Micro-RNAs (miRNAs) have been suggested to play pivotal roles in multifarious diseases associated with the posttranscriptional regulation of protein-coding genes. In this study, we aimed to investigate the function of miRNAs in type 2 diabetes mellitus. The miRNAs expression profiles were examined by miRNA microarray analysis of skeletal muscles from healthy and Goto-Kakizaki rats. We identified four up-regulated miRNAs, and 11 miRNAs that were down-regulated relative to normal individuals. Among induced miRNAs were three paralogs of miR-29, miR-29a, miR-29b, and miR-29c. Northern blotting further confirmed their elevated expression in three important target tissues of insulin action: muscle, fat, and liver of diabetic rats. Adenovirus-mediated overexpression of miR-29a/b/c in 3T3-L1 adipocytes could largely repress insulin-stimulated glucose uptake, presumably through inhibiting Akt activation. The increase in miR-29 level caused insulin resistance, similar to that of incubation with high glucose and insulin in combination, which, in turn, induced miR-29a and miR-29b expression. In this paper, we demonstrate that Akt is not the direct target gene of miR-29 and that the negative effects of miR-29 on insulin signaling might be mediated by other unknown intermediates. Taken together, these data reveal the crucial role of miR-29 in type 2 diabetes.

Toxoplasma gondii , as an obligate intracellular and promiscuous pathogen of mammalian cells, utilizes host sugars for energy and to generate glycoconjugates that are important to its survival and virulence. Here, we report that T. gondii glucose transporter (TgGT1) is proficient in transporting mannose, galactose, and fructose besides glucose, and serves as a major hexose transporter at its plasma membrane. Toxoplasma harbors 3 additional putative sugar transporters (TgST1–3), of which TgST2 is expressed at its surface, whereas TgST1 and TgST3 are intracellular. Surprisingly, TgGT1 and TgST2 are nonessential to the parasite as their ablations inflict only a 30% or no defect in its intracellular growth, respectively. Indeed, Toxoplasma can also tolerate the deletion of both genes while incurring no further growth phenotype. Unlike Δ tgst2 , the modest impairment in Δ tggt1 and Δ tggt1 /Δ tgst2 mutants is because of a minor delay in their intracellular replication, which is a direct consequence of the abolished import of glucose. The Δ tggt1 displays an attenuated motility in defined minimal media that is rescued by glutamine. TgGT1-complemented parasites show an entirely restored growth, motility, and sugar import. The lack of exogenous glucose in Δ tggt1 culture fails to accentuate its intrinsic growth defect and prompts it to procure glutamine to sustain its metabolism. Unexpectedly, in vivo virulence of Δ tggt1 in mice remains unaffected. Taken together, our data demonstrate that glucose is nonessential for T. gondii tachyzoites, underscore glutamine is a complement substrate, and provide a basis for understanding the adaptation of T. gondii to diverse host cells.

Toxoplasma gondii is an intracellular protozoan parasite capable of causing devastating infections in immunocompromised and immunologically immature individuals. In this report, we demonstrate the relative independence of T. gondii from its host cell for aminoglycerophospholipid synthesis. The parasite can acquire the lipid precursors serine, ethanolamine, and choline from its environment and use them for the synthesis of its major lipids, phosphatidylserine (PtdSer), phosphatidylethanolamine (PtdEtn), and phosphatidylcholine (PtdCho), respectively. Dimethylethanolamine (Etn(Me)(2)), a choline analog, dramatically interfered with the PtdCho metabolism of T. gondii and caused a marked inhibition of its growth within human foreskin fibroblasts. In tissue culture medium supplemented with 2 mm Etn(Me)(2), the parasite-induced lysis of the host cells was dramatically attenuated, and the production of parasites was inhibited by more than 99%. The disruption of parasite growth was paralleled by structural abnormalities in its membranes. In contrast, no negative effect on host cell growth and morphology was observed. The data also reveal that the Etn(Me)(2)-supplemented parasite had a time-dependent decrease in its PtdCho content and an equivalent increase in phosphatidyldimethylethanolamine, whereas other major lipids, PtdSer, PtdEtn, and PtdIns, remained largely unchanged. Relative to host cells, the parasites incorporated more than 7 times as much Etn(Me)(2) into their phospholipid. These findings reveal that Etn(Me)(2) selectively alters parasite lipid metabolism and demonstrate how selective inhibition of PtdCho synthesis is a powerful approach to arresting parasite growth.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects warm-blooded vertebrates, including humans. Asexual reproduction in T. gondii allows it to switch between the rapidly replicating tachyzoite and quiescent bradyzoite life cycle stages. A transient cyclic AMP (cAMP) pulse promotes bradyzoite differentiation, whereas a prolonged elevation of cAMP inhibits this process. We investigated the mechanism(s) by which differential modulation of cAMP exerts a bidirectional effect on parasite differentiation. There are three protein kinase A (PKA) catalytic subunits ( Tg PKAc1 to -3) expressed in T. gondii . Unlike Tg PKAc1 and Tg PKAc2, which are conserved in the phylum Apicomplexa, Tg PKAc3 appears evolutionarily divergent and specific to coccidian parasites. Tg PKAc1 and Tg PKAc2 are distributed in the cytomembranes, whereas Tg PKAc3 resides in the cytosol. Tg PKAc3 was genetically ablated in a type II cyst-forming strain of T. gondii (Pru Δku80Δhxgprt ) and in a type I strain (RH Δku80Δhxgprt ), which typically does not form cysts. The Δ pkac3 mutant exhibited slower growth than the parental and complemented strains, which correlated with a higher basal rate of tachyzoite-to-bradyzoite differentiation. 3-Isobutyl-1-methylxanthine (IBMX) treatment, which elevates cAMP levels, maintained wild-type parasites as tachyzoites under bradyzoite induction culture conditions (pH 8.2/low CO ₂ ), whereas the Δ pkac3 mutant failed to respond to the treatment. This suggests that Tg PKAc3 is the factor responsible for the cAMP-dependent tachyzoite maintenance. In addition, the Δ pkac3 mutant had a defect in the production of brain cysts in vivo , suggesting that a substrate of Tg PKAc3 is probably involved in the persistence of this parasite in the intermediate host animals. IMPORTANCE Toxoplasma gondii is one of the most prevalent eukaryotic parasites in mammals, including humans. Parasites can switch from rapidly replicating tachyzoites responsible for acute infection to slowly replicating bradyzoites that persist as a latent infection. Previous studies have demonstrated that T. gondii cAMP signaling can induce or suppress bradyzoite differentiation, depending on the strength and duration of cAMP signal. Here, we report that Tg PKAc3 is responsible for cAMP-dependent tachyzoite maintenance while suppressing differentiation into bradyzoites, revealing one mechanism underlying how this parasite transduces cAMP signals during differentiation.