Treatment with the antileukemic agent asparaginase can induce acute pancreatitis, but the pathophysiology remains obscure. In the liver of mice, eukaryotic initiation factor 2 (eIF2) kinase general control nonderepressible 2 (GCN2) is essential for mitigating metabolic stress caused by asparaginase. We determined the consequences of asparaginase treatment on the pancreata of wild-type (WT, GCN2-intact) and GCN2-deleted (ΔGcn2) mice. Mean pancreas weights in ΔGcn2 mice treated with asparaginase for 8 days were increased (P < 0.05) above all other groups. Histological examination revealed acinar cell swelling and altered staining of zymogen granules in ΔGcn2, but not WT, mice. Oil Red O staining and measurement of pancreas triglycerides excluded lipid accumulation as a contributor to acini appearance. Instead, transmission electron microscopy revealed dilatation of the endoplasmic reticulum (ER) and accumulation of autophagic vacuoles in the pancreas of ΔGcn2 mice treated with asparaginase. Consistent with the idea that loss of GCN2 in a pancreas exposed to asparaginase induced ER stress, phosphorylation of protein kinase R-like ER kinase (PERK) and its substrate eIF2 was increased in the pancreas of asparaginase-treated ΔGcn2 mice. In addition, mRNA expression of PERK target genes, activating transcription factors 4, 3, and 6 (Atf4, Atf3, and Atf6), fibroblast growth factor 21 (Fgf21), heat shock 70-kDa protein 5 (Hspa5), and spliced Xbp1 (sXbp1), as well as pancreas mass, was elevated in the pancreas of asparaginase-treated ΔGcn2 mice. Furthermore, genetic markers of oxidative stress [sirtuin (Sirt1)], inflammation [tumor necrosis factor-α (Tnfα)], and pancreatic injury [pancreatitis-associated protein (Pap)] were elevated in asparaginase-treated ΔGcn2, but not WT, mice. These data indicate that loss of GCN2 predisposes the exocrine pancreas to a maladaptive ER stress response and autophagy during asparaginase treatment and represent a genetic basis for development of asparaginase-associated pancreatitis.

Phytoecdysteroids such as 20-hydroxyecdysone (20HE) are nutritional supplements marketed as enhancers of lean body mass. In this study the impact of 20HE ingestion on protein kinase B/Akt-mechanistic target of rapamycin complex 1 signaling in the skeletal muscle and liver of male rats was found to be limited. Bioavailability of 20HE, whether consumed alone or with leucine, also remained low at all doses ingested. Additional work is necessary to clarify 20HE mechanism of action in vivo.

Phytoecdysteroids such as 20HE are ingredients in various nutritional supplements marketed to enhance physical performance and lean body mass. Recent in vitro studies suggest 20HE may activate protein synthesis via a phosphoinositide‐3‐kinase‐Akt signaling mechanism. To confirm these findings in vivo and evaluate downstream activation of mammalian target of rapamycin complex 1 (mTORC1) signaling, overnight fasted 4‐6 week old male rats (n=5‐6 per group) were randomized to one of three study designs: 1) gavaged with 0, 10, 50, 200 mg/kg 20HE and euthanized 30 min post‐gavage; 2) gavaged with 200 mg/kg 20HE or excipient and euthanized at 30 min, 60 min, 120 min, 240 min post‐gavage, and 3) administered excipient or 200 mg/kg 20HE alone or in combination with 1.35 g/kg L‐leucine and euthanized 30 min post‐gavage. Independent evaluation of 20HE confirmed purity as reported by Sigma before solubilization in 3% DMSO or 70% Labrasol® as excipient. Phosphorylation of Akt at Thr308 and Ser473, mTOR at Ser2448, ribosomal S6 kinase (S6K1) at Thr389, and eukaryotic initiation factor 4E binding protein 1 (4E‐BP1) at Thr 37/46 were evaluated by immunoblot. Leucine administered in 3% DMSO or Labrasol robustly increased phosphorylation of mTOR, S6K1 and 4E‐BP1 but not Akt in muscle and liver. In contrast, at all doses and times, 20HE did not significantly increase phosphorylation of Akt, mTOR, S6K1 or 4E‐BP1 as compared to excipient controls. Furthermore, 20HE dampened mTORC1 signaling by leucine in both muscle and liver. These data do not support the idea that anabolic effects of 20HE are acutely mediated by mTORC1. Grant Funding Source: Supported by NJAES and USDA Multistate NC1184

L‐asparaginase (ASNase) is widely used to treat leukemia in children but it causes metabolic complications and liver toxicity. ASNase activates the amino acid response (AAR), increasing gene‐specific translation of the transcription factor ATF4 to regain homeostasis via altered gene expression. We hypothesized that ATF4 deficiency would compromise the hepatic AAR to ASNase and augment hepatotoxicity. Intact (Atf4wt), heterozygous (Atf4het) and homozygous null (Atf4null) mice were administered ASNase (3 IU/g BW i.p.) or saline once daily for 8d. Blood and tissues were collected 8h after final injection. Atf4null mice treated with ASNase lost body weight and experienced premature mortality while all other strains appeared healthy. ASNase did not alter liver mass but promoted lipid accumulation in all genetic strains of mice to a similar degree. Phosphorylation of eIF2 was increased in the liver of all mice treated with ASNase and in saline‐injected Atf4null mice. Examination of the hepatic AAR showed many genes including Asns, Atf5, Chop, Fgf21, 4ebp1 and Gadd34 required ATF4 for full induction. Furthermore, inflammatory stress was evident in the liver of Atf4null but not Atf4het or Atf4wt mice treated with ASNase, similar to that previously reported in Gcn2null mice (AJP 305:E1124, 2013). These results suggest that ATF4 mediates GCN2‐driven hepatic adaptive responses to ASNase and prevents liver inflammation but not lipid accumulation. HD070487 (TGA); GM49164 (RCW)

Asparaginase is a chemotherapy agent used in the treatment of acute lymphoblastic leukemia. Asparaginase can cause severe pancreatitis but the molecular basis is unknown. In liver of mice, the eIF2 kinase GCN2 is essential for mitigating metabolic stress caused by asparaginase. This study examined the role of GCN2 in the pancreas of mice treated with asparaginase. Eight week old wild type or GCN2 KO mice were injected once daily for 8 d with either 3 IU/g BW of saline or asparaginase. Eight hours after final injection, mice were sacrificed and pancreata were weighed and harvested. In GCN2 KO mice treated with asparaginase, pancreas weights were significantly increased (P<0.05) and the organs visibly enlarged. Histological examination revealed ductal dilatation and swollen acinar cells in GCN2 KO only. Oil red O staining and measurement of pancreas triglycerides ruled out lipid accumulation as a contributing factor. No sign of cell death by TUNEL stain were detected in the pancreas, and serum amylase activity did not differ among treatment groups. However, Pancreatitis Associated Protein (PAP) mRNA expression was elevated in livers of asparaginase‐treated GCN2 KO mice only. Phosphorylation of eIF2 and pancreatic expression of asparagine synthetase were similar among treatment groups, but mTORC1 signaling was decreased to the greatest extent in the pancreata of asparaginase‐treated GCN2 KO mice. These data suggest that loss of GCN2 predisposes the pancreas toward the development of asparaginase‐associated pancreatitis. Funded by NIH HD070487