A ketogenic diet reduces metabolic syndrome-induced allodynia and promotes peripheral nerve growth in mice

Abstract

Current experiments investigated whether a ketogenic diet impacts neuropathy associated with obesity and prediabetes. Mice challenged with a ketogenic diet were compared to mice fed a high-fat diet or a high-fat diet plus exercise. Additionally, an intervention switching to a ketogenic diet following 8 weeks of high-fat diet was performed to compare how a control diet, exercise, or a ketogenic diet affects metabolic syndrome-induced neural complications. When challenged with a ketogenic diet, mice had reduced bodyweight and fat mass compared to high-fat-fed mice, and were similar to exercised, high-fat-fed mice. High-fat-fed, exercised and ketogenic-fed mice had mildly elevated blood glucose; conversely, ketogenic diet-fed mice were unique in having reduced serum insulin levels. Ketogenic diet-fed mice never developed mechanical allodynia contrary to mice fed a high-fat diet. Ketogenic diet fed mice also had increased epidermal axon density compared all other groups. When a ketogenic diet was used as an intervention, a ketogenic diet was unable to reverse high-fat fed-induced metabolic changes but was able to significantly reverse a high-fat diet-induced mechanical allodynia. As an intervention, a ketogenic diet also increased epidermal axon density. In vitro studies revealed increased neurite outgrowth in sensory neurons from mice fed a ketogenic diet and in neurons from normal diet-fed mice given ketone bodies in the culture medium. These results suggest a ketogenic diet can prevent certain complications of prediabetes and provides significant benefits to peripheral axons and sensory dysfunction.

Keywords: DRG; Diabetes; Exercise; High-fat; Ketogenic; Ketones; Mice; Pain.

Figure 1. Experimental Timeline of Cohorts

a) Challenge cohort: All mice remained on a control diet until baseline testing was completed, then mice were changed to experimental diets and exercise for 12 weeks. b) Intervention cohort: all mice remained on a high fat diet for 8 weeks, at which point baseline testing was completed, mice were divided into different intervention groups and provided different diets and exercise for 12 additional weeks.

Figure 2. A Ketogenic Diet Leads to a Different Metabolic Profile than a High-fat Diet

(a) A high-fat diet causes weight gain relative to all groups, whereas exercised or ketogenic diet-fed mice displayed significant weight gain relative to standard diet controls (n=18 for all groups). (b) High-fat exercised and ketogenic-diet fed mice displayed increased energy intake relative to CF-Sed or HF-Sed mice (n=18 for all groups) (c) Body composition analysis performed by EchoMRI of fat mass following 12 weeks of diet and exercise revealed that a high-fat diet causes increased fat mass in mice relative to all groups, while exercised or a ketogenic diet-fed mice display fat mass levels more similar to CF-Sed control mice. Ketogenic diet-fed mice displayed increased fat mass compared to HF-Ex mice (n=18 for all groups) (d) Ketogenic diet-fed mice had increased serum ketone levels after 1 week, with a decrease over time. However, ketone levels remained slightly elevated compared to all other groups All data presented as mean ± SEM *:CF-Sed vs. HF-Sed #: Keto vs. HF-Sed ^: HF-Sed vs. HF-Ex +: HF-Ex vs. CF-Sed °: Keto vs. CF-Sed $: Keto vs. HF-Ex * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001

Figure 3. A Ketogenic Diet Does Not Alter Glucose Tolerance and Fasting Insulin Similar to A High-fat Diet

(a) Compared to control-diet fed mice, moderate increases in blood glucose levels were evident in mice fed a high-fat diet, a high-fat diet plus exercise and mice fed a ketogenic diet. Frank hyperglycemia never developed in any group (n=18 for all groups). (b) Following 12 weeks of dietary challenge, high-fat diet fed sedentary mice had a reduced IPGTT relative to control-diet fed mice, while ketogenic-diet fed mice displayed no significant changes in IPGTT compared to control diet-fed mice (n=5 for all groups). (c) After 12 weeks, a high-fat diet increased serum insulin levels compared to both ketogenic-diet fed and control-diet fed mice. (d) A high-fat diet, even with exercise increased HOMA-IR values compared to both control- and ketogenic-diet fed mice following 12 weeks of diet or exercise. All data presented as mean ± SEM *:CF-Sed vs. HF-Sed #: Keto vs. HF-Sed ^: HF-Sed vs. HF-Ex +: HF-Ex vs. CF-Sed °: Keto vs. CF-Sed $: Keto vs. HF-Ex * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001

