Limited forensic and clinical experience and the lack of confirmatory testing strategies for synthetic cannabinoids (SC) prevent adequate characterization of SC toxicity and the potential impact on public health. A statewide surveillance system identified a fatality involving a 23‐year‐old man found with a large stab wound to the neck following use of a SC product suspected of containing AM2201. Analytical testing for common SCs, SC metabolites, routine drugs of abuse, and over‐the‐counter medications was performed on heart blood obtained at autopsy. Additionally, assays were performed on the SC raw material and drug paraphernalia found on the decedent. High concentrations of AM2201 were detected in all samples. AM2201 metabolites were detected in postmortem blood. Other than a trace amount of JWH‐073 found in smoke residue, no other substances were detected. Psychiatric complications including self‐induced, lethal trauma can occur after the use of SC products.
Myostatin is a TGFβ family member and negative regulator of muscle size. Due to the complexity of the molecular pathway between myostatin mRNA/protein and changes in transcription, it has been difficult to understand whether myostatin plays a role in resistance exercise-induced skeletal muscle hypertrophy. To circumvent this problem, we determined the expression of a unique myostatin target gene, Mighty, following resistance exercise. Mighty mRNA increased by 6 h (82.9±24.21%) and remained high out to 48 h (56.5±19.67%) after resistance exercise. Further examination of the soleus, plantaris and tibialis anterior muscles showed that the change in Mighty mRNA at 6 h correlated with the increase in muscle size associated with this protocol (R2 = 0.9996). The increase in Mighty mRNA occurred both independent of Smad2 phosphorylation and in spite of an increase in myostatin mRNA (341.8±147.14% at 3 h). The myostatin inhibitor SKI remained unchanged. However, activated Notch, another potential inhibitor of TGFβ signaling, increased immediately following resistance exercise (83±11.2%) and stayed elevated out to 6 h (78±16.6%). Electroportion of the Notch intracellular domain into the tibialis anterior resulted in an increase in Mighty mRNA (63±13.4%) that was equivalent to the canonical Notch target HES-1 (94.4±7.32%). These data suggest that acute resistance exercise decreases myostatin signaling through the activation of the TGFβ inhibitor Notch resulting in a decrease in myostatin transcriptional activity that correlates well with muscle hypertrophy.
BackgroundAnabolic resistance is the inability to increase protein synthesis in response to an increase in amino acids following a meal. One potential mediator of anabolic resistance is endoplasmic reticulum (ER) stress. The purpose of the present study was to test whether ER stress impairs the response to growth factors and leucine in muscle cells.MethodsMuscle cells were incubated overnight with tunicamycin or thapsigargin to induce ER stress and the activation of the unfolded protein response, mTORC1 activity at baseline and following insulin and amino acids, as well as amino acid transport were determined.ResultsER stress decreased basal phosphorylation of PKB and S6K1 in a dose-dependent manner. In spite of the decrease in basal PKB phosphorylation, insulin (10–50 nM) could still activate both PKB and S6K1. The leucine (2.5–5 mM)-induced phosphorylation of S6K1 on the other hand was repressed by low concentrations of both tunicamycin and thapsigargin. To determine the mechanism underlying this anabolic resistance, several inhibitors of mTORC1 activation were measured. Tunicamycin and thapsigargin did not change the phosphorylation or content of either AMPK or JNK, both increased TRB3 mRNA expression and thapsigargin increased REDD1 mRNA. Tunicamycin and thapsigargin both decreased the basal phosphorylation state of PRAS40. Neither tunicamycin nor thapsigargin prevented phosphorylation of PRAS40 by insulin. However, since PKB is not activated by amino acids, PRAS40 phosphorylation remained low following the addition of leucine. Blocking PKB using a specific inhibitor had the same effect on both PRAS40 and leucine-induced phosphorylation of S6K1.ConclusionER stress induces anabolic resistance in muscle cells through a PKB/PRAS40-induced blockade of mTORC1.
The goal of the current work was to profile positive (mTORC1 activation, autocrine/paracrine growth factors) and negative [AMPK, unfolded protein response (UPR)] pathways that might regulate overload-induced mTORC1 (mTOR complex 1) activation with the hypothesis that a number of negative regulators of mTORC1 will be engaged during a supraphysiological model of hypertrophy. To achieve this, mTORC1-IRS-1/2 signaling, BiP/CHOP/IRE1α, and AMPK activation were determined in rat plantaris muscle following synergist ablation (SA). SA resulted in significant increases in muscle mass of ∼4% per day throughout the 21 days of the experiment. The expression of the insulin-like growth factors (IGF) were high throughout the 21st day of overload. However, IGF signaling was limited, since IRS-1 and -2 were undetectable in the overloaded muscle from day 3 to day 9. The decreases in IRS-1/2 protein were paralleled by increases in GRB10 Ser501/503 and S6K1 Thr389 phosphorylation, two mTORC1 targets that can destabilize IRS proteins. PKB Ser473 phosphorylation was higher from 3–6 days, and this was associated with increased TSC2 Thr939 phosphorylation. The phosphorylation of TSC2 Thr1345 (an AMPK site) was also elevated, whereas phosphorylation at the other PKB site, Thr1462, was unchanged at 6 days. In agreement with the phosphorylation of Thr1345, SA led to activation of AMPKα1 during the initial growth phase, lasting the first 9 days before returning to baseline by day 12. The UPR markers CHOP and BiP were elevated over the first 12 days following ablation, whereas IRE1α levels decreased. These data suggest that during supraphysiological muscle loading at least three potential molecular brakes engage to downregulate mTORC1.
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