An In Vivo Study Of Bone Microstructure And Metabolism Changes Under The Combined Intervention Of Ketogenic Diet With Intermittent Fasting

Ketogenic diet(KD)is characterized by low carbohydrate and high fat.As an effective regimen,it has been used in paediatric intractable epilepsy for decades.Every-other-day ketogenic diet(EODKD),the combination of KD with intermittent fasting,showed encouraging potential for better control of seizures somehow.However,there is no concordance in the literature about the chronic side effects of EODKD,especially it’s indistinct if EODKD would lead to severer bone loss than KD,which merit consideration when EODKD is used clinically.This study aims to establish the Sprague-Dawley(SD)rat model of KD and EODKD so as to compare the integrated effects of different ketogenic condition on the microstructure and mechanical strength of long bones in rats,as well as the potential mechanisms of bone loss.Part I Establishment of Sprague-Dawley(SD)rats models of KD and EODKD1.Backgrounds and objectives There are strong clinical evidence indicating that KD is effective in diseases like epilepsy,obesity,diabetes and even spinal cord injury.Previously,there were plenty of researches which have set up the animal models of high-fat and low-carbohydrate(HFLC)diet.As a special form of HFLC,it is necessary to establish a model of KD for further study.Besides,by combining KD with intermittent fasting,we found EODKD could induce a higher level of ketone body in both serum and cerebral fluid of rats.Meanwhile,it could have better control of seizures than KD clinically.The study aimed to establish the animal models of KD and EODKD using SD rats in order to compare the metabolic changes in different ketogenic status,.2.Methods Thirty 8-week old male SD rats were randomly and equally divided into the KD group,the EODKD group and the Control group based on the principle of weight proximity.They were fed with continuous,intermittent ketogenic fodder and persistent standard diet respectively for totally 12 weeks.The ketogenic fodder used in our study contained a ratio of fat to carbohydrate plus protein nearly 3:1,and for the EODKD group,it was added the first day at 8 a.m.and removed the next 8 a.m.(with only water left)and so on.During the whole experimental period,the body weights of all rats were obtained weekly,while the blood ketone and glucose levels were tested fortnightly by tail vein puncture.For the EODKD group,the blood samples were always collected at the end of fasting days.The curves of body weight,blood ketone and glucose levels of each week were made after experiment.One-Way ANOVA with subsequent Tukey post hoc test was used to compare the initial data between groups,while the data during the entire experiment was compared using ANOVA for repeated measurement with Tukey post hoc test.3.Results The initial data between all groups showed similar level at body weight,blood ketone and blood glucose.Compared with Control group,both KD and EODKD groups showed significantly decreased body weight throughout the 12-week experimental period(p=0.042 and p=0.013 respectively),but no difference were found between them.Averagely the ketone levels in three groups are 0.3±0.1mmol/L,1.7±0.6mmol/L and 2.7±1.0mmol/L respectively for the Control,KD and EODKD group.Both KD and EODKD group were significantly higher than the Control group during the experiment(both p<0.001),and EODKD was even higher than the KD group(p=0.016).On the contrary,the Control group had a 7.0±0.6mmol/L blood glucose on average during the study,which was the highest in three groups(p=0.005 and p=0.011 respectively compared with KD and EODKD).And no difference was found in glucose levels between the rest two groups(KD:5.9±0.5mmol/L;EODKD:5.8±0.6mmol/L).4.Conclusions This study successfully established rat models of KD and EODKD,which were characterized by sustained high ketone le-vels and relatively declined glucose levels.Compared with the KD group,the EODKD could induce a higher state of ketogenesis but with a similar level of glucose.Besides,compared with standard diet,12-week feeding of KD may lead to significant decline in body weight,but it may not demonstrate further reduction under EODKD.Part II Comparison of KD and EODKD on bone microarchitecture and metabolism1.Backgrounds and objective The decline of bone mass and deterioration of trabecular architecture may lead to increased bone fragility and risks of fractures,thus early prevention is obviously important.As KD is getting popular in clinical application,there are repeatedly clinical reports about the metabolic disorders in mineral content,as well as impaired bone mineral density(BMD)and bone growth in patients under KD.On the other hand,EODKD could induce a higher ketone level and have better seizure control than KD,it could somehow replace KD in the future.However,it’s indistinct about the side effects of EODKD,especially on bone loss.The study aims to clarify the effects of KD in bone mass of rats,and compare EODKD with it in aspects of bone metabolism and microstructure.2.Methods After 12 weeks differential feeding,total BMD and body fat percentage were obtained by dual energy X-ray absorptiometry(DEXA),after which the proximal tibia were analyzed using high resolution micro-CT,and mechanical strength of femur were evaluated by three-point bending test.Serum Calcium,phosphate,25-OH vitamin D,alkaline phosphatase(ALP)and tartrate-resistant acid phosphatase(TRAP)were measured,with histologic staining of collagen type I(Col I),osteocalcin(OCN)and TRAP.Besides,osteogenic capabilities of bone marrow stromal cells(BMSCs)in each group were determined by ALP activity and alizarin red staining of calcium nodules.3.Results Compared with Control group,KD and EODKD led to higher body fat percentage and lower BMD.They also compromised both cancellous and cortical bone mass,with enhanced osteoclastic and alleviated osteogenic process as well.Compared with KD group,EODKD group showed both significantly suppressed osteoclastic and osteogenic process,but no significance in bone microstructures or mechanical properties were found between them.4.Conclusion Both KD and EODKD may lead to osteoporotic effects in long bones,potential mechanisms may include inhibited bone formation and activated bone resorption.Compared with KD,EODKD may not induce aggravated bone loss and mechanical properties,paving a basis for its application in better seizure control.