Irene K. Moore

Prevention of fat-induced insulin resistance by salicylate

Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and may involve fat-induced activation of a serine kinase cascade involving IKK-beta. To test this hypothesis, we first examined insulin action and signaling in awake rats during hyperinsulinemic-euglycemic clamps after a lipid infusion with or without pretreatment with salicylate, a known inhibitor of IKK-beta. Whole-body glucose uptake and metabolism were estimated using [3-(3)H]glucose infusion, and glucose uptake in individual tissues was estimated using [1-(14)C]2-deoxyglucose injection during the clamp. Here we show that lipid infusion decreased insulin-stimulated glucose uptake and activation of IRS-1-associated PI 3-kinase in skeletal muscle but that salicylate pretreatment prevented these lipid-induced effects. To examine the mechanism of salicylate action, we studied the effects of lipid infusion on insulin action and signaling during the clamp in awake mice lacking IKK-beta. Unlike the response in wild-type mice, IKK-beta knockout mice did not exhibit altered skeletal muscle insulin signaling and action following lipid infusion. In summary, high-dose salicylate and inactivation of IKK-beta prevent fat-induced insulin resistance in skeletal muscle by blocking fat-induced defects in insulin signaling and action and represent a potentially novel class of therapeutic agents for type 2 diabetes.

Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance

Insulin resistance in skeletal muscle and liver may play a primary role in the development of type 2 diabetes mellitus, and the mechanism by which insulin resistance occurs may be related to alterations in fat metabolism. Transgenic mice with muscle- and liver-specific overexpression of lipoprotein lipase were studied during a 2-h hyperinsulinemic–euglycemic clamp to determine the effect of tissue-specific increase in fat on insulin action and signaling. Muscle–lipoprotein lipase mice had a 3-fold increase in muscle triglyceride content and were insulin resistant because of decreases in insulin-stimulated glucose uptake in skeletal muscle and insulin activation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity. In contrast, liver–lipoprotein lipase mice had a 2-fold increase in liver triglyceride content and were insulin resistant because of impaired ability of insulin to suppress endogenous glucose production associated with defects in insulin activation of insulin receptor substrate-2-associated phosphatidylinositol 3-kinase activity. These defects in insulin action and signaling were associated with increases in intracellular fatty acid-derived metabolites (i.e., diacylglycerol, fatty acyl CoA, ceramides). Our findings suggest a direct and causative relationship between the accumulation of intracellular fatty acid-derived metabolites and insulin resistance mediated via alterations in the insulin signaling pathway, independent of circulating adipocyte-derived hormones.

n-3 Fatty Acids Preserve Insulin Sensitivity In Vivo in a Peroxisome Proliferator–Activated Receptor-α–Dependent Manner

Recent studies have suggested that n-3 fatty acids, abundant in fish oil, protect against high-fat diet–induced insulin resistance through peroxisome proliferator–activated receptor (PPAR)-α activation and a subsequent decrease in intracellular lipid abundance. To directly test this hypothesis, we fed PPAR-α null and wild-type mice for 2 weeks with isocaloric high-fat diets containing 27% fat from either safflower oil or safflower oil with an 8% fish oil replacement (fish oil diet). In both genotypes the safflower oil diet blunted insulin-mediated suppression of hepatic glucose production (P < 0.02 vs. genotype control) and PEPCK gene expression. Feeding wild-type mice a fish oil diet restored hepatic insulin sensitivity (hepatic glucose production [HGP], P < 0.002 vs. wild-type mice fed safflower oil), whereas in contrast, in PPAR-α null mice failed to counteract hepatic insulin resistance (HGP, P = NS vs. PPAR-α null safflower oil–fed mice). In PPAR-α null mice fed the fish oil diet, safflower oil plus fish oil, hepatic insulin resistance was dissociated from increases in hepatic triacylglycerol and acyl-CoA but accompanied by a more than threefold increase in hepatic diacylglycerol concentration (P < 0.0001 vs. genotype control). These data support the hypothesis that n-3 fatty acids protect from high-fat diet–induced hepatic insulin resistance in a PPAR-α–and diacylglycerol-dependent manner.

