The contribution of visceral fat to improved insulin signaling in Ames dwarf mice

Ames dwarf (Prop1^df, df/df) mice are characterized by growth hormone (GH), prolactin, and thyrotropin deficiency, remarkable extension of longevity and increased insulin sensitivity with low levels of fasting insulin and glucose. Plasma levels of anti‐inflammatory adiponectin are increased in df/df mice, while pro‐inflammatory IL‐6 is decreased in plasma and epididymal fat. This represents an important shift in the balance between pro‐ and anti‐inflammatory adipokines in adipose tissue, which was not exposed to GH signals during development or adult life. To determine the role of adipose tissue in the control of insulin signaling in these long‐living mutants, we examined the effects of surgical removal of visceral (epididymal and perinephric) adipose tissue. Comparison of the results obtained in df/df mice and their normal (N) siblings indicated different effects of visceral fat removal (VFR) on insulin sensitivity and glucose tolerance. The analysis of the expression of genes related to insulin signaling indicated that VFR improved insulin action in skeletal muscle in N mice. Interestingly, this surgical intervention did not improve insulin signaling in df/df mice skeletal muscle but caused suppression of the signal in subcutaneous fat. We conclude that altered profile of adipokines secreted by visceral fat of Ames dwarf mice may act as a key contributor to increased insulin sensitivity and extended longevity of these animals.     

Differential expression and functional analysis of the tristetraprolin family during early differentiation of 3T3-L1 preadipocytes

The tristetraprolin (TTP) family comprises zinc finger-containing AU-rich element (ARE)-binding proteins consisting of three major members: TTP, ZFP36L1, and ZFP36L2. The present study generated specific antibodies against each TTP member to evaluate its expression during differentiation of 3T3-L1 preadipocytes. In contrast to the inducible expression of TTP, results indicated constitutive expression of ZFP36L1 and ZFP36L2 in 3T3-L1 preadipocytes and their phosphorylation in response to differentiation signals. Physical RNA pull-down and functional luciferase assays revealed that ZFP36L1 and ZFP36L2 bound to the 3' untranslated region (UTR) of MAPK phosphatase-1 (MKP-1) mRNA and downregulated Mkp-1 3'UTR-mediated luciferase activity. Mkp-1 is an immediate early gene for which the mRNA is transiently expressed in response to differentiation signals. The half-life of Mkp-1 mRNA was longer at 30 min of induction than at 1 h and 2 h of induction. Knockdown of TTP or ZFP36L2 increased the Mkp-1 mRNA half-life at 1 h of induction. Knockdown of ZFP36L1, but not ZFP36L2, increased Mkp-1 mRNA basal levels via mRNA stabilization and downregulated ERK activation. Differentiation induced phosphorylation of ZFP36L1 through ERK and AKT signals. Phosphorylated ZFP36L1 then interacted with 14-3-3, which might decrease its mRNA destabilizing activity. Inhibition of adipogenesis also occurred in ZFP36L1 and TTP knockdown cells. The findings indicate that the differential expression of TTP family members regulates immediate early gene expression and modulates adipogenesis.     

The phosphatonins and the regulation of phosphate transport and vitamin D metabolism

Phosphate homeostasis is preserved during variations in phosphate intake by short-term intrinsic renal and intestinal adaptations in transport processes, and by more long-term hormonal mechanisms, which regulate the efficiency of phosphate transport in the kidney and intestine. Recently, several phosphaturic peptides such as fibroblast growth factor 23 (FGF-23), secreted frizzled-related protein-4 (sFRP-4), extracellular phosphoglycoprotein (MEPE) and fibroblast growth factor 7 (FGF-7) have been shown to play a pathogenic role in several hypophosphatemic disorders such as tumor-induced osteomalacia (TIO), autosomal dominant hypophosphatemic rickets (ADHR), X-linked hypophosphatemic rickets (XLH), the McCune-Albright syndrome (MAS) and fibrous dysplasia (FD). These proteins induce phosphaturia and hypophosphatemia in vivo, and inhibit sodium-dependent renal phosphate transport in cultured renal epithelial cells. Interestingly, despite the induction of hypophosphatemia by FGF-23 and sFRP-4 in vivo, serum 1, 25-dihydroxyvitamin D (1alpha,25(OH)(2)D) concentrations are decreased or remain inappropriately normal, suggesting an inhibitory effect of these proteins on 25-hydroxyvitamin D 1alpha-hydroxylase activity. In FGF-23 knockout mice, 25-hydroxyvitamin D 1alpha-hydroxylase expression is increased and elevated serum 1alpha,25(OH)(2)D levels cause significant hypercalcemia and hyperphosphatemia. MEPE, however, increases circulating 1alpha,25(OH)(2)D. Circulating or local concentrations of these peptides/proteins may regulate 25-hydroxyvitamin D 1alpha-hydroxylase activity in renal tissues under physiologic circumstances.     

