How does 5-Amino-1MQ (and JBSNF) increase NAD+ and Metabolic Rate while Regenerating Aging Skeletal Muscle?

[01dragonslayer]

“Aging is accompanied by progressive declines in skeletal muscle mass and strength and impaired regenerative capacity, predisposing older adults to debilitating age-related muscle deteriorations and severe morbidity.

Muscle stem cells (muSCs) that proliferate, differentiate to fusion-competent myoblasts, and facilitate muscle regeneration are increasingly dysfunctional upon aging, impairing muscle recovery after injury. While regulators of muSC activity can offer novel therapeutics to improve recovery and reduce morbidity among aged adults, there are no known muSC regenerative small molecule therapeutics.

We recently developed small molecule inhibitors of nicotinamide N- methyltransferase (NNMT), an enzyme overexpressed with aging in skeletal muscles and linked to impairment of the NAD+ salvage pathway, dysregulated sirtuin 1 activity, and increased muSC senescence. We hypothesized that NNMT inhibitor (NNMTi) treatment will rescue age-related deficits in muSC activity to promote superior regeneration post-injury in aging muscle.

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Results revealed that muscle stem cell proliferation and subsequent fusion were elevated in NNMTi-treated mice, supporting nearly 2-fold greater CSA and shifts in fiber size distribution to greater proportions of larger sized myofibers and fewer smaller sized fibers in NNMTi-treated mice compared to controls. Prolonged NNMTi treatment post-injury further augmented myofiber regeneration evinced by increasingly larger fiber CSA. Importantly, improved muSC activity translated not only to larger myofibers after injury but also to greater contractile function, with the peak torque of the TA increased by ~70% in NNMTi-treated mice compared to controls. Similar results were recapitulated in vitro with C2C12 myoblasts, where NNMTi treatment promoted and enhanced myoblast differentiation with supporting changes in the cellular NAD+/NADH redox states. Taken together, these results provide the first clear evidence that NNMT inhibitors constitute a viable pharmacological approach to enhance aged muscle regeneration by rescuing muSC function, supporting the development of NNMTi as novel mechanism-of-action therapeutic to improve skeletal muscle regenerative capacity and functional recovery after musculoskeletal injury in older adults.” (1)

Risk of Muscle Wasting in Elderly:​

"The population of older (60+ years of age) adults is rapidly expanding in the United States and throughout the world, placing ever-increasing strains on health care resources and an urgent need for improved approaches to elder care. One of the most significant impacts of aging is the progressive decline in skeletal muscle mass and strength, with concomitant deteriorations in physical function and mobility that are strongly associated with numerous chronic diseases and increased mortality. While all older individuals experience muscle degeneration, approximately 30% of adults over 60 years of age and 50% of adults over 80 years of age develop sarcopenia, a geriatric disease characterized by significant and objective defects in muscle mass, strength, and function. Sarcopenic elderly individuals are at a 2-to 5-fold increased risk for permanent disability and greatly diminished quality of life arising from progressive muscle degeneration, decreased muscle function, and poor muscle quality that predispose them to debilitating falls and substantial disease burden. Furthermore, as muscle regenerative capacity of older adults becomes increasingly compromised, it leads to delayed and impaired recovery following muscle injury, decreased mobility and independence, increased hospitalization costs, and higher mortality rates."

What are Muscle Stem Cells?​

"Muscle stem cells (muSCs; also termed satellite cells) are responsible for mediating skeletal muscle repair by proliferating and differentiating into fusion-competent myoblasts and facilitating myofiber regeneration following injury. The major driving factor for delayed and impaired recovery of aged muscle following injury appears to be a significant decrease in muSC regenerative capacity and function. These events occur independent of sarcopenia, resulting in the formation of dysfunctional senescent muSCs that are no longer activated by muscle injury or turnover stimuli and present compromised ability to proliferate, differentiate, fuse, and promote the repair and replacement of myofibers. Additionally, muSC abundance has been observed to decline in some aged skeletal muscle tissues as muSCs develop diminished intrinsic capacity for reversible quiescence, which further reduces the population of muSCs supporting the post-injury repair cascade. Applications of muSCs in promoting muscle repair have been preclinically investigated; however, there are no approved pharmacological therapeutics that can safely and effectively enhance muSC activation and regenerative capacity to promote recovery from injury among aging adults." (1)

Excess NNMT/NAD+ Deficit Linked to Decline of Muscle Stem Cells:​

"Nicotinamide adenine dinucleotide (NAD+) is a fundamental cellular regulator of energy metabolism, mitochondrial function, and bioenergetics that declines with age. Age-related diminished NAD+ contributes to altered skeletal muscle metabolism and mitochondrial function, progressive muscle degeneration and loss of function, which further link to increased muSC senescence, reduced muSC function, dysfunctional muscle regeneration, and impaired muscle repair. Recently, several studies have observed that nutraceutical supplements such as nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide analogues (e.g., acipimox), which increase intracellular NAD+ levels and stimulate muscle NAD+ biosynthetic pathways (e.g., NAD+ salvage pathway), can improve muscle metabolic, mitochondrial, and muSC function, and accelerate muscle regeneration in aged mice. Thus, pharmacologically enhancing NAD+ levels in aging skeletal muscle tissues may provide a viable approach to improve the regenerative function of aged muSCs." (1)

