A greatly extended PPARGC1A genomic locus encodes several new brain-specific isoforms and influences Huntington disease age of onset.

PGC-1α has been implicated in the pathogenesis of neurodegenerative disorders. Several single-nucleotide polymorphisms (SNPs) located in two separate haplotype blocks of PPARGC1A have shown associations with Huntington's disease (HD) and Parkinson's disease, but causative SNPs have not been identified. One SNP (rs7665116) was located in a highly conserved 233 bp region of intron 2. To determine whether rs7665116 is located in an alternative exon, we performed 5'-RLM-RACE from exon 3 and discovered multiple new transcripts that initiated from a common novel promoter located 587 kb upstream of exon 2, but did not contain the conserved region harboring rs7665116. Using real-time polymerase chain reaction, RNase protection assays and northern blotting, we show that the majority of these transcripts are brain specific and are at least equally or perhaps more abundant than the reference sequence PPARGC1A transcripts in whole brain. Two main transcripts containing independent methionine start codons encode full-length brain-specific PGC-1α proteins that differ only at their N-termini (NTs) from PGC-1α, encoded by the reference sequence. Additional truncated isoforms containing these NTs that are similar to NT-PGC-1α exist. Other transcripts may encode potential dominant negative forms, as they are predicted to lack the second LXXLL motif that serves as an interaction site for several nuclear receptors. Furthermore, we show that the new promoter is active in neuronal cell lines and describe haplotypes encompassing this region that are associated with HD age of onset. The discovery of such a large PPARGC1A genomic locus and multiple isoforms in brain warrants further functional studies and may provide new tissue-specific targets for treating neurodegenerative diseases.

Potential role of upstream stimulatory factor 1 gene variant in familial combined hyperlipidemia and related disorders.

OBJECTIVE: Genetic studies implicated upstream stimulatory factor 1 (USF1) in familial combined hyperlipidemia because the rs2073658 minor allele was associated with reduced risk of familial combined hyperlipidemia and related disorders. The molecular mechanisms whereby rs2073658 influences trait expression have remained elusive. METHODS AND RESULTS: Plasma lipids, rs2073658 genotypes (N=372), and hepatic transcript levels (N=96) of USF1 and genes involved in hepatic lipoprotein production were determined in obese subjects. The rs2073658 minor allele was associated with reduced plasma triglycerides (TGs) (P<0.001), hepatic USF1 (P<0.01), and microsomal TG transfer protein transcript levels (P<0.05). Functional studies in human hepatocellular carcinoma cells showed that rs2073658 is located in a forkhead box A2 (FOXA2) binding site and that major allele constructs displayed higher transcriptional activity than minor allele constructs. Knockdown of FOXA2 reduced the activity of major, but not minor allele constructs. Furthermore, an interaction between hepatic FOXA2 transcript levels and rs2073658 minor allele carrier status on hepatic USF1 transcript levels was observed in vivo (P<0.05). USF1 activated the transcription of FOXA2 and FOXA2 strongly activated the transcription of microsomal TG transfer protein. CONCLUSIONS: A feed-forward loop comprising activation of USF1 transcription by FOXA2 and activation of FOXA2 transcription by USF1, driving microsomal TG transfer protein expression, is modulated by rs2073658. Hence, rs2073658 likely influences hepatic TG secretion.

Characterization of novel peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) isoform in human liver.

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a transcriptional coactivator that contributes to the regulation of numerous transcriptional programs including the hepatic response to fasting. Mechanisms at transcriptional and post-transcriptional levels allow PGC-1α to support distinct biological pathways. Here we describe a novel human liver-specific PGC-1α transcript that results from alternative promoter usage and is induced by FOXO1 as well as glucocorticoids and cAMP-response element-binding protein signaling but is not present in other mammals. Hepatic tissue levels of novel and wild-type transcripts were similar but were only moderately associated (p < 0.003). Novel mRNA levels were associated with a polymorphism located in its promoter region, whereas wild-type transcript levels were not. Furthermore, hepatic PCK1 mRNA levels exhibited stronger associations with the novel than with the wild-type transcript levels. Except for a deletion of 127 amino acids at the N terminus, the protein, termed L-PGC-1α, is identical to PGC-1α. L-PGC-1α was localized in the nucleus and showed coactivation properties that overlap with those of PGC-1α. Collectively, our data support a role of L-PGC-1α in gluconeogenesis, but functional differences predicted from the altered structure suggest that L-PGC-1α may have arisen to adapt PGC-1α to more complex metabolic pathways in humans.