Serum-free culture of Japanese quail kidney cells regulation of vitamin D metabolism

Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 · 10⁶ cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25–30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromotographically separated on Sephadex LH-20. Three have been identified as 1α,25-dihydroxyvitamin D-3 (1,25(OH)₂D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)₂D-3) and 1α,24,25-trihhydroxyvitamin D-3 (1,24,25(OH)₃D-3). The activities of the 25-OH-D-3:1α- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)₂D-3 decreased 1,25(OH)₂D-3 synthesis, and increased both 24,25(OH)₂D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1α-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25-(OH)₂D-3 synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.     

The molecular mechanisms of vitamin C on cell cycle regulation in B16F10 murine melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose-dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53-p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C-induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53-p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K-family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C-induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2-p53-p21Waf1/Cip1 pathway.     

The stromal‐vascular fraction of adipose tissue contributes to major differences between subcutaneous and visceral fat depots

Adipose tissue represents a complex tissue both in terms of its cellular composition, as it includes mature adipocytes and the various cell types comprising the stromal‐vascular fraction (SVF), and in relation to the distinct biochemical, morphological and functional characteristics according to its anatomical location. Herein, we have characterized the proteomic profile of both mature adipocyte and SVF from human visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) fat depots in order to unveil differences in the expression of proteins which may underlie the distinct association of VAT and SAT to several pathologies. Specifically, 24 proteins were observed to be differentially expressed between SAT SVF versus VAT SVF from lean individuals. Immunoblotting and RT‐PCR analysis confirmed the differential regulation of the nuclear envelope proteins lamin A/C, the membrane‐cytoskeletal linker ezrin and the enzyme involved in retinoic acid production, aldehyde dehydrogenase 1A2, in the two fat depots. In sum, the observation that proteins with important cell functions are differentially distributed between VAT and SAT and their characterization as components of SVF or mature adipocytes pave the way for future research on the molecular basis underlying diverse adipose tissue‐related pathologies such as metabolic syndrome or lipodystrophy.     

Nongenomic regulation of extracellular matrix events by vitamin D metabolites

Vitamin D metabolites appear to regulate chondrocytes and osteoblasts via a combination of genomic and nongenomic mechanisms. Specificity of the nongenomic response to either 1,25-(OH)₂D₃ or 24, 25-(OH)₂D₃ may be conferred by the chemical composition of the target membrane and its fluid mosaic structure, by the presence of specific membrane receptors, or by the interaction with classic Vitamin D receptors. Nongenomic effects have been shown to include changes in membrane fluidity, fatty acid acylation and reacylation, arachidonic acid metabolism and prostaglandin production, calcium ion flux, and protein kinaase C activity. Chondrocytes metabolize 25-(OH)D₃ to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃; production of these metabolites is regulated by both growth factors and hormones and is dependent on the state of cell maturation. 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ may interact directly with extracellular matix vesicles to regulate their function in the matrix, including protease activity, resulting in matrix modefication and calcification. Isolated matrix vesicles, produced by growth zone chondrocytes, can activate latent transforming growth factor-β when incubated with exogenous 1,25-(OH)₂D₃. These observations suggest that nongenomic regulation of martix vesicle structure and function may be a mechanism by which mesenchymal cells, like osteoblasts and chndrocytes, may modulate events in the extracellular matrix at sites distant from the cell surace.     

