Functional differences in visceral and subcutaneous fat pads originate from differences in the adipose stem cell

Metabolic pathologies mainly originate from adipose tissue (AT) dysfunctions. AT differences are associated with fat-depot anatomic distribution in subcutaneous (SAT) and visceral omental (VAT) pads. We address the question whether the functional differences between the two compartments may be present early in the adipose stem cell (ASC) instead of being restricted to the mature adipocytes. Using a specific human ASC model, we evaluated proliferation/differentiation of ASC from abdominal SAT-(S-ASC) and VAT-(V-ASC) paired biopsies in parallel as well as the electrophysiological properties and functional activity of ASC and their in vitro-derived adipocytes. A dramatic difference in proliferation and adipogenic potential was observed between the two ASC populations, S-ASC having a growth rate and adipogenic potential significantly higher than V-ASC and giving rise to more functional and better organized adipocytes. To our knowledge, this is the first comprehensive electrophysiological analysis of ASC and derived-adipocytes, showing electrophysiological properties, such as membrane potential, capacitance and K(+)-current parameters which confirm the better functionality of S-ASC and their derived adipocytes. We document the greater ability of S-ASC-derived adipocytes to secrete adiponectin and their reduced susceptibility to lipolysis. These features may account for the metabolic differences observed between the SAT and VAT. Our findings suggest that VAT and SAT functional differences originate at the level of the adult ASC which maintains a memory of its fat pad of origin. Such stem cell differences may account for differential adipose depot susceptibility to the development of metabolic dysfunction and may represent a suitable target for specific therapeutic approaches.     

Effect of conjugated linoleic acid on body fat,tumor necrosis factor alpha and resistin secretion in spontaneously hypertensive rats

Conjugated linoleic acid (CLA) is a naturally occurring group of dienoic derivaties of linoleic acid found mainly in beef and dairy products. CLA has been reported to reduce body fat, as well as to possess anticarcinogenic, antiatherogenic and procatabolic activities in animals. The objective of this study was to evaluate the effect of CLA supplementation to spontaneously hypertensive rats (SHR) on body fat, biochemical parameters of serum related tumor necrosis factor alpha (TNF-α) and resistin secretion. Thirty rats were divided in three groups, the first group of spontaneously hypertensive rats received a standard diet (V-SHR group, n=10), a second group of SHR was fed 1.5% of conjugated linoleic acid (CLA-SHR group, n=10) and the third was the control, non-hypertensive group (KW, n=10) also on a standard diet including 7.5% of sunflower oil during eight weeks.After CLA diet administration, spontaneously hypertensive rats showed a significant reduction in blood pressure, serum glucose, cholesterol and triacylglycerols, together with reduction of index of body fat, pericardic, abdominal and epididymal adipose tissue. These effects were accompanied by a decrease in the secretion of TNF-α and resistin.     

Relation of leptin and tumor necrosis factor alpha to body weight changes in patients with pulmonary tuberculosis

In this study we investigated whether leptin and TNFalpha levels change with improvement in body weight with antituberculotic therapy in active tuberculosis patients. 30 patients (8 females and 22 males) with active pulmonary tuberculosis formed the patient group, and 25 sex- and age-matched healthy subjects (8 females and 17 males) served as the control group. Body weight, body mass index (BMI) and serum leptin and plasma TNFalpha levels are measured before and in the sixth month of therapy in all patients. Before the initiation of therapy, BMI of the patients was significantly lower than BMI of the controls (20.2 +/- 1.6 vs. 25.2 +/- 2.7 kg/m(2), respectively; p < 0.05). After treatment, BMI of the patients increased significantly to 21.4 +/- 1.9 kg/m(2) (p < 0.05), but was still lower than that of the controls (p < 0.05). Pretreatment serum leptin (4.5 +/- 0.9 vs. 2.1 +/- 0.2 ng/ml, respectively; p < 0.05) and plasma TNFalpha (27.9 +/- 3.4 vs. 23.9 +/- 3.0 pg/ml, respectively; p < 0.05) levels of the patients were significantly higher than those of the controls. After treatment, serum leptin levels increased to 6.7 +/- 2.2 ng/ml, but this rise was not statistically significant (p > 0.05). Treatment did not result in any significant change in TNFalpha levels, either. Delta leptin was highly related to Delta BMI in patients with tuberculosis (r = 0.68, p = 0.02). In the pretreatment period, there was a significant correlation between leptin and TNFalpha levels in the whole patient group (r = 0.78, p < 0.001), and in female (r = 0.74, p < 0.001) and male patients separately (r = 0.74, p = 0.035). In conclusion, leptin and TNFalpha may be responsible for the weight loss in pulmonary tuberculosis patients, but their levels do not change with improvement in body weight with antituberculotic treatment.     

