Peptide hormones in infants with feeding disorders.

The prevalence of eating problems in otherwise healthy infants is a common problem in Western countries. Peptide hormones such as adiponectin, ghrelin and resistin have been shown to play an important role in the regulation of satiety and hunger in several diseases and states. The aim of this study was to evaluate the peptide hormone levels in children with eating problems. In this study, 12 otherwise healthy infants (mean age 10.4 months) with eating problems and 12 healthy controls were studied. At their first hospital visit samples for analysis of adiponectin, ghrelin and resistin were obtained and a careful physical examination was carried out. To exclude any possible anatomic or metabolic reason for eating problems necessary investigations were also performed. Adiponectin levels were significantly higher in the cases than in the controls (p = 0.033), and the difference was still significant after adjustment for weight (p < 0.05). Resistin and ghrelin concentrations showed no significant differences. Conclusions. For the first time we were able to show in this pilot study that adiponectin concentrations were elevated in the infants with eating problems. Cross-sectional association does not necessarily imply causal relationship. Thus, further studies with larger number of cases will be needed to clarify the role of adiponectin in the eating problems in infants.

Resistin is an indicator of the metabolic syndrome according to five different definitions in the Finnish Health 2000 survey.

BACKGROUND: Resistin is a peptide hormone secreted mainly from human monocytes and macrophages. It has an unclear association with the metabolic syndrome, which is a cluster of cardiovascular risk factors such as glucose intolerance, central obesity, insulin resistance, dyslipidemia, and hypertension. We examined the association of resistin with metabolic syndrome and its components in a population-based cohort. METHODS: A subsample of a large Finnish cross-sectional health examination survey (the Health 2000 Survey) was studied. Resistin was measured using an in-house assay based on the DELFIA® technique in 1,508 Finnish men and women aged 45-74 years. Metabolic syndrome was defined according to five different definitions. RESULTS: Resistin levels were higher in the subjects with metabolic syndrome when compared to the subjects without metabolic syndrome (P < 0.05 for every metabolic syndrome criterion). In logistic regression analysis, a high resistin level was an independent predictor of the prevalence of metabolic syndrome (P < 0.05 for every criterion). Resistin was positively associated with waist circumference, tumor necrosis factor-α, and insulin resistance assessed by the homeostasis model and inversely with total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol after adjusting for age, gender, and body mass index (P < 0.05 for all). CONCLUSIONS: These results clarify the controversial association of resistin in obesity and metabolic syndrome, suggesting that a high resistin level is associated with clustering of metabolic disturbances.

Ghrelin and obestatin modulate early atherogenic processes on cells: enhancement of monocyte adhesion and oxidized low-density lipoprotein binding.

Emerging evidence indicates the potential involvement of ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, in low-grade inflammatory diseases such as obesity and atherosclerosis. The goal of the present study was to use cell culture models to investigate the influences of ghrelin and obestatin in processes participating in atherogenesis. We studied monocyte adhesion, monocyte chemoattractant protein-1, and adhesion molecule expression on endothelial cells as well as binding of oxidized low-density lipoprotein (LDL) and acetylated LDL to macrophages. Ghrelin treatment increased adhesion of calcein-labeled THP-1 monocytes to EA.hy 926 endothelial cells. Simultaneously, ghrelin increased the expression of intercellular adhesion molecule-1 measured by quantitative reverse transcriptase polymerase chain reaction. Tumor necrosis factor-alpha stimulation together with ghrelin treatment decreased both monocyte adhesion and vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1 expression and, together with obestatin treatment, decreased vascular cell adhesion molecule-1 expression. Finally, ghrelin and obestatin increased binding of oxidized LDL to thioglycollate-elicited mouse peritoneal macrophages. No changes were observed in the uptake of acetylated LDL by mouse J774.A1 macrophages after exposure to ghrelin or obestatin. In conclusion, we found 3 lines of in vitro evidence supporting proatherogenic properties of ghrelin in the early stages of the disease. However, in the presence of tumor necrosis factor-alpha stimulation, opposite effects of ghrelin were observed, suggesting that ghrelin may also have an anti-inflammatory role in the presence of increased inflammation, for example, during the more progressed phases of atherogenesis.

