Observational study of bone accretion during successful weight loss in obese adolescents

Objective: To assess bone mineral content (BMC) among obese adolescents who lose weight during a critical period for bone accretion. Methods and Procedures: Whole body, lumbar spine, lower, and upper limb BMC were measured in 62 obese adolescents who completed an intensive 12‐month weight loss trial. BMC was adjusted for height (z ‐scores) using data from a reference group of 66 adolescents (who were 18% overweight). Results: At baseline, the BMC of the obese group was higher than the reference group. During the 12‐month weight loss program, unadjusted BMC increased among the obese adolescents, despite successful weight loss. After adjustment for height, whole body BMC did not change significantly from baseline to 12 months (mean ± s.d.: 1.08 ± 0.67 to 1.06 ± 0.67, P = 0.7). Region‐specific BMC‐for‐height however decreased for the lower (1.07 ± 0.57 to 0.95 ± 0.59, P < 0.001) and upper (1.29 ± 0.56 to 1.18 ± 0.57, P = 0.01) limbs, but lumbar spine BMC‐for‐height increased (0.14 ± 1.06 to 0.40 ± 0.94, P < 0.001). These changes were largely and independently explained by changes in lean and fat mass. Discussion: This study confirms that obese adolescents have high BMC for height and suggests that, unlike adults, their BMC continues to increase during weight loss and remains higher than the BMC of a reference group. After adjustment for growth‐related changes, lower and upper limb BMC appears to decrease, while lumbar spine BMC appears to increase. These results suggest that to optimize the health benefits of weight loss among obese adolescents, their bone health should be better understood and addressed.     

HCV NS3 serine protease-neutralizing single-chain antibodies isolated by a novel genetic screen

Hepatitis C virus (HCV) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer. The high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for HCV. The HCV NS3 is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. Here, we report the development of a novel bacterial genetic screen for inhibitors of NS3 catalysis and its application for the isolation of single-chain antibody-inhibitors. Our screen is based on the concerted co-expression of a reporter gene, of recombinant NS3 protease and of fusion-stabilized single-chain antibodies (scFvs) in Escherichia coli. The reporter system had been constructed by inserting a short peptide corresponding to the NS5A/B cleavage site of NS3 into a permissive site of the enzyme beta-galactosidase. The resulting engineered lacZ gene, coding for an NS3-cleavable beta-galactosidase, is carried on a low copy plasmid that also carried the NS3 protease-coding sequence. The resultant beta-galactosidase enzyme is active, conferring a Lac+ phenotype (blue colonies on indicator 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal) plates), while induction of NS3 expression results in loss of beta-galactosidase activity (transparent colonies on X-gal plates). The identification of inhibitors, as shown here by isolating NS3-inhibiting single-chain antibodies, expressed from a compatible high copy number plasmid, is based on the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active). Our source of inhibitory scFvs was an scFv library that we prepared from spleens of NS3-immunized mice and subjected to limited affinity selection. Once isolated, the inhibitors were validated as genuine and specific NS3 binders by an enzyme-linked immunosorbent assay and as bone fide NS3 serine protease inhibitors by an in vitro catalysis assay. We further show that upon expression as cytoplasmic intracellular antibodies (intrabodies) in NS3-expressing mammalian cells, three of the scFvs inhibit NS3-mediated cell proliferation. Although applied here for the isolation of antibody-based inhibitors, our genetic screen should be applicable for the identification of candidate inhibitors from other sources.     

Calcium and 1,25-dihydroxyvitamin D3 regulation of adipokine expression

Objective: Obesity is associated with elevated oxidative stress and low‐grade systemic inflammation. We have demonstrated recently that 1α,25‐(OH)₂‐D₃ promotes reactive oxygen species production in cultured adipocytes, whereas suppression of 1α,25‐(OH)₂‐D₃ by increasing dietary calcium down‐regulates diet‐induced oxidative stress in aP2‐agouti transgenic mice. However, whether the anti‐obesity effect of dietary calcium plays a role in regulation of obesity‐associated inflammation is not clear. Research Methods and Procedures: We investigated the role of dietary calcium in the regulation of inflammatory cytokine production in aP2‐agouti transgenic mice fed low‐ and high‐calcium obesigenic diets and in the modulation of cytokine production by 1α,25‐(OH)₂‐D₃ in cultured murine and human adipocytes. Results: The high‐calcium diet inhibited the expression of pro‐inflammatory factors tumor necrosis factor α and interleukin (IL)‐6 by 64% and 51%, respectively (p < 0.001), in visceral fat, stimulated the expression of the anti‐inflammatory factors IL‐15 and adiponectin by 52% (p = 0.001) and 54% (p = 0.025), respectively, in visceral fat, and induced a 2‐fold increase in IL‐15 expression in soleus muscle (p = 0.01) compared with litter mate controls on a low‐calcium diet. 1α,25‐(OH)₂‐D₃ also markedly stimulated the expression of tumor necrosis factor α (p < 0.001) and IL‐6 (p = 0.016) in differentiated 3T3‐L1 adipocytes and increased IL‐6 (p = 0.004) and IL‐8 (p < 0.001) production in differentiated human adipocytes. These effects were blocked by calcium channel antagonism with nifedipine. Discussion: These data demonstrate that 1α,25‐(OH)₂‐D₃ favors inflammatory cytokine expression and inhibits anti‐inflammatory cytokine expression; accordingly, suppression of 1α,25‐(OH)₂‐D₃ by dietary calcium inhibits adipocyte‐derived inflammation associated with obesity.     

