Gheorghe Stoian

Exocrine cell-derived microparticles in response to lipopolysaccharide promote endocrine dysfunction in cystic fibrosis.

BACKGROUND Diabetes in cystic fibrosis (CF) is a result of exocrine pancreas alteration followed by endocrine dysfunction at a later stage. Microparticles (MPs) are plasma membrane fragments shed from stimulated or damaged cells that act as cellular effectors. Our aim was to identify a new form of interaction between exocrine and endocrine pancreatic cells mediated by exocrine MPs, in the context of recurrent infection in CF. METHODS MPs from either human exocrine CFTRΔF508-mutated (CFPAC-1) cells or exocrine normal pancreatic (PANC-1) cells were collected after treatment by LPS from Pseudomonas aeruginosa and applied to rat endocrine normal insulin-secreting RIN-m5F cells. MP membrane integration in target cells was established by confocal microscopy and flow cytometry using PKH26 lipid probe. Apoptosis, lysosomal activity, insulin secretion were measured after 18 h. MP-mediated NF-κB activation was measured in HEK-Blue reporter cells by SEAP reporter gene system and in RIN-m5F cells by Western blot. In endocrine normal cells, CFTR inhibition was achieved using Inhibitor-172. RESULTS Compared to PANC-1, MPs from CFPAC-1 significantly reduced insulin secretion and lysosomal activity in RIN-m5F. MPs induced NF-κB activation by increasing the level of IκB phosphorylation. Moreover, the inhibition of NF-κB activation using specific inhibitors was associated with a restored insulin secretion. Interestingly, CFTR inhibition in normal RIN-m5F cells promoted apoptosis and decreased insulin secretion. CONCLUSIONS During recurrent infections associated with CF, exocrine MPs may contribute to endocrine cell dysfunction via NF-κB pathways. Membrane CFTR dysfunction is associated with decreased insulin secretion.

Spin-spin relaxation times in myocardial hypertrophy induced by endocrine agents in rat

Magnetic resonance techniques afford a significant advantage for noninvasive diagnosis of cardiovascular pathology. The purpose of our present study was to assay the proton nuclear magnetic resonance (1H-NMR) sensitivity in the differential diagnosis of certain endocrine cardiovascular complications. In this context, we investigated the water state and content in the hypertrophied myocardium. Male and female Wistar rats were treated with different hormones (hydrocortisone acetate, testosterone, estradiol, thyroid hormones) in combination with isoproterenol (a synthetic catecholamine that induces myocardial ischemia and hypertrophy). The animals were sacrificed after 20 days of treatment and samples of integral myocardium and left ventricular myocardium were analyzed on a1H-NMR AREMI spectrometer (0.6 T; proton resonance at 25 MHz). The estimation ofT2 was made by Carr-Purcell-Meiboom-Gill pulse sequence. The data were fitted to a bi-exponential curve, yielding short (T21) values forbound water and long (T22) values forfree water. In order to evaluate the myocardial hypertrophy, the following ratios were calculated: integral myocardium to body weight; left ventricle to body weight; left ventricle to integral myocardium. The first two ratios were also calculated for dried tissue, in order to estimate its contribution to myocardial hypertrophy. Our findings demonstrate that myocardial hypertrophy is associated with a decrease ofT22, as a consequence of the increase in the dried component (i.e. proteins) of the tissue, while the total tissue, while the total tissue water (H2Ot%, measured by gravimetry) was not significantly modified. Nevertheless, it is reasonable that the increase in the protein content would be proportional with the increase in H2Ot%. The decrease ofT21 seems to be proportional with the level of left ventricle hypertrophy in female groups. The1H-NMR measurements were much sensitive for the differential diagnosis of myocardial hypertrophy in the case of left ventricle.

β-nicotinamide mononucleotide (NMN) production in Escherichia coli

Diabetes is a chronic and progressive disease with continuously increasing prevalence, rising financial pressure on the worldwide healthcare systems. Recently, the insulin resistance, hallmark of type 2 diabetes, was cured in mice treated with NAD+ precursor β-nicotinamide mononucleotide (NMN), no toxic effects being reported. However, NMN has a high price tag, more cost effective production methods are needed. This study proposes a biotechnological NMN production method in Escherichia coli. We show that bicistronic expression of recombinant nicotinamide phosphoribosyl transferase (Nampt) and phosphoribosyl pyrophosphate (PRPP) synthetase in the presence of nicotinamide (NAM) and lactose may be a successful strategy for cost effective NMN production. Protein expression vectors carrying NAMPT gene from Haemophilus ducreyi and PRPP synthetase from Bacillus amyloliquefaciens with L135I mutation were transformed in Escherichia coli BL21(DE3)pLysS. NMN production reached a maximum of 15.42 mg per L of bacterial culture (or 17.26 mg per gram of protein) in these cells grown in PYA8 medium supplemented with 0.1% NAM and 1% lactose.