Serum metabolome analysis reveals medicinal fungi Phellinus igniarius ameliorated type 2 diabetes mellitus indications in rats via modulation of amino acid and carbohydrate metabolism.

Medicinal fungi Phellinus igniarius exhibited hypoglycemic effects; however, the protective mechanisms of P. igniarius on type 2 diabetes are not yet fully understood. Herein, the anti-diabetic effect of P. igniarius was investigated via gas chromatography-mass spectrometry (GC/MS)-based metabolome analysis. The rats were divided into normal group; model group; positive group; and groups treated with low, medium, and high dose of P. igniarius. After the treatments, a significant decrease in blood glucose concentration was observed. The levels of total cholesterol and triglyceride were dramatically decreased, whereas the level of insulin was increased. Multivariate statistical analysis revealed 31 differential endogenous metabolites between model group and normal group. A total of 14, 28, and 31 biomarkers were identified for low, medium, and high dose of P. igniarius treated groups, respectively. Twenty-one of the biomarkers were validated by using standard substances. The linear correlation coefficients ranged from 0.9990 to 1.0000. The methodology exhibited good repeatability, recoveries, and stability. The major intervened metabolic pathways covered glyoxylate and dicarboxylic acid metabolism; alanine, aspartate, and glutamate metabolism; and glycine, serine, and threonine metabolism. Our metabolome analysis has provided insights into the underlying mechanism of P. igniarius on type 2 diabetes.

The Role of Calmodulin vs. Synaptotagmin in Exocytosis.

Exocytosis is a Ca2+-regulated process that requires the participation of Ca2+ sensors. In the 1980s, two classes of Ca2+-binding proteins were proposed as putative Ca2+ sensors: EF-hand protein calmodulin, and the C2 domain protein synaptotagmin. In the next few decades, numerous studies determined that in the final stage of membrane fusion triggered by a micromolar boost in the level of Ca2+, the low affinity Ca2+-binding protein synaptotagmin, especially synaptotagmin 1 and 2, acts as the primary Ca2+ sensor, whereas calmodulin is unlikely to be functional due to its high Ca2+ affinity. However, in the meantime emerging evidence has revealed that calmodulin is involved in the earlier exocytotic steps prior to fusion, such as vesicle trafficking, docking and priming by acting as a high affinity Ca2+ sensor activated at submicromolar level of Ca2+. Calmodulin directly interacts with multiple regulatory proteins involved in the regulation of exocytosis, including VAMP, myosin V, Munc13, synapsin, GAP43 and Rab3, and switches on key kinases, such as type II Ca2+/calmodulin-dependent protein kinase, to phosphorylate a series of exocytosis regulators, including syntaxin, synapsin, RIM and Ca2+ channels. Moreover, calmodulin interacts with synaptotagmin through either direct binding or indirect phosphorylation. In summary, calmodulin and synaptotagmin are Ca2+ sensors that play complementary roles throughout the process of exocytosis. In this review, we discuss the complementary roles that calmodulin and synaptotagmin play as Ca2+ sensors during exocytosis.

The M domain in UNC-13 regulates the probability of neurotransmitter release.

Synapses exhibit multiple forms of short-term plasticities, which have been attributed to the heterogeneity of neurotransmitter release probability. However, the molecular mechanisms that underlie the differential release states remain to be fully elucidated. The Unc-13 proteins appear to have key roles in synaptic function through multiple regulatory domains. Here, we report that deleting the M domain in Caenorhabditis elegans UNC-13MR leads to a significant increase in release probability, revealing an inhibitory function of this domain. The inhibitory effect of this domain is eliminated when the C1 and C2B domains are absent or activated, suggesting that the M domain inhibits release probability by suppressing the activity of C1 and C2B domains. When fused directly to the MUNC2C fragment of UNC-13, the M domain greatly enhances release probability. Thus, our findings reveal a mechanism by which the UNC-13 M domain regulates synaptic transmission and provides molecular insights into the regulation of release probability.

A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release.

Ca2+-dependent neurotransmitter release requires synaptotagmins as Ca2+ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains-C2A and C2B-to Ca2+. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca2+ sensor in Caenorhabditis elegans. Here, we report a new Ca2+ sensor, SNT-3, which triggers delayed Ca2+-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca2+ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca2+ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca2+ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini-mediated membrane tethering. Our findings therefore reveal a novel dual Ca2+ sensor system in C. elegans and provide significant insights into Ca2+-regulated exocytosis.