Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research

Redirection of SKN-1 abates the negative metabolic outcomes of a perceived pathogen infection

Early host responses toward pathogens are essential for defense against infection. In Caenorhabditis elegans, the transcription factor, SKN-1, regulates cellular defenses during xenobiotic intoxication and bacterial infection. However, constitutive activation of SKN-1 results in pleiotropic outcomes, including a redistribution of somatic lipids to the germline, which impairs health and shortens lifespan. Here, we show that exposing C. elegans to Pseudomonas aeruginosa similarly drives the rapid depletion of somatic, but not germline, lipid stores. Modulating the epigenetic landscape refines SKN-1 activity away from innate immunity targets, which alleviates negative metabolic outcomes. Similarly, exposure to oxidative stress redirects SKN-1 activity away from pathogen response genes while restoring somatic lipid distribution. In addition, activating p38/MAPK signaling in the absence of pathogens, is sufficient to drive SKN-1-dependent loss of somatic fat. These data define a SKN-1- and p38-dependent axis for coordinating pathogen responses, lipid homeostasis, and survival and identify transcriptional redirection, rather than inactivation, as a mechanism for counteracting the pleiotropic consequences of aberrant transcriptional activity

A MicroRNA Linking Human Positive Selection and Metabolic Disorders

Postponed access: the file will be accessible after 2021-10-14Positive selection in Europeans at the 2q21.3 locus harboring the lactase gene has been attributed to selection for the ability of adults to digest milk to survive famine in ancient times. However, the 2q21.3 locus is also associated with obesity and type 2 diabetes in humans, raising the possibility that additional genetic elements in the locus may have contributed to evolutionary adaptation to famine by promoting energy storage, but which now confer susceptibility to metabolic diseases. We show here that the miR-128-1 microRNA, located at the center of the positively selected locus, represents a crucial metabolic regulator in mammals. Antisense targeting and genetic ablation of miR-128-1 in mouse metabolic disease models result in increased energy expenditure and amelioration of high-fat-diet-induced obesity and markedly improved glucose tolerance. A thrifty phenotype connected to miR-128-1-dependent energy storage may link ancient adaptation to famine and modern metabolic maladaptation associated with nutritional overabundance.acceptedVersio

β-Aminoisobutyric Acid Induces Browning of White Fat and Hepatic β-Oxidation and Is Inversely Correlated with Cardiometabolic Risk Factors

The transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) regulates metabolic genes in skeletal muscle and contributes to the response of muscle to exercise. Muscle PGC-1α transgenic expression and exercise both increase the expression of thermogenic genes within white adipose. How the PGC-1α-mediated response to exercise in muscle conveys signals to other tissues remains incompletely defined. We employed a metabolomic approach to examine metabolites secreted from myocytes with forced expression of PGC-1α, and identified β-aminoisobutyric acid (BAIBA) as a small molecule myokine. BAIBA increases the expression of brown adipocyte-specific genes in white adipocytes and β-oxidation in hepatocytes both in vitro and in vivo through a PPARα-mediated mechanism, induces a brown adipose-like phenotype in human pluripotent stem cells, and improves glucose homeostasis in mice. In humans, plasma BAIBA concentrations are increased with exercise and inversely associated with metabolic risk factors. BAIBA may thus contribute to exercise-induced protection from metabolic diseases

Surviving starvation simply without TFEB.

Starvation is among the most ancient of selection pressures, driving evolution of a robust arsenal of starvation survival defenses. In order to survive starvation stress, organisms must be able to curtail anabolic processes during starvation and judiciously activate catabolic pathways. Although the activation of metabolic defenses in response to nutrient deprivation is an obvious component of starvation survival, less appreciated is the importance of the ability to recover from starvation upon re-exposure to nutrients. In order for organisms to successfully recover from starvation, cells must be kept in a state of ready so that upon the return of nutrients, activities such as growth and reproduction can be resumed. Critical to this state of ready is the lysosome, an organelle that provides essential signals of nutrient sufficiency to cell growth-activating pathways in the fed state. In this issue, Murphy and colleagues provide evidence that exposure of Caenorhabditis elegans roundworms to 2 simple nutrients, glucose and the polyunsaturated fatty acid linoleate, is able to render lysosomal function competent to activate key downstream starvation recovery pathways, bypassing the need for a master transcriptional regulator of lysosomes. These findings provide a quantum leap forward in our understanding of the cellular determinants that permit organisms to survive cycles of feast and famine

