The relationships between the levels of transcripts and the levels of the proteins they encode have not been examined comprehensively in mammals, although previous work in plants and yeast suggest a surprisingly modest correlation. We have examined this issue using a genetic approach in which natural variations were used to perturb both transcript levels and protein levels among inbred strains of mice. We quantified over 5,000 peptides and over 22,000 transcripts in livers of 97 inbred and recombinant inbred strains and focused on the 7,185 most heritable transcripts and 486 most reliable proteins. The transcript levels were quantified by microarray analysis in three replicates and the proteins were quantified by Liquid Chromatography–Mass Spectrometry using O(18)-reference-based isotope labeling approach. We show that the levels of transcripts and proteins correlate significantly for only about half of the genes tested, with an average correlation of 0.27, and the correlations of transcripts and proteins varied depending on the cellular location and biological function of the gene. We examined technical and biological factors that could contribute to the modest correlation. For example, differential splicing clearly affects the analyses for certain genes; but, based on deep sequencing, this does not substantially contribute to the overall estimate of the correlation. We also employed genome-wide association analyses to map loci controlling both transcript and protein levels. Surprisingly, little overlap was observed between the protein- and transcript-mapped loci. We have typed numerous clinically relevant traits among the strains, including adiposity, lipoprotein levels, and tissue parameters. Using correlation analysis, we found that a low number of clinical trait relationships are preserved between the protein and mRNA gene products and that the majority of such relationships are specific to either the protein levels or transcript levels. Surprisingly, transcript levels were more strongly correlated with clinical traits than protein levels. In light of the widespread use of high-throughput technologies in both clinical and basic research, the results presented have practical as well as basic implications.

To understand pathways mediating the inflammatory responses of human aortic endothelial cells to oxidized phospholipids, we previously used a combination of genetics and genomics to model a coexpression network encompassing >1000 genes. CHAC1 (cation transport regulator-like protein 1), a novel gene regulated by ox-PAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycerophosphorylcholine), was identified in a co-regulated group of genes enriched for components of the ATF4 (activating transcription factor 4) arm of the unfolded protein response pathway. Herein, we characterize the role of CHAC1 and validate the network model. We first define the activation of CHAC1 mRNA by chemical unfolded protein response-inducers, but not other cell stressors. We then define activation of CHAC1 by the ATF4-ATF3-CHOP (C/EBP homologous protein), and not parallel XBP1 (X box-binding protein 1) or ATF6 pathways, using siRNA and/or overexpression plasmids. To examine the subset of genes downstream of CHAC1, we used expression microarray analysis to identify a list of 227 differentially regulated genes. We validated the activation of TNFRSF6B (tumor necrosis factor receptor superfamily, member 6b), a FASL decoy receptor, in cells treated with CHAC1 small interfering RNA. Finally, we showed that CHAC1 overexpression enhanced apoptosis, while CHAC1 small interfering RNA suppressed apoptosis, as determined by TUNEL, PARP (poly(ADP-ribose) polymerase) cleavage, and AIF (apoptosis-inducing factor) nuclear translocation.

OBJECTIVE-In diabetes, glucose toxicity affects different organ systems, including pancreatic islets where it leads to ␤-cell apoptosis, but the mechanisms are not fully understood. Recently, we identified thioredoxin-interacting protein (TXNIP) as a proapoptotic ␤-cell factor that is induced by glucose, raising the possibility that TXNIP may play a role in ␤-cell glucose toxicity.RESEARCH DESIGN AND METHODS-To assess the effects of glucose on TXNIP expression and apoptosis and define the role of TXNIP, we used INS-1 ␤-cells; primary mouse islets; obese, diabetic BTBR.ob mice; and a unique mouse model of TXNIP deficiency (HcB-19) that harbors a natural nonsense mutation in the TXNIP gene. RESULTS-Incubation of INS-1 cells at 25 mmol/l glucose for24 h led to an 18-fold increase in TXNIP protein, as assessed by immunoblotting. This was accompanied by increased apoptosis, as demonstrated by a 12-fold induction of cleaved caspase-3. Overexpression of TXNIP revealed that TXNIP induces the intrinsic mitochondrial pathway of apoptosis. Islets of diabetic BTBR.ob mice also demonstrated increased TXNIP and apoptosis as did isolated wild-type islets incubated at high glucose. In contrast, TXNIP-deficient HcB-19 islets were protected against glucose-induced apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and caspase-3, indicating that TXNIP is a required causal link between glucose toxicity and ␤-cell death.CONCLUSIONS-These findings shed new light onto the molecular mechanisms of ␤-cell glucose toxicity and apoptosis, demonstrate that TXNIP induction plays a critical role in this vicious cycle, and suggest that inhibition of TXNIP may represent a novel approach to reduce glucotoxic ␤-cell loss. Diabetes 57: 938-944, 2008

Pancreatic beta-cell loss through apoptosis represents a key factor in the pathogenesis of diabetes; however, no effective approaches to block this process and preserve endogenous beta-cell mass are currently available. To study the role of thioredoxin-interacting protein (TXNIP), a proapoptotic beta-cell factor we recently identified, we used HcB-19 (TXNIP nonsense mutation) and beta-cell-specific TXNIP knockout (bTKO) mice. Interestingly, HcB-19 mice demonstrate increased adiposity, but have lower blood glucose levels and increased pancreatic beta-cell mass (as assessed by morphometry). Moreover, HcB-19 mice are resistant to streptozotocin-induced diabetes. When intercrossed with obese, insulin-resistant, and diabetic mice, double-mutant BTBRlep(ob/ob)txnip(hcb/hcb) are even more obese, but are protected against diabetes and beta-cell apoptosis, resulting in a 3-fold increase in beta-cell mass. Beta-cell-specific TXNIP deletion also enhanced beta-cell mass (P<0.005) and protected against diabetes, and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) revealed a approximately 50-fold reduction in beta-cell apoptosis in streptozotocin-treated bTKO mice. We further discovered that TXNIP deficiency induces Akt/Bcl-xL signaling and inhibits mitochondrial beta-cell death, suggesting that these mechanisms may mediate the beta-cell protective effects of TXNIP deficiency. These results suggest that lowering beta-cell TXNIP expression could serve as a novel strategy for the treatment of type 1 and type 2 diabetes by promoting endogenous beta-cell survival.

Background: CHAC1 is associated with the stress response in atherosclerosis. Results: ATF4, ATF3, and CEBP␤ regulate CHAC1 transcription. Human CHAC1 protein overexpression depletes glutathione. Conclusion: CHAC1 is induced following multiple cell stress signals and leads to depletion of glutathione. Significance: CHAC1 may be an essential link between stress signaling and the oxidative status of the cell, contributing to multiple diseases.