Hepatic gluconeogenesis is absolutely required for survival during prolonged fasting or starvation, but is inappropriately activated in diabetes mellitus. Glucocorticoids and glucagon have strong gluconeogenic actions on the liver. In contrast, insulin suppresses hepatic gluconeogenesis. Two components known to have important physiological roles in this process are the forkhead transcription factor FOXO1 (also known as FKHR) and peroxisome proliferative activated receptor-gamma co-activator 1 (PGC-1alpha; also known as PPARGC1), a transcriptional co-activator; whether and how these factors collaborate has not been clear. Using wild-type and mutant alleles of FOXO1, here we show that PGC-1alpha binds and co-activates FOXO1 in a manner inhibited by Akt-mediated phosphorylation. Furthermore, FOXO1 function is required for the robust activation of gluconeogenic gene expression in hepatic cells and in mouse liver by PGC-1alpha. Insulin suppresses gluconeogenesis stimulated by PGC-1alpha but co-expression of a mutant allele of FOXO1 insensitive to insulin completely reverses this suppression in hepatocytes or transgenic mice. We conclude that FOXO1 and PGC-1alpha interact in the execution of a programme of powerful, insulin-regulated gluconeogenesis.

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We attempted to test the idea that the relative abundance of each individual tRNA isoacceptor in Escherichia coli can be altered by varying its cognate codon concentration. In order to change the overall codon composition of the messenger pool, we have expressed in E. coli lacZ with the aid of T7 RNA polymerase and, separately, we have expressed a truncated tufB gene (designated ⌬tufB) with the aid of E. coli RNA polymerase so that their respective gene products individually accounted for 30% of the total bacterial protein. Unexpectedly, the maximum expression of either test gene has no specific effect on the relative rates of synthesis of the tRNA species that we studied. Instead, we find that there is a cumulative breakdown of rRNAs, which results in a loss of ribosomes and protein synthetic capacity. After either of the test genes is maximally induced, there is a growing fraction of protein synthesis invested in ␤-galactosidase or ⌬tufB that is matched by a comparable decrease of the fraction of normal protein synthesis. We have also observed enhanced accumulation of two heat shock proteins during overexpression. Finally, after several hours of overexpression of either test protein, the bacteria are no longer viable. These results are relevant to the practical problems of obtaining high expression levels for cloned proteins.There is a good correlation between the frequencies of codon usage in the mRNA pools and the abundance of the corresponding tRNA species for bacteria growing in rich laboratory media (10). Furthermore, when the quality of the culture media is systematically varied, the tRNA abundance changes in expected ways. In particular, the relative fraction of major tRNA species that translate major codons increases as the growth rates of the bacteria increase (4, 6). This variation is thought to be parallel to an increased usage of major codons in the mRNA pool at the higher growth rates (3,5,14). The present experiments were initiated to study the influence of the codon frequencies in the mRNA pool on the synthesis of individual tRNA species by bacteria.We have followed the consequences for Escherichia coli of overexpressing to very high levels genes whose protein products have no function for the cells, so-called gratuitous proteins. In this way, we have manipulated the codon composition of the mRNA pool, and we have measured the influence of this manipulation on the abundance of a number of tRNA species. The two gratuitous proteins that we have used were chosen because they are encoded by mRNA species with very different codon frequencies. One such protein is ␤-galactosidase, which is metabolically useless under the growth conditions we studied and which has an mRNA with a predominantly minor codon composition (21). The other is a truncated version of elongation factor Tu, called ⌬EF-Tu, which is functionally inactive and is encoded primarily by major codons (21). The results of overexpressing these two proteins were similar and not at all the expected ones.Expression of either ␤-galactosidase or ...

Advanced glycation end products (AGEs), known promoters of diabetic complications, form abundantly in heated foods and are ingested in bioreactive forms. To test whether dietary AGEs play a role in the progression of insulin resistance, C57/BL/KsJ db/db mice were randomly placed for 20 weeks on a diet with either a low AGE content (LAD) or a 3.4-fold higher content of AGE (high AGE diet [HAD]), including N-carboxymethyllysine (CML) and methylglyoxal (MG). LAD-fed mice showed lower fasting plasma insulin levels throughout the study (P ‫؍‬ 0.01). Body weight was reduced by ϳ13% compared with HAD-fed mice (P ‫؍‬ 0.04) despite equal food intake. LAD-fed mice exhibited significantly improved responses to both glucose (at 40 min, P ‫؍‬ 0.003) and insulin (at 60 min, P ‫؍‬ 0.007) tolerance tests, which correlated with a twofold higher glucose uptake by adipose tissue (P ‫؍‬ 0.02). Compared with the severe hypertrophy and morphological disorganization of islets from HAD-fed mice, LAD-fed mice presented a better-preserved structure of the islets. LAD-fed mice demonstrated significantly increased plasma HDL concentrations (P < 0.0001). Consistent with these observations, LAD-fed mice exhibited twofold lower serum CML and MG concentrations compared with HAD-fed mice (P ‫؍‬ 0.02). These results demonstrate that reduced AGE intake leads to lower levels of circulating AGE and to improved insulin sensitivity in db/db mice.

Excessive production of triglyceride-rich VLDL is attributable to hypertriglyceridemia. VLDL production is facilitated by microsomal triglyceride transfer protein (MTP) in a rate-limiting step that is regulated by insulin. To characterize the underlying mechanism, we studied hepatic MTP regulation by forkhead box O1 (FoxO1), a transcription factor that plays a key role in hepatic insulin signaling. In HepG2 cells, MTP expression was induced by FoxO1 and inhibited by exposure to insulin. This effect correlated with the ability of FoxO1 to bind and stimulate MTP promoter activity. Deletion or mutation of the FoxO1 target site within the MTP promoter disabled FoxO1 binding and resulted in abolition of insulin-dependent regulation of MTP expression. We generated mice that expressed a constitutively active FoxO1 transgene and found that increased FoxO1 activity was associated with enhanced MTP expression, augmented VLDL production, and elevated plasma triglyceride levels. In contrast, RNAi-mediated silencing of hepatic FoxO1 was associated with reduced MTP and VLDL production in adult mice. Furthermore, we found that hepatic FoxO1 abundance and MTP production were increased in mice with abnormal triglyceride metabolism. These data suggest that FoxO1 mediates insulin regulation of MTP production and that augmented MTP levels may be a causative factor for VLDL overproduction and hypertriglyceridemia in diabetes.