Louise Cathrine Braun Olsen

Statins inhibit protein lipidation and induce the unfolded protein response in the non-sterol producing nematode Caenorhabditis elegans.

Statins are compounds prescribed to lower blood cholesterol in millions of patients worldwide. They act by inhibiting HMG-CoA reductase, the rate-limiting enzyme in the mevalonate pathway that leads to the synthesis of farnesyl pyrophosphate, a precursor for cholesterol synthesis and the source of lipid moieties for protein prenylation. The nematode Caenorhabditis elegans possesses a mevalonate pathway that lacks the branch leading to cholesterol synthesis, and thus represents an ideal organism to specifically study the noncholesterol roles of the pathway. Inhibiting HMG-CoA reductase in C. elegans using statins or RNAi leads to developmental arrest and loss of membrane association of a GFP-based prenylation reporter. The unfolded protein response (UPR) is also strongly activated, suggesting that impaired prenylation of small GTPases leads to the accumulation of unfolded proteins and ER stress. UPR induction was also observed upon pharmacological inhibition of farnesyl transferases or RNAi inhibition of a specific isoprenoid transferase (M57.2) and found to be dependent on both ire-1 and xbp-1 but not on pek-1 or atf-6, which are all known regulators of the UPR. The lipid stores and fatty acid composition were unaffected in statin-treated worms, even though they showed reduced staining with Nile red. We conclude that inhibitors of HMG-CoA reductase or of farnesyl transferases induce the UPR by inhibiting the prenylation of M57.2 substrates, resulting in developmental arrest in C. elegans. These results provide a mechanism for the pleiotropic effects of statins and suggest that statins could be used clinically where UPR activation may be of therapeutic benefit.

Bioactive components from flowers of Sambucus nigra L. increase glucose uptake in primary porcine myotube cultures and reduce fat accumulation in Caenorhabditis elegans.

Obesity and insulin resistance in skeletal muscles are major features of type 2 diabetes. In the present study, we examined the potential of Sambucus nigra flower (elderflowers) extracts to stimulate glucose uptake (GU) in primary porcine myotubes and reduce fat accumulation (FAc) in Caenorhabditis elegans. Bioassay guided chromatographic fractionations of extracts and fractions resulted in the identification of naringenin and 5-O- caffeoylquinic acid exhibiting a significant increase in GU. In addition, phenolic compounds related to those found in elderflowers were also tested, and among these, kaempferol, ferulic acid, p-coumaric acid, and caffeic acid increased GU significantly. FAc was significantly reduced in C. elegans, when treated with elderflower extracts, their fractions and the metabolites naringenin, quercetin-3-O-rutinoside, quercetin-3-O-glucoside, quercetin-3-O-5″-acetylglycoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, and isorhamnetin-3-O-glucoside and the related phenolic compounds kaempferol and ferulic acid. The study indicates that elderflower extracts contain bioactive compounds capable of modulating glucose and lipid metabolism, suitable for nutraceutical and pharmaceutical applications.

The Adiponectin Receptor Homologs in C. elegans Promote Energy Utilization and Homeostasis

Adiponectin is an adipokine with insulin-sensitising actions in vertebrates. Its receptors, AdipoR1 and AdipoR2, are PAQR-type proteins with 7-transmembrane domains and topologies reversed that of GPCRs, i.e. their C-termini are extracellular. We identified three adiponectin receptor homologs in the nematode C. elegans, named paqr-1, paqr-2 and paqr-3. These are differently expressed in the intestine (the main fat-storing tissue), hypodermis, muscles, neurons and secretory tissues, from which they could exert systemic effects. Analysis of mutants revealed that paqr-1 and -2 are novel metabolic regulators in C. elegans and that they act redundantly but independently from paqr-3. paqr-2 is the most important of the three paqr genes: mutants grow poorly, fail to adapt to growth at low temperature, and have a very high fat content with an abnormal enrichment in long (C20) poly-unsaturated fatty acids when combined with the paqr-1 mutation. paqr-2 mutants are also synthetic lethal with mutations in nhr-49, sbp-1 and fat-6, which are C. elegans homologs of nuclear hormone receptors, SREBP and FAT-6 (a Δ9 desaturase), respectively. Like paqr-2, paqr-1 is also synthetic lethal with sbp-1. Mutations in aak-2, the C. elegans homolog of AMPK, or nhr-80, another nuclear hormone receptor gene, suppress the growth phenotype of paqr-2 mutants, probably because they restore the balance between energy expenditure and storage. We conclude that paqr-1 and paqr-2 are receptors that regulate fatty acid metabolism and cold adaptation in C. elegans, that their main function is to promote energy utilization rather than storage, and that PAQR class proteins have regulated metabolism in metazoans for at least 700 million years.

Tissue- and paralogue-specific functions of acyl-CoA-binding proteins in lipid metabolism in Caenorhabditis elegans.

