Adipocyte Ceramides Regulate Subcutaneous Adipose Browning, Inflammation, and Metabolism.

Adipocytes package incoming fatty acids into triglycerides and other glycerolipids, with only a fraction spilling into a parallel biosynthetic pathway that produces sphingolipids. Herein, we demonstrate that subcutaneous adipose tissue of type 2 diabetics contains considerably more sphingolipids than non-diabetic, BMI-matched counterparts. Whole-body and adipose tissue-specific inhibition/deletion of serine palmitoyltransferase (Sptlc), the first enzyme in the sphingolipid biosynthesis cascade, in mice markedly altered adipose morphology and metabolism, particularly in subcutaneous adipose tissue. The reduction in adipose sphingolipids increased brown and beige/brite adipocyte numbers, mitochondrial activity, and insulin sensitivity. The manipulation also increased numbers of anti-inflammatory M2 macrophages in the adipose bed and induced secretion of insulin-sensitizing adipokines. By comparison, deletion of serine palmitoyltransferase from macrophages had no discernible effects on metabolic homeostasis or adipose function. These data indicate that newly synthesized adipocyte sphingolipids are nutrient signals that drive changes in the adipose phenotype to influence whole-body energy expenditure and nutrient metabolism.

Extensive diversity in circadian regulation of plasma lipids and evidence for different circadian metabolic phenotypes in humans.

The circadian system regulates daily rhythms in lipid metabolism and adipose tissue function. Although disruption of circadian clock function is associated with negative cardiometabolic end points, very little is known about interindividual variation in circadian-regulated metabolic pathways. Here, we used targeted lipidomics-based approaches to profile the time course of 263 lipids in blood plasma in 20 healthy individuals. Over a span of 28 h, blood was collected every 4 h and plasma lipids were analyzed by HPLC/MS. Across subjects, about 13% of lipid metabolites showed circadian variation. Rhythmicity spanned all metabolite classes examined, suggesting widespread circadian control of lipid-mediated energy storage, transport, and signaling. Intersubject agreement for lipids identified as rhythmic was only about 20%, however, and the timing of lipid rhythms ranged up to 12 h apart between individuals. Healthy subjects therefore showed substantial variation in the timing and strength of rhythms across different lipid species. Strong interindividual differences were also observed for rhythms of blood glucose and insulin, but not cortisol. Using consensus clustering with iterative feature selection, subjects clustered into different groups based on strength of rhythmicity for a subset of triglycerides and phosphatidylcholines, suggesting that there are different circadian metabolic phenotypes in the general population. These results have potential implications for lipid metabolism disorders linked to circadian clock disruption.

Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice.

Obesity is associated with an enhanced inflammatory response that exacerbates insulin resistance and contributes to diabetes, atherosclerosis, and cardiovascular disease. One mechanism accounting for the increased inflammation associated with obesity is activation of the innate immune signaling pathway triggered by TLR4 recognition of saturated fatty acids, an event that is essential for lipid-induced insulin resistance. Using in vitro and in vivo systems to model lipid induction of TLR4-dependent inflammatory events in rodents, we show here that TLR4 is an upstream signaling component required for saturated fatty acid-induced ceramide biosynthesis. This increase in ceramide production was associated with the upregulation of genes driving ceramide biosynthesis, an event dependent of the activity of the proinflammatory kinase IKKß. Importantly, increased ceramide production was not required for TLR4-dependent induction of inflammatory cytokines, but it was essential for TLR4-dependent insulin resistance. These findings suggest that sphingolipids such as ceramide might be key components of the signaling networks that link lipid-induced inflammatory pathways to the antagonism of insulin action that contributes to diabetes.

Muscle wasted: a novel component of the Drosophila histone locus body required for muscle integrity.

Skeletal muscles arise by cellular differentiation and regulated gene expression. Terminal differentiation programmes such as muscle growth, extension and attachment to the epidermis, lead to maturation of the muscles. These events require changes in chromatin organization as genes are differentially regulated. Here, we identify and characterise muscle wasted (mute), a novel component of the Drosophila histone locus body (HLB). We demonstrate that a mutation in mute leads to severe loss of muscle mass and an increase in levels of normal histone transcripts. Importantly, Drosophila Myocyte enhancer factor 2 (Mef2), a central myogenic differentiation factor, and how, an RNA binding protein required for muscle and tendon cell differentiation, are downregulated. Mef2 targets are, in turn, misregulated. Notably, the degenerating muscles in mute mutants show aberrant localisation of heterochromatin protein 1 (HP1). We further show a genetic interaction between mute and the Stem-loop binding protein (Slbp) and a loss of muscle striations in Lsm11 mutants. These data demonstrate a novel role of HLB components and histone processing factors in the maintenance of muscle integrity. We speculate that mute regulates terminal muscle differentiation possibly through heterochromatic reorganisation.

