Toll-like receptor 2-deficient mice are protected from insulin resistance and beta cell dysfunction induced by a high-fat diet.

AIMS/HYPOTHESIS: Inflammation contributes to both insulin resistance and pancreatic beta cell failure in human type 2 diabetes. Toll-like receptors (TLRs) are highly conserved pattern recognition receptors that coordinate the innate inflammatory response to numerous substances, including NEFAs. Here we investigated a potential contribution of TLR2 to the metabolic dysregulation induced by high-fat diet (HFD) feeding in mice. METHODS: Male and female littermate Tlr2(+/+) and Tlr2(-/-) mice were analysed with respect to glucose tolerance, insulin sensitivity, insulin secretion and energy metabolism on chow and HFD. Adipose, liver, muscle and islet pathology and inflammation were examined using molecular approaches. Macrophages and dendritic immune cells, in addition to pancreatic islets were investigated in vitro with respect to NEFA-induced cytokine production. RESULTS: While not showing any differences in glucose homeostasis on chow diet, both male and female Tlr2(-/-) mice were protected from the adverse effects of HFD compared with Tlr2(+/+) littermate controls. Female Tlr2(-/-) mice showed pronounced improvements in glucose tolerance, insulin sensitivity, and insulin secretion following 20 weeks of HFD feeding. These effects were associated with an increased capacity of Tlr2(-/-) mice to preferentially burn fat, combined with reduced tissue inflammation. Bone-marrow-derived dendritic cells and pancreatic islets from Tlr2(-/-) mice did not increase IL-1beta expression in response to a NEFA mixture, whereas Tlr2(+/+) control tissues did. CONCLUSION/INTERPRETATION: These data suggest that TLR2 is a molecular link between increased dietary lipid intake and the regulation of glucose homeostasis, via regulation of energy substrate utilisation and tissue inflammation.

Basal lipolysis, not the degree of insulin resistance, differentiates large from small isolated adipocytes in high-fat fed mice.

AIMS/HYPOTHESIS: Adipocytes in obesity are characterised by increased cell size and insulin resistance compared with adipocytes isolated from lean patients. However, it is not clear at present whether hypertrophy actually does drive adipocyte insulin resistance. Thus, the aim of the present study was to metabolically characterise small and large adipocytes isolated from epididymal fat pads of mice fed a high-fat diet (HFD). METHODS: C57BL/6J mice were fed normal chow or HFD for 8 weeks. Adipocytes from epididymal fat pads were isolated by collagenase digestion and, in HFD-fed mice, separated into two fractions according to their size by filtration through a nylon mesh. Viability was assessed by lactate dehydrogenase and 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium assays. Basal and insulin-stimulated D-[U-(14)C]glucose incorporation and lipolysis were measured. Protein levels and mRNA expression were determined by western blot and real-time RT-PCR, respectively. RESULTS: Insulin-stimulated D-[U-(14)C]glucose incorporation into adipocytes isolated from HFD-fed mice was reduced by 50% compared with adipocytes from chow-fed mice. However, it was similar between small (average diameter 60.9 +/- 3.1 microm) and large (average diameter 83.0 +/- 6.6 microm) adipocytes. Similarly, insulin-stimulated phosphorylation of protein kinase B and AS160 were reduced to the same extent in small and large adipocytes isolated from HFD-mice. In addition, insulin failed to inhibit lipolysis in both adipocyte fractions, whereas it decreased lipolysis by 30% in adipocytes of chow-fed mice. In contrast, large and small adipocytes differed in basal lipolysis rate, which was twofold higher in the larger cells. The latter finding was associated with higher mRNA expression levels of Atgl (also known as Pnpla2) and Hsl (also known as Lipe) in larger adipocytes. Viability was not different between small and large adipocytes. CONCLUSIONS/INTERPRETATION: Rate of basal lipolysis but not insulin responsiveness is different between small and large adipocytes isolated from epididymal fat pads of HFD-fed mice.

Functional expression of human HMG-CoA reductase in Saccharomyces cerevisiae: a system to analyse normal and mutated versions of the enzyme in the context of statin treatment.

