups (Fig. 1d).
2. KD impaired perfusion recovery and revascularization in chronic hind limb ischemia
To examine the effect of KD on perfusion recovery ability in chronic ischemic injury, a mouse ischemic limb model was used in mice fed both the KD and ND. Perfusion was assayed by laser Doppler perfusion imaging on days 0, 7, and 21 following femoral artery ligation surgery. We observed that the KD reduced perfusion signals associated with more pronounced non-perfusion signals in ischemic limbs than in mice fed the ND on days 7 and 21 (Figure 2a). Quantitatively, we calculated the ratio of perfusion between ischemic and non-ischemic limbs for each mouse and found that the perfusion ratio was 86.2% at the 3-week point in the ND group, whereas it was only 52.4% in the KD group. There were no significant differences between the two groups on day 0, indicating slow recovery of limb circulation caused by KD (Figure 2b). To assess the angiogenic effect, capillary density was measured. Using immunofluorescence staining, capillary density was detected using anti-CD31 antibody in the hindlimb (Figure 2c). The micrographs showed that ischemic limbs of KD-fed mice displayed reduced capillary density (less red CD31 (+) signal), indicating reduced angiogenesis (Figure 2d). This was confirmed by western blot (WB) and quantitative PCR (qPCR) results of CD31 and vascular endothelial growth factor A (VEGFA), two common indicators of tissue revascularization . By performing qPCR, we found that the mRNA levels of both CD31 (platelet endothelial cell adhesion molecule-1) and VEGFA were significantly reduced in the ischemic hind limb tissue of mice in the KD group compared with those in the ND group (Figure 2e). Further WB tests showed that both CD31 and VEGFA protein expression levels significantly decreased in the hind limb tissues of KD mice following ischemic surgery compared with mice in the ND group, but no significant difference was found in limb tissues between the KD and ND groups before ischemic surgery, indicating that KD reduced revascularization in hind limb tissue after ischemia (Figure 2f). Overall, these data indicate that KD impeded revascularization and blood perfusion of hind limb tissue after ischemia.
3. KD induced muscle atrophy
To assess the effect of KD on muscle regeneration and recovery following hind limb ischemia, we observed gastrocnemius muscle shape and weighed gastrocnemius muscle mass of both legs of mice fed KD and ND 28 days after surgery. We observed obvious atrophy of the gastrocnemius muscle on both legs of KD mice compared with those of mice in the ND group (Fig. 3a). To further confirm muscle atrophy in KD mice, we then assessed gastrocnemius muscle mass and found that the net weight of both the ischemic and lateral gastrocnemius muscles of mice in the KD group significantly decreased compared with that of mice in the ND group, indicating that KD not only induced muscle atrophy of the ischemic limb but also caused atrophy in non-ischemic muscle (Fig. 3b). We analyzed the muscle recovery rate by calculating the ratio of ischemic gastrocnemius muscle weight to lateral gastrocnemius muscle weight of each mouse and found that the recovery ability of the gastrocnemius muscle after ischemia was reduced in KD mice compared with mice in the ND group (Fig. 3c). We further assessed the effect of KD on muscle regeneration ability by H&E staining of the gastrocnemius muscle of the ischemic hind limb. We observed irregular and small muscle fibers in the KD group compared to the ND group (Fig. 3d) and found that the calculated regenerating area decreased in mice fed KD based on H&E staining (Fig. 3e). Since we found muscle atrophy both in ischemic and nonischemic limb tissues, we hypothesized that there may be other causes for muscle atrophy besides reduced blood flow, thus we further examined whether muscle atrophy-related genes (FOXO3 and LC3) were involved in this process. We found mRNA expression levels of both FOXO3 and LC3 genes were significantly decreased in ischemic hind limb tissues of mice in the KD group compared with mice in the ND group, indicating that induction of muscle atrophy-related gene expression may be one of the reasons for muscle atrophy caused by KD.
4. KD delayed wound healing and increased toe necrosis rate
We observed wound healing at the surgical site in each mouse and found that surgical wounds of mice in the ND group healed much faster than those of mice in the KD group. Images of wound closure in mice 28 days after hind limb surgery showed that the surgical wound of each mouse in the ND group healed completely when inflammatory and purulent exudation was observed at the surgical wound of mice in the KD group, indicating that KD significantly delayed wound healing and caused inflammation around the surgical sites (Fig. 4a). We also observed severe toe necrosis in KD mice and analyzed the necrosis ratio of toes in the two groups using necrosis score (1 point for toenail blackening, 3 points for toe necrosis, and 5 points for foot having fallen off) and found a higher ratio of toe necrosis in the KD group than in the ND group (Fig. 4b). H&E staining also revealed massive inflammatory cell infiltration in ischemic hind limb tissue in the KD group, while there was no sign of inflammation in the ND group (Fig. 4c). The ratio of necrotic area analysis based on H&E staining showed a significantly higher ratio of necrotic area in the KD group than in the ND group (Fig. 4d). According to a previous study, KD has anti-inflammatory effects by reducing the inflammasome (NLRP3) and inflammatory gene expression, such as IL-β and IL-6 . Therefore, we further examined the expression of inflammation-related genes (IL-β, IL-6, and IL-18) in the ischemic hind limb tissue of mice in the ND and KD groups and found that KD significantly decreased inflammatory gene expression in ischemic limb tissues, further explaining the aggravated inflammation in KD mice (Fig. 4e).
