Effects of dietary carbohydrate on the development of obesity in heterozygous Zucker rats

Rats carrying one copy of the fa allele are predisposed to diet-induced metabolic disturbances which contribute to hyperinsulinemia, obesity and dyslipidemia. To investigate the role of dietary carbohydrate and fat in the development of these conditions, we fed 6-week old male heterozygous (fa/+) lean rats carbohydrate-free diets containing primarily saturated fat either ad libitum or pair-fed. These diets were compared to standard chow and to a high saturated fat mixed diet containing 10% energy from sucrose for 4 weeks. The carbohydrate-free diet resulted in significantly lower circulating glucose levels compared to all other groups (p = 0.006). Weight gain was negligible in the carbohydrate free groups compared to standard diet and 10% sucrose diet (p = 0.03). This was reflected in energy efficiency which was markedly reduced (90%) in the carbohydrate-free groups compared to the other groups (p = 0.04). Corresponding changes were noted in fat pad mass. The subscapular and epididymal fat pads were increased 42% and 44%, respectively, in animals consuming the 10% sucrose diet compared to all other groups (p < 0.01). Comparable changes in fatty acid synthase (FAS) mRNA were observed in response to the carbohydrate-free diet, which resulted in a 53% decrease in adipocyte FAS mRNA (p < 0.001). Addition of 10% sucrose to the diet completely reversed this effect resulting in a 69% increase in adipocyte FAS mRNA compared to the carbohydrate-free groups (p = 0.01). Similarly, hepatic FAS mRNA was elevated by 51% and 66% in the 10% sucrose and standard diet groups respectively, compared to the carbohydrate-free groups. Therefore, diets that contain minimal carbohydrate may minimize net lipid storage and adiposity.     

Selection of side-chain carbons in a high-molecular-weight, hydrophobic peptide using solid-state spectral editing methods

Solid-state spectral editing techniques have been used by others to simplify ¹³C CPMAS spectra of small organic molecules, synthetic organic polymers, and coals. One approach utilizes experiments such as cross-polarization-with-polarization-inversion and cross-polarization-with-depolarization to generate subspectra. This work shows that this particular methodology is also applicable to natural-abundance ¹³C CPMAS NMR studies of high-molecular-weight biopolymers. The editing experiments are demonstrated first with model peptides and then with -elastin, a high-molecular-weight peptidyl preparation obtained from the elastic fibers in mammalian tissue. The latter has a predominance of small, nonpolar residues, which is evident in the crowded aliphatic region of typical ¹³C CPMAS spectra. Spectral editing is particularly useful for simplifying the aliphatic region of the NMR spectrum of this elastin preparation.     

β-Adrenergic receptor population is up-regulated by increased cyclic adenosine monophosphate concentration in chicken skeletal muscle cells in culture

Summary Skeletal muscle hypertrophy is promoted in vivo by administration of β-adrenergic receptor (βAR) agonists. Chicken skeletal muscle cells were treated with 1 μM isoproterenol, a strong βAR agonist, between days 7 and 10 in culture. βAR population increased by approximately 40% during this treatment; however, the ability of the cells to synthesize cyclic adenosine monophosphate (cAMP) was diminished by twofold. Neither the basal concentration of cAMP nor the quantity of myosin heavy chain (MHC) was affected by the 3-d exposure to isoproterenol. To understand further the relationship between intracellular cAMP levels, βAR population, and muscle protein accumulation, intracellular cAMP levels were artificially elevated by treatment with 0–10 μM forskolin for 3 d. The basal concentration of cAMP in forskolintreated cells increased up to sevenfold in a dose-dependent manner. Increasing concentrations of forskolin also led to an increase in βAR population, with a maximum increase of approximately 40–60% at 10 μM forskolin. A maximum increase of 40–50% in the quantity of MHC was observed at 0.2 μM forskolin, but higher concentrations of forskolin reduced the quanity of MHC back to control levels. At 0.2 μM forskolin, intracellular levels of cAMP were higher by approximately 35%, and the βAR population was higher by approximately 30%. Neither the number of muscle nuclei fused into myotubes nor the percentage of nuclei in myotubes was affected by forskolin at any of the concentrations studied.     

Computer-Aided Resolution of an Experimental Paradox in Bacterial Chemotaxis

Escherichia coli responds to its environment by means of a network of intracellular reactions which process signals from membrane-bound receptors and relay them to the flagellar motors. Although characterization of the reactions in the chemotaxis signaling pathway is sufficiently complete to construct computer simulations that predict the phenotypes of mutant strains with a high degree of accuracy, two previous experimental investigations of the activity remaining upon genetic deletion of multiple signaling components yielded several contradictory results (M. P. Conley, A. J. Wolfe, D. F. Blair, and H. C. Berg, J. Bacteriol. 171:5190–5193, 1989; J. D. Liu and J. S. Parkinson, Proc. Natl. Acad. Sci. USA 86:8703–8707, 1989). For example, “building up” the pathway by adding back CheA and CheY to a gutted strain lacking chemotaxis genes resulted in counterclockwise flagellar rotation whereas “breaking down” the pathway by deleting chemotaxis genes except cheA and cheY resulted in alternating episodes of clockwise and counterclockwise flagellar rotation. Our computer simulation predicts that trace amounts of CheZ expressed in the gutted strain could account for this difference. We tested this explanation experimentally by constructing a mutant containing a new deletion of the che genes that cannot express CheZ and verified that the behavior of strains built up from the new deletion does in fact conform to both the phenotypes observed for breakdown strains and computer-generated predictions. Our findings consolidate the present view of the chemotaxis signaling pathway and highlight the utility of molecularly based computer models in the analysis of complex biochemical networks.     

Simple high-performance liquid chromatography method for the simultaneous determination of serum caffeine and paraxanthine following rapid sample preparation

A simple reversed-phase high-performance liquid chromatography (HPLC) method for the simultaneous determination of caffeine and paraxanthine in human serum is described. Serum proteins are precipitated with perchloric acid and the resulting supernatant neutralized for direct injection onto an HPLC column. The method uses a phosphate–methanol mobile phase (85:15, v/v) at pH 4.9 with a flow-rate of 1.75 ml/min and quantitation is by UV absorbance at 274 nm. Elution times are approximately 18 min for caffeine and 8 min for paraxanthine. Theobromine and theophylline have elution times of 5.4 and 9.4 min and do not interfere in the assay. The intra-assay and between-assay means for precision and accuracy for both drugs are: 4.5% C.V. and 3.3% deviation. The sensitivity of the method is 50 ng/ml for each drug.