Agonist-induced activation of peroxisome proliferator-activated receptor gamma (PPAR gamma) is known to cause adipocyte differentiation and insulin sensitivity. The biological role of PPAR gamma was investigated by gene targeting. Homozygous PPAR gamma-deficient embryos died at 10.5-11.5 dpc due to placental dysfunction. Quite unexpectedly, heterozygous PPAR gamma-deficient mice were protected from the development of insulin resistance due to adipocyte hypertrophy under a high-fat diet. These phenotypes were abrogated by PPAR gamma agonist treatment. Heterozygous PPAR gamma-deficient mice showed overexpression and hypersecretion of leptin despite the smaller size of adipocytes and decreased fat mass, which may explain these phenotypes at least in part. This study reveals a hitherto unpredicted role for PPAR gamma in high-fat diet-induced obesity due to adipocyte hypertrophy and insulin resistance, which requires both alleles of PPAR gamma.

Insulin receptor substrate-1 (IRS-1) is the major substrate of insulin receptor and IGF-1 receptor tyrosine kinases; it has an apparent relative molecular mass of 160-190,000 (M(r), 160-190K) on SDS polyacrylamide gel. Tyrosine-phosphorylated IRS-1 binds the 85K subunit of phosphatidylinositol 3-kinase which may be involved in the translocation of glucose transporters and the abundant src homology protein (ASH)/Grb2 which may be involved in activation of p21ras and MAP kinase cascade. IRS-1 also has binding sites for Syp and Nck and other src homology 2 (SH2) signalling molecules. To clarify the physiological roles of IRS-1 in vivo, we made mice with a targeted disruption of the IRS-1 gene locus. Mice homozygous for targeted disruption of the IRS-1 gene were born alive but were retarded in embryonal and postnatal growth. They also had resistance to the glucose-lowering effects of insulin, IGF-1 and IGF-2. These data suggest the existence of both IRS-1-dependent and IRS-1-independent pathways for signal transduction of insulin and IGFs.

To investigate the role of insulin receptor substrate (IRS)-2 in vivo, we generated IRS-2-deficient mice by gene targeting. Although homozygous IRS-2-deficient mice (IRS-2 -/-mice) had a body weight similar to wildtype mice, they progressively developed type 2 diabetes at 10 weeks. IRS-2 -/-mice showed insulin resistance and a defect in the insulin-stimulated signaling pathway in liver but not in skeletal muscle. Despite insulin resistance, the amount of ␤-cells was reduced to 83% of that in wild-type mice, which was in marked contrast to the 85% increase in the amount of ␤-cells in IRS-1-deficient mice (IRS-1 -/-mice) to compensate for insulin resistance. Thus, IRS-2 plays a crucial role in the regulation of ␤-cell mass. On the other hand, insulin secretion by the same number of cells in response to glucose measured ex vivo was significantly increased in IRS-2 -/-mice compared with wild-type mice but was decreased in IRS-1 -/-mice. These results suggest that IRS-1 and IRS-2 may play different roles in the regulation of ␤-cell mass and the function of individual ␤-cells.

The hallmark of type 2 diabetes, the most common metabolic disorder, is a defect in insulin-stimulated glucose transport in peripheral tissues. Although a role for phosphoinositide-3-kinase (PI3K) activity in insulin-stimulated glucose transport and glucose transporter isoform 4 (Glut4) translocation has been suggested in vitro, its role in vivo and the molecular link between activation of PI3K and translocation has not yet been elucidated. To determine the role of PI3K in glucose homeostasis, we generated mice with a targeted disruption of the gene encoding the p85alpha regulatory subunit of PI3K (Pik3r1; refs 3-5). Pik3r1-/- mice showed increased insulin sensitivity and hypoglycaemia due to increased glucose transport in skeletal muscle and adipocytes. Insulin-stimulated PI3K activity associated with insulin receptor substrates (IRSs) was mediated via full-length p85 alpha in wild-type mice, but via the p50 alpha alternative splicing isoform of the same gene in Pik3r1-/- mice. This isoform switch was associated with an increase in insulin-induced generation of phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P3) in Pik3r1-/- adipocytes and facilitation of Glut4 translocation from the low-density microsome (LDM) fraction to the plasma membrane (PM). This mechanism seems to be responsible for the phenotype of Pik3r1-/- mice, namely increased glucose transport and hypoglycaemia. Our work provides the first direct evidence that PI3K and its regulatory subunit have a role in glucose homeostasis in vivo.

Intercellular attachment is an essential process in the morphogenesis of multicellular organisms. A unique mutant, nolac-H18 (nonorganogenic callus with loosely attached cells), generated by T-DNA transformation using leaf-disk cultures of haploid Nicotiana plumbaginifolia, lost the ability to form tight intercellular attachments and adventitious shoots. The gene tagged with T-DNA, named NpGUT1 (glucuronyltransferase 1), was similar to the gene for the catalytic domains of animal glucuronyltransferases and was expressed predominantly in shoot and root apical meristems. The transformation of NpGUT1 complemented the nolac-H18 mutation, and the expression of antisense NpGUT1 RNA produced crumbled shoots. The mutation caused defects in the glucuronic acid of rhamnogalacturonan II of pectin, which drastically reduced the formation of borate cross-linking of rhamnogalacturonan II. NpGUT1, which encodes a unique glucuronyltransferase, is a glycosyltransferase gene identified in pectin biosynthesis and is essential for intercellular attachment in plant meristems and tissues. S patially and temporally controlled intercellular attachment and communication are indispensable for the organization of plant tissues, making them critical for normal development and morphogenesis in every multicellular organism. Plant cell walls are composed primarily of cellulose microfibrils, hemicellulose, pectic polysaccharides, and small amounts of structural proteins (1-3). Pectin is believed to be involved in intercellular attachment because it is localized mainly in the primary cell wall, middle lamella, and cell corners. Pectin consists mostly of three structurally well-characterized polysaccharides: homogalacturonans (HGs) and highly branched rhamnogalacturonans I and II (RG-I and RG-II). Compared with cellulose and hemicellulose, little is known about the synthesis and assembly of pectins. The biosynthesis of HG, RG-I, and RG-II likely requires at least 41 unique glycosyltransferases (1). The activities of several transferases involved in the biosynthesis of pectin have been identified (1). However, none of these enzymes have been purified, and their genes have never been identified.Recently, we established a system for producing mutants called nolac (nonorganogenic callus with loosely attached cells) by T-DNA transformation, which involves in vitro cultures of leaf disks of haploid Nicotiana plumbaginifolia (4). These mutants are defective in intercellular attachment, which results in the failure of organogenesis. Haploid N. plumbaginifolia plants (5) are suitable for generating and studying such mutants, because mutations have a direct effect on phenotype and because cells with embryo-lethal mutations can be maintained in tissue culture as unorganized callus, which enables us to analyze mutant cell walls. We identified 199 lines of callus with loosely attached cells from cultures of 2,970 leaf disks that had been transformed with T-DNA. Although, only 25 of these continued to grow on the medium, nolac-H18 had a growth rate that was s...