Apelin, a cytokine mainly secreted by adipocytes, is closely related with insulin resistance. The underlying molecular mechanisms of how apelin affects insulin resistance, however, are poorly understood. This study aimed to investigate the effect of apelin on glucose metabolism and insulin resistance in 3T3-L1 adipocytes. After 10 ng/ml TNF-α treatment for 24 h, insulin-stimulated glucose uptake was reduced by 47% in 3T3-L1 adipocytes. Apelin treatment improved glucose uptake in a time- and dose-dependent manner. Treatment of 1,000 nM apelin for 60 min maximally augmented glucose uptake in insulin-resistant 3T3-L1 adipocytes. Furthermore, apelin pre-incubation also increased adipocytes' insulin-stimulated glucose uptake, and PI3K/Akt pathway were involved in these effects. In addition, immunocytochemistry staining and western blotting analysis indicated that apelin could increase glucose transporter 4 translocation from the cytoplasm to the plasma membrane. Apelin also increased the anti-inflammatory adipokine adiponectin mRNA expression while reducing that of pro-inflammatory adipokine interleukin-6 in insulin-resistant 3T3-L1 adipocytes. These results suggest that apelin stimulates glucose uptake through the PI3K/Akt pathway, promotes GLUT4 translocation from the cytoplasm to the plasma membrane, and modulates inflammatory responses in insulin-resistant 3T3-L1 adipocytes.

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The peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending in d-alanyl-d-lactate and binds with high affinity to peptidoglycan ending in d-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a native d-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genus Lactobacillus. Finally, our system represents the first “plug and play” counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium.IMPORTANCE The genus Lactobacillus contains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selected Lactobacillus species. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)—an essential enzyme involved in peptidoglycan synthesis—increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance in Lactobacillus; 81% of Lactobacillus species are intrinsically resistant to vancomycin, which makes our tool broadly applicable.

Bacteriophages derived from lysogens are abundant in gut microbiomes. Currently, mechanistic knowledge is lacking on the ecological ramifications of prophage carriage yet is essential to explain the abundance of lysogens in the gut. An extensive screen of the bacterial gut symbiont Lactobacillus reuteri revealed that biologically active prophages are widely distributed in this species. L. reuteri 6475 produces phages throughout the mouse intestinal tract, but phage production is associated with reduced fitness of the lysogen. However, phage production provides a competitive advantage in direct competition with a nonlysogenic strain of L. reuteri that is sensitive to these phages. This combination of increased competition with a fitness trade-off provides a potential explanation for the domination of lysogens in gut ecosystem and how lysogens can coexist with sensitive hosts.