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.     

Structure of human tumor necrosis factor alpha derived from recombinant DNA

Recombinant DNA derived tumor necrosis factor alpha, when expressed at a high level in Escherichia coli, appeared in the pellet and soluble fractions of disrupted cells. The protein was purified from the pellet fraction by solubilizing it in urea and reducing agent and was refolded into a buffer without these additives. The structure of the protein was identical with that purified from the soluble fraction without exposure to both reducing and denaturing agents, as demonstrated by circular dichroism, gel filtration, and sulfhydryl titration. As a reflection of the structural similarity, both purified proteins showed identical cytolytic activity on mouse L929 cells. The protein was characterized as an essentially nonhelical and beta-sheet-rich structure and possibly as a noncovalently associating oligomer. Two cysteine residues form an intrapolypeptide disulfide bond.     

Ethnic and sex differences in visceral,subcutaneous, and total body fat in children and adolescents

Objective: This study investigated ethnic and sex differences in the distribution of fat during childhood and adolescence. Design and Methods : A cross‐sectional sample (n = 382), aged 5–18 years, included African American males (n = 84), White males (n = 96), African American females (n = 118), and White females (n = 84). Measures for total body fat (TBF) mass and abdominal adipose tissue (total volume and L4‐L5 cross‐sectional area) for both subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) depots were assessed by dual‐energy X‐ray absorptiometry and magnetic resonance image, respectively. Analyses of covariance (ANCOVAs) were used to determine ethnic and sex differences in TBF (adjusted for age) and ethnic and sex differences in SAT and VAT (adjusted for both age and TBF). Results: Age‐adjusted TBF was greater in African Americans (P = 0.017) and females (P < 0.0001) compared with Whites and males, respectively. In age‐ and TBF‐adjusted ANCOVAs, no differences were found in the SAT. The VAT volume was, however, greater in Whites (P < 0.0001) and males (P < 0.0001) compared with African Americans and females, respectively. Similar patterns were observed in SAT and VAT area at L4‐L5. Conclusions: The demonstrated ethnic and sex differences are important confounders in the prevalence of obesity and in the assignment of disease risk in children and adolescents.     

Stable Escherichia coli-Clostridium acetobutylicum shuttle vector for secretion of murine tumor necrosis factor alpha.

Recombinant plasmids were constructed to secrete mouse tumor necrosis factor alpha (mTNF-alpha) from Clostridium acetobutylicum. The shuttle plasmids contained the clostridial endo-beta1, 4-glucanase (eglA) promoter and signal sequence that was fused in frame to the mTNF-alpha cDNA. The construction was first tested in Escherichia coli and then introduced in C. acetobutylicum DSM792 by electroporation. Controls confirmed the presence and stability of the recombinant plasmids in this organism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an in vitro cytotoxic assay were used to monitor expression and secretion of mTNF-alpha during growth. Significant levels of biologically active mTNF-alpha were measured in both lysates and supernatants. The present report deals with investigations on the elaboration of a gene transfer system for cancer treatment using anaerobic bacteria.     