AIMS: Statins - inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase - are known to reduce blood cholesterol levels. In this paper, we present a Saccharomyces cerevisiae expression system, which enables quick evaluation of the sensitivity of the wild-type and/or mutant forms of human HMG-CoA reductase towards statins or other drugs. METHODS AND RESULTS: We analysed the sequence of the HMG-CoA reductase gene in DNA extracted from blood samples of 16 patients with cardiovascular disorders. We applied the yeast system to examine the sensitivity of the wild-type and mutated versions of the hHMG-CoA reductase to different types of statins. CONCLUSION: The yeast and mammalian HMG-CoA reductases demonstrate structural and functional conservation, and expression of human HMG-CoA reductase in yeast complements the lethal phenotype of strains lacking the HMG1 and HMG2 genes. SIGNIFICANCE AND IMPACT OF THE STUDY: These data indicate that a yeast expression system can serve to study the influence of selected mutations in human HMG-CoA reductase on the sensitivity of the enzyme to commonly prescribed statins. Our results suggest that this model system is suitable for the development and selection of lipid-lowering drugs as well as for the examination of DNA sequence variations in the context of statin therapy.

Isolation of the catalase T structural gene of Saccharomyces cerevisiae by functional complementation.

The catalase T structural gene of Saccharomyces cerevisiae was cloned by functional complementation of a mutation causing specific lack of the enzyme (cttl). Catalase T-deficient mutants were obtained by UV mutagenesis of an S. cerevisiae strain bearing the cas1 mutation, which causes insensitivity of catalase T to glucose repression. Since the second catalase protein of S. cerevisiae, catalase A, is completely repressed on 10% glucose, catalase T-deficient mutant colonies could be detected under such conditions. A cttl mutant was transformed with an S. cerevisiae gene library in plasmid YEp13. Among the catalase T-positive clones, four contained overlapping DNA fragments according to restriction analysis. Hybridization selection of yeast mRNA binding specifically to one of the cloned DNAs, translation of this mRNA in cell-free protein synthesis systems, and demonstration of catalase T protein formation by specific immunoadsorption showed that the catalase T structural gene had been cloned. By subcloning, the gene was located within a 3.5-kilobase S. cerevisiae DNA fragment. As in wild-type cells, catalase T synthesis in cttl mutant cells transformed with plasmids containing this fragment is sensitive to glucose repression. By DNA-RNA hybridization, catalase T transcripts were shown to be present in oxygen-adapting cells but absent from heme-deficient cells.

The product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, specifically functions in mitochondria.

We reported previously that the product of the DNA damage-inducible gene of Saccharomyces cerevisiae, DIN7, belongs to a family of proteins that are involved in DNA repair and replication. The family includes S. cerevisiae proteins Rad2p and its human homolog XPGC, Rad27p and its mammalian homolog FEN-1, and Exonuclease I (Exo I). Here, we report that Din7p specifically affects metabolism of mitochondrial DNA (mtDNA). We have found that dun1 strains, defective in the transcriptional activation of the DNA damage-inducible genes RNR1, RNR2, and RNR3, exhibit an increased frequency in the formation of the mitochondrial petite (rho(-)) mutants. This high frequency of petites arising in the dun1 strains is significantly reduced by the din7::URA3 allele. On the other hand, overproduction of Din7p from the DIN7 gene placed under control of the GAL1 promoter dramatically increases the frequency of petite formation and the frequency of mitochondrial mutations conferring resistance to erythromycin (E(r)). The frequencies of chromosomal mutations conferring resistance to canavanine (Can(r)) or adenine prototrophy (Ade(+)) are not affected by enhanced synthesis of Din7p. Experiments using Din7p fused to the green fluorescent protein (GFP) and cell fractionation experiments indicate that the protein is located in mitochondria. A possible mechanism that may be responsible for the decreased stability of the mitochondrial genome in S. cerevisiae cells with elevated levels of Din7p is discussed.

A novel cross-phylum family of proteins comprises a KRR1 (YCL059c) gene which is essential for viability of Saccharomyces cerevisiae cells.

We demonstrate here that the open reading frame (ORF) YCL059c, discovered during the systematic sequencing of chromosome III [Oliver et al., Nature 357 (1992) 38-46], codes for a protein essential for yeast: neither spore germination nor cell division occur in strains deleted for this gene. We have cloned the wild-type (wt) gene and shown that it complements the deletion. A relatively abundant RNA transcript corresponds to the gene. The protein has no similarity to proteins of known function. Interestingly, however, it is homologous to several expressed sequence tags (EST) of unknown function from Caenorhabditis elegans, Oryza sativa and Homo sapiens. Thus, a novel family of proteins of presumably nuclear localization, with a characteristic highly basic motif, KRR-R, transcends various phyla, and plays an important role in cellular processes. We propose to call this essential gene KRR1.