ACBP (acyl-CoA-binding protein) is a small primarily cytosolic protein that binds acyl-CoA esters with high specificity and affinity. ACBP has been identified in all eukaryotic species, indicating that it performs a basal cellular function. However, differential tissue expression and the existence of several ACBP paralogues in many eukaryotic species indicate that these proteins serve distinct functions. The nematode Caenorhabditis elegans expresses seven ACBPs: four basal forms and three ACBP domain proteins. We find that each of these paralogues is capable of complementing the growth of ACBP-deficient yeast cells, and that they exhibit distinct temporal and tissue expression patterns in C. elegans. We have obtained loss-of-function mutants for six of these forms. All single mutants display relatively subtle phenotypes; however, we find that functional loss of ACBP-1 leads to reduced triacylglycerol (triglyceride) levels and aberrant lipid droplet morphology and number in the intestine. We also show that worms lacking ACBP-2 show a severe decrease in the β-oxidation of unsaturated fatty acids. A quadruple mutant, lacking all basal ACBPs, is slightly developmentally delayed, displays abnormal intestinal lipid storage, and increased β-oxidation. Collectively, the present results suggest that each of the ACBP paralogues serves a distinct function in C. elegans.

Screening for Bioactive Metabolites in Plant Extracts Modulating Glucose Uptake and Fat Accumulation

Dichloromethane and methanol extracts of seven different food and medicinal plants were tested in a screening platform for identification of extracts with potential bioactivity related to insulin-dependent glucose uptake and fat accumulation. The screening platform included a series of in vitro bioassays, peroxisome proliferator-activated receptor (PPAR) γ-mediated transactivation, adipocyte differentiation of 3T3-L1 cell cultures, and glucose uptake in both 3T3-L1 adipocytes and primary porcine myotubes, as well as one in vivo bioassay, fat accumulation in the nematode Caenorhabditis elegans. We found that dichloromethane extracts of aerial parts of golden root (Rhodiola rosea) and common elder (Sambucus nigra) as well as the dichloromethane extracts of thyme (Thymus vulgaris) and carrot (Daucus carota) were able to stimulate insulin-dependent glucose uptake in both adipocytes and myotubes while weekly activating PPARγ without promoting adipocyte differentiation. In addition, these extracts were able to decrease fat accumulation in C. elegans. Methanol extracts of summer savory (Satureja hortensis), common elder, and broccoli (Brassica oleracea) enhanced glucose uptake in myotubes but were not able to activate PPARγ, indicating a PPARγ-independent effect on glucose uptake.

Chemical Genomics and Emerging DNA Technologies in the Identification of Drug Mechanisms and Drug Targets

Chemical genomics combines chemistry with molecular biology as a means of exploring the function of unknown proteins or identifying the proteins responsible for a particular phenotype induced by a small cell-permeable bioactive molecule. Chemical genomics therefore has the potential to identify and validate therapeutic targets and to discover drug candidates for rapidly and effectively generating new interventions for human diseases. The recent emergence of genomic technologies and their application on genetically tractable model organisms like Drosophila melanogaster, Caenorhabditis elegans and Saccharomyces cerevisiae have provided momentum to cell biological and biomedical research, particularly in the functional characterization of gene functions and the identification of novel drug targets. We therefore anticipate that chemical genomics and the vast development of genomic technologies will play critical roles in the genomic age of biological research and drug discovery. In the present review we discuss how simple biological model organisms can be used as screening platforms in combination with emerging genomic technologies to advance the identification of potential drugs and their molecular mechanisms of action.

StUbEx PLUS—A Modified Stable Tagged Ubiquitin Exchange System for Peptide Level Purification and In-Depth Mapping of Ubiquitination Sites

Modulation of protein activities by reversible post-translational modifications (PTMs) is a major molecular mechanism involved in the control of virtually all cellular processes. One of these PTMs is ubiquitination, which regulates key processes including protein degradation, cell cycle, DNA damage repair, and signal transduction. Because of its importance for numerous cellular functions, ubiquitination has become an intense topic of research in recent years, and proteomics tools have greatly facilitated the identification of many ubiquitination targets. Taking advantage of the StUbEx strategy for exchanging the endogenous ubiquitin with an epitope-tagged version, we created a modified system, StUbEx PLUS, which allows precise mapping of ubiquitination sites by mass spectrometry. Application of StUbEx PLUS to U2OS cells treated with proteasomal inhibitors resulted in the identification of 41 589 sites on 7762 proteins, which thereby revealed the ubiquitous nature of this PTM and demonstrated the utility of the approach for comprehensive ubiquitination studies at site-specific resolution.

C. elegans: A Model for Understanding Lipid Accumulation

Regulation and coordination of lipid metabolism involve complex interactions between the feeding regulatory centres in the nervous system and the regulated uptake, intracellular transport, storage, and utilization of stored lipids. As energy is essential to all cellular processes, it is thought that complex networks have evolved to ensure survival by maintaining adequate energy reservoirs. However, in times of nutrient abundance and imbalance, improper regulation and coordination of these networks can lead to obesity and other metabolic diseases and syndromes. Obesity genes must be considered as molecular components of such networks which function at an organismal level to orchestrate energy intake and expenditure. Thus, the functions of obesity genes must be understood within the context of these networks in intact animals. Since the majority of genes required for lipid homeostasis are evolutionarily conserved, much information can be obtained relevant to complex organisms by studying simple eukaryotes like C. elegans. Its genetic tractability makes C. elegans a highly attractive platform for identifying lipid regulatory pathways, drugs, and their molecular targets which ultimately will help us to understand the origin of metabolic diseases such as obesity and diabetes. Here we briefl y present some central aspects of lipid accumulation in C. elegans and discuss its merits as a platform for identifi cation and development of novel bioactive compounds regulating lipid storage.