Novel muteins of human tumor necrosis factor alpha

For chemical synthesis of a gene coding for human tumor necrosis factor alpha (TNF-alpha), DNA sequence predicted by the amino acid sequence of human TNF molecule was prepared. Codons were chosen according to the codon usage in Escherichia coli (E. coli). The 490 bp gene was assembled by enzymic ligation of 42 oligonucleotides and was cloned into a vector (pKK223-3) for high expression of active TNF-alpha in E. coli. With use of site-directed mutagenesis on this DNA, five different muteins of TNF-alpha were synthesized. TNF-M1 and TNF-M4 have deletions of His-73 and Gln-102, respectively. These deletions didn't cause loss of the cytotoxic activity against L929 cells. TNF-M5, which has a substitution of Asp-10 to Arg, had the similar cytotoxic activity to that of TNF-alpha. The cytotoxic spectra against several tumor cells were not changed by this substitution. TNF-M3 has an amino acid substitution of Glu-116 to His which occupies this position in human TNF-beta. This substitution didn't change the cytotoxicity. In addition, evidence was presented that the change of the carboxyl terminal residue doesn't always influence the cytotoxic activity of TNF-alpha. Many different muteins were also isolated by random mutagenesis with hydroxylamine-HCl. One of the muteins, which carries a mutation of His-15 to Tyr, lost the cytotoxic activity almost completely.     

Dual role of tumor necrosis factor alpha in brain injury

Brain injury (ischemia, trauma) is among the leading cause of mortality and disability in the western world. It induces increased production of tumor necrosis factor (TNF alpha) by brain resident cells. There is conflicting evidence on the role of this response in the injured brain, showing its potential effect in both processes of repair and of damage. This review presents data from clinical and experimental studies on the stimulation of TNF alpha production in brain injury and on the deleterious consequence of this acute response. Its inhibition by pharmacologic agents, neutralizing antibodies or soluble receptors has protective effects. In contrast, there are reports (from in-vitro studies or knock-out mice) on the beneficial effects of TNF alpha. To reconcile these apparently conflicting reports, the exact timing and extent of TNF alpha activation must be taken into account, as well as the presence of other mediators such as reactive oxygen species. It is suggested that the appropriate context of mediators, at any given time after brain injury may well determine whether the effect of TNF alpha is protective or toxic.     

Protein secretion biotechnology using Streptomyces lividans: large-scale production of functional trimeric tumor necrosis factor alpha.

We evaluated the feasibility of large-scale production of biopharmaceuticals expressed as heterologous polypeptides from the Gram-positive bacterium Streptomyces lividans. As a model protein we used murine tumor necrosis factor alpha (mTNFalpha). mTNFalpha fused C-terminally to the secretory signal peptide of the subtilisin-inhibitor protein from Streptomyces venezuelae. Under appropriate fermentation conditions, significant amounts of mature mTNFalpha (80-120 mg/L) can be recovered from spent growth media. Efficient downstream processing allowing rapid purification of mTNFalpha from culture supernatants was developed. Importantly, the protein is recovered from the spent growth medium in its native trimeric state as judged by biophysical analysis. Further, mTNFalpha secreted by S. lividans is significantly more active in an in vitro apoptosis tissue culture assay than a corresponding polypeptide produced in Escherichia coli. This pilot study provides the first validation of S. lividans protein secretion as an alternative bioprocess for large-scale production of oligomeric proteins of potential therapeutic value.     

Canarypox virus-induced maturation of dendritic cells is mediated by apoptotic cell death and tumor necrosis factor alpha secretion

Recombinant avipox viruses are being widely evaluated as vaccines. To address how these viruses, which replicate poorly in mammalian cells, might be immunogenic, we studied how canarypox virus (ALVAC) interacts with primate antigen-presenting dendritic cells (DCs). When human and rhesus macaque monocyte-derived DCs were exposed to recombinant ALVAC, immature DCs were most susceptible to infection. However, many of the infected cells underwent apoptotic cell death, and dying infected cells were engulfed by uninfected DCs. Furthermore, a subset of DCs matured in the ALVAC-exposed DC cultures. DC maturation coincided with tumor necrosis factor alpha (TNF-alpha) secretion and was significantly blocked in the presence of anti-TNF-alpha antibodies. Interestingly, inhibition of apoptosis with a caspase 3 inhibitor also reduced some of the maturation induced by exposure to ALVAC. This indicates that both TNF-alpha and the presence of primarily apoptotic cells contributed to DC maturation. Therefore, infection of immature primate DCs with ALVAC results in apoptotic death of infected cells, which can be internalized by noninfected DCs driving DC maturation in the presence of the TNF-alpha secreted concomitantly by exposed cells. This suggests an important mechanism that may influence the immunogenicity of avipox virus vectors.