Age reprogramming: cell rejuvenation by partial reprogramming.

'Age reprogramming' refers to the process by which the molecular and cellular pathways of a cell that are subject to age-related decline are rejuvenated without passage through an embryonic stage. This process differs from the rejuvenation observed in differentiated derivatives of induced pluripotent stem cells, which involves passage through an embryonic stage and loss of cellular identity. Accordingly, the study of age reprogramming can provide an understanding of how ageing can be reversed while retaining cellular identity and the specialised function(s) of a cell, which will be of benefit to regenerative medicine. Here, we highlight recent work that has provided a more nuanced understanding of age reprogramming and point to some open questions in the field that might be explored in the future.

The ORMDL3 asthma susceptibility gene regulates systemic ceramide levels without altering key asthma features in mice.

BACKGROUND: Genome-wide association studies in asthma have repeatedly identified single nucleotide polymorphisms in the ORM (yeast)-like protein isoform 3 (ORMDL3) gene across different populations. Although the ORM homologues in yeast are well-known inhibitors of sphingolipid synthesis, it is still unclear whether and how mammalian ORMDL3 regulates sphingolipid metabolism and whether altered sphingolipid synthesis would be causally related to asthma risk. OBJECTIVE: We sought to examine the in vivo role of ORMDL3 in sphingolipid metabolism and allergic asthma. METHODS: Ormdl3-LacZ reporter mice, gene-deficient Ormdl3-/- mice, and overexpressing Ormdl3Tg/wt mice were exposed to physiologically relevant aeroallergens, such as house dust mite (HDM) or Alternaria alternata, to induce experimental asthma. Mass spectrometry-based sphingolipidomics were performed, and airway eosinophilia, TH2 cytokine production, immunoglobulin synthesis, airway remodeling, and bronchial hyperreactivity were measured. RESULTS: HDM challenge significantly increased levels of total sphingolipids in the lungs of HDM-sensitized mice compared with those in control mice. In Ormdl3Tg/wt mice the allergen-induced increase in lung ceramide levels was significantly reduced, whereas total sphingolipid levels were not affected. Conversely, in liver and serum, levels of total sphingolipids, including ceramides, were increased in Ormdl3-/- mice, whereas they were decreased in Ormdl3Tg/wt mice. This difference was independent of allergen exposure. Despite these changes, all features of asthma were identical between wild-type, Ormdl3Tg/wt, and Ormdl3-/- mice across several models of experimental asthma. CONCLUSION: ORMDL3 regulates systemic ceramide levels, but genetically interfering with Ormdl3 expression does not result in altered experimental asthma.

Disturbed sphingolipid metabolism with elevated 1-deoxysphingolipids in glycogen storage disease type I - A link to metabolic control.

BACKGROUND: 1-Deoxysphingolipids (1-deoxySLs) are atypical sphingolipids. They are formed during sphingolipid de novo synthesis by the enzyme serine palmitoyltransferase, due to the alternate use of alanine over its canonical substrate serine. Pathologically elevated 1-deoxySL are involved in several neurological and metabolic disorders. The objective of this study was to investigate the role of 1-deoxySL in glycogen storage disease type I (GSDI). METHODS: In this prospective, longitudinal observational study (median follow-up 1.8y), the plasma 1-deoxySL profile was analyzed in 15 adult GSDI patients (12 GSDIa, 3 GSDIb), and 31 healthy controls, along with standard parameters for monitoring GSDI. RESULTS: 1-Deoxysphinganine (1-deoxySA) concentrations were elevated in GSDI compared to controls (191 ±â€¯129 vs 35 ±â€¯14 nmol/l, p < 0.0001). Concordant with the mechanism of 1-deoxySL synthesis, plasma alanine was higher (625 ±â€¯182 vs 398 ±â€¯90 µmol/l, p < 0.0001), while serine was lower in GSDI than in controls (88 ±â€¯22 vs 110 ±â€¯18 µmol/l. p < 0.001). Accordingly, serine, alanine and triglycerides were determinants of 1-deoxySA in the longitudinal analysis of GSDIa. 1-deoxySA concentrations correlated with the occurrence of low blood glucose (area under the curve below 4 mmol/l) in continuous glucose monitoring. The 1-deoxySL profile in GSDIb was distinct from GSDIa, with a different ratio of saturated to unsaturated 1-deoxySL. CONCLUSION: In addition to the known abnormalities of lipoproteins, GSDI patients also have a disturbed sphingolipid metabolism with elevated plasma 1-deoxySL concentrations. 1-DeoxySA relates to the occurrence of low blood glucose, and may constitute a potential new biomarker for assessing metabolic control. GSDIa and Ib have distinct 1-deoxySL profiles indicating that both GSD subtypes have diverse phenotypes regarding lipid metabolism.

ORMDL3 expression levels have no influence on the activity of serine palmitoyltransferase.