5. KD indued ischemic limb tissue fibrosis
We evaluated the effect of KD on limb tissue fibrosis after ischemia using Masson staining and examination of fibrosis-related gene expression. Masson staining images showed severe fibrosis in the ischemic hind limb tissue of mice in the KD group compared to that of mice in the ND group (Fig. 5a). Additionally, analysis of the fibrotic area in the two groups based on Masson staining showed a significant increase in the fibrotic area of ischemic hind limb tissue of mice in the KD group compared with that of those in the ND group (Fig. 5b). We then examined the gene expression levels of Cola2 and α-SMA to further evaluate fibrosis, and found that both Cola2 and α-SMA mRNA expression levels increased in the ischemic limb tissue of mice in the KD group compared with those in the ND group (Fig. 5c). Moreover, this was consistent with the finding of increased α-SMA protein expression in ischemic tissue of mice in the KD group (Fig. 5d). We also found α-SMA protein expression was slightly increased in non-ischemic tissue of mice in the KD group compared to that of mice in the ND group, indicating that KD can also trigger fibrosis without an ischemic condition.
6. KD affected hind limb tissue metabolism both before and after ischemia at the genetic level
To understand the cellular impact of the KD, we investigated the metabolic status of hind limb tissues by examining metabolism-related genes, including those present during glycolysis (represented by GLUT4, GLUT1, HK2, and PDK1), fatty acid oxidation (represented by CD36 and CPT1), and ketone body metabolism (represented by HMGCS2, BDH1, and SCOT), before and after ischemic surgery. qPCR analysis of hind limb tissues before ischemia in the two groups of mice showed that KD significantly decreased glycolysis by decreasing GLUT4, GLUT1, and HK2 gene expression and increasing PDK1 gene expression (Fig. 6a), while it increased fatty acid utilization by increasing CD36 and CPT1 gene expression compared with ND mice (Fig. 6b). Ketolysis was simultaneously reduced by decreased BHD1 and SCOT gene expression in KD mice, while there was no significant difference in ketogenesis between the two groups represented by HMGCS2 gene expression (Fig. 6c). qPCR performed subsequently in ischemic limb tissues of mice in the two groups on day 7 after ischemic surgery showed that KD further decreased glycolysis in limb tissue after ischemia (Fig. 6d). In contrast to the result of increased fatty acid oxidation found in non-ischemic tissue of mice fed with KD (Fig. 6e), fatty acid oxidation was decreased in limb tissue after ischemia. The effect of KD on ketone metabolism in ischemic tissue was observed by further decreased ketolysis and increased ketogenesis, which also differed from the results observed in non-ischemic tissue (Fig. 6f).
7. KD affected hind limb tissue metabolism both before and after ischemia at the protein level
We further evaluated the metabolic changes caused by KD at the protein level in the hind limb tissues before and after ischemia. We performed WB (Fig. 7a) and found that KD decreased glucose uptake in the hind limb tissue of mice both before and after ischemia, as represented by GLUT4 and GLUT1, while a decrease was more significant in ischemic tissue (Fig. 6b, c). We further investigated how KD affects glycolysis, and found that expression of the HK2 protein, a glycolytic enzyme, was also decreased in the hind limb tissue of KD mice both before and after ischemia, but with a more significant decrease in ischemic tissue than that in mice in the ND group (Fig. 7d). However, PDK1 protein, an inhibitor of glycolysis, showed increased expression in hind limb tissue both before and after ischemia in KD mice, with a greater increase in ischemic tissue than that in mice in the ND group (Fig. 7e). The above results indicated that KD decreased glycolysis in hind limb tissue at the protein level, both before and after ischemia, and that it produced a greater decrease after ischemia. We examined CPT1 protein expression, representing fatty acid uptake, and found that it was increased in hind limb tissue of KD mice before ischemia, but decreased after ischemia compared with ND mice, indicating that KD increased fatty acid utilization of limb tissue before ischemia, and decreased its utilization under ischemic conditions (Fig. 7f). We also observed BDH1 and SCOT protein expression level, representing ketolysis of limb tissue; these were decreased in limb tissue of mice both before and after surgery in the KD group, while the decrease was more significant in limb tissue after ischemia compared to that of mice in the ND group (Fig. 7g, h).