Effects of fatty (fa) allele and high-fat diet on adipose tissue leptin and lipid metabolism.

Leptin is an adipocyte-secreted hormone that binds hypothalamic receptors and potently decreases food intake. Leptin receptor defects in homozygous mutant Zucker fatty ( fa/fa) rats lead to massive obesity, hyperphagia, decreased energy expenditure, and insulin resistance, while the phenotype of heterozygous ( Fa/fa) lean rats lies between lean ( Fa/Fa) and obese ( fa/fa) rats. Whether heterezygotes exhibit specific changes in lipid metabolism in a diet-responsive manner is not clear. Thus, the specific aim of this study was to test whether the presence of one fa allele modulates lipid metabolism and leptin, and whether these effects are exacerbated by high-fat diet. We demonstrate that the presence of one fa allele significantly increases lipogenesis in adipose tissue assessed by glycerol-3-phosphate dehydrogenase (GPDH) and fatty acid synthase (FAS) activities. FAS is more responsive to high-fat diets than GPDH in Fa/fa rats. Adipose tissue leptin levels are significantly higher in fat pads of Fa/fa compared to Fa/Fa rats. Moreover, Fa/fa rats fed high-fat diet show an additional two-fold increase in leptin levels compared to wild type rats on the same diet. Collectively, these results indicate that the presence of one fa allele increase adipocyte lipogenic enzyme activities, which results in hyperleptinemia concurrent with increased adiposity.     

Chromatin protein methylation in proliferating liver and hepatoma cells.

Chromatin protein methylation in proliferating liver cells and hepatomas was examined in vivo and in vitro. Methylation in vivo was estimated using 2-14 C-L-methionine and 3H-methyl-L-methionine. 3H/14C ratios were calculated for crude histone and nonhistone chromatin protein fractions and compared to those calculated for serum albumin. From this, determination of a methylation index was calculated using serum albumin as a standard. Results of this study indicate that both histones and nonhistone chromatin proteins are methylated to a greater extent in proliferating liver cells and in hepatomas than in control preparations. These observations are generally supported by our in vitro methylation studies. The results are discussed in terms of chromatin protein methylation and its possible relationship to DNA replication.     

Streptozotocin and Alloxan-based Selection Improves Toxin Resistance of Insulin-Producing RINm Cells

The aim of our study was to develop a method for selection of subpopulations of insulin producing RINm cells with higher resistance to beta cell toxins. Cells, resistant to streptozotocin (RINmS) and alloxan (RINmA), were obtained by repeated exposure of parental RINm cells to these two toxins, while the defense capacity, was estimated by the MTT colorimetric method, and [³H]-thymidine incorporation assay. We found that RINmS and RINmA displayed higher resistance to both streptozotocin (STZ) and alloxan (AL) when compared to the parental RINm cells. In contrast, no differences in sensitivity to hydrogen peroxide were found between toxin selected and parental cells. Partial protection from the toxic effect of STZ and AL was obtained only in the parental RINm cells after preincubation of cells with the unmetabolizable 3- O-methyl-glucose. The possibility that GLUT-2 is involved in cell sensitivity to toxins was confirmed by Western blot analysis, which showed higher expression of GLUT-2 in parental RINm compared to RINmS and RINmA cells. In addition to the higher cell defense property evidenced in the selected cells, we also found higher insulin content and insulin secretion in both RINmS and RINmA cells when compared to the parental RINm cells. In conclusion, STZ and AL treatment can be used for selection of cell sub-populations with higher cell defense properties and hormone production. The different GLUT-2 expression in parental and re sistant cells suggest involvement of GLUT-2 in mechanisms of cell response to different toxins.     

The contribution of adrenal and gonadal androgens to the growth in height of adolescent males

Gonadal androgens are known to regulate the rate of growth in height during adolescence, particularly in males, but little is known of the role of adrenal androgens in this process. In a prospective multiple regression model we show that both adrenal and gonadal androgens contribute to the increasing rate of growth in height prior to peak height velocity and the decreasing rate of height growth in later adolescence. Since adrenal androgen secretion begins in mid-childhood, long before gonadal maturation and the secretion of testicular androgens, these findings may prove helpful in explaining population variation in adolescent growth.     

A role for amino acids in the induction of deoxyribonucleic acid synthesis in liver

Nuclear DNA synthesis and mitosis are induced in the parenchymal liver cells of unoperated rats after a shift from a protein-free to a protein- or amino acid-containing diet. Protein or amino acids in the preparatory mash block the responses of the liver to the second diet. The formation of liver DNA after the nutritional shift begins several hours earlier than after partial hepatectomy. A small number of changes in the hepatic levels of amino acids and other ninhydrin-positive compounds result from the nutritional shift and some of these changes occur as well after 70% hepatectomy and the infusion of intact rats with the TAGH solution.