<i>kat-1</i> mRNA is not transcriptionally regulated by serotonin, and <i>tub-1</i> regulates LRO Nile red in parallel with serotonin and <i>kat-1</i>.

<p>(A) <i>kat</i>-1 mRNA abundance does not change with exogenous serotonin treatment. (B) <i>kat-1</i> mRNA abundance is identical in <i>tph-1</i> and <i>tub-1</i> mutants. (A and B, N = 3; significance by ANOVA with Bonferroni correction.)</p

Gene-gene interaction network underlying LRO accumulation of Nile red and autofluorescence.

<p>(A) Seventy-nine genes affecting LRO Nile red were inactivated by RNAi in wild type worms and 6 genetic mutants with altered LRO Nile red phenotypes to determine gene-gene interactions. Genes were clustered by <i>k</i>-means clustering, with the heatmap indicating genes that increase LRO Nile red in yellow and decrease LRO Nile red in blue. No inference can be made on the absolute magnitude of the effect from the heatmap as data from each mutant for all 79 genes are normalized and scaled (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003908#s4" target="_blank">methods</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003908#pgen.1003908.s007" target="_blank">table S3</a> for absolute fold change differences). Genes knocked down by RNAi are along the vertical axis and mutant backgrounds are along the horizontal axis. Two clusters of genes that show decreases in all mutants tested are indicated by the black bars to the immediate right of the heatmap. Mutants are organized by hierarchical clustering, indicating that overall <i>kat-1</i> and <i>tph-1</i> which lacks serotonin cluster most closely to each other (dendrogram on top of the heatmap). (B) The same 79 genes were inactivated in the same seven genetic backgrounds, imaged for blue autofluorescence and organized by cluster analysis as in A. Three clusters of genes that have known regulators of LRO biogenesis are indicated by the black bars along the vertical axis to the right of the heatmap (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003908#pgen.1003908.s008" target="_blank">table S4</a>). (C) The solute transporter <i>K09C4.5</i> RNAi increases LRO Nile red in all strains tested except the TOR complex 2 mutant <i>rict-1</i>. (D) RNAi to the catalytic subunit of the protein phosphatase calcineurin, <i>tax-6</i>, led to a decrease in LRO Nile red in all strains tested except <i>rict-1</i> in which there was a small but measurable increase. (E) RNAi to the Kelch domain protein <i>kel-1</i> increases blue autofluorescence only in a <i>rict-1</i> mutant. (F) RNAi to acyl-coenzyme A synthetase <i>acs-6</i> increases blue autofluorescence in a manner genetically dependent upon TOR complex 2 mutant <i>rict-1</i>. (C–F, N as in table S3 and S4; significance by unequal variance, two-tailed Student's t-test with Bonferroni step-down correction.)</p

Rictor/TORC2 regulates fat metabolism, feeding, growth, and life span in Caenorhabditis elegans

Rictor is a component of the target of rapamycin complex 2 (TORC2). While TORC2 has been implicated in insulin and other growth factor signaling pathways, the key inputs and outputs of this kinase complex remain unknown. We identified mutations in the Caenorhabditis elegans homolog of rictor in a forward genetic screen for increased body fat. Despite high body fat, rictor mutants are developmentally delayed, small in body size, lay an attenuated brood, and are short-lived, indicating that Rictor plays a critical role in appropriately partitioning calories between long-term energy stores and vital organismal processes. Rictor is also necessary to maintain normal feeding on nutrient-rich food sources. In contrast to wild-type animals, which grow more rapidly on nutrient-rich bacterial strains, rictor mutants display even slower growth, a further reduced body size, decreased energy expenditure, and a dramatically extended life span, apparently through inappropriate, decreased consumption of nutrient-rich food. Rictor acts directly in the intestine to regulate fat mass and whole-animal growth. Further, the high-fat phenotype of rictor mutants is genetically dependent on akt-1, akt-2, and serum and glucocorticoid-induced kinase-1 (sgk-1). Alternatively, the life span, growth, and reproductive phenotypes of rictor mutants are mediated predominantly by sgk-1. These data indicate that Rictor/TORC2 is a nutrient-sensitive complex with outputs to AKT and SGK to modulate the assessment of food quality and signal to fat metabolism, growth, feeding behavior, reproduction, and life span

