N-acetylcarnosine attenuates age-associated declines in multi-organ systems to improve survival

Abstract

Histidine containing dipeptides (HCDs) such as N-acetylcarnosine are endogenous metabolites that are ergogenic and mitigate metabolic dysfunction. We previously demonstrated that short-term N-acetylcarnosine treatment is highly efficacious in protecting muscle atrophy induced by disuse. Here we demonstrate that a 6-months treatment of N-acetylcarnosine attenuates a broad spectrum of age-associated maladies and improved survival by ~50% in female mice. A comprehensive survey of organ systems revealed that N-acetylcarnosine prevents decline in adiposity, diastolic function, vasodilation, muscle strength, and bone density. Together, N-acetylcarnosine substantially delays the onset of system-wide end-stage pathology to prolong lifespan. As an endogenously present metabolite, treatment with N-acetylcarnosine may be a safe and promising intervention to promote healthy aging in humans.

Figure 1 –. N-acetylcarnosine supplementation preferentially improves female mouse survival.

A) Schematic of the preclinical trial design. C57Bl/6 mice from the NIA aging colony were acquired at 17 months of age. After acclimation, mice underwent baseline testing before being randomized to receive either vehicle or N-acetylcarnosine-supplemented water for 6 months. Mice were weekly monitored for body weight and food and water consumption. Various physiologic tests were conducted during the 5^th month of the intervention. Mice were euthanized and tissues were harvested after 6 months of intervention period. B) Schematic representation of HCD metabolism. Radar plots of C) N-acetylcarnosine, D) protein carbonyls, E) Carnosine, F) Histidine, G) Anserine, and H) Homocarnosine demonstrate enrichment of N-acetylcarnosine. Values in radar plots are Z-scores. Corresponding concentrations and statistical analyses are in Supplemental Figure 1. Vehicle treated female (I) and male (J) mice experienced typical survival rates for this strain and colony (approximately 50% and 75% respectively) at 24 months. Female mice (starting n=45 vehicle, n=46 N-acetylcarnosine) treated with N-acetylcarnosine improved their survival probability by ~50%. Statistical testing for HCD enrichment was performed via two-way repeated measures ANOVA with Bonferroni post-hoc test where appropriate. Survival was assessed via generating Kaplan-Meier survival curves and testing the disparity of the curves via Mantel-Cox test. Comparisons were considered statistically significant if p<0.05. HCD; Histidine Containing Dipeptide, GABA; gamma-aminobutyric acid, CN1; carnosinase 1, CARNS; carnosine synthase. Schematic images were generated via BioRender.

Figure 2 –. N-acetylcarnosine supplementation preserves body mass in female mice.

Body weights in the female (starting n=39 vehicle, n=40 N-acetylcarnosine) (A) and male (starting n=20 per group) (B) mice fell precipitously over the course of the trial regardless of treatment. Female mice treated with N-acetylcarnosine were able to delay the decrease in bodyweight. Bodyweights are reported as one-month averages and are presented as Mean ± SEM. N-acetylcarnosine did not affect lean mass in female (C) or male (D) mice. N-acetylcarnosine preserved fat mass in female mice (E) (n=57 per group) but not male (F) (n=20 per group) mice. G) Preservation of fat mass in female mice was attributed to larger gWAT not iWAT or BAT fat pad (n=16 vehicle, n=20 N-acetylcarnosine). H) H&E stain of gWAT tissues did not reveal differences in adipocyte size between groups. Despite increased adiposity, liver weight (I) was not altered by N-acetylcarnosine (n=15 vehicle, n=20 N-acetylcarnosine. J) H&E and trichrome stains did not indicate altered steatosis or fibrosis in livers. Statistical testing for bodyweight and body composition comparisons was performed via one-way repeated measures ANOVA with Bonferroni post-hoc test where appropriate. Statistical testing for comparison of tissue weights was performed via unpaired student t-test. Comparisons were considered statistically significant if p<0.05. gonadal white adipose tissue; gWAT, inguinal white adipose tissue; iWAT, Brown Adipose Tissue; BAT.

Figure 3 –. N-acetylcarnosine supplementation does not alter metabolic or neuroendocrine system in female mice.

A) Glucose tolerance via intraperitoneal glucose tolerance test (IPGTT) was not different between treatment and vehicle mice (n=6 vehicle, n=9 N-acetylcarnosine). 72-hour indirect calorimetry revealed suppressed energy expenditure (VO₂) (B) and respiratory exchange ratio (RER) (C) during both light and dark phases (n=5 vehicle, n=8 N-acetylcarnosine). Respiratory exchange ratio is the volume of CO₂ consumed (VCO₂) proportional to the O₂ consumed (VO₂) and indicates substrate utilization whereby a value closer to 0.75 indicates complete reliance on fat oxidation and a value closer to 1 indicates complete reliance on glucose oxidation. Ambulatory frequency was also suppressed in N-acetylcarnosine mice (D) (n=5 vehicle, n=8 N-acetylcarnosine). E) Brain weights were not different between treatment groups (n=14 vehicle, n=19 N-acetylcarnosine). F) Change in time spent freezing before and after an audible tone that proceeded with a shock on the first (condition day) but not the second (follow up) day was not different between groups indicating no effect of N-acetylcarnosine on conditional memory formation (n=5 vehicle, n=9 N-acetylcarnosine). G) Frequency of novel, non-consecutive exploration (alterations) between arms in the Y-maze was not different between groups indicating no effect of N-acetylcarnosine on short-term memory (n=20/14 pre/post vehicle, n=20/16 pre/post N-acetylcarnosine). H) Peripheral pain tolerance assessed via Von Frey hair filaments were not different between groups (n=5 vehicle, n=9 N-acetylcarnosine). Data are represented as Mean ± SEM. Statistical significance was assessed via two-way repeated measures ANOVA with Bonferroni correction, or unpaired t-test where appropriate. Comparisons were considered statistically significant if p<0.05. RER; respiratory exchange ratio, CLAMS; comprehensive lab animal monitoring system.

