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Excessive alcohol (EtOH) consumption causes an imbalance in protein metabolism. EtOHimpairs protein synthesis in C2C12 myoblasts via a FoxO1‐AMPK‐TSC2‐mTORC1 pathway and also induces protein degradation. As the underlying regulatory signaling cascades for these processes are currently poorly defined, we tested the hypothesis that alcohol‐induced autophagy is mediated via activation of the PIK3C3 complex that is regulated by FoxO1‐AMPK. C2C12 myoblasts were incubated with EtOHfor various periods of time, and autophagy pathway‐related proteins were assessed by Western blotting and immunoprecipitation. Expression of targeted genes was suppressed using electroporation of specific siRNAs and chemical inhibitors. Incubation of C2C12 myoblasts with 100 mMEtOHincreased the autophagy markers LC3B‐IIand ATG7, whereas levels of SQSTM1/p62 decreased. The lysosomal inhibitor bafilomycin A1 caused a similar response, although there was no additive effect when combined with EtOH. EtOHaltered ULK1 S555 and S757 phosphorylation in a time‐ and AMPK‐dependent manner. The activation of AMPKand ULK1 was associated with increased BECN1 (S93, S14) and PIK3C3/VPS34 (S164) phosphorylation as well as increased total ATG14 and PIK3C3. These changes promoted formation of the ATG14‐AMBRA1‐BECN1‐PIK3C3 proautophagy complex that is important in autophagosome formation. EtOH‐induced changes were not associated with increased production of PtdIns3P, which may be due to enhanced PIK3C3 complex binding with 14‐3‐3θ. Reduction of AMPKusing siRNAsuppressed the stimulatory effect of EtOHon BECN1 S93, BECN1 S14, and PIK3C3 S164 phosphorylation in a time‐dependent manner. Likewise, knockdown of AMPKor chemical inhibition of FoxO1 attenuated phosphorylation of ULK1 at both residues. Knockdown of ULK1 or BECN1 antagonized the effect of EtOHon LC3B‐II,SQSTM1, and ATG7 protein expression. EtOH‐induced autophagy is mediated through changes in phosphorylation and interaction of various PIK3C3 complex components. This, in turn, is regulated either directly via FoxO1‐AMPKor indirectly via the FoxO1‐AMPK‐ULK1 signaling cascade in a mTORC1‐independent or mTORC1‐dependent manner. In muscle, alcohol activates the pro‐autophagy ATG14‐PIK3C3 complex by increasing BECN1 phosphorylation (S14 and S93) and ATG14. In parallel, alcohol inhibits the non‐autophagy PIK3C3 complex by increasing total and phosphorylated (S164) PIK3C3. Alcohol‐induced phosphorylation and dephosphorylation of ULK1 at S555 and S757 alters the interaction between AMPK and ULK1, and increases association of ULK1 with BECN1, ATG14 and PIK3C3. Stimulation of autophagy by alcohol is regulated by FoxO1‐AMPK signaling that is mediated via activation of ULK1 and ATG14‐containing PIK3C3 complexes.

Background: The mammalian target of rapamycin (mTOR) kinase controls cell growth, proliferation, and metabolism through 2 distinct multiprotein complexes, mTORC1 and mTORC2. We reported that alcohol (EtOH) inhibits mTORC1 activity and protein synthesis in C2C12 myoblasts. However, the role that mTORC2 plays in this process has not been elucidated. In this study, we investigated whether mTORC2 functions as part of a feedback regulator in response to EtOH, acting to maintain the balance between the functions of Akt, mTORC2, and mTORC1.

Background: Alcohol and the antiretroviral drug indinavir (Ind) decrease protein synthesis in skeletal muscle under in vivo and in vitro conditions. The goal of the present study was to identify signaling mechanisms responsible for the inhibitory effect of ethanol (EtOH) and Ind on protein synthesis.

Alcohol and the antiretroviral drug indinavir (Ind) decrease protein synthesis in skeletal muscle under in vivo and in vitro conditions. The goal of the present study was to identify signaling mechanisms responsible for the inhibitory effect of ethanol (EtOH) and Ind on protein synthesis.

Combined antiretroviral drug regimens have promoted clinical, immunologic, and virologic improvements in AIDS patients. Nevertheless, these therapies are associated with derangements in lipid and carbohydrate metabolism. In this study, we examined the effects of a representative protease inhibitor (nelfinavir), a nonnucleoside reverse transcriptase inhibitor (nevirapine), and a nucleoside reverse transcriptase inhibitor (zidovudine) on protein synthesis in skeletal muscle cells. To examine these processes, C2C12 myocytes were treated with increasing concentrations of nelfinavir, nevirapine, or zidovudine for 1 or 2 days, and rates of protein synthesis were determined by measuring [35S]methionine/cysteine incorporation into cellular proteins. Treatment of myocytes with therapeutic concentrations of nelfinavir, nevirapine, or zidovudine for 48 hr decreased protein synthesis by 14–20%. An ∼60% decline was observed in cells treated with higher concentrations of nevirapine or nelfinavir. In contrast, the basal rate of protein synthesis was not affected when cells were incubated with these compounds for 24 hr. Therapeutic concentrations of nelfinavir and nevirapine did not impair the anabolic effect of insulin on protein synthesis. However, zidovudine suppressed the stimulatory effect of insulin. The decreased protein synthesis induced by nelfinavir and zidovudine was associated with decreases in the phosphorylation of the S6 ribosomal protein (rpS6) and the repressor binding protein 4EBP1, while the inhibitory effect of nevirapine was mainly associated with a decline in phosphorylated 4EBP1. In conclusion, nelfinavir, nevirapine, and zidovudine treatments decreased protein synthesis in myocytes and this effect was correlated with a reduction in the phosphorylation level of proteins that regulate translation initiation.