The yeast gene YJR025c encodes a 3-hydroxyanthranilic acid dioxygenase and is involved in nicotinic acid biosynthesis.

We have deleted the yeast gene YJR025c and shown that this leads to an auxotrophy for nicotinic acid. The deduced protein sequence of the gene product is homologous to the human 3-hydroxyanthranilic acid dioxygenase (EC 1.13.11.6) which is part of the kynurenine pathway for the degradation of tryptophan and the biosynthesis of nicotinic acid. In cell-free extracts the 3-hydroxyanthranilic acid dioxygenase activity is proportional to the copy number of the YJR025c gene. As YJR025c encodes the yeast 3-hydroxyanthranilic acid dioxygenase, we have named this gene BNA1 for biosynthesis of nicotinic acid.

Maintenance of the peroxisomal compartment in glucose-repressed and anaerobically grown Saccharomyces cerevisiae cells.

According to the current model of peroxisome biogenesis, the inheritance of this compartment requires the growth and division of pre-existing organelles followed by their distribution between mother and daughter cells. However, no known peroxisomal functions are present nor required for Saccharomyces cerevisiae cells grown under glucose repression and in anaerobiosis and the peroxisomal compartment becomes virtually indistinguishable under such conditions. This raised the question of the fate of this compartment in such cells. Is it maintained throughout prolonged growth under glucose repression or does it disappear from the cell and then reassemble on demand? To study the maintenance of putatively functional peroxisomes in S cerevisiae cells grown under conditions of glucose repression and anaerobiosis, we applied the vector-mediated overexpression of peroxisome matrix enzyme's catalase A and acyl-CoA oxidase. Evidence is presented that in S cerevisiae the peroxisomal import machinery responsible for targeting of matrix enzymes into this compartment is preserved under glucose repression and in the absence of oxygen.

Isolation and functional characterization of mutant ferrochelatases in Saccharomyces cerevisiae.

Ferrochelatase is a mitochondrial inner membrane-bound enzyme that catalyzes the incorporation of ferrous iron into protoporphyrin, the last step in protoheme biosynthesis. It is encoded by the HEM15 gene in the yeast Saccharomyces cerevisiae. Five hem15 mutants causing defective heme synthesis and protoporphyrin accumulation were investigated. The mutations were identified by sequencing the mutant hem15 alleles amplified in vitro from mutant genomic DNA. A single nucleotide change, causing an amino acid substitution, was found in each mutant. The substitution L62F caused a five-fold increase in Vmax and 32-fold and four-fold increases in the KM's for protoporphyrin and metal. Replacements of the conserved G47 by S and S102 by F increased the KM for protoporphyrin 10-fold without affecting the affinity for metal or enzyme activity. Two amino acid changes, L205P and P221L, produced a thermosensitive phenotype. In vivo heme synthesis, the amount of immunodetected protein, and ferrochelatase activity measured in vitro were more affected in cells grown at 37 degrees C than at 30 degrees C. The effects of these mutations on the enzyme function are discussed with respects to ferrochelatase structure and mechanism of action.

The deficiency of sterol biosynthesis in Saccharomyces cerevisiae affects the synthesis of glycosyl derivatives of dolichyl phosphates.

Mutants deficient in sterol (thermosensitive ergosterol auxotrophs) erg 8, 9, 12 and heme synthesis hem 1, 12 were screened for the level of free dolichol and dolichyl phosphate synthesized in the mevalonate pathway as well as for the activity of dolichyl phosphate-dependent glycosyl transferases. The amount of DolP synthesized via CTP-dependent phosphorylation was the same in mutants and parental strains. However, mannosylation and glucosylation of endogenous dolichyl phosphates in ergosterol mutants was about four times lower compared to parental strains, while the same reactions carried out with exogenous Dol24P reached 80% of the level observed in parental strains indicating that activities of DolPMan and DolPGlc synthases are not the rate-limiting factors. It is postulated that the de novo synthesis of DolP is impaired in the ergosterol mutants. Moreover, a block in the ergosterol branch of the metabolic pathway (erg 9) causes an increase in the de novo synthesis of dolichyl phosphate.

The KRR1 gene encodes a protein required for 18S rRNA synthesis and 40S ribosomal subunit assembly in Saccharomyces cerevisiae.

