Aging is now at the forefront of major challenges faced globally, creating an immediate need for safe, widescale interventions to reduce the burden of chronic disease and extend human healthspan. Metformin and rapamycin are two FDA-approved mTOR inhibitors proposed for this purpose, exhibiting significant anti-cancer and anti-aging properties beyond their current clinical applications. However, each faces issues with approval for off-label, prophylactic use due to adverse effects. Here, we initiate an effort to identify nutraceuticals—safer, naturally-occurring compounds—that mimic the anti-aging effects of metformin and rapamycin without adverse effects. We applied several bioinformatic approaches and deep learning methods to the Library of Integrated Network-based Cellular Signatures (LINCS) dataset to map the gene- and pathway-level signatures of metformin and rapamycin and screen for matches among over 800 natural compounds. We then predicted the safety of each compound with an ensemble of deep neural network classifiers. The analysis revealed many novel candidate metformin and rapamycin mimetics, including allantoin and ginsenoside (metformin), epigallocatechin gallate and isoliquiritigenin (rapamycin), and withaferin A (both). Four relatively unexplored compounds also scored well with rapamycin. This work revealed promising candidates for future experimental validation while demonstrating the applications of powerful screening methods for this and similar endeavors.
The G protein b subunit Gb5 uniquely forms heterodimers with R7 family regulators of G protein signaling (RGS) proteins (RGS6, RGS7, RGS9, and RGS11) instead of Gg. Although the Gb5-RGS7 complex attenuates Ca 21 signaling mediated by the muscarinic M3 receptor (M3R), the route of Ca 21 entry (i.e., release from intracellular stores and/or influx across the plasma membrane) is unknown. Here, we show that, in addition to suppressing carbachol-stimulated Ca 21 release, Gb5-RGS7 enhanced Ca 21 influx. This novel effect of Gb5-RGS7 was blocked by nifedipine and 2-aminoethoxydiphenyl borate. Experiments with pertussis toxin, an RGS domain-deficient mutant of RGS7, and UBO-QIC {L-threonine,}, a novel inhibitor of Gq, showed that Gb5-RGS7 modulated a Gq-mediated pathway. These studies indicate that Gb5-RGS7, independent of RGS7 GTPase-accelerating protein activity, couples M3R to a nifedipine-sensitive Ca 21 channel. We also compared the action of Gb5-RGS7 on M3R-induced Ca 21 influx and release elicited by different muscarinic agonists. Responses to Oxo-M [oxotremorine methiodide N,N,N,-trimethyl-4-(2-oxo-1-pyrrolidinyl)-2-butyn-1-ammonium iodide] were insensitive to Gb5-RGS7. Pilocarpine responses consisted of a large release and modest influx components, of which the former was strongly inhibited whereas the latter was insensitive to Gb5-RGS7. McN-A-343 [(4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride] was the only compound whose total Ca 21 response was enhanced by Gb5-RGS7, attributed to, in part, by the relatively small Ca 21 release this partial agonist stimulated. Together, these results show that distinct agonists not only have differential M3R functional selectivity, but also confer specific sensitivity to the Gb5-RGS7 complex.
A gradual decline in insulin response is known to precede the onset of type 1 diabetes (T1D). To track age-related changes in the β-cell function of non-obese diabetic (NOD) mice, the most commonly used animal model for T1D, and to establish differences between those who do and do not become hyperglycemic, we performed a long-term longitudinal oral glucose tolerance test (OGTT) study (10–42 weeks) in combination with immunofluorescence imaging of islet morphology and cell proliferation. We observed a clear biphasic decline in insulin secretion (AUC0–30min) even in euglycemic animals. A first phase (10–28 weeks) consisted of a relatively rapid decline and paralleled diabetes development in the same cohort of animals. This was followed by a second phase (29–42 weeks) during which insulin secretion declined much slower while no additional animals became diabetic. Blood glucose profiles showed a corresponding, but less pronounced change: the area under the concentration curve (AUC0–150min) increased with age, and fit with a bilinear model indicated a rate-change in the trendline around 28 weeks. In control NOD scids, no such changes were observed. Islet morphology also changed with age as islets become surrounded by mononuclear infiltrates, and, in all mice, islets with immune cell infiltration around them showed increased β-cell proliferation. In conclusion, insulin secretion declines in a biphasic manner in all NOD mice. This trend, as well as increased β-cell proliferation, is present even in the NODs that never become diabetic, whereas, it is absent in control NOD scid mice.
