Although obesity is a risk factor for development of type 2 diabetes and chemical modification of proteins by advanced glycoxidation and lipoxidation end products is implicated in the development of diabetic complications, little is known about the chemical modification of proteins in adipocytes or adipose tissue. In this study we show that S-(2-succinyl)cysteine (2SC), the product of chemical modification of proteins by the Krebs cycle intermediate, fumarate, is significantly increased during maturation of 3T3-L1 fibroblasts to adipocytes. Fumarate concentration increased >5-fold during adipogenesis in medium containing 30 mM glucose, producing a >10-fold increase in 2SC-proteins in adipocytes compared with undifferentiated fibroblasts grown in the same high glucose medium. The elevated glucose concentration in the medium during adipocyte maturation correlated with the increase in 2SC, whereas the concentration of the advanced glycoxidation and lipoxidation end products, N ⑀ -(carboxymethyl)lysine and N ⑀ -(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one-and two-dimensional electrophoresis and ϳ60 2SC-proteins were detected using an anti-2SC polyclonal antibody. Several of the prominent and well resolved proteins were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. These include cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein. We propose that the increase in fumarate and 2SC is the result of mitochondrial stress in the adipocyte during adipogenesis and that 2SC may be a useful biomarker of mitochondrial stress in obesity, insulin resistance, and diabetes.The adipocyte is increasingly recognized as a dynamic cell that readily adapts to the changing nutritional status of the body. In a state of over-nutrition, the adipocyte responds by synthesizing and storing triglycerides, a process that may eventually lead to obesity and insulin resistance and then to diabetes.Chemical modification of proteins by advanced glycoxidation and lipoxidation end products (AGE/ALEs) 3 is increased in diabetes and is strongly implicated in the development of diabetic complications (1-3). However, relatively little is known about the chemical modification of proteins in adipocytes or adipose tissue or the possible role of chemical modifications of proteins in the regulation of adipocyte metabolism during adipogenesis or diabetogenesis.In general, AGE/ALEs are derivatives of lysine and arginine residues, formed by reaction of the amino or guanidino groups on protein with electrophilic intermediates in carbohydrate and lipid autoxidation or metabolism (4). In contrast to extracellular proteins, the lower pK a sulfhydryl group of cysteine residues is a more likely target for modification by electrophiles on intracellular proteins. We recently described S-(2-succinyl)cysteine (2SC) as a novel chemical modification of cysteine residues in proteins, formed by a...

Hemoglobin gene expression in non-erythroid cells has been previously reported in activated macrophages from adult mice and lens cells, and recent studies indicate that alveolar epithelial cells can be derived from hematopoietic stem cells. Our laboratory has now produced strong evidence that hemoglobin is expressed by alveolar type II (ATII) cells and Clara cells, the primary producers of pulmonary surfactant. ATII cells are also closely involved in innate immunity within the lung and are stem cells that differentiate into alveolar type I cells. Reverse transcriptase-PCR was used to measure the expression of transcripts from the ␣-and ␤-globin gene clusters in several human and rodent pulmonary epithelial cells. Surprisingly, the two major globin mRNAs characteristic of adult erythroid precursor cells were clearly expressed in human A549 and H441 cell lines, mouse MLE-15 cells, and primary ATII cells isolated from normal rat and mouse lungs. DNA sequencing verified that these PCR products were indeed the result of specific amplification of globin gene cDNAs. These alveolar epithelial cells also expressed the corresponding hemoglobin protein subunits as determined by Western blotting, and tandem mass spectrometry sequencing was used to verify the presence of both ␣-and ␤-globin polypeptides in rat primary ATII cells. The function of hemoglobin expression by cells of the pulmonary epithelium will be determined by future studies, but this novel finding could potentially have important implications for the physiology and pathology of the lung.

The effects of bovine pulmonary surfactant-associated protein B (SP-B) on molecular packing of model membrane lipids (7:1 DPPC/DPPG) were studied by fluorescence anisotropy. The bilayer surface was markedly ordered by SP-B below the gel to fluid phase transition temperature (Tc) while it was only slightly ordered above this temperature as indicated by surface-sensitive probes 6-NBD-PC and 6-NBD-PG. The effects of SP-B on fluorescence anisotropy were concentration dependent, reaching maximal activity at 1-2% protein to phospholipid by weight. Anisotropy measurements of interior-selective fluorescent probes (cis-parinaric acid and DPH) imply that addition of SP-B into the phospholipid shifted the Tc of the model membrane but did not alter lipid order at the membrane interior. Since fluorescence anisotropy studies with trans-parinaric acid, an interior-sensitive probe with high affinity for gel-phase lipids, did not detect any changes in lipid packing or Tc, it is likely that SP-B resides primarily in fluid-phase domains. Fluorescence lifetime measurements indicated that two conformers of the NBD-PC probe exist in this DPPC/DPPG model membrane system. Fluorescence intensity measurements generated with NBD-PC and NBD-PG, in conjunction with information from lifetime measurements, support the concept that SP-B increases the distribution of the short-lifetime conformer in the gel phase. In addition, the anisotropy and intensity profiles of NBD-PG in the model membrane indicate that bovine SP-B interacts selectively with phosphatidylglycerol.

