Mechanisms underlying hypertriglyceridemia in rats with monosodium l-glutamate-induced obesity: Evidence of XBP-1/PDI/MTP axis activation

Non-alcoholic fatty liver disease (NAFLD) is intimately associated with insulin resistance and hypertriglyceridemia, whereas many of the mechanisms underlying this association are still poorly understood. In the present study, we investigated the relationship between microsomal triglyceride transfer protein (MTP) and markers of endoplasmic reticulum (ER) stress in the liver of rats subjected to neonatal monosodium l-glutamate (MSG)-induced obesity. At age 120 days old, the MSG-obese animals exhibited hyperglycemia, hypertriglyceridemia, insulin resistance, and liver steatosis, while the control (CTR) group did not. Analysis using fast protein liquid chromatography of the serum lipoproteins revealed that the triacylglycerol content of the very low-density lipoprotein (VLDL) particles was twice as high in the MSG animals compared with the CTR animals. The expression of ER stress markers, GRP76 and GRP94, was increased in the MSG rats, promoting a higher expression of X-box binding protein 1 (XBP-1), protein disulfide isomerase (PDI), and MTP. As the XBP-1/PDI/MTP axis has been suggested to represent a significant lipogenic mechanism in the liver response to ER stress, our data indicate that hypertriglyceridemia and liver steatosis occurring in the MSG rats are associated with increased MTP expression.     

Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies intracellularly generated cholest‐4‐en‐3‐one

The infectivity and persistence of Mycobacterium tuberculosis requires the utilization of host cell cholesterol. We have examined the specific role of cytochrome P450 CYP125A1 in the cholesterol degradation pathway using genetic, biochemical and high‐resolution mass spectrometric approaches. The analysis of lipid profiles from cells grown on cholesterol revealed that CYP125A1 is required to incorporate the cholesterol side‐chain carbon atoms into cellular lipids, as evidenced by an increase in the mass of the methyl‐branched phthiocerol dimycocerosates. We observed that cholesterol‐exposed cells lacking CYP125A1 accumulate cholest‐4‐en‐3‐one, suggesting that this is a physiological substrate for this enzyme. Reconstitution of enzymatic activity with spinach ferredoxin and ferredoxin reductase revealed that recombinant CYP125A1 indeed binds both cholest‐4‐en‐3‐one and cholesterol, efficiently hydroxylates both of them at C‐27, and then further oxidizes 27‐hydroxycholest‐4‐en‐3‐one to cholest‐4‐en‐3‐one‐27‐oic acid. We determined the X‐ray structure of cholest‐4‐en‐3‐one‐bound CYP125A1 at a resolution of 1.58 Å. CYP125A1 is essential for growth of CDC1551 in media containing cholesterol or cholest‐4‐en‐3‐one. In its absence, the latter compound is toxic for both CDC1551 and H37Rv when added with glycerol as a second carbon source. CYP125A1 is a key enzyme in cholesterol metabolism and plays a crucial role in circumventing the deleterious effect of cholest‐4‐en‐3‐one.     

Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy

Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Here we report that Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, we show a high conservation in Dg-Dys interaction between human and Drosophila. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, we now report that Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR.     

Deletion of a xenobiotic metabolizing gene in mice affects folate metabolism

The mouse arylamine N-acetyltransferase 2 (Nat2) and its homologue (NAT1) in humans are known to detoxify xenobiotic arylamines and are also thought to play a role in endogenous metabolism. Human NAT1 is highly over-expressed in estrogen receptor positive breast tumours and is implicated in susceptibility to neural tube defects. In vitro assays have suggested an endogenous role for human NAT1 in folate metabolism, but in vivo evidence to support this hypothesis has been lacking. Mouse Nat2 provides a good model to study human NAT1 as it shows similar expression profiles and substrate specificities. We have generated transgenic mice lacking a functional Nat2 gene and compared the urinary levels of acetylated folate metabolite para-aminobenzoylglutamate in Nat2 knockout and Nat2 wild-type mice. These results support an in vivo role for mouse Nat2/human NAT1 in folate metabolism. In addition, effects of the Nat2 deletion on sex ratios and neural tube development are described.     

Hyperglycemia and loss of ovarian hormones mediate atheroma formation through endothelial layer disruption and increased permeability

The overall goal of this project was to examine the interactions of hyperglycemia and loss of ovarian hormones on the artery wall in a type I diabetic mouse model. Intact or ovariectomized (OVX) female BALB/C mice were fed a high-cholesterol diet. Half the animals were treated with steptozotocin to induce insulin-deficient diabetes mellitus, generating four treatment groups: control, intact; control, ovariectomized; diabetic, intact; diabetic, ovariectomized (DOVX). We examined arterial structure and function and found that 1) diabetes and ovariectomy additively increased endothelial layer permeability, 2) arterial stiffening was increased in DOVX, 3) DOVX synergistically increased atheroma formation, and 4) ultrastructural evaluation revealed that the basal lamina was often multilayered and formed convoluted aggregates separating endothelium from the internal elastic lamina in diabetic, but not control arteries or arteries from OVX mice. Endothelium overlying these regions formed thin cytoplasmic extensions between these aggregates and was often separated from the basal lamina by electron lucent spaces. Our studies showed that diabetes and loss of ovarian function have additive and synergistic effects to worsen arterial pathophysiology by disrupting the arterial endothelial layer with increased permeability and increased atheroma formation.     

Lack of effect of dopaminergic antagonists in a rodent model of peritoneal sepsis

Central nervous system dopaminergic mechanisms have been implicated in the cytokine response to stress and sepsis. We here describe the effects of haloperidol or clozapine in the treatment of sepsis induced by cecal ligation and puncture. Male Wistar rats were subjected to the CLP procedure were treated with haloperidol or clozapine and plasma cytokines, myeloperoxidase activity, markers of organ injury and survival was analyzed. The addition of haloperidol or clozapine to basic support did not diminished hepatic, renal, pancreatic or muscular damage observed after sepsis. Neither haloperidol, nor clozapine, modulates pro and antiinflammatory cytokines after sepsis induction. In addition, haloperidol treatment did not diminished myeloperoxidase activity in the kidney, lung or liver, or altered BALF markers of lung damage or inflammatory infiltration. Our data did not support a role of haloperidol or clozapine as an immunomodulator agent in the treatment of sepsis in an animal model of peritonitis.     

From the deep sea to the stars: human life support through minimal communities

Support of human life during long-distance exploratory space travel or in the creation of human habitats in extreme environments can be accomplished using the action of microbial consortia inhabiting interconnected bioreactors, designed for the purpose of reconversion of solid, liquid and gaseous wastes produced by the human crew or by one of the compartments of the bioregenerative loop, into nutritional biomass, oxygen and potable water. The microorganisms responsible for bioregenerative life support are part of Earth's own geomicrobial reconversion cycle. Depending on the resources and conditions available, minimal life support systems can be assembled using appropriately selected microorganisms that possess metabolic routes for each specific purpose in the transformation cycle. Under control of an engineered system, a reliable life-support system can hence be provided for.