Figure 4. A Ketogenic Diet Obesity Intervention Does Not Reverse Obesity Measures but Decreases Insulin

(a) Following 8 weeks of a high-fat diet, mice remaining on a high-fat diet or those switched to a ketogenic diet continued to gain weight for the remainder of the study. Mice given access to a running wheel maintained their weight, while those switched to a control diet lost weight (b) Following 8 weeks of a high-fat diet, mice switched to a ketogenic diet still consumed the most energy per day (c) Mice given access to a voluntary running wheel or a control diet had decreased fat mass (n=10 for all groups) d) Following 8 weeks of a high-fat diet, only a control-fed sedentary intervention reduced blood glucose levels (n=10 for all groups) (e) Insulin levels were normalized by switching mice back to a control diet, whereas maintenance on a high fat diet (exercised or not) kept insulin levels elevated. Mice switched to a ketogenic diet fell in between levels in control-diet and high fat diet-fed mice. (f) Following 8 weeks of a high-fat diet, only an intervention using control-fed diet was able to normalize HOMA-IR. Ketogenic-diet fed mice again fell between mice fed a control-diet or a high fat diet. All data presented as mean ± SEM #: HF-Sed vs. Keto ^: HF-Sed vs. HF-Ex °: CF-Sed vs. Keto ∇: HF-Ex vs. Keto * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001

Figure 5. A Ketogenic Diet Prevents and Reverses Diet-Induced Mechanical Allodynia

(a) Mechanical paw withdrawal thresholds reveal that mechanical allodynia develops in mice challenged with a high-diet compared to mice fed a control diet. (b) Thermal sensitivity was unaffected by diet or exercise in any group. (c) As an intervention, mice fed a ketogenic diet has significant reversal of mechanical allodynia. All data presented as mean ± SEM *: CF-Sed vs. HF-Sed #: Keto vs. HF-Sed * $: Keto vs. HF-Ex * p<0.05; ** p<0.01; *** p<0.001

Figure 6. IENFD Is Increased By A Ketogenic Diet

(a) A ketogenic-fed mouse footpad stained with the panaxonal antibody protein gene product 9.5 (PGP) (red) and NGF receptor TrkA (green) following 12 weeks of diet in the challenge cohort (b) ketogenic-fed mouse PGP fibers 12 weeks following the intervention from a high-fat diet in the intervention cohort (c) In the challenge cohort, ketogenic-fed mice have increased epidermal nerve fiber density compared to all other groups. High-fat-fed exercised mice displayed decreased nerve fiber density compared to all groups (d) in the intervention cohort, ketogenic-fed mice also displayed increased epidermal nerve fiber density compared to all other groups. High-fat-exercised mice displayed decreased nerve fiber density compared to control-fed sedentary mice. All data presented as mean ± SEM * p<0.05; ** p<0.01; *** p<0.001, **** p<0.0001

Figure 7. Neurite Outgrowth Is Increased By Ketone Supplementation

(a) Neurite outgrowth of primary DRG from ketogenic-fed mice following 4 days of culture in 10mM glucose Nutrient Hams F12 media (b) Neurite outgrowth of primary DRG from control-fed mice cultured in Nutrient Hams F12 media with 5mM glucose and 5mM ketone (c) Mice fed a ketogenic diet one month prior to culture display improved neurite outgrowth as compared to mice fed a control or high-fat diet when all placed in 10mM glucose F12 media (d) DRG cultured in F12 media supplemented with 5mM ketones and 5mM glucose or 10mM ketones display improved neurite outgrowth as compared to traditional 10mM glucose media. All data presented as mean ± SEM * p<0.05; *** p<0.001