NNMT Enzyme Can Decrease NAD+ and its Precursor Niacin (Vitamin B3):​

"Nicotinamide N-methyltransferase (NNMT) was originally identified as the enzyme responsible for the methylation of nicotinamide (NAM), one of the forms of vitamin B3. Methylated NAM (MNAM) is eventually excreted from the body. Recent evidence has expanded the role of NNMT beyond clearance of excess vitamin B3." (3)

"The cytosolic enzyme nicotinamide N-methyltransferase (NNMT) has been newly discovered to modulate the levels of nicotinamide precursors required for NAD+ biosynthesis, and thus plays a crucial role in regulating the NAD+ salvage pathway and cellular metabolism. NNMT is expressed in skeletal muscle, with progressively greater expression associated with aging in muscle tissues. Importantly, NNMT is a dominant component of the gene expression signature for sarcopenia, overexpression of which could lead to significantly reduced levels of NAD+ and associated declines in muscle mass, strength, and function that accompany aging." (1)

Excess NNMT Linked to Body Weight:​

"Body weight predicted NNMT activity in white fat, but not in brown fat or any other tissue, and only in incipient obesity." (2)

Excess NNMT Linked to Aged-Muscles:​

"Immunolabeling of NNMT revealed NNMT-specific staining both in the TA muscle obtained from 24-month-old and 4-month-old mice... Consistent with the differential immunohistochemical NNMT results in aged versus young TA tissue, the expression level of NNMT protein in aged TA muscle was ~3-fold higher compared to the level of NNMT protein in young TA tissue. Taken together, these results support increased NNMT protein expression in the skeletal muscle as a function of age, suggesting likely enhanced NNMT activity in aged muscle tissue." (1)

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NNMT Inhibitor Enhanced Muscle Regeneration in Aged Mice:​

"Our results indicated that treatment of aged mice with a small molecule NNMT inhibitor (NNMTi) enhanced proliferation, fusion, and regenerative capacity of muSCs, subsequently increasing functional performance of skeletal muscle. Similar results were demonstrated in vitro with C2C12 cells that closely mimic muSCs, where NNMTi treatment promoted myoblast differentiation and supporting metabolic changes in NAD+ salvage pathway-related cellular metabolites. To the best of our knowledge, this is the first study to convincingly demonstrate that NNMT inhibition rescues age-related muSC deficits and serves as a suitable therapeutic approach to reactivate skeletal muSCs in aging skeletal muscle, thus mitigating age-associated impaired muscle regeneration and improving skeletal muscle remodeling and function following injury."

"Fig. 2A and 2B present representative images that highlight muSC proliferation and fusion into damaged myofibers of aged TA muscle in control and NNMTi (10 mg/kg, bid)-treated mice. As indicated by white arrowheads, NNMT inhibition increased the number of proliferating muSCs. However, the total number of muSCs (i.e., Pax7+/DAPI+ cell counts per unit area) were unchanged in the control, low NNMTi dose, and high NNMTi dose groups... NNMT inhibition promoted greater fusion of muSCs into damaged myofibers, suggesting improved muscle regeneration following injury.

"White arrowheads denote muSCs that have proliferated post-injury (EdU+/Pax7+/DAPI+). Yellow arrowheads indicate myonuclei fused into damaged myofibers post-injury (EdU+/Pax7−/DAPI+)..." (1)

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"Consistent with these results, extensive quantification of numerous images revealed a dose-related increase in the relative abundance of proliferating muSCs and fibers with integrated myonuclei via muSC fusion following 1-week post-injury NNMTi treatment. The 5 mg/kg and 10 mg/kg doses of NNMTi tested resulted in 60% and 75% higher incidence of proliferating/active muSCs, respectively, relative to control. The odds ratio of active muSCs for control was 0.54 and 0.46 times the odds at the low and high doses, respectively. Although the higher treatment dose produced ~11% higher incidence of activated muSC compared to the lower treatment dose, this observed difference did not rise to the level of being statistically significant."

"Similarly, dose-dependent trends were observed with NNMTi treatment in the incidence of newly acquired myonuclei via muSC fusion into damaged myofibers (Fig. 2D). The relative numbers of fibers with an EdU+ myonucleus increased 40% and 48% with NNMTi treatment at 5 mg/kg and 10 mg/kg, respectively, relative to control."

