Sphingoid bases and the serine catabolic enzyme CHA1 define a novel feedforward/feedback mechanism in the response to serine availability.

Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.

Differential regulation of dihydroceramide desaturase by palmitate versus monounsaturated fatty acids: implications for insulin resistance.

Much data implicate saturated fatty acids in deleterious processes associated with obesity, diabetes, and the metabolic syndrome. Many of these changes may be due to aberrant generation of bioactive lipids when saturated fatty acid availability to tissues is increased. On the other hand, studies are emerging that implicate the monounsaturated fatty acid oleate in protection from saturated fat mediated toxicity; however, the mechanisms are not well understood. Our data demonstrate a novel role for palmitate in increasing mRNA encoding DES1, which is the enzyme responsible for generating ceramide from its precursor dihydroceramide and thus controls synthesis of the bioactive lipid ceramide. Moreover, co-treatment with oleate prevented the increase in ceramide, and this occurred through attenuation of the increase in message and activity of DES1. Knockdown of DES1 also protected from palmitate-induced insulin resistance, and overexpression of this enzyme ameliorated the protective effect of oleate. Together, these findings provide insight into the mechanisms of oleate-mediated protection against metabolic disease and provide novel evidence for fatty acid-mediated regulation of a key enzyme of ceramide biosynthesis.

Regulation of yeast nutrient permease endocytosis by ATP-binding cassette transporters and a seven-transmembrane protein, RSB1.

Ceramide is produced by the condensation of a long chain base with a very long chain fatty acid. In Saccharomyces cerevisiae, one of the two major long chain bases is called phytosphingosine (PHS). PHS has been shown to cause toxicity in tryptophan auxotrophic strains of yeast because this bioactive ceramide precursor causes diversion of the high affinity tryptophan permease Tat2 to the vacuole rather than the plasma membrane. Loss of the integral membrane protein Rsb1 increased PHS sensitivity, which was suggested to be due to this protein acting as an ATP-dependent long chain base efflux protein. More recent experiments demonstrated that loss of the genes encoding the ATP-binding cassette transporter proteins Pdr5 and Yor1 elevated PHS tolerance. This increased resistance was suggested to be due to increased expression of RSB1. Here, we provide an alternative view of PHS resistance influenced by Rsb1 and Pdr5/Yor1. Rsb1 has a seven-transmembrane domain topology more consistent with that of a regulatory protein like a G-protein-coupled receptor rather than a transporter. Importantly, an rsb1Δ cell does not exhibit higher internal levels of PHS compared with isogenic wild-type cells. However, tryptophan transport is increased in pdr5Δ yor1 strains and reduced in rsb1Δ cells. Localization and vacuolar degradation of Tat2 are affected in these genetic backgrounds. Finally, internalization of FM4-64 dye suggests that loss of Pdr5 and Yor1 slows normal endocytic rates. Together, these data argue that Rsb1, Pdr5, and Yor1 regulate the endocytosis of Tat2 and likely other membrane transporter proteins.

Sphingolipid metabolism and analysis in metabolic disease.

Sphingolipids are an important class of structural and signaling molecules within the cell. As sphingolipids have been implicated in the development and pathogenesis of insulin resistance and the metabolic syndrome, it is important to understand their regulation and metabolism. Although these lipids are initially produced through a common pathway, there is no "generic" sphingolipid. Indeed, the biophysical and signaling properties of lipids may be manipulated by the subunit composition or isoform of their synthetic enzymes, via regulation of substrate integration. Functionally distinct pools of chemically-equivalent lipids may also be generated by de novo synthesis and recycling of existing complex sphingolipids. The highly integrated metabolism of the many bioactive sphingolipids means that manipulation of one enzyme or metabolite can result in a ripple effect, causing unforeseen changes in metabolite levels, enzyme activities, and cellular programmes. Fortunately, a suite of techniques, ranging from thin-layer chromatography to liquid chromatography-mass spectrometry approaches, allows investigators to undertake a functional characterization of all or part of the sphingolipidome in their systems of interest.

A pigmented octogenarian.

An 81-year-old woman presented with cachexia, malaise, deranged liver function tests (LFTs), hepatomegaly and hyperpigmentation. Hereditary haemochromatosis (HH) was ultimately diagnosed on genetic testing. Haemochromatosis is a difficult diagnosis in older people because not only are the symptoms protean and occur commonly in other geriatric conditions, but HH is also considered an unlikely new diagnosis in the over 80s; it is generally felt to present in the 5th decade for men or 6th decade for women. However, older people can present with only mild disease, so diagnosis is important not just for family screening but also since treatment can reduce unnecessary morbidity and mortality in a population with increasing longevity.

Modulation of sphingolipid metabolism by the phosphatidylinositol-4-phosphate phosphatase Sac1p through regulation of phosphatidylinositol in Saccharomyces cerevisiae.

Sphingolipids and phosphoinositides both play signaling roles in Saccharomyces cerevisiae. Although previous data indicate independent functions for these two classes of lipids, recent genetic studies have suggested interactions between phosphatidylinositol (PtdIns) phosphate effectors and sphingolipid biosynthetic enzymes. The present study was undertaken to further define the effects of phosphatidylinositol 4-phosphate (PtdIns(4)P) metabolism on cell sphingolipid metabolism. The data presented indicate that deletion of SAC1, a gene encoding a PtdIns(4)P phosphatase, increased levels of most sphingolipid species, including sphingoid bases, sphingoid base phosphates, and phytoceramide. In contrast, sac1Delta dramatically reduced inositol phosphosphingolipids, which result from the addition of a PtdIns-derived phosphoinositol head group to ceramides through Aur1p. Deletion of SAC1 decreased PtdIns dramatically in both steady-state and pulse labeling studies, suggesting that the observed effects on sphingolipids may result from modulation of the availability of PtdIns as a substrate for Aur1p. Supporting this hypothesis, acute attenuation of PtdIns(4)P production through Stt4p immediately increased PtdIns and subsequently reduced sphingoid bases. This reduction was overcome by the inhibition of Aur1p. Moreover, modulation of sphingoid bases through perturbation of PtdIns(4)P metabolism initiated sphingolipid-dependent biological effects, supporting the biological relevance for this route of regulating sphingolipids. These findings suggest that, in addition to potential signaling effects of PtdInsP effectors on sphingolipid metabolism, PtdIns kinases may exert substantial effects on cell sphingolipid profiles at a metabolic level through modulation of PtdIns available as a substrate for complex sphingolipid synthesis.