The muscarinic M3 receptor (M3R) is a Gq-coupled receptor and is known to interact with many intracellular regulatory proteins. One of these molecules is Gβ5-RGS7, the permanently associated heterodimer of G protein β-subunit Gβ5 and RGS7, a regulator of G protein signaling. Gβ5-RGS7 can attenuate M3R-stimulated release of Ca2+ from intracellular stores or enhance the influx of Ca2+ across the plasma membrane. Here we show that deletion of amino acids 304–345 from the central portion of the i3 loop renders M3R insensitive to regulation by Gβ5-RGS7. In addition to the i3 loop, interaction of M3R with Gβ5-RGS7 requires helix 8. According to circular dichroism spectroscopy, the peptide corresponding to amino acids 548–567 in the C-terminus of M3R assumes an α-helical conformation. Substitution of Thr553 and Leu558 with Pro residues disrupts this α-helix and abolished binding to Gβ5-RGS7. Introduction of the double Pro substitution into full-length M3R (M3RTP/LP) prevents trafficking of the receptor to the cell surface. Using atropine or other antagonists as pharmacologic chaperones, we were able to increase the level of surface expression of the TP/LP mutant to levels comparable to that of wild-type M3R. However, M3R-stimulated calcium signaling is still severely compromised. These results show that the interaction of M3R with Gβ5-RGS7 requires helix 8 and the central portion of the i3 loop.
Background/objectives Type 2 diabetes (T2D) is a global pandemic, and contributes significantly to the increasing incidence of conditions such as cardiovascular disease (CVD). Postprandial plasma glucose measured 2-h after the start of a meal is a good indicator of the overall status of glucose homeostasis. Clove ( Syzygium aromaticum L. ) and its essential oils (eugenol and acetyl eugenol) have been shown in preclinical studies to modulate pathways involved in glucose homeostasis. In addition, a water-soluble polyphenolic extract of unopened clove buds was recently shown to benefit liver function and redox status. Therefore, we conducted an open-label pilot study to test whether this polyphenolic clove extract (PCE) could influence glucose metabolism. Methods We evaluated the effect of PCE supplementation (250 mg once daily for 30 days) on preprandial glucose levels and 2-h postprandial glucose levels in 13 otherwise healthy volunteers who were stratified into two groups according to their initial preprandial glucose levels: Group I ( n = 7) ≤100 mg/dL, Group II ( n = 6) – between 101 and 125 mg/dL. In an effort to elucidate the molecular mechanisms of PCE action, we tested in vitro the effects of PCE on glucose uptake, hepatocyte glucose production, and carbohydrate hydrolyzing enzymes. Results At day 12 of supplementation, we observed statistically significant reductions in mean postprandial glucose levels in both groups [(Group I: Initial - Day 12 PPG = 13.29 mg/dL, 95% CI: 3.329–23.24) (Group II: Initial – Day 12 PPG = 16.67 mg/dL, 95% CI: 4.687–28.65, P = 0.0159)], which continued through study completion at day 30. PCE supplementation significantly decreased mean preprandial glucose levels only in Group II at Days 24 (Initial – Day 24 = 13.00 mg/dL, 95% CI: 1.407–24.59, P = 0.0345) and 30 (Initial – Day 30 = 13.67 mg/dL, 95% CI: 5.766–21.57, P = 0.0067). In cell-based assays, PCE enhanced glucose uptake in L6 myocytes and inhibited hepatocyte glucose production HepG2 cells. In cell-free assays, PCE inhibited α-amylase activity and α-glucosidase activity. Conclusions These findings underscore the therapeutic utility of PCE for maintaining healthy glucose metabolism and warrant further larger-scale clinical trials. Trial registration This trial was retrospectively registered in the ISRCTN registry on September 29, 2018 ( ISRCTN15680985 ).
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