Overexpression of uncoupling protein 3 in skeletal muscle protects against fat-induced insulin resistance

Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and is strongly associated with obesity. Increased concentrations of intracellular fatty acid metabolites have been postulated to interfere with insulin signaling by activation of a serine kinase cascade involving PKCtheta in skeletal muscle. Uncoupling protein 3 (UCP3) has been postulated to dissipate the mitochondrial proton gradient and cause metabolic inefficiency. We therefore hypothesized that overexpression of UCP3 in skeletal muscle might protect against fat-induced insulin resistance in muscle by conversion of intramyocellular fat into thermal energy. Wild-type mice fed a high-fat diet were markedly insulin resistant, a result of defects in insulin-stimulated glucose uptake in skeletal muscle and hepatic insulin resistance. Insulin resistance in these tissues was associated with reduced insulin-stimulated insulin receptor substrate 1- (IRS-1-) and IRS-2-associated PI3K activity in muscle and liver, respectively. In contrast, UCP3-overexpressing mice were completely protected against fat-induced defects in insulin signaling and action in these tissues. Furthermore, these changes were associated with a lower membrane-to-cytosolic ratio of diacylglycerol and reduced PKCtheta activity in whole-body fat-matched UCP3 transgenic mice. These results suggest that increasing mitochondrial uncoupling in skeletal muscle may be an excellent therapeutic target for type 2 diabetes mellitus.

Muscle-Specific IRS-1 Ser→Ala Transgenic Mice Are Protected From Fat-Induced Insulin Resistance in Skeletal Muscle

OBJECTIVE—Insulin resistance in skeletal muscle plays a critical role in the pathogenesis of type 2 diabetes, yet the cellular mechanisms responsible for insulin resistance are poorly understood. In this study, we examine the role of serine phosphorylation of insulin receptor substrate (IRS)-1 in mediating fat-induced insulin resistance in skeletal muscle in vivo. RESEARCH DESIGN AND METHODS—To directly assess the role of serine phosphorylation in mediating fat-induced insulin resistance in skeletal muscle, we generated muscle-specific IRS-1 Ser302, Ser307, and Ser612 mutated to alanine (Tg IRS-1 Ser→Ala) and IRS-1 wild-type (Tg IRS-1 WT) transgenic mice and examined insulin signaling and insulin action in skeletal muscle in vivo. RESULTS—Tg IRS-1 Ser→Ala mice were protected from fat-induced insulin resistance, as reflected by lower plasma glucose concentrations during a glucose tolerance test and increased insulin-stimulated muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. In contrast, Tg IRS-1 WT mice exhibited no improvement in glucose tolerance after high-fat feeding. Furthermore, Tg IRS-1 Ser→Ala mice displayed a significant increase in insulin-stimulated IRS-1–associated phosphatidylinositol 3-kinase activity and Akt phosphorylation in skeletal muscle in vivo compared with WT control littermates. CONCLUSIONS—These data demonstrate that serine phosphorylation of IRS-1 plays an important role in mediating fat-induced insulin resistance in skeletal muscle in vivo.

Formation of circular amplifications in Saccharomyces cerevisiae by a breakage‐fusion‐bridge mechanism

Primary gene amplification, the mutation from one gene copy per genome to two or more copies per genome, is a major mechanism of oncogene overexpression in human cancers. Analysis of the structures of amplifications can provide important evidence about the mechanism of amplification formation. We report here the analysis of the structures of four independent spontaneous circular amplifications of ADH4:CUP1 in the yeast Saccharomyces cerevisiae. The structures of all four amplifications are consistent with their formation by a breakage‐fusion‐bridge (BFB) mechanism. All four of these amplifications include a centromere as predicted by the BFB model. All four of the amplifications have a novel joint located between the amplified DNA and the telomere, which results in a dicentric chromosome, and is adjacent to all the copies of the amplified DNA as predicted by the BFB model. In addition we demonstrated that two of the amplifications contain most of chromosome VII in an unrearranged form in a 1:1 ratio with the normal copy of chromosome VII, again consistent with the predictions of the BFB model. Finally, all four amplifications are circular, one stable endpoint for molecules after breakage‐ fusion‐bridge. Environ. Mol. Mutagen. 36:113–120, 2000.

Telomere sequences at the novel joints of four independent amplifications in Saccharomyces cerevisiae.

Primary gene amplification, the mutation from one copy of a gene per genome to two or more genes per genome is a major mechanism of oncogene overexpression. We previously developed a system in the yeast Saccharomyces cerevisiae to phenotypically detect primary amplifications of a reporter cassette, ADH4:CUP1. We present here the sequence analysis of novel joints from four independent, spontaneous circular amplifications identified by the ADH4:CUP1 system. All four novel joints consist of C1–3 A telomeric repeats joined to short (14‐ to 16‐bp) CA‐rich tracts between ADH4 and the telomere of chromosome VII. In three of the four amplifications, the telomeric sequence and the CA‐rich tract that are joined in the amplification are normally located in inverted orientation to each other on chromosome VII. In the fourth amplification, the CA‐rich tract on chromosome VII is joined to telomere sequences from another chromosome. We suggest that formation of these amplifications was initiated by recombination between these CA‐rich tracts and a telomere. The resulting dicentric chromosome could start a breakage‐fusion‐bridge cycle that could be resolved by the formation of a circular amplification structure. Environ. Mol. Mutagen. 36:105–112, 2000.