SWAP-70 identifies a transitional subset of actin filaments in motile cells.

Functionally different subsets of actin filament arrays contribute to cellular organization and motility. We report the identification of a novel subset of loose actin filament arrays through regulated association with the widely expressed protein SWAP-70. These loose actin filament arrays were commonly located behind protruding lamellipodia and membrane ruffles. Visualization of these loose actin filament arrays was dependent on lamellipodial protrusion and the binding of the SWAP-70 PH-domain to a 3'-phosphoinositide. SWAP-70 with a functional pleckstrin homology-domain lacking the C-terminal 60 residues was targeted to the area of the loose actin filament arrays, but it did not associate with actin filaments. The C-terminal 60 residues were sufficient for actin filament association, but they provided no specificity for the subset of loose actin filament arrays. These results identify SWAP-70 as a phosphoinositide 3-kinase signaling-dependent marker for a distinct, hitherto unrecognized, array of actin filaments. Overexpression of SWAP-70 altered the actin organization and lamellipodial morphology. These alterations were dependent on a proper subcellular targeting of SWAP-70. We propose that SWAP-70 regulates the actin cytoskeleton as an effector or adaptor protein in response to agonist stimulated phosphatidylinositol (3,4)-bisphosphate production and cell protrusion.

No Concentration Decrease of House Dust Mite Allergens With Rising Altitude in Alpine Regions.

PURPOSE: Several studies over the past 4 decades have indicated a significant reduction in house dust mite (HDM) and HDM allergen concentration in areas higher than 1,500 m above sea level. These have served as basis of allergen avoidance therapies for HDM allergy and asthma. However, modern construction techniques used in the insulation, heating, and glazing of buildings as well as global warming have changed the environmental parameters for HDM living conditions. The present study revisits the paradigm of decreasing HDM allergen concentrations with increasing altitude in the alpine region of Germany and Austria. METHODS: A total of 122 dust samples from different abodes (hotels, privates and mountain huts) at different altitudes (400-2,600 m) were taken, and concentrations of HDM allergens were analyzed. Humidity and temperature conditions, and numerous indoor environmental parameters such as fine dust, type of flooring, age of building, and frequency of cleaning were determined. RESULTS: HDM allergen concentrations did not significantly change with increasing altitude or relative humidity. At the level of indoor parameters, correlations could be found for different flooring types and the concentration of HDM allergens. CONCLUSIONS: In contrast to the widespread view of the relationship between altitude and HDM allergen concentrations, clinically relevant concentrations of HDM allergens could be detected in high-lying alpine regions in Austria and Germany. These results indicate that improvement in conditions of asthmatic patients sensitized against HDMs during a stay at high altitude can no longer be ascribed to decreased levels of HDM allergens, instead, other mechanisms may trigger the beneficial effect.

Glucose acts as a regulator of serum iron by increasing serum hepcidin concentrations.

Mutual clinical and molecular interactions between iron and glucose metabolism have been reported. We aimed to investigate a potential effect of glucose on iron homeostasis. We found that serum iron concentrations gradually decreased over 180 min after the administration of 75 g of glucose from 109.8 ± 45.4 mg/L to 94.4 ± 40.4 mg/L (P<.001; N=40) but remained unchanged in control subjects receiving tap water (N=21). Serum hepcidin, the key iron regulatory hormone which is mainly derived from hepatocytes but also expressed in pancreatic ß-cells, increased within 120 min after glucose ingestion from 19.7 ± 9.9 nmol/L to 31.4 ± 21.0 nmol/L (P<.001). In cell culture, glucose induced the secretion of hepcidin and insulin into the supernatant of INS-1E cultures, but did not change the amount of hepcidin detectable in the hepatocyte cell culture HepG2. We additionally confirmed the expression of hepcidin in a human islet cell preparation. These results suggest that glucose acts as a regulator of serum iron concentrations, most likely by triggering the release of hepcidin from ß-cells.