The intracellular domain of Dumbfounded affects myoblast fusion efficiency and interacts with Rolling pebbles and Loner.

Drosophila body wall muscles are multinucleated syncytia formed by successive fusions between a founder myoblast and several fusion competent myoblasts. Initial fusion gives rise to a bi/trinucleate precursor followed by more fusion cycles forming a mature muscle. This process requires the functions of various molecules including the transmembrane myoblast attractants Dumbfounded (Duf) and its paralogue Roughest (Rst), a scaffold protein Rolling pebbles (Rols) and a guanine nucleotide exchange factor Loner. Fusion completely fails in a duf, rst mutant, and is blocked at the bi/trinucleate stage in rols and loner single mutants. We analysed the transmembrane and intracellular domains of Duf, by mutating conserved putative signaling sites and serially deleting the intracellular domain. These were tested for their ability to translocate and interact with Rols and Loner and to rescue the fusion defect in duf, rst mutant embryos. Studying combinations of double mutants, further tested the function of Rols, Loner and other fusion molecules. Here we show that serial truncations of the Duf intracellular domain successively compromise its function to translocate and interact with Rols and Loner in addition to affecting myoblast fusion efficiency in embryos. Putative phosphorylation sites function additively while the extreme C terminus including a PDZ binding domain is dispensable for its function. We also show that fusion is completely blocked in a rols, loner double mutant and is compromised in other double mutants. These results suggest an additive function of the intracellular domain of Duf and an early function of Rols and Loner which is independent of Duf.

The recombination activation gene 1 (Rag1) is expressed in a subset of zebrafish olfactory neurons but is not essential for axon targeting or amino acid detection.

BACKGROUND: Rag1 (Recombination activation gene-1) mediates genomic rearrangement and is essential for adaptive immunity in vertebrates. This gene is also expressed in the olfactory epithelium, but its function there is unknown. RESULTS: Using a transgenic zebrafish line and immunofluorescence, we show that Rag1 is expressed and translated in a subset of olfactory sensory neurons (OSNs). Neurons expressing GFP under the Rag1 promoter project their axons to the lateral region of the olfactory bulb only, and axons with the highest levels of GFP terminate in a single glomerular structure. A subset of GFP-expressing neurons contain Galphao, a marker for microvillous neurons. None of the GFP-positive neurons express Galphaolf, Galphaq or the olfactory marker protein OMP. Depletion of RAG1, by morpholino-mediated knockdown or mutation, did not affect axon targeting. Calcium imaging indicates that amino acids evoke chemotopically organized glomerular activity patterns in a Rag1 mutant. CONCLUSION: Rag1 expression is restricted to a subpopulation of zebrafish olfactory neurons projecting to the lateral olfactory bulb. RAG1 catalytic activity is not essential for axon targeting, nor is it likely to be required for regulation of odorant receptor expression or the response of OSNs to amino acids.

Dynamic spatiotemporal expression of LIM genes and cofactors in the embryonic and postnatal cerebral cortex.

LIM-homeodomain (LIM-HD) genes encode a family of transcription factors known to be involved in development and patterning in several systems. Previously, we have shown that LIM-HD gene Lhx2 is required for the formation of a crucial boundary in the dorsal telencephalon (Bulchand et al. [2001] Mech Dev 100:165-175). To further explore the role of LIM-HD genes as well as the broader LIM gene family in dorsal telencephalic development, we examined the expression pattern of the members of this gene family and their cofactors in the developing mouse cerebral cortex. Transcription factor activity of the LIM-HD proteins requires the formation of a tetrameric complex consisting of two LIM-HD molecules linked by a dimer of cofactor (Clim) molecules. LIM-only (Lmo) proteins can interfere with this process by competing for the cofactors. LIM-HD protein function, thus, can be modulated by the presence of the appropriate Clim or Lmo molecules. At least 13 LIM-HD, 4 Lmo, and 2 Clim genes have been identified in the mouse. Several of these genes exhibit complex spatiotemporal patterns spanning different stages of cortical development, from embryonic to postnatal ages. Noteworthy features of the expression patterns include delineation of boundaries within the developing cortex, up- or down-regulation during formation of selected cortical layers, and a striking complementarity of expression of several members consistent with specific functions in cortical development. Significantly, in some cases, Lmo or Clim gene expression is robust where no LIM-HD gene expression is detectable. These results suggest multiple and distinct roles for LIM-HD, Lmo, and Clim genes in cortical development, and also support a LIM-HD-independent role for some Lmo and Clim members.