A 66-year-old woman was admitted with diarrhea, weight loss, slight recurrent abdominal pain, and raised serum amylase and lipase. Lactose intolerance was diagnosed, and treatment was begun. The symptoms diminished. However, slight back pain and elevated serum amylase and lipase levels persisted. A pancreatic tumor was then suspected. Ultrasound, spiral computed tomography (CT), magnetic resonance imaging (MRI), and magnetic resonance cholangiopancreatography (MRCP) examinations were inconclusive. Endoscopic retrograde cholangiopancreatography (ERCP) showed a slight narrowing of the pancreatic duct within the pancreatic body, and endoscopic ultrasound (EUS) revealed a 10 mm intrapancreatic lesion. Finally, intraductal ultrasonography (IDUS) reliably identified a small pancreatic tumor. The tumor was resected, and histology confirmed a well-differentiated adenocarcinoma in situ (UICC stage 0, TisN0M0). This case shows that using high-resolution imaging techniques such as EUS plus IDUS, small malignant pancreatic lesions can be detected at an early stage, when curative action is possible.

Background:Hepatic cysts are detected incidentally in 2.5–5% of the population. Only about 15% of such cysts are symptomatic. Since laparoscopic deroofing for liver cysts was first described in 1991 there have been a number of reports of successful laparoscopic management of hepatic cysts, including the laparoscopic management of complex and parasitic cysts. Methods:A systematic review of English-language articles on the subject appearing in journals through May 2002 was conducted using the Medline database. Results:Only a minority of cystic liver lesions need treatment, and the therapeutic approach is guided by the type of cyst. Laparoscopic deroofing (combined with omentoplasty and/or oversewing) of uncomplicated liver cysts is associated with a recurrence rate of 10–25%, with less morbidity and mortality as compared to open surgery. On the other hand, treatment of complex liver cysts and hydatid cysts by laparoscopy is not considered a standard approach. Conclusion:The laparoscopic approach in uncomplicated but symptomatic liver cysts is effective, with low morbidity and mortality. Additional omentoplasty or oversewing appears to reduce the recurrence rate.

Background: Acute and chronic alcohol intoxication decreases skeletal muscle protein synthesis under in vivo conditions. We investigated whether ethanol (EtOH) and its major metabolites, acetaldehyde and acetate, can directly modulate protein balance under in vitro conditions.

Abstract. Estrogens regulate the production of angiotensinogen (AOG) in liver and other tissues. However, evidence suggests that the effect is tissue specific and that it may depend on a variety of factors. We have tested the effects of ethynylestradiol (EE) on liver AOG mRNA and plasma AOG in intact male and ovariectomized female rats, as well as in hypophysectomized male rats. EE stimulated both variables to a comparable extent and in a dose-dependent manner. However, its effect on plasma AOG was significantly higher in female than in male rats. In addition, there was no response in hypophysectomized male rats; however, responsiveness was restored by pretreatment of the animals with prolactin. AOG mRNA concentration was also affected by EE in brain, but there were striking time- and region-specific differences. These results indicate that cell-specific factors also modulate the response of the AOG gene to EE in extrahepatic tissues.

In the accompanying paper (Brown, C. R., Doxsey, S. J., Hong-Brown, L. W., Martin, R. L., and Welch, W. J.(1996) J. Biol. Chem.271, 824-832) two molecular chaperones, hsp 73 and TCP-1, were shown to be integral components of the centrosome. Here we show that heat shock treatment adversely affects both the structure and function of the centrosome, and that hsp 73 plays a role in the repair of the organelle. After heat shock treatment, the centrosome could not be identified via indirect immunofluorescence and cells were unable to support microtubule regrowth. During recovery from heat shock, a strong correlation between the return of staining of three centrosomal antigens (hsp 73, TCP-1, and pericentrin) and the recovery of microtubule regrowth properties was found. Incubation of cells with glycerol, a protein protective agent, prevented the heat induced alterations in the structure/function of the centrosome. Likewise, the recovery of the structure and function of the centrosome after heat shock treatment was significantly accelerated in cells first made thermotolerant. We provide evidence that this process is related to the levels of hsp 73 since: 1) microinjection of hsp 73 antibody blocked centrosomal reassembly and microtubule regrowth abilities following heat shock; and 2) microinjection of purified hsp 73 protein prior to heat shock treatment accelerated both the repair and function of the organelle, similar to that observed for thermotolerant cells.

Molecular chaperones play an important role in facilitating the proper maturation of many newly synthesized proteins. Here we provide evidence that molecular chaperones also participate in regulating the assembly of the microtubule cytoskeleton. Via indirect immunofluorescence analysis, both hsp 73 and TCP-1 localized within the centrosome in interphase and mitotic cells. These proteins, along with the centrosome-specific protein, pericentrin, were also present within an enriched preparation of centrosomes. Because the centrosome serves as an initiation site for microtubule growth, we examined the ability of cells to regrow their microtubule network in the presence of hsp 73 or TCP-1 specific antibodies. Purified tubulin and GTP were added to cells following the depolymerization and extraction of cellular microtubules. Microtubules were observed to nucleate off the centrosome using this system, even in the presence of anti-hsp 73 antibodies. Incubation with anti-TCP-1 antibodies, however, blocked microtubule regrowth off the centrosome. Similarly, anti-TCP-1 antibodies microinjected into living cells first treated with nocodazole also inhibited the regrowth of the microtubule network following removal of the microtubule poison. Our results complement earlier genetic studies in yeast implicating a role for TCP-1 in microtubule mediated processes, and may help to explain the previously reported mitotic and meiotic abnormalities associated with TCP-1 mutations.