The expression of human resistin in different leucocyte lineages is modulated by LPS and TNFalpha.

OBJECTIVE: Human resistin has been linked to several inflammatory diseases such as atherosclerosis. This study aimed to clarify the expression of resistin in different inflammatory cells and its effect on endothelial cells. RESULTS: In this study, RNA and protein expression of resistin were detected in human primary neutrophils, monocytes, and T cells as well as in human Jurkat T cells, RPMI-8226 B cells, monocytic U937, and myeloblastic HL-60 cell lines. The highest resistin protein and mRNA level were detected in neutrophils, primary monocytes, and monocytic U937 cells. The RNA expression of resistin was upregulated both in neutrophils and in U937 cells after exposure to LPS. Also TNFalpha induced resistin RNA expression in neutrophils, U937, T-lymphocytic Jurkat cells, and B-lymphocytic RPMI-8226 cells. The RNA and protein expression of resistin decreased as the monocytic U937 cells differentiated into macrophage-like cells. In endothelial EA.hy 926 cells, resistin increased the expression of MCP-1 and PECAM-1 and adhesion of monocytes to endothelial cells. CONCLUSIONS: The wide-ranging expression of resistin in white blood cells and the upregulation of its expression by inflammatory reagents LPS and TNFalpha support the fact that increased resistin could be involved in several inflammatory diseases.

The effect of energy restriction during pregnancy on obesity-related peptide hormones in rat offspring.

It has been proposed that fetal exposure to environmental stressors, such as undernutrition, during critical periods of development may lead to adaptations that permanently change the structure and function of the body. These adaptations may be important for immediate survival during fetal development, but can predispose to disease in later life. We designed a pilot study investigating the effect of fetal undernutrition on the obesity-related peptides adiponectin, ghrelin, leptin and resistin levels in rat. We also wanted to explore changes in lipid and insulin metabolism. Sprague-Dawley rats were randomly assigned to three dietary treatment groups on day 4 of gestation. The control group was fed ad libitum and the food-restricted rats received either 75% or 50% of ad libitum food intake until parturition. Serum levels of obesity-related peptides as well as lipid and insulin levels were measured from 1-month-old pups. Serum resistin concentrations were higher in both food-restricted groups and serum adiponectin concentration was lower in the 50% food-restricted group compared to the control group. Serum total cholesterol levels were significantly higher in both food-restricted groups. These results indicate that undernutrition during fetal development may lead to unfavorable changes in obesity-related peptide hormones as well as evoking adverse changes in serum cholesterol levels. The observed changes may predispose to insulin resistance and significantly increase the risk of developing cardiovascular disease in later life.

Gene expression profiles in fetal and neonatal rat offspring of energy-restricted dams.

BACKGROUND/AIMS: Nutrition during fetal and early postnatal development can have permanent effects on physiology resulting in an increased risk for disease in later life. The aim of this study was to explore changes in gene expression related to maternal energy restriction during pregnancy in rat fetuses and in neonatal rat offspring. METHODS: From day 4 of gestation until parturition, energy-restricted dams received either 75 or 50% of ad libitum food intake. Microarray analyses were performed on whole 13- and 17-day fetuses and 1-day-old pups. Protein and fat contents of the dams' milk were analyzed in the different feeding groups. RESULTS: A surprisingly small number of genes were differentially expressed between the groups, probably due to the strict control of fetal development. Interestingly, the expressions of many pancreatic digestion enzymes were reduced in the 1-day-old pups of the energy-restricted dams. A statistically significant difference in milk protein content was observed on day 1 post-partum between the gestationally food-restricted groups. CONCLUSIONS: The expressions of several genes that may have an important role in the normal development of organs were affected by undernutrition during fetal development. In addition, undernutrition may have affected the function of the exocrine pancreas.

Estrogen replacement therapy decreases plasma adiponectin but not resistin in postmenopausal women.