The gut peptide Reg3g links the small intestine microbiome to the regulation of energy balance,glucose levels,and gut function

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Local hyperthermia therapy induces browning of white fat and treats obesity

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Hypoxia stimulates the autocrine regulation of migration of vascular smooth muscle cells via HIF-1alpha-dependent expression of thrombospondin-1

The migration of vascular smooth muscle cells from the media to intima and their subsequent proliferation are critical causes of arterial wall thickening. In atherosclerotic lesions increases in the thickness of the vascular wall and the impairment of oxygen diffusion capacity result in the development of hypoxic lesions. We investigated the effect of hypoxia on the migration of human coronary artery smooth muscle cells (CASMCs) via HIF-1alpha-dependent expression of thrombospondin-1 (TSP-1). When the cells were cultured under hypoxic conditions, mRNA and protein levels of TSP-1, and mRNA levels of integrin beta(3) were increased with the increase in HIF-1alpha protein. DNA synthesis and migration of the cells were stimulated under the conditions, and a neutralizing anti-TSP-1 antibody apparently suppressed the migration, but not DNA synthesis. The migration was also inhibited by RGD peptide that binds to integrin beta(3). Furthermore, the migration was completely suppressed in HIF-1alpha-knockdown cells exposed to hypoxia, while it was significantly enhanced in HIF-1alpha-overexpressing cells. These results suggest that the hypoxia induces the migration of CASMCs, and that the migration is elicited by TSP-1 of which induction is fully dependent on the stabilization of HIF-1alpha, in autocrine regulation. Thus we suggest that HIF-1alpha plays an important role in the pathogenesis of atherosclerosis.     

The Rates of Postmortem Proteolysis of Glutamate Transporters Differ Dramatically between Cells and between Transporter Subtypes

Glutamate transporters (GLT-1, GLAST, EAAC1) limit the actions of excitatory amino acids. Because a disturbed transporter operation can cause or aggravate neurological diseases, transporters are of considerable neuropathological interest. Human samples, however, are seldom obtained fresh. Here, we used mice brains to study how fast glutamate transporters are degraded after death. Immunoblots showed that terminal GLT-1 epitopes (within residues 1–26 and 518–573) had mostly disappeared after 24 hr. GLAST termini (1–25 and 522–543) degraded slightly slower. In contrast, epitopes within central parts of GLT-1 (493–508) and the EAAC1 C-terminus (510–523) were readily detectable after 72 hr. The decline in immunoreactivity of the GLT-1 and GLAST termini was also seen in tissue sections, but proteolysis did not happen synchronously in all cells. At 24 hr, scattered cells remained strongly immunopositive, while the majority of cells were completely immunonegative. GLAST and GLT-1 co-localized in neocortical tissue, but at 12 hr, many GLAST-positive cells had lost the GLT-1 termini. The uneven disappearance of labeling was not observed with the antibodies to GLT-1 residues 493–508. The immunoreactivity to this epitope correlated better with the reported glutamate uptake activity. Thus, postmortem delay may affect epitopes differently, possibly causing erroneous conclusions about relative expression levels.     

The regulation of the proton conductance of brown fat mitochondria. Identification of functional and non-functional nucleotide-binding sites

The binding of purine nucleotides to intact brown fat mitochondria is re-examined. In addition to the previously reported high affinity binding site, a low-affinity site is found, which requires several minutes to saturate. Only the high affinity site is functional in regulating the proton and halide permeabilities of the mitochondria. The low affinity site can introduce errors in the use of nucleotide binding to quantitate the Mr 32000 uncoupling protein unique to these mitochondria.     

Omental 11beta-hydroxysteroid dehydrogenase 1 correlates with fat cell size independently of obesity

