Enhanced Lipid Oxidation and Maintenance of Muscle Insulin Sensitivity Despite Glucose Intolerance in a Diet-Induced Obesity Mouse Model

Background

Diet-induced obesity is a rising health concern which can lead to the development of glucose intolerance and muscle insulin resistance and, ultimately, type II diabetes mellitus. This research investigates the associations between glucose intolerance or muscle insulin resistance and tissue specific changes during the progression of diet-induced obesity.

Methodology

C57BL/6J mice were fed a normal or high-fat diet (HFD; 60% kcal fat) for 3 or 8 weeks. Disease progression was monitored by measurements of body/tissue mass changes, glucose and insulin tolerance tests, and ex vivo glucose uptake in intact muscles. Lipid metabolism was analyzed using metabolic chambers and ex vivo palmitate assays in intact muscles. Skeletal muscle, liver and adipose tissues were analyzed for changes in inflammatory gene expression. Plasma was analyzed for insulin levels and inflammatory proteins. Histological techniques were used on muscle and liver cryosections to assess metabolic and morphological changes.

Principal Findings/Conclusions

A rapid shift in whole body metabolism towards lipids was observed with HFD. Following 3 weeks of HFD, elevated total lipid oxidation and an oxidative fiber type shift had occurred in the skeletal muscle, which we propose was responsible for delaying intramyocellular lipid accumulation and maintaining muscle’s insulin sensitivity. Glucose intolerance was present after three weeks of HFD and was associated with an enlarged adipose tissue depot, adipose tissue inflammation and excess hepatic lipids, but not hepatic inflammation. Furthermore, HFD did not significantly increase systemic or muscle inflammation after 3 or 8 weeks of HFD suggesting that early diet-induced obesity does not cause inflammation throughout the whole body. Overall these findings indicate skeletal muscle did not contribute to the development of HFD-induced impairments in whole-body glucose tolerance following 3 weeks of HFD.     

Sequences within Both the 5′ UTR and Gag Are Required for Optimal In Vivo Packaging and Propagation of Mouse Mammary Tumor Virus (MMTV) Genomic RNA

Background

This study mapped regions of genomic RNA (gRNA) important for packaging and propagation of mouse mammary tumor virus (MMTV). MMTV is a type B betaretrovirus which preassembles intracellularly, a phenomenon distinct from retroviruses that assemble the progeny virion at cell surface just before budding such as the type C human and feline immunodeficiency viruses (HIV and FIV). Studies of FIV and Mason-Pfizer monkey virus (MPMV), a type D betaretrovirus with similar intracellular virion assembly processes as MMTV, have shown that the 5′ untranslated region (5′ UTR) and 5′ end of gag constitute important packaging determinants for gRNA.

Methodology

Three series of MMTV transfer vectors containing incremental amounts of gag or 5′ UTR sequences, or incremental amounts of 5′ UTR in the presence of 400 nucleotides (nt) of gag were constructed to delineate the extent of 5′ sequences that may be involved in MMTV gRNA packaging. Real time PCR measured the packaging efficiency of these vector RNAs into MMTV particles generated by co-transfection of MMTV Gag/Pol, vesicular stomatitis virus envelope glycoprotein (VSV-G Env), and individual transfer vectors into human 293T cells. Transfer vector RNA propagation was monitored by measuring transduction of target HeLaT4 cells following infection with viral particles containing a hygromycin resistance gene expression cassette on the packaged RNA.

Principal Findings

MMTV requires the entire 5′ UTR and a minimum of ∼120 nucleotide (nt) at the 5′ end of gag for not only efficient gRNA packaging but also propagation of MMTV-based transfer vector RNAs. Vector RNAs without the entire 5′ UTR were defective for both efficient packaging and propagation into target cells.

Conclusions/Significance

These results reveal that the 5′ end of MMTV genome is critical for both gRNA packaging and propagation, unlike the recently delineated FIV and MPMV packaging determinants that have been shown to be of bipartite nature.     

Iron transport in the genus Marinobacter

Marinobacter belong to the class of Gammaproteobacteria and these motile, halophilic or halotolerent bacteria are widely distributed throughout the world’s oceans having been isolated from a wide variety of marine environments. They have also been identified as members of the bacterial flora associated with other marine organisms. Here, using a combination of natural products chemistry and genomic analysis, we assess the nature of the siderophores produced by this genus and their potential relationship to phylogeny and lifestyle/ecological niche of this diverse group of organisms. Our analysis shows a wide level of diversity in siderophore based iron uptake systems among this genus with three general strategies: (1) production and utilization of native siderophores in addition to utilization of a variety of exogenous ones, (2) production and utilization of native siderophores only, (3) lack of siderophore production but utilization of exogenous ones. They all share the presence of at least one siderophore-independent iron uptake ABC transport systems of the FbpABC iron metal type and lack the ability for direct transport of ferrous iron. Siderophore production and utilization can be correlated with phylogeny and thus it forms a type of chemotaxonomic marker for this genus.     