The effects of estrogen replacement therapy (ERT) to cardiovascular disease risk are still unclear. Low adiponectin and high resistin plasma concentrations are reported to be associated with atherosclerosis. However, it is not known how ERT affects plasma adiponectin and resistin concentrations. Seventy-three hysterectomized, nondiabetic, postmenopausal women were randomized in a double-blind, double-dummy study to receive either peroral estradiol valerate or transdermal 17beta-estradiol gel for 6 months. Biochemical measurements were determined from samples taken before and after the therapy. Peroral estradiol valerate therapy decreased adiponectin concentrations from 13.6 to 11.6 mg/L (P = .008), whereas transdermal 17beta-estradiol gel had no effect (12.7 vs 12.2 mg/L). Neither treatment changed the resistin concentrations significantly. Plasma concentrations of estradiol and estrone did not correlate with adiponectin or resistin concentrations before or after therapy. The change in adiponectin concentration correlated significantly with the changes in waist-hip ratio, very low-density lipoprotein triglycerides, and insulin-like growth factor 1 in the peroral estradiol valerate group. The changes in these variables and the change in estradiol concentration explained 43.1% (P = .001) of the variability in the change of plasma adiponectin, the change in very low-density lipoprotein triglycerides being the strongest determinant (beta = -.407, P = .011). The results show that peroral ERT can decrease plasma adiponectin levels. However, ERT does not seem to influence plasma resistin concentrations.

DNA polymerase epsilon associates with the elongating form of RNA polymerase II and nascent transcripts.

DNA polymerase epsilon co-operates with polymerases alpha and delta in the replicative DNA synthesis of eukaryotic cells. We describe here a specific physical interaction between DNA polymerase epsilon and RNA polymerase II, evidenced by reciprocal immunoprecipitation experiments. The interacting RNA polymerase II was the hyperphosphorylated IIO form implicated in transcriptional elongation, as inferred from (a) its reduced electrophoretic mobility that was lost upon phosphatase treatment, (b) correlation of the interaction with phosphorylation of Ser5 of the C-terminal domain heptapeptide repeat, and (c) the ability of C-terminal domain kinase inhibitors to abolish it. Polymerase epsilon was also shown to UV crosslink specifically alpha-amanitin-sensitive transcripts, unlike DNA polymerase alpha that crosslinked only to RNA-primed nascent DNA. Immunofluorescence microscopy revealed partial colocalization of RNA polymerase IIO and DNA polymerase epsilon, and immunoelectron microscopy revealed RNA polymerase IIO and DNA polymerase epsilon in defined nuclear clusters at various cell cycle stages. The RNA polymerase IIO-DNA polymerase epsilon complex did not relocalize to specific sites of DNA damage after focal UV damage. Their interaction was also independent of active DNA synthesis or defined cell cycle stage.

The screening of expression and purification conditions for replicative DNA polymerase associated B-subunits, assignment of the exonuclease activity to the C-terminus of archaeal pol D DP1 subunit.

The B-subunits of replicative DNA polymerases belong to the superfamily of calcineurin-like phosphoesterases and are conserved from Archaea to humans. Recently we and others have shown that the B-subunit (DP1) of the archaeal family D DNA polymerase is responsible for proofreading 3'-5' exonuclease activity. The similarity of B-subunit sequences implies a common fold, but since the key catalytic and metal binding residues of the phosphoesterase domain are disrupted in the eukaryotic B-subunits, their common function has not been identified. To study the structure and activities of B-subunits in more detail, we expressed 13 different recombinant B-subunits in Escherichia coli. We found that the solubility of a protein could be predicted from the calculated GRAVY score. These scores were useful for the selection of proteins for successful expression. We optimized the expression and purification of Methanocaldococcus (Methanococcus) jannaschii DP1 of DNA polymerase D (MjaDP1) and show that the protein co-purifies with a thermostable nuclease activity. Truncation of the protein indicates that the N-terminus (aa 1-134) is not needed for catalysis. The C-terminal part of the protein containing both the calcineurin-like phosphoesterase domain and the OB-fold is sufficient for the nuclease activity.

Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D.

The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3'-5' proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn2+-dependent 3'-5' exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.