"One-week post-injury treatment with NNMTi at the high dose (10 mg/kg) produced a significant 1.8-fold increase in the mean CSA of damaged TA muscle fibers relative to control. These results demonstrate that small molecule-mediated in vivo NNMT inhibition improves muscle regeneration and growth, consistent with the enhanced muSC activation and fusion observed following injury of the aged muscle (Fig. 2)."

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"As shown in Fig. 3C, the mean CSA of damaged muscle fibers from aged mice was significantly (2-to 3-fold) larger at 3-weeks post-injury compared to 1-week post-injury, indicating progressive longitudinal recovery of damaged muscle in aged mice. Consistent with CSA results observed with 1-week post-injury treatment, three-week post-injury treatment with 10 mg/kg NNMTi produced a statistically significant and physiologically relevant 1.5-fold increase in the mean CSA of damaged TA muscle fibers relative to control."

NNMT Inhibition Improved Aged-Muscle Contraction Strength:​

"Peak torque output of the damaged TA muscle, when normalized to body weight of each animal, was 67% higher in NNMTi-treated group compared to control group. This increase was functionally and statistically significant (p = 0.033 vs. control; Fig. 4A), and consistent with the increased fiber size observed in the treated cohort."

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NNMT Inhibition Produced No Systemic Toxicity:​

"...a complete plasma chemistry panel including major organ enzymes (liver, pancreas, gastrointestinal, renal, endocrine), total lipid, plasma proteins, and major electrolytes were profiled and compared between control and high dose (10 mg/kg) NNMTi-treated plasma samples. No significant differences were noted in the levels of the tested enzymes, glucose, cholesterol, plasma proteins, and electrolytes between control and NNMTi-treated samples (Table 1)...Taken together, 1-week post-injury daily repeat-dosing of NNMTi was well tolerated with no untoward systemic toxicity or behavioral implications in aged mice."

5-Amino-1MQ and JBSNF-000088 are Potent NNMT Inhibitors:​

"Here we report a small molecule, JBSNF-000088 which is a substrate analog of nicotinamide, that inhibits NNMT activity across species, reduces plasma MNA levels by ~50% and drives insulin sensitization, glucose metabolism and body weight reduction in DIO mice." (4)

"NNMT inhibition using 5-amino-1MQ (30µM concentration) in both the pre-adipocytes (P < 0.01, treated pre-adipocytes vs. untreated controls) and the adipocytes (P < 0.05, treated adipocytes vs. untreated controls) resulted in significant reduction in the intracellular levels of 1-MNA..." (5)

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"Relative NNMT protein expression and reaction product 1-MNA levels in the pre-adipocytes vs adipocytes, and the mechanistic validation of NNMT inhibitors in adipocytes." (5)

Summary:​

"To our knowledge, this is the first study to demonstrate that systemic treatment of aged animals with a small molecule drug-like NNMT inhibitor can rejuvenate muSCs, thereby robustly promoting muSC activation and fusion which are critical to support de novo myofiber regeneration and repair following injury. At 1-week post-injury, control aged mice showed poor myofiber growth, minimal muSC activity, and a compromised skeletal muscle repair profile; these observations were consistent with impaired muSC regenerative capacity previously reported in skeletal muscles from aged mice. In contrast, aged mice treated with NNMTi demonstrated dramatically increased frequency of activated muSCs and greatly enhanced muscle repair via muSC fusion and subsequent myonuclear accrual. Both the number of activated muSC and the average size of regenerating myofibers on the injured limb had nearly doubled at 1-week post-injury with treatment facilitating the development of myofibers with much larger cross-sectional areas, similar to youthful myogenic growth response. Consistent with increased muscle fiber growth, NNMTi treatment nearly doubled peak dorsiflexor torque output on the injured limb, suggesting clinically meaningful overall improvements in muscle strength and function. Additionally, at 3-weeks post-injury, when the damaged muscles of the control animals had undergone additional recovery, prolonged NNMTi treatment resulted in further enhancement of myofiber growth compared to controls. These results suggest that prolonged NNMTi treatment enables a more complete recovery of the injured aged muscles, as average myofiber sizes were comparable between injured TA muscles of the treated aged mice and uninjured TA muscles of similar aged (24-mo-old) mice.

As muscles age, NNMT expression and activity increases in the skeletal muscles, which impairs NAD+ flux through the salvage pathway and dysregulates SIRT-1 activity, thereby interfering with the capacity of muSC to maintain quiescence as well as the efficiency to proliferate, differentiate, and promote efficient muscle regeneration following injury. Given that NNMT appears to be uniformly expressed in skeletal muscles independent of fiber type distribution (i.e., type I, IIa, IIb) as previously demonstrated, suggests its dynamic involvement in the regulation of both oxidative and glycolytic metabolism."

 

[R2D2]

too bad it's so expensive

 

[01dragonslayer]

too bad it's so expensive

Definitely is pricey.