Adiponectin is an adipose tissue-specific protein that is abundantly present in the circulation and suggested to be involved in insulin sensitivity and development of atherosclerosis. Because cytokines are suggested to regulate adiponectin, the aim of the present study was to investigate the interaction between adiponectin and three adipose tissue-derived cytokines (IL-6, IL-8, and TNF-alpha). The study was divided into three substudies as follows: 1) plasma adiponectin and mRNA levels in adipose tissue biopsies from obese subjects [mean body mass index (BMI): 39.7 kg/m2, n = 6] before and after weight loss; 2) plasma adiponectin in obese men (mean BMI: 38.7 kg/m2, n = 19) compared with lean men (mean BMI: 23.4 kg/m2, n = 10) before and after weight loss; and 3) in vitro direct effects of IL-6, IL-8, and TNF-alpha on adiponectin mRNA levels in adipose tissue cultures. The results were that 1) weight loss resulted in a 51% (P < 0.05) increase in plasma adiponectin and a 45% (P < 0.05) increase in adipose tissue mRNA levels; 2) plasma adiponectin was 53% (P < 0.01) higher in lean compared with obese men, and plasma adiponectin was inversely correlated with adiposity, insulin sensitivity, and IL-6; and 3) TNF-alpha (P < 0.01) and IL-6 plus its soluble receptor (P < 0.05) decreased adiponectin mRNA levels in vitro. The inverse relationship between plasma adiponectin and cytokines in vivo and the cytokine-induced reduction in adiponectin mRNA in vitro suggests that endogenous cytokines may inhibit adiponectin. This could be of importance for the association between cytokines (e.g., IL-6) and insulin resistance and atherosclerosis.
Bacterial growth is crucially dependent on protein synthesis and thus on the cellular abundance of ribosomes and related proteins. Here, we show that the slow diffusion of the bulky tRNA complexes in the crowded cytoplasm imposes a physical limit on the speed of translation, which ultimately limits the rate of cell growth. To study the required allocation of ancillary translational proteins to alleviate the effect of molecular crowding, we develop a model for cell growth based on a coarse-grained partitioning of the proteome. We find that coregulation of ribosome-and tRNA-affiliated proteins is consistent with measured growth-rate dependencies and results in near-optimal allocation over a broad range of growth rates. The analysis further resolves a long-standing controversy in bacterial growth physiology concerning the growth-rate dependence of translation speed and serves as a caution against premature identification of phenomenological parameters with mechanistic processes. Bacterial cell growth and protein synthesis are tightly coupled as proteins account for a large fraction of the cellular biomass (1). In the model organism Escherichia coli, over half of the biomass is protein (2), and protein synthesis accounts for more than two-thirds of the cell's ATP budget during rapid growth (3). Therefore, the machinery of protein synthesis, i.e., ribosomes, tRNAs, and ribosome-affiliated factors, plays a central role in maintaining exponential growth (1, 4). This is manifested by an increased ribosome content in rapidly growing cells (2,5,6), by direct observations that protein synthesis is limited by the availability of free ribosomes (7), and by considerations that link evolutionary selective pressure to the cost of protein synthesis (8).The most striking evidence for the central role of ribosomes in cell growth is provided by the linear relation between the ribosome mass fraction and the growth rate for bacteria grown in media containing different nutrients. This linear relation, which emerged from the systematic characterization of bacterial cells growing at different rates (5, 9), is illustrated in Fig. 1A with data for E. coli (2, 10, 11). It can be interpreted as reflecting the intrinsically autocatalytic activity of ribosomes synthesizing ribosomal proteins (9, 12) and identifies the fraction of ribosomes allocated to making ribosomal proteins as a key determinant of the growth rate (11). The picture that emerges from such considerations has formed the basis of a systematic theory of bacterial growth, based on empirical "growth laws", similar to the phenomenological laws of physics (11,13). The theory provides a successful framework for the analysis of the interdependence of cell growth and gene expression, of the effects of antibiotics, and of protein overexpression (11) without the need to characterize how the individual steps of synthesis and degradation are affected by the global state of the cell (14).In addition to their high ribosome content, rapidly growing bacteria also contain large amounts of othe...
ClpB is thought to be involved in proteolysis because of its sequence similarity to the ClpA subunit of the ClpA-ClpP protease. It has recently been shown that ClpP is a heat shock protein. Here we show that ClpB is the Escherichia coli heat shock protein F84.1. The F84.1 protein was overproduced in strains containing the clpB gene on a plasmid and was absent from two-dimensional gels from a clpB null mutation. Besides possessing a slower growth rate at 44 degrees C, the null mutant strain had a higher rate of death at 50 degrees C. We used reverse transcription of in vivo mRNA to show that the clpB gene was expressed from a sigma 32-specific promoter consensus sequence at both 37 and 42 degrees C. We noted that the clpB+ gene also caused the appearance of a second protein spot, F68.5, on two-dimensional gels. This spot was approximately 147 amino acids smaller than F84.1 and most probably is the result of a second translational start on the clpB mRNA. F68.5 can be observed on many published two-dimensional gels of heat-induced E. coli proteins, but the original catalog of 17 heat shock proteins did not include this spot.
