Effect of high-fat diet on glucose homeostasis and gene expression in glucokinase knockout mice.

AIM: We have generated a heterozygous glucokinase knockout mouse (gk(del/wt)), upon which we investigated the effect of high-fat diet (HFD) with respect to metabolic control and both hepatic and beta-cell gene expression. We also investigated the in vitro efficacy of a glucokinase activator (GKA) on glucose-stimulated insulin secretion (GSIS) in gk(del/wt)mouse islets. METHODS: Male gk(del/wt)and gk(wt/wt)mice were grouped (n = 8-10) at 10 weeks of age and fed HFD or chow diet (CD) for 10 weeks. Multiple parameters including blood glucose, plasma insulin and glucose tolerance were assessed. Further animal groups were used for in vitro GSIS and islet and liver gene expression analysis. RESULTS AND CONCLUSIONS: gk(del/wt)mice showed early-onset persistent hyperglycaemia, raised glycated haemoglobin levels, impaired GSIS and glucose tolerance but no change in plasma cholesterol, non-esterified fatty acids or triglyceride levels. After HFD feeding, insulin levels of gk(del/wt)mice were less than half that of gk(wt/wt)mice, although they were equivalent to gk(wt/wt)mice on CD. While gk(wt/wt)mice maintained moderate hyperglycaemia, gk(del/wt)mice became overtly diabetic, with worsened glucose tolerance. A GKA (GKA50) increased GSIS, at 10 mM glucose, in gk(del/wt)mice to an extent at least as great as that seen in gk(wt/wt)mice on both CD and HFD. gk(del/wt)mice showed only a small number of changes in gene expression compared with gk(wt/wt)mice. We propose the high fat-fed gk(del/wt)mouse as a model of type 2 diabetes and report retained efficacy of a GKA on in vitro GSIS.

A selective peroxisome proliferator-activated receptor delta agonist promotes reverse cholesterol transport.

The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of the fibrate drugs and the antidiabetic effects of the glitazone drugs in humans are due to activation of the alpha (NR1C1) and gamma (NR1C3) subtypes, respectively. By contrast, the therapeutic potential of the delta (NR1C2) subtype is unknown, due in part to the lack of selective ligands. We have used combinatorial chemistry and structure-based drug design to develop a potent and subtype-selective PPARdelta agonist, GW501516. In macrophages, fibroblasts, and intestinal cells, GW501516 increases expression of the reverse cholesterol transporter ATP-binding cassette A1 and induces apolipoprotein A1-specific cholesterol efflux. When dosed to insulin-resistant middle-aged obese rhesus monkeys, GW501516 causes a dramatic dose-dependent rise in serum high density lipoprotein cholesterol while lowering the levels of small-dense low density lipoprotein, fasting triglycerides, and fasting insulin. Our results suggest that PPARdelta agonists may be effective drugs to increase reverse cholesterol transport and decrease cardiovascular disease associated with the metabolic syndrome X.

Genomic structure and chromosomal mapping of the mouse nov gene.

The nov gene encodes a cysteine-rich protein that is overexpressed in avian nephroblastomas. It is a member of the CCN family of proteins, all of which are involved in cell growth. Genomic and cDNA clones encompassing the mouse nov gene have been isolated and characterized. The mouse nov gene is highly conserved with the human and chick nov genes at the level of nucleotide sequence and genomic organization. The exon structure reflects the modular organization of the NOV protein in a number of structural domains. These are highly conserved with other members of the CCN family, as is the distribution of 38 of its 40 cysteine residues. The nov gene maps to chromosome 15, between D15 Mit 153 and D15 Mit 183, in a region of conserved synteny with human chromosome 8.

An Escherichia coli system for assay of F1p site-specific recombination on substrate plasmids.

We have developed an Escherichia coli system for testing the behaviour of plasmids carrying target sites for the F1p site-specific recombinase. The E. coli strain BL-FLP is described, which carries a chromosomally integrated bacteriophage T7 RNA polymerase gene expressed from a lac promoter, and harbours the plasmid pMS40.pMS40 has the features: (i) it carries the FLP recombinase gene under the control of a bacteriophage T7 promoter, (ii) it confers kanamycin resistance, and (iii) it uses an R6K origin of replication; these two latter features make it compatible with most conventional cloning vectors. Substrate plasmids carrying F1p-recognition targets (FRT) are transformed into BL-FLP, and the consequences of F1p-mediated recombination can be analysed after subsequent extraction of plasmid DNA. We show that this system is capable of base-perfect F1p-mediated recombination on plasmid substrates. We also present a corrected sequence of the commonly used F1p substrate plasmid, pNEO beta GAL (O'Gorman et al. (1991) Science 251, 1351-1355).

Multiple cloning sites carrying loxP and FRT recognition sites for the Cre and Flp site-specific recombinases.

Plasmids were constructed carrying the loxP and FRT recognition sites for the Cre and Flp site-specific recombinases, respectively, within multiple cloning sites. Vectors carrying single and tandemly repeated targets are available with various flanking restriction enzyme sites. In addition, a series of plasmids carrying both loxP and FRT sites is available. These vectors facilitate construction of target molecules for these site-specific recombinases which are becoming increasingly important tools for the in vivo manipulation of DNA.

FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis.

FLP site-specific recombinase was expressed in stably transformed tobacco and Arabidopsis. FLP-expressing tobacco lines were crossed with other transformed tobacco lines that contained a stably integrated FLP recognition target construct(s). The target construct consisted of two directly-oriented FLP recognition targets (FRTs), flanking a hygromycin resistance cassette located between a GUS coding region and an upstream 35S CaMV promoter. Excision of the hygromycin resistance cassette by FLP-mediated recombination between FRTs brings the GUS coding region under the transcriptional control of the CaMV 35S promoter. In the absence of FLP-mediated recombination, the GUS gene is transcriptionally silent. GUS activity was observed in the progeny of all crosses made between FLP recombinase-expressing and target-containing tobacco lines, but not in the selfs of parents. The predicted recombination product remaining after excision was confirmed by PCR and Southern analysis. In Arabidopsis, inducible expression of FLP recombinase was achieved from the soybean Gmhsp 17.6L heat-shock promoter. Heat-shock induction of FLP expression in plants containing the target construct led to activation of constitutive GUS expression in a subset of cells, whose progeny, therefore, were GUS-positive. A variety of clonal sectors were produced in plants derived from seed that was heat-shocked during germination. The ability to control the timing of GUS activation was demonstrated by heat-shock of unopened flower heads which produced large sectors. It was concluded that heat-shock-induced expression of FLP recombinase provides a readily controllable method for generating marked clonal sectors in Arabidopsis, the size and distribution of which reflects the timing of applied heat-shock.

Site-specific recombinases: tools for genome engineering.

Site-specific recombinases from bacteriophage and yeasts have been developed as novel tools for manipulating DNA both in the test-tube and in living organisms. We discuss the characteristics of these enzyme systems, review their application in genetic and developmental studies and speculate on their future potential for large-scale directed modifications of eukaryotic genomes.