Figure 4 –. N-acetylcarnosine supplementation improves cardiovascular function in female mice.

A) Heart weight normalized to tibial length was not different between groups of female mice. B) H&E staining of heart tissues was also indiscernible between groups. C) Heart rate measured via electrocardiogram was performed on anesthetized mice during echocardiography and was not different between groups. Metrics of systolic function including cardiac output (D) and ejection fraction (E) were not different between groups whereas fractional shortening was reduced by N-acetylcarnosine treatment (F). Measures of diastolic function including E/A (ventricular filling) (G) and E/e’ (marker of left ventricular filling pressures) (H) were not different between groups. Isolated femoral arteries (n=8 vehicle, n=15 N-acetylcarnosine, 2 segments per mouse) were pre-contracted to 65% of maximal developed tension, and vasorelaxation to cumulative doses of Ach was recorded as percent (%) vasorelaxation. No differences were observed between groups. A second Ach dose-response curve was completed (30-min later) in arteries from both groups that incubated with LNMMA for 30-min. While L-NMMA attenuated vasorelaxation in arteries from vehicle and N-acetylcarnosine-treated mice, the contribution from NO to vasorelaxation was greater in the latter group (I), an observation that was substantiated by calculating NO bioavailability (J). Data are represented as Mean ± SEM. Statistical significance was assessed via two-way repeated measures ANOVA with Bonferroni correction, or unpaired t-test where appropriate. Comparisons were considered statistically significant if p<0.05. Heart Rate; HR.

Figure 5 –. N-acetylcarnosine supplementation does not alter renal function in female mice.

Kidney weight was not different between groups (A). H&E and Trichrome stains of kidney sections were unremarkable and indistinguishable between groups (B). Mice were placed in single housed, urine collection chambers for 24 hours to collect 24-hour urine volume which was not different between groups (C). 24-hour urine and plasma and urine creatinine concentration were used to determine creatinine clearance (D) which also was not affected by N-acetylcarnosine. Glomerular filtration determined by monitoring the filtration of a FIT-c labeled senestrin under anesthesia was also not different between groups in female mice (n=5 vehicle, n=7 N-acetyl carnosine) (E). Data are represented as Mean ± SEM. Statistical significance was assessed via two-way repeated measures ANOVA with Bonferroni correction, or unpaired t-test where appropriate. Comparisons were considered statistically significant if p<0.05.

Figure 6 –. N-acetylcarnosine supplementation improves muscle function in female mice.

A) Lower hind limb weights were not different between groups (n=23–24 vehicle, n=35–37 N-acetylcarnosine) with the exception of gastrocnemius (Gast). C) Histogram of the frequency of fibers sizes was not different between groups in EDL (n=6 vehicle, n=8 N-acetylcarnosine). D) Representative image of immunofluorescent stain for laminin used to determine muscle cross sectional area in the EDL. Ex vivo specific force measured in EDL (E) (n=8 vehicle, n=8 N-acetylcarnosine) and SOL (F) (n=8 vehicle, n=9 N-acetylcarnosine) were elevated in N-acetylcarnosine-treated mice across stimulation frequencies ranging from 10–200 Hz. In vivo functional data as quantified by rotarod (G), grip strength (H), and treadmill work capacity (I) were unaffected by N-acetylcarnosine treatment. Data are represented as Mean ± SEM. Statistical significance was assessed via two-way repeated measures ANOVA or two-way ANOVA with Bonferroni correction, or unpaired t-test where appropriate. Comparisons were considered statistically significant if p<0.05. Tibialis anterior; TA, plantaris; Plant, gastrocnemius; Gast, extensor digitorum longus; EDL, soleus; SOL.

Figure 7 –. N-acetylcarnosine supplementation improves bone density in female mice.

A) Representative μCT image of femur analyzed for bone density in the distal femur. μCT of femurs revealed increased trabecular number (B) and decreased trabecular spacing (C) without a change in trabecular thickness (D) which contributed to a near-significant increase in the proportion of bone volume (E) in the ROI. Data are represented as Mean ± SEM. Statistical significance was assessed via unpaired t-test where appropriate. Comparisons were considered statistically significant if p<0.05. Cross sectional area; CSA, micro computed tomography; μCT.