Objectives: In ideopathic obesity, there is evidence that enhanced cortisol regeneration within abdominal subcutaneous adipose tissue may contribute to adiposity and metabolic disease. Whether the cortisol regenerating enzyme, 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1), or glucocorticoid receptor (GRα) levels are altered in other adipose depots remains uncertain. Our objective was to determine the association between 11βHSD1 and GRα mRNA levels in four distinct adipose depots and measures of obesity and the metabolic syndrome. Research Methods and Procedures: Adipose tissue biopsies were collected from subcutaneous (abdominal, thigh, gluteal) and intra‐abdominal (omental) adipose depots from 21 women. 11βHSD1 and GRα mRNA levels were measured by real‐time polymerase chain reaction. Body composition, fat distribution, fat cell size, and blood lipid, glucose, and insulin levels were measured. Results: 11βHSD1 mRNA was highest in abdominal subcutaneous (p < 0.001) and omental (p < 0.001) depots and was positively correlated with BMI and visceral adiposity in all depots. Omental 11βHSD1 correlated with percent body fat (R = 0.462, p < 0.05), fat cell size (R = 0.72, p < 0.001), and plasma triglycerides (R = 0.46, p < 0.05). Conversely, GRα mRNA was highest in omental fat (p < 0.001). GRα mRNA was negatively correlated with BMI in the abdominal subcutaneous (R = ?0.589, p < 0.05) and omental depots (R = ?0.627, p < 0.05). Omental GRα mRNA was inversely associated with visceral adiposity (R = ?0.507, p < 0.05), fat cell size (R = ?0.52, p < 0.01), and triglycerides (R = ?0.50, p < 0.05). Discussion: Obesity was associated with elevated 11βHSD1 mRNA in all adipose compartments. GRα mRNA is reduced in the omental depot with obesity. The novel correlation of 11βHSD1 with omental fat cell size, independent of obesity, suggests that intracellular cortisol regeneration is a strong predictor of hypertrophy in the omentum.     

Embryogenic staging of fugu, Takifugu rubripes, and expression profiles of aldh1a2, aldh1a3 and cyp26a1

Fugu (Takifugu rubripes) has contributed as an ideal model organism for understanding the structure and evolution of vertebrate genomes, but also has potential as a good model organism for developmental biology because of the availability of the genome information. However, there is no comprehensive report describing the developmental stages, which is fundamental data for developmental biology. Here we describe a series of stages of the embryonic development of fugu during the first 8 days after fertilization, i.e. from fertilization to hatching. We define seven periods of embryogenesis – the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. Stages subdividing these periods are defined based on morphological characteristics. In addition, as a model experiment of gene expression analysis using this staging series, we performed in situ hybridization of aldh1a2, aldh1a3 and cyp26a1 that play regulatory roles in retinoic acid (RA) metabolism essential for embryogenesis. This report provides fundamental information on fugu embryogenesis, which is anticipated to facilitate the use of fugu as a model organism for developmental studies.     

Backbone makes a significant contribution to the electrostatics of alpha/beta-barrel proteins.

The electrostatic properties of seven alpha/beta-barrel enzymes selected from different evolutionary families were studied: triose phosphate isomerase, fructose-1,6-bisphosphate aldolase, pyruvate kinase, mandelate racemase, trimethylamine dehydrogenase, glycolate oxidase, and narbonin, a protein without any known enzymatic activity. The backbone of the alpha/beta-barrel has a distinct electrostatic field pattern, which is dipolar along the barrel axis. When the side chains are included in the calculations the general effect is to modulate the electrostatic pattern so that the electrostatic field is generally enhanced and is focused into a specific area near the active site. We use the electrostatic flux through a square surface near the active site to gauge the functionally relevant magnitude of the electrostatic field. The calculations reveal that in six out of the seven cases the backbone itself contributes greater than 45% of the total flux. The substantial electrostatic contribution of the backbone correlates with the known preference of alpha/beta-barrel enzymes for negatively charged substrates.     

A new active vitamin D analog,ED-71, causes increase in bone mass with preferential effects on bone in osteoporotic patients

As a candidate for active vitamin D analogs that have selective effects on bone, 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71) has been synthesized and is currently under clinical trials. In ovariectomized rat model for osteoporosis, ED-71 caused an increase bone mass at the lumbar vertebra to a greater extent than 1alpha-hydroxyvitamin D3 (alfacalcidol), while enhancing calcium absorption and decreasing serum parathyroid hormone levels to the same degree as alfacalcidol. ED-71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers.An early phase II clinical trial was conducted with 109 primary osteoporotic patients. The results indicate that oral daily administration of ED-71 (0.25, 0.5, 0.75, and 1.0 microgram) for 6 months increased lumbar bone mineral density in a dose-dependent manner without causing hypercalcemia and hypercalciuria. ED-71 also exhibited a dose-dependent suppression of urinary deoxypyridinoline with no significant reduction in serum osteocalcin. These results demonstrate that ED-71 has preferential effects on bone with diminished effects on intestinal calcium absorption. ED-71 offers potentially a new modality of therapy for osteoporosis with selective effects on bone.     

Time-dependent regulation of cytokine production by RNA binding proteins defines T cell effector function

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