Impaired Macrophage and Satellite Cell Infiltration Occurs in a Muscle-Specific Fashion Following Injury in Diabetic Skeletal Muscle

Background

Systemic elevations in PAI-1 suppress the fibrinolytic pathway leading to poor collagen remodelling and delayed regeneration of tibialis anterior (TA) muscles in type-1 diabetic Akita mice. However, how impaired collagen remodelling was specifically attenuating regeneration in Akita mice remained unknown. Furthermore, given intrinsic differences between muscle groups, it was unclear if the reparative responses between muscle groups were different.

Principal Findings

Here we reveal that diabetic Akita muscles display differential regenerative responses with the TA and gastrocnemius muscles exhibiting reduced regenerating myofiber area compared to wild-type mice, while soleus muscles displayed no difference between animal groups following injury. Collagen levels in TA and gastrocnemius, but not soleus, were significantly increased post-injury versus controls. At 5 days post-injury, when degenerating/necrotic regions were present in both animal groups, Akita TA and gastrocnemius muscles displayed reduced macrophage and satellite cell infiltration and poor myofiber formation. By 10 days post-injury, necrotic regions were absent in wild-type TA but persisted in Akita TA. In contrast, Akita soleus exhibited no impairment in any of these measures compared to wild-type soleus. In an effort to define how impaired collagen turnover was attenuating regeneration in Akita TA, a PAI-1 inhibitor (PAI-039) was orally administered to Akita mice following cardiotoxin injury. PAI-039 administration promoted macrophage and satellite cell infiltration into necrotic areas of the TA and gastrocnemius. Importantly, soleus muscles exhibit the highest inducible expression of MMP-9 following injury, providing a mechanism for normative collagen degradation and injury recovery in this muscle despite systemically elevated PAI-1.

Conclusions

Our findings suggest the mechanism underlying how impaired collagen remodelling in type-1 diabetes results in delayed regeneration is an impairment in macrophage infiltration and satellite cell recruitment to degenerating areas; a phenomena that occurs differentially between muscle groups.     

Influence of Ficus carica and Olea europaea leaves extracts on the mycelial growth of mushrooms in vitro

The use of 20% plant leaves extracts included fig (Ficus carica) and olive (Olea europaea) and their mixture 1:1 as an amendment in the solid agar medium (PDA) is beneficial to promote the growth of four mycelial mushrooms. These are Pleurotus ostreatus (Grey oyster mushroom), Pleurotus cornucopiae (Yellow oyster mushroom), Coriolus versicolor (Turkey Tail mushroom), and Ganoderma lucidum (Reishi mushroom). C. versicolor showed better growth reached 67?mm significantly (p?<?0.05) on OC medium after five days. While, P. cornucopiae recorded the lowest growth on FC medium reached 35.3?mm. Induction percentage of mycelial growth is changing according to the type of medium and species of fungus. In general, FOH medium exhibited the best percentage of induction was 14.89%, followed 12.48% and 9.43% by OH and OC media, while the lower percentages were 5.02% and 5.12% on FH and FC media, respectively. FC medium did not induce growth of P. cornucopiae and C. versicolor. The sterilization by Autoclave and Millipore filter showed different induction percentages. Finally, the extracts of fig and olive were useful to add in the culture media to improve the growth of mycelial mushroom in vitro.     

Molecular Cloning and Genetic Analysis of Functional merB Gene from Indian Isolates of Escherichia coli

Studies were carried out to characterize organomercurial lyase genes from wild type mercury-resistant Escherichia coli isolates, previously collected from five geographically distinct regions of the Indian subcontinent. PCR amplification followed by DNA sequencing of amplified fragments showed three merB identical to the previously characterized mer B from E. coli pR831b that were thus considered as the same gene. The remaining two genes derived from E. coli isolates of an almost mercury-free site (Dal lake, Kashmir) and designated as pIAAD₃ merB and pIAAD₁₄ merB showed slight variation (2%) at base. However, this variation in pIAAD₃ due to the absence of base “T” at 479 position results in complete frame shift and the predicted MerB-like polypeptide derived from it showed 21.53% divergent at its C terminal end from the previously characterized pR831b MerB. The expression profile of pIAAD₃ merB in pQE30 and pUC18 vectors each demonstrated 22.2 kDa proteins. The induced DH5α E. coli cells possessing pIAAD₃ merB cloned in pUC18 vector split phenyl mercuric acetate (PMA) into benzene and inorganic mercury efficiently, thus giving a clue that the expressed gene product is biologically active. The current study suggests that such genetic changes may take place in the continued absence of mercury pressure, and with such modifications, they finally break down to act as vestigial remnants. Further work is going on in our lab to exploit pIAAD₃ merB for the bioremediation of mercury-polluted sites.