Objective Recently vitamin D deficiency has been associated with increased risks for preeclampsia and diagnosis of early-onset, severe preeclampsia (EOSPE). The purpose of this investigation was to examine the association between vitamin D levels and small for gestational age (SGA) in patients with EOSPE. Study Design Patients with EOSPE were recruited and demographics, outcomes, and plasma were collected. 25-hydroxy-vitamin D (25-OH-D) was assessed by radioimmunoassay and reported in ng/mL. Results were analyzed by Mann Whitney U test and Spearman correlation and reported as median (Q1–Q3). Results In patients with EOSPE (n=56), 25-OH-D was lower in patients with SGA (16.8 ng/mL [8.9–23]) verses normal fetal growth (25.3 ng/mL [16–33]) (p=0.02). 25-OH-D was significantly correlated with percentile growth at delivery (ρ = 0.31, p=0.02). Conclusions Vitamin D is lower among patients with SGA in EOSPE than those without growth retardation. We suspect that vitamin D may impact fetal growth through placental mechanisms.

Alveolar epithelial cells are directly exposed to acute and chronic fluctuations in alveolar oxygen tension. Previously, we found that the oxygen-binding protein hemoglobin is expressed in alveolar Type II cells (ATII). Here, we report that ATII cells also express a number of highly specific transcription factors and other genes normally associated with hemoglobin biosynthesis in erythroid precursors. Because hypoxia-inducible factors (HIFs) were shown to play a role in hypoxia-induced changes in ATII homeostasis, we hypothesized that the hypoxia-induced increase in intracellular HIF exerts a concomitant effect on ATII hemoglobin expression. Treatment of cells from the ATII-like immortalized mouse lung epithelial cell line-15 (MLE-15) with hypoxia for 20 hours resulted in dramatic increases in cellular levels of HIF-2a protein and parallel significant increases in hemoglobin messenger RNA (mRNA) and protein expression, as compared with that of control cells cultured in normoxia. Significant increases in the mRNA of globin-associated transcription factors were also observed, and RNA interference (RNAi) experiments demonstrated that the expression of hemoglobin is at least partially dependent on the cellular levels of globin-associated transcription factor isoform 1 (GATA-1). Conversely, levels of prosurfactant proteins B and C significantly decreased in the same cells after exposure to hypoxia. The treatment of MLE-15 cells cultured in normoxia with prolyl 4-hydroxylase inhibitors, which mimic the effects of hypoxia, resulted in increases of hemoglobin and decreases of surfactant proteins. Taken together, these results suggest a relationship between hypoxia, HIFs, and the expression of hemoglobin, and imply that hemoglobin may be involved in the oxygen-sensing pathway in alveolar epithelial cells.Keywords: hemoglobin; alveolar epithelial cells; hypoxia; hypoxiainducible factor Pulmonary hypoxia occurs under both physiologic and pathologic conditions. In fact, a hypoxic environment is necessary for proper embryonic lung development, by promoting the formation of microvasculature and epithelial branching morphogenesis (the average fetal blood O 2 fraction is z2-5%) (1-3). However, postnatal decreases in alveolar oxygen tension as a result of pulmonary disease disrupt alveolar homeostasis. High-altitude ascent, pathologic conditions resulting in inadequate respiration, pulmonary edema after acute lung injury, or congestive heart failure may all result in decreased oxygen tension. Alveolar Type II (ATII) cells represent approximately two thirds of epithelial cell numbers, and are of special clinical interest because of their role in the production, secretion, and recycling of pulmonary surfactant (4). In addition, ATII cells differentiate into Type I (ATI ) cells upon epithelial injury, and also act to clear fluid from the alveolar space. Although numerous studies evaluated the effects of hypoxia on the pulmonary endothelium, few sought to identify hypoxia-regulated genes in alveolar epithelial cells.Our previous stu...