Mutations in the ketothiolase <i>kat-1</i> lead to resistance to exogenous serotonin treatment.

<p>(A) Six mutations in <i>kat-1</i> were identified in a forward genetic screen for resistance to the Nile red accumulation reduction induced by exogenous serotonin. Two mutations (<i>mg449</i> and <i>mg448</i>) led to predicted truncated proteins and therefore the mutations are predicted strong loss of functions. Nucleotide numbers refer to the spliced RNA of <i>kat-1</i> except for <i>mg449</i> where the unspliced <i>kat-1</i> RNA is used as a reference to indicate the loss of a splice donor site. (B) In the absence of exogenous serotonin, <i>kat-1</i> mutants show a more than doubling of LRO Nile red on day 1 of adulthood. (N>25, significance by unpaired, equal variance Student's t-test.) (C) <i>kat-1</i> mutants show attenuated loss of LRO Nile red in response to 55 hours of exogenous serotonin treatment. Wild type worms lose essentially all LRO Nile red, and while <i>kat-1</i> mutants lose approximately the same quantitative amount of LRO Nile red, they preserve 50% of their LRO Nile red level. (N>25, significance by ANOVA with Bonferroni correction.) (D) Wild type and <i>tub-1</i> mutants have full response to serotonin, reducing LRO Nile red to low levels. On the other hand, the <i>kat-1;tub-1</i> double mutant, like the <i>kat-1</i> single mutant, has significant resistance to exogenous serotonin treatment.</p

Suppression of the high LRO Nile red phenotype of <i>kat-1</i> by mutations in the proton coupled amino acid transmembrane transporter <i>skat-1</i>.

<p>(A) a <i>kat-1</i> mutant has a doubling of LRO Nile red. A <i>skat-1</i> single mutant has a 30% reduction in LRO Nile red. The <i>kat-1;skat-1</i> double mutant show a near complete absence of LRO Nile red, indicating that <i>skat-1</i> completely suppresses the high LRO Nile red of a <i>kat-1</i> mutant. Representative images of individual animals are shown in the right panel. (B) As with the <i>kat-1;skat-1</i> double mutant, loss of <i>skat-1</i> in mutants lacking serotonin (<i>tph-1</i> and <i>cat-4</i>) show a synergistic decrease in LRO Nile red. The dramatic increase in LRO Nile red in the <i>kat-1;tub-1</i> double mutant is strongly suppressed in the <i>kat-1;skat-1;tub-1</i> triple mutant (representative image shown at right). (A and B, N>25, significance by ANOVA with Bonferroni correction.) (C) <i>skat-1</i> acts in the intestine in a cell autonomous manner to regulate LRO Nile red. Expression of <i>skat-1</i> under the intestine specific promoter <i>vha-6</i> in a <i>kat-1;skat-1</i> double mutant fully restores the high LRO Nile red phenotype of a <i>kat-1</i> mutant, whereas expression in the nervous system under the <i>rab-3</i> promoter does not. (D) SKAT-1::GFP expressed under the intestine-specific vha-6 promoter highlights hollow vesicles which are also positive for autofluorescent material, likely representing lysosome-related organelles (<i>arrows</i>). A large amount of SKAT-1::GFP is localized to smaller, more punctate cytoplasmic structures and does not co-localize with autofluorescent material (<i>angle brackets</i>).</p