ORMDL proteins are believed to be negative regulators of serine palmitoyltransferase (SPT), which catalyzes the first and rate limiting step in sphingolipid (SL) de novo synthesis. Several single-nucleotide polymorphisms (SNPs) that are close to the ORMDL3 locus have been reported to increase ORMDL3 expression and to be associated with an elevated risk for early childhood asthma; however, the direct effect of ORMDL3 expression on SPT activity and its link to asthma remains elusive. In this study, we investigated whether ORMDL3 expression is associated with changes in SPT activity and total SL levels. Ormdl3-knockout (Ormdl3-/-) and transgenic (Ormdl3Tg/wt) mice were generated to study the effect of ORMDL3 on total SL levels in plasma and tissues. Cellular SPT activity was measured in mouse embryonic fibroblasts from Ormdl3-/- mice, as well as in HEK293 cells in which ORMDL3 was overexpressed and silenced. Furthermore, we analyzed the association of the reported ORMDL3 asthma SNPs with plasma sphingoid bases in a population-based cohort of 971 individuals. Total C18-long chain bases were not significantly altered in the plasma and tissues of Ormdl3-/- mice, whereas C18-sphinganine showed a small and significant increase in plasma, lung, and liver tissues. Mouse embryonic fibroblast cells from Ormdl3-/- mice did not show an altered SPT activity compared with Ormdl3+/- and Ormdl3+/+ mice. Overexpression or knockdown of ORMDL3 in HEK293 cells did not alter SPT activity; however, parallel knockdown of all 3 ORMDL isoforms increased enzyme activity significantly. A significant association of the annotated ORMDL3 asthma SNPs with plasma long-chain sphingoid base levels could not be confirmed. ORMDL3 expression levels seem not to be directly associated with changes in SPT activity. ORMDL3 might influence de novo sphingolipid metabolism downstream of SPT.-Zhakupova, A., Debeuf, N., Krols, M., Toussaint, W., Vanhoutte, L., Alecu, I., Kutalik, Z., Vollenweider, P., Ernst, D., von Eckardstein, A., Lambrecht, B. N., Janssens, S., Hornemann, T. ORMDL3 expression levels have no influence on the activity of serine palmitoyltransferase.

Novel Mouse Models of Methylmalonic Aciduria Recapitulate Phenotypic Traits with a Genetic Dosage Effect.

Methylmalonic aciduria (MMAuria), caused by deficiency of methylmalonyl-CoA mutase (MUT), usually presents in the newborn period with failure to thrive and metabolic crisis leading to coma or even death. Survivors remain at risk of metabolic decompensations and severe long term complications, notably renal failure and neurological impairment. We generated clinically relevant mouse models of MMAuria using a constitutive Mut knock-in (KI) allele based on the p.Met700Lys patient mutation, used homozygously (KI/KI) or combined with a knockout allele (KO/KI), to study biochemical and clinical MMAuria disease aspects. Transgenic Mut(ki/ki) and Mut(ko/ki) mice survive post-weaning, show failure to thrive, and show increased methylmalonic acid, propionylcarnitine, odd chain fatty acids, and sphingoid bases, a new potential biomarker of MMAuria. Consistent with genetic dosage, Mut(ko/ki) mice have lower Mut activity, are smaller, and show higher metabolite levels than Mut(ki/ki) mice. Further, Mut(ko/ki) mice exhibit manifestations of kidney and brain damage, including increased plasma urea, impaired diuresis, elevated biomarkers, and changes in brain weight. On a high protein diet, mutant mice display disease exacerbation, including elevated blood ammonia, and catastrophic weight loss, which, in Mut(ki/ki) mice, is rescued by hydroxocobalamin treatment. This study expands knowledge of MMAuria, introduces the discovery of new biomarkers, and constitutes the first in vivo proof of principle of cobalamin treatment in mut-type MMAuria.

Propofol (Diprivan®) and Intralipid® exacerbate insulin resistance in type-2 diabetic hearts by impairing GLUT4 trafficking.

BACKGROUND: The IV anesthetic, propofol, when administered as fat emulsion-based formulation (Diprivan) promotes insulin resistance, but the direct effects of propofol and its solvent, Intralipid, on cardiac insulin resistance are unknown. METHODS: Hearts of healthy and type-2 diabetic rats (generated by fructose feeding) were aerobically perfused for 60 minutes with 10 µM propofol in the formulation of Diprivan or an equivalent concentration of its solvent Intralipid (25 µM) ± insulin (100 mU•L). Glucose uptake, glycolysis, and glycogen metabolism were measured using [H]glucose. Activation of Akt, GSK3ß, AMPK, ERK1/2, p38MAPK, S6K1, JNK, protein kinase Cθ (PKCθ), and protein kinase CCßII (PKCßII) was determined using immunoblotting. GLUT4 trafficking and phosphorylations of insulin receptor substrate-1 (IRS-1) at Ser307(h312), Ser1100(h1101), and Tyr608(hTyr612) were measured. Mass spectrometry was used to determine acylcarnitines, phospholipids, and sphingolipids. RESULTS: Diprivan and Intralipid reduced insulin-induced glucose uptake and redirected glucose to glycogen stores in diabetic hearts. Reduced glucose uptake was accompanied by lower GLUT4 trafficking to the sarcolemma. Diprivan and Intralipid inactivated GSK3ß but activated AMPK and ERK1/2 in diabetic hearts. Only Diprivan increased phosphorylation of Akt(Ser473/Thr308) and translocated PKCθ and PKCßII to the sarcolemma in healthy hearts, whereas it activated S6K1 and p38MAPK and translocated PKCßII in diabetic hearts. Furthermore, only Diprivan phosphorylated IRS-1 at Ser1100(h1101) in healthy and diabetic hearts. JNK expression, phosphorylation of Ser307(h312) of IRS-1, and PKCθ expression and translocation were increased, whereas GLUT4 expression was reduced in insulin-treated diabetic hearts. Phosphatidylglycerol, phosphatidylethanolamine, and C18-sphingolipids accumulated in Diprivan-perfused and Intralipid-perfused diabetic hearts. CONCLUSIONS: Propofol and Intralipid promote insulin resistance predominantly in type-2 diabetic hearts.