The newly discovered Saccharomyces cerevisiae gene KRR1 (YCL059c) encodes a protein essential for cell viability. Krr1p contains a motif of clustered basic amino acids highly conserved in the evolutionarly distant species from yeast to human. We demonstrate that Krr1p is localized in the nucleolus. The KRR1 gene is highly expressed in dividing cells and its expression ceases almost completely when cells enter the stationary phase. In vivo depletion of Krr1p leads to drastic reduction of 40S ribosomal subunits due to defective 18S rRNA synthesis. We propose that Krr1p is required for proper processing of pre-rRNA and the assembly of preribosomal 40S subunits.

Saccharomyces cerevisiae--a model organism for the studies on vacuolar transport.

The role of the yeast vacuole, a functional analogue of the mammalian lysosome, in the turnover of proteins and organelles has been well documented. This review provides an overview of the current knowledge of vesicle mediated vacuolar transport in the yeast Saccharomyces cerevisiae cells. Due to the conservation of the molecular transport machinery S. cerevisiae has become an important model system of vacuolar trafficking because of the facile application of genetics, molecular biology and biochemistry.

Sequencing and functional analysis of the yeast genome.

The genome of the yeast Saccharomyces cerevisiae was sequenced by an international consortium of laboratories from Europe, Canada, the U.S.A. and Japan. This project is now finished and the complete sequence of the first eukaryotic genome was released to the public data bases in April 1996. An overview and preliminary analysis of the entire genome sequence was presented in a special issue of Nature in May 1997, entitled "The yeast genome directory". At its origin the Yeast Genome Sequencing Project provoked much debate and controversy; however, the final results obtained and the insights this has given us into the organisation and content of a eukaryotic genome have more than justified the expectations of the supporters of the project. The importance of genomic sequencing and analysis, especially of model organisms, is now widely accepted and this has resulted in the birth of the new science of genomics (Botstein & Cherry, 1997, Proc. Natl. Acad. Sci. U.S.A. 94, 5506). The information from gene and protein sequences ultimately lead to functional description of all genes. The main strategies describing possible ways to analyse the function of new genes that have been identified by systematic sequencing of Saccharomyces cerevisiae genome are described.

Anaerobic growth of Saccharomyces cerevisiae alleviates the lethal effect of phosphotyrosyl phosphatase activators depletion.

Saccharomyces cerevisiae homologues of phosphotyrosyl phosphatase activator (PTPA) are encoded byRRD1 and RRD2, genes whose combined deletion is synthetic lethal. Previously we have shown that the lethality of rrd1,2delta can be suppressed by increasing the osmolarity of the medium. Here we show that the lethality of rrd1,2delta is also suppressed under oxygen-limited conditions. The absence of respiration per se is not responsible for the suppression since elimination of the mitochondrial genome or a block in heme biosynthesis fail to rescue the rrd1,2delta double mutation.

Saccharomyces cerevisiae mutants defective in heme biosynthesis as a tool for studying the mechanism of phototoxicity of porphyrins.

Mutants of Saccharomyces cerevisiae accumulating uroporphyrin (UP) or protoporphyrin (PP) were used as a model for the in vivo phototoxic effect of porphyrins observed in the human skin photosensitivity associated with porphyrias (porphyria cutanea tarda and erythropoietic protoporphyria). We have found that UP is localized in vacuoles and PP is present in all compartments except vacuoles in yeast cells. Endogenous PP is much more effective as a photosensitizer of yeast cells than UP. Protoporphyrin action is strictly dependent on the presence of oxygen. In contrast, UP displays a phototoxic effect even if oxygen is not present in the suspension, implicating a free radical mechanism that operates in anaerobiosis upon photosensitization by UP. Catalase or superoxide dismutase deficiency affects photosensitization by UP. A possible mechanism of UP photosensitizing activity is discussed.

Diamino acid derivatives of porphyrins penetrate into yeast cells, induce photodamage, but have no mutagenic effect.

The yeast Saccharomyces cerevisiae was used as a model eukaryotic organism to study the uptake of diamino acid derivatives of porphyrins and their phototoxicity with particular emphasis on possible mutagenic effects. The water-soluble hematoporphyrin derivatives diarginate (HpD[Arg]2) and 1-arginin di(N-amino acid)-protoporphyrinate used in this study are effective photosensitizers in tumor photodynamic therapy. Depending on the amino acid substituent, the porphyrin derivatives differ in their affinity for yeast cells. It is shown that HpD(Arg)2 and PP(Met)2 (Arg)2 penetrate into the yeast cell and are metabolized. Both compounds sensitize yeast cells to photodamage but have no mutagenic effect on nuclear or mitochondrial genomes.