Whole-body R2∗ mapping to quantify tissue iron in iron storage organs: reference values and a genotype.

AIM: To define reference values for the transverse relaxation rate (R2∗) in iron storage organs and to investigate the role of human haemochromatosis protein (HFE) genotype on iron storage. MATERIALS AND METHODS: Whole-body magnetic resonance imaging (MRI) including a five-echo gradient-echo sequence was performed in 483 volunteers (269 men, mean age 59.3 ± 12.2 years) without clinical evidence of an iron storage disease at 1.5 T. R2∗ values were assessed for liver, spleen, pancreas, heart, bones, and brain parenchyma. The HFE genotype was determined regarding the single nucleotide polymorphisms (SNPs) rs74315324, rs1799945, rs41303501, rs1800562, rs1800730. R2∗ values were compared among participants without and with at least one mutation. R2∗ reference values were defined using volunteers without any mutation. RESULTS: Three hundred and one participants had no mutations in any HFE SNP, 182 had at least one mutation. HFE gene mutations were distributed as (heterozygous/homozygous) rs1799945:132/9, rs1800562:33/1, and rs1800730:11/0. Mean R2∗ values ± SD (per second) in the group without mutation were: liver: 33.4 ± 12.7, spleen: 24.1 ± 13.8, pancreas: 27.2 ± 6.6, heart: 32.7 ± 11.8, bone: 69.3 ± 21.0, brain parenchyma: 13.9 ± 1.2. No significant difference in R2∗ values were found between participants with and without the HFE gene mutation for any examined iron storage organ (pliver=0.09, pspleen=0.36, ppancreas = 0.08, pheart = 0.36, pbone = 0.98, pbrain=0.74). CONCLUSION: Reference values of R2∗ in iron storage organs are feasible to support the diagnosis of iron storage diseases. Non-specific mutations in HFE SNPs appear not to affect the phenotype of tissue iron accumulation.

Overexpression of glutamine: fructose-6-phosphate amidotransferase in the liver of transgenic mice results in enhanced glycogen storage, hyperlipidemia, obesity, and impaired glucose tolerance.

To examine the effect of increased hexosamine flux in liver, the rate-limiting enzyme in hexosamine biosynthesis (glutamine:fructose-6-phosphate amidotransferase [GFA]) was overexpressed in transgenic mice using the PEPCK promoter. Liver from random-fed transgenic mice had 1.6-fold higher GFA activity compared with nontransgenic control littermates (276 +/- 24 pmol x mg(-1) x min(-1) in transgenic mice vs. 176 +/- 18 pmol x mg(-1) x min(-1) in controls, P < 0.05) and higher levels of the hexosamine end product UDP-N-acetyl glucosamine (288 +/- 11 pmol/g in transgenic mice vs. 233 +/- 10 pmol/g in controls, P < 0.001). Younger transgenic mice compared with control mice had lower fasting serum glucose (4.8 +/- 0.5 mmol/l in transgenic mice vs. 6.5 +/- 0.8 mmol/l in controls, P < 0.05) without higher insulin levels (48.0 +/- 7.8 pmol/l in transgenic mice vs. 56.4 +/- 5.4 pmol/l in controls, P = NS); insulin levels were significantly lower in transgenic males (P < 0.05). At 6 months of age, transgenic animals had normal insulin sensitivity by the hyperinsulinemic clamp technique. Hepatic glycogen content was higher in the transgenic mice (108.6 +/- 5.2 pmol/g in transgenic mice vs. 32.8 +/- 1.3 micromol/g in controls, P < 0.01), associated with an inappropriate activation of glycogen synthase. Serum levels of free fatty acids (FFAs) and triglycerides were also elevated (FFAs, 0.67 +/- 0.03 mmol/l in transgenic mice vs. 0.14 +/- 0.01 in controls; triglycerides, 1.34 +/- 0.15 mmol/l in transgenic mice vs. 0.38 +/- 0.01 in controls, P < 0.01). Older transgenic mice became heavier than control mice and exhibited relative glucose intolerance and insulin resistance. The glucose disposal rate at 8 months of age was 154 +/- 5 mg x kg(-1) x min(-1) in transgenic mice vs. 191 +/- 6 mg x kg(-1) x min(-1) in controls (P < 0.05). We conclude that hexosamines are mediators of glucose sensing for the regulation of hepatic glycogen and lipid metabolism. Increased hexosamine flux in the liver signals a shift toward fuel storage, resulting ultimately in obesity and insulin resistance.

Regulation of glutamine:fructose-6-phosphate amidotransferase by cAMP-dependent protein kinase.

Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme in hexosamine biosynthesis, an important pathway for cellular glucose sensing. Human GFA has two potential sites for phosphorylation by cAMP-dependent protein kinase A (PKA). To test whether GFA activity is regulated by cAMP-dependent phosphorylation, rat aortic smooth muscle cells were treated in vivo with cAMP-elevating agents, 10 micromol/l forskolin, 1 mmol/l 8-Br-cAMP, or 3-isobutyl-1-methylxanthine. All treatments resulted in rapid and significant increases (2- to 2.4-fold) in GFA activity assayed in cytosolic extracts. Maximal effects of forskolin were observed at 10 micromol/l and 60 min. Preincubation of cells with cycloheximide did not abolish the effect of forskolin. Incubation of cytosolic extracts at 37 degrees C for 10 min in a buffer without phosphatase inhibitors led to a 79% decrease of GFA activity. This loss of activity was inhibited by the addition of phosphatase inhibitors (5 mmol/l sodium orthovanadate, 50 mmol/l sodium fluoride, or 5 mmol/l EDTA, but not 100 nmol/l okadaic acid), suggesting that GFA undergoes rapid dephosphorylation by endogenous phosphatases. Purified GFA is phosphorylated in vitro by purified PKA, resulting in a 1.7-fold increase in GFA activity. Treatment of GFA with purified protein kinase C had no effect. We conclude that GFA activity may be modulated by cAMP-dependent phosphorylation.

Extraordinary conservation of cysteines among homologous Chironomus silk proteins sp185 and sp220.

Aquatic larvae of the midge, Chironomus tentans, synthesize a 185-kDa silk protein (sp185) with the cysteine-containing motif Cys-X-Cys-X-Cys (where X is any residue) every 20-28 residues. We report here the cloning and full-length sequence of cDNAs encoding homologous silk proteins from Chironomus pallidivittatus (sp185) and Chironomus thummi (sp220). Deduced amino acid sequences reveal proteins of nearly identical mass composed of 72 blocks of 20-28 residues, 61% of which can be described by the motif X5-8-Cys-X5-(Trp/Phe/Tyr)-X4-Cys-X-Cys-X-Cys. Spatial arrangement of these residues is preserved more than surrounding sequences. cDNA clones enabled us to map the genes on polytene chromosomes and identify for the first time the homolog of the Camptochironomus Balbiani ring 3 locus in Chironomus thummi. The apparent molecular weight difference between these proteins (185 vs 220 kDa) is not attributable to primary structure and may be due to differential N-linked glycosylation. DNA distances and codon substitutions indicate that the C. tentans and C. pallidivittatus genes are more related to each other than either is to C. thummi; however, substitution rates for the 5'- and 3'-halves of these genes are different. Blockwise sequence comparisons suggest intragenic variation in that some regions evolved slower or faster than the mean and may have been subjected to different selective pressures.

Identification of divergent homologs of Chironomus tentans sp185 and its Balbiani ring 3 gene in Australasian species of Chironomus and Kiefferulus.

A 185-kDa silk protein (sp185) from Chironomus tentans, present in both larval and prepupal silks, contains a striking amino acid sequence motif, Cys-X-Cys-X-Cys, which occurs about every 22-26 residues. Homologous proteins have been found in Chironomus pallidivittatus (sp185) and Chironomus thummi (sp220), which apparently differ in size but are very similar in overall composition and sequence. While surveying Australasian species of Chironomus and Kefferulus we obtained evidence for immunologically related silk protein having similar size and amino acid composition, but noticeably less Cys. Interspecies in situ hybridization to polytene chromosomes with C. tentans and C. pallidivittatus cDNA probes indicated that each species had a related gene. One pair of C. tentans cDNA-derived primers enabled polymerase chain reaction amplification of a discrete fragment of this gene from Kiefferulus 'cornishi'. Preliminary sequence information for this fragment confirmed the presence of an encoded Cys-X-Cys-X-Cys motif in what appeared to be a similar protein region containing less Cys. We conclude that homologs of C. tentans sp185 and its gene have been identified which may contain significant deviations in structure. Once suitable libraries are available, probes described here will be useful for selecting cDNA and genomic clones for detailed study.

A cell-specific glycosylated silk protein from Chironomus thummi salivary glands. Cloning, chromosomal localization, and characterization of cDNA.

Chironomid salivary glands contain 40 cells dedicated to the synthesis of a relatively small ensemble of silk proteins. Glands in some species contain a special lobe composed of 4 cells distinguishable from the others. We have cloned a special lobe-specific cDNA from Chironomus thummi salivary glands. Northern blots of salivary gland RNA demonstrated that the cDNA hybridizes to a 2.5-kilobase transcript present only in the special lobe. In situ hybridization mapped the gene encoding this cDNA to region A2b on polytene chromosome IV, the locus of the special lobe-specific Balbiani ring a. The deduced amino acid sequence encodes a protein with a calculated molecular mass of 77 kDa and numerous potential glycosylation sites; it appears unrelated to other known chironomid silk proteins. Polyclonal antibody, raised against a cDNA-encoded fusion protein, reacted exclusively with a special lobe-specific 160-kDa silk protein. Lectin binding studies indicate that the immunoreactive 160-kDa protein contains both N- and O-linked glycan moieties. We conclude that glycosylation most likely contributes to the difference between calculated and apparent molecular masses and that this cDNA encodes the special lobe-specific silk protein previously described as ssp160 (Kolesnikov, N. N., Karakin, E. I., Sebeleva, T. E., Meyer, L., and Serfling, E. (1981) Chromosoma 83, 661-677).