Cholesteryl ester transfer protein and hepatic lipase gene polymorphisms: effects on hepatic mRNA levels, plasma lipids and carotid atherosclerosis.

OBJECTIVE: HDL modifying effects of cholesteryl ester transfer protein (CETP) and hepatic lipase (LIPC) depend in part on each other. We studied associations of CETP-Taq1B and -514C>T-LIPC polymorphisms with hepatic mRNA levels, and their combined effects on plasma lipids and carotid atherosclerosis. METHODS: We genotyped the CETP-Taq1B and the -514C>T-LIPC polymorphisms in 67 obese women in whom hepatic CETP and LIPC transcript levels were determined as well as in 1549 participants of the Salzburg Atherosclerosis Prevention Program in Subjects at High Individual Risk (SAPHIR). Carotid atherosclerosis was assessed by intima-media thickness and extent of plaques (B-score) of the carotid arteries. RESULTS: In obese women, CETP-Taq1B and -514C>T-LIPC variant alleles were associated with reduced hepatic levels of CETP and LIPC mRNA, respectively. The CETP and LIPC polymorphisms accounted for 12.9 and 14.4% of the variability in respective transcripts. In the SAPHIR population, CETP-Taq1B showed independent effects on LDL diameter, HDL and LDL cholesterol, apolipoproteins AI and B and cholesterol/HDL cholesterol, while -514C>T-LIPC revealed independent effects on HDL cholesterol and apolipoprotein AI. The two polymorphisms displayed interactions at the level of HDL cholesterol. Compared to subjects carrying wild-type alleles at both loci, subjects homozygous for the CETP wild-type allele, but heterozygous for the LIPC polymorphism and subjects heterozygous for the CETP polymorphism, but homozygous for the LIPC wild-type allele showed an increased risk of carotid atherosclerosis (both P<0.05). CONCLUSIONS: CETP and LIPC polymorphisms influence the respective hepatic transcript levels, demonstrate interactions on HDL cholesterol and suggest that imbalances between CETP and LIPC activities may modulate the risk of carotid atherosclerosis.

Conventional protein kinase C mediates phorbol-dibutyrate-induced cytoskeletal remodeling in a7r5 smooth muscle cells.

Phorbol dibutyrate (PDBu) induced the formation of podosome-like structures together with partial disassembly of actin stress fibers in A7r5 smooth muscle cells. These podosomes contained alpha-actinin, F-actin, and vinculin and exhibit a tubular, column-like structure arising perpendicularly from the bottom of PDBu-treated cells. The conventional protein kinase C (PKC) antagonist, GO6976, inhibited PDBu-induced cytoskeletal remodeling at 0.1 microM, whereas the novel PKC antagonist, rottlerin, was ineffective at 10 microM. PDBu induced the translocation of the conventional PKC-alpha but not the novel PKC-delta to the sites of podosome formation in A7r5 cells. Although partial disassembly of actin stress fibers was observed in both Y-27632- and PDBu-treated cells, focal adhesions were much reduced in number and size only in Y-27632-treated cells. Furthermore, PDBu restored focal adhesions in Y-27632-treated cells. Live video fluorescence microscopy of alpha-actinin GFP revealed a lag phase of about 20 min prior to the rapid formation and dynamic reorganization of podosomes during PDBu treatment. These findings suggest that conventional PKCs mediate PDBu-induced formation of dynamic podosome-like structures in A7r5 cells, and Rho-kinase is unlikely to be the underlying mechanism. The podosome columns could represent molecular scaffolds where PKC-alpha phosphorylates regulatory proteins necessary for Ca(2+) sensitization in smooth muscle cells.