Suppression of de novo sphingolipid biosynthesis mitigates sarcopenia

Abstract

There are no current standard-of-care treatments for sarcopenia, an age-associated decline in muscle mass and strength. A new study shows that genetically or pharmacologically countering the age-associated accumulation of sphingolipids in skeletal muscle can ameliorate sarcopenia in mice. The authors also identify genetic variants linked to sphingolipid biosynthesis that associate with muscle function in aged humans.

Aging comes with the loss of muscle mass and strength leading to a condition called sarcopenia. Muscle mass decreases 3-8% per year after 30 years of age and accelerates further to 6-15% after 65 years of age.¹ Sarcopenia is the primary cause of decline in our ability to perform daily tasks and thus quality of life. Currently no pharmacologic intervention exists to prevent or treat this debilitating condition in part due lack of clarity on the mechanisms that drive sarcopenia. In the current issue of Nature Aging, Laurila et. al. implicate age-related accumulation of dihydroceramides, an intermediate metabolite in the de novo sphingolipid synthesis pathway, in the sarcopenic process in mice and humans.² Strikingly, the authors found that pharmacologic inhibition of dihydroceramide or ceramide accumulation ameliorated sarcopenia in mice.

Sphingolipids comprise a large family (>4,000 species) of lipids whose de novo synthesis is catalysed by serine palmitoyl-transferase-1 (SPT1) that acylates amino acid serine with palmitate.³ Subsequently, a second acyl chain is added via one of the six isoforms of ceramide synthase (CERS1-6) to produce dihydroceramides, which is then converted to ceramides by dihydroceramide desaturase 1 or 2 (DES1/2) by insertion of a double bond (Figure 1). Accumulation of skeletal muscle ceramides is known to occur in muscle under states of excess fatty acid influx such as with obesity.⁴

Figure 1 -. Inhibiting de novo sphingolipid synthesis protects muscles from sarcopenia.

Laurila and colleagues found that aging is accompanied by an increase in the expression of SPT1 and CERS2 that leads to the elevation of muscle ceramides (see metabolic pathway on the left of the figure). In mice, inhibition of de novo sphingolipid synthesis pathway using myriocin or AAV9-shSPT1 attenuated age-associated muscle atrophy and weakness. In vitro evidence using CERS2 and DES1 deletion suggested that the beneficial effects of SPT1 inhibition may be due to reduced content of dihydroceramides, rather than ceramides.

Laurila et. al. demonstrated that aging increases skeletal muscle expression of genes involved in sphingolipid biosynthesis in mice and in humans. In mice, these changes coincided with an increase in ceramides in skeletal muscle. To study the potential role of ceramide accumulation on aging muscle, the authors used myriocin, a potent inhibitor of SPT1. Aging is classically characterized by preferential loss of type II (fast twitch) muscle fibers⁵ which the authors confirmed. Treating old mice with myriocin for three days per week for 6 months effectively lowered total muscle ceramides, and prevented the decrease in muscle mass and myofiber diameter. In vivo functional tests showed that these mice were also protected from age-related decline in strength and function. These pharmacologic studies were complemented by experiments using AAV9-induced silencing of skeletal muscle SPT1 leading to similar results, suggesting that the effects of myriocin on ameliorating sarcopenia is likely due to its effect on sphingolipid biosynthesis.

Inhibition of SPT is predicted to lower abundance of all metabolites in the de novo sphingolipid synthesis pathway, including ceramides. To answer the question of whether ceramides or other molecules produced by the SPT pathway are involved in sarcopenia, the authors turned to a C2C12 cell line, a murine myotube model. Analogous to the findings in vivo, myriocin treatment in C2C12 cells decreased total ceramides and dihydroceramides and resulted in increased myotube area, concomitant with an increase in the rate of protein synthesis. These findings were recapitulated with CRISPR knockout of SPT1 and CERS2. In contrast, CRISPR knockout of DES1, an enzyme that converts dihydroceramides to ceramides by insertion of a double bond, had the opposite effect, reducing myotube area. Like SPT1 and CERS2 deletion, DES1 deletion lowered ceramides, but unlike SPT1 or CERS2 deletion, DES1 deletion elevated dihydroceramides in these cells. Authors interpreted these findings to indicate that dihydroceramides, not ceramides, contribute to sarcopenia. In support of these findings, pharmacological inhibition of DES1/2 by GT-11 neutralized the beneficial effects of SPT1 deletion in vitro.

These findings are in contrast to growing findings by Scott Summers and other groups that genetic or pharmacologic inhibition of DES1 ameliorate metabolic dysfunction across multiple organ systems including improving skeletal muscle insulin sensitivity.⁶ The exact mechanism of how a double bond present in ceramides confer differential physiological consequences to dihydroceramides remains unclear. The double bond alters the biophysical properties of the molecule, modifying membrane packing.⁷ Desaturation may leave ceramides more susceptible to becoming oxidized, which may propagate oxidative stress to impair insulin signalling.⁸ Through their actions on PP2A and PKCζ, ceramides are known to upregulate intracellular lipid metabolism and downregulate glucose metabolism by acting on SREBP1, Akt, HSL, and CD36.⁹ On the other hand, much less is known regarding the downstream actions of dihydroceramides. Loss of DES1 attenuates adipogenesis, providing an example that dihydroceramide accumulation can lead to potentially negative outcomes.¹⁰ Together, because there are interests to inhibit DES1/2 to promote favourable health outcomes, it would be important to confirm whether muscle DES1 inhibition would promote skeletal muscle atrophy in vivo. Further mechanistic insights on how dihydroceramides, instead of ceramides, promote sarcopenia is also needed.

Regardless of the uncertainty in dihydroceramides being one of the metabolites involved in sarcopenia, upstream inhibition of muscle de novo sphingolipid synthesis by myriocin or SPT1 deletion appears to be a promising strategy in ameliorating sarcopenia. It is exciting that SPT1 expression was higher in a relatively small cohort of older adults (n=21, mean age 78y) compared to younger (n=15, mean age 25y) individuals, suggesting that this pathway is potentially highly relevant for muscle aging in humans. To further support the potential translatability, the authors show that myriocin enhances myotube areas in human primary muscle cells. Moreover, in datasets from the UK Biobank (UKBB, n=93,211) and the Helsinki Birth Cohort Study (HBCS, n=13,345) , the authors found that a SNP near the SPTLC1 gene (rs10820914) was associated with reduced skeletal muscle gene expression of SPT1 and positively associated with grip strength and improved fitness score, two physiological measures that are predictive of frailty, an age-related condition often associated with sarcopenia.¹¹ Conversely, a SNP near the DEGS1 gene was associated with decreased muscle DES1 expression, and was inversely associated with grip strength.

This new evidence supports the notion that de novo synthesis of sphingolipids is upregulated in skeletal muscle with age. Pharmacologic or genetic inhibition of SPT1 confers protection from age-associated loss in muscle mass and function in mice, suggesting a potential to target this pathway to treat sarcopenia. Because of some degree of uncertainty on the identity of the molecules and mechanisms by which SPT1 inhibition leads to protection against muscle atrophy and weakness, additionally in vivo studies are warranted, in particular to target muscle DES1 specifically.