The amount of 140 individual proteins of E. coli B/r was measured during balanced growth in five different media. The abundance of each protein was determined from its absolute amount in 14C-glucose-minimal medium and a measurement of its relative amount at each growth rate using a double labeling technique. Separation of the proteins was carried out by two-dimensional gel electrophoresis. This catalog of proteins, combined with 50 additional ribosomal proteins already studied, comprises about 5% of the coding capacity of the genome, but accounts for two thirds of the cell's protein mass. The behavior of most of these proteins could be described by a relatively small number of patterns. 102 of the 140 proteins exhibited nearly linear variations with growth rate. The remaining 38 proteins exhibited levels which seemed to depend more on the chemical nature of the medium than on growth rate. Proteins, including the ribosomal proteins, that increase in amount with increasing growth rate account for 20% of total cell protein by weight during growth on acetate, 32% on glucose-minimal medium and 55% on glucose-rich medium. Proteins with invariant levels in the various media comprise about 4% of the cell's total protein.
Individual mRNAs are translated by multiple ribosomes that initiate translation with an interval of a few seconds. The ribosome speed is codon dependent, and ribosome queuing has been suggested to explain specific data for translation of some mRNAs in vivo. By modeling the stochastic translation process as a traffic problem, we here analyze conditions and consequences of collisions and queuing. The model allowed us to determine the on-rate (0.8 to 1.1 initiations/s) and the time (1 s) the preceding ribosome occludes initiation for Escherichia coli lacZ mRNA in vivo. We find that ribosome collisions and queues are inevitable consequences of a stochastic translation mechanism that reduce the translation efficiency substantially on natural mRNAs. The cells minimize collisions by having its mRNAs being unstable and by a highly selected codon usage in the start of the mRNA. The cost of mRNA breakdown is offset by the concomitant increase in translation efficiency.
The insulin resistance syndrome is characterized by several risk factors for cardiovascular disease. Chronic chemical activation of AMP-activated protein kinase by the adenosine analog 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside (AICAR) has been shown to augment insulin action, upregulate mitochondrial enzymes in skeletal muscles, and decrease the content of intraabdominal fat. Furthermore, acute AICAR exposure has been found to reduce sterol and fatty acid synthesis in rat hepatocytes incubated in vitro as well as suppress endogenous glucose production in rats under euglycemic clamp conditions. To investigate whether chronic AICAR administration, in addition to the beneficial effects on insulin sensitivity, is capable of improving other phenotypes associated with the insulin resistance syndrome, obese Zucker (fa/fa) rats (n ؍ 6) exhibiting insulin resistance, hyperlipidemia, and hypertension were subcutaneously injected with AICAR (0.5 mg/g body wt) daily for 7 weeks. Obese control rats were either pair-fed (PF) (n ؍ 6) or ad libitum-fed (AL) (n ؍ 6). Lean Zucker rats (fa/؊) (n ؍ 8) served as a reference group. AICAR administration significantly reduced plasma triglyceride levels (P < 0.01 for AICAR vs. AL, and P ؍ 0.05 for AICAR vs. PF) and free fatty acids (P < 0.01 for AICAR vs. AL, and P < 0.05 for AICAR vs. PF) and increased HDL cholesterol levels (P < 0.01 for AICAR vs. AL and PF). AICAR treatment also lowered systolic blood pressure by 14.6 ؎ 4.3 mmHg (P < 0.05), and AICAR-treated animals exhibited a tendency toward decreased intra-abdominal fat content. Furthermore, AICAR administration normalized the oral glucose tolerance test and decreased fasting concentrations of glucose and insulin close to the level of the lean animals. Finally, in line with previous findings, AICAR treatment was also found to enhance GLUT4 protein expression and to increase maximally insulin-stimulated glucose transport in primarily white fast-twitch muscles. Our data provide strong evidence that longterm administration of AICAR improves glucose tolerance, improves the lipid profile, and reduces systolic blood pressure in an insulin-resistant animal model. The present study gives additional support to the hypothesis that AMPK activation might be a potential future pharmacological strategy for treating the insulin resistance syndrome.
The question of whether or not ‘rare’ codons are translated with the same rate as ‘common’ codons was investigated by measuring the translation time for two genes, lacI and bla, rich in rare codons, and comparing the results with the translation times measured on fus, tsf, tuf and rpsA which have very few rare codons. The rate of synthesis of the lac repressor was first measured with the up‐promoter mutation lacIq1 present on the high copy number plasmid pBR322. In such a strain the average translation times for lacI and bla were 50% slower than the rate calculated from the translation time for the four ribosomal proteins. In a strain having lacIq1 on an F'lac episome this difference was much smaller, thus slow translation of genes rich in rare codons is exaggerated in strains with increased drain on the rare codon tRNAs. The data do not exclude that only a subset of the rare codons is translated more slowly. Translation times were also measured in cells growing in different media, and the translation chain growth rate was found to increase by approximately 40% going from acetate medium to a fully supplemented medium.
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