leupeptins and Body-Weight

To establish the therapeutic potential of proteasome inhibition, we examined the therapeutic effects of MG132 (Z-Leu-Leu-Leu-aldehyde) in an experimental model of acute pancreatitis.. Pancreatitis was induced in rats by two hourly intraperitoneal (ip) injections of cholecystokinin octapeptide (CCK; 2 x 100 microg/kg) and the proteasome inhibitor MG132 (10 mg/kg ip) was administered 30 min after the second CCK injection. Animals were sacrificed 4 h after the first injection of CCK.. Administering the proteasome inhibitor MG132 (at a dose of 10 mg/kg, ip) 90 min after the onset of pancreatic inflammation induced the expression of cell-protective 72 kDa heat shock protein (HSP72) and decreased DNA-binding of nuclear factor-kappaB (NF-kappaB). Furthermore MG132 treatment resulted in milder inflammatory response and cellular damage, as revealed by improved laboratory and histological parameters of pancreatitis and associated oxidative stress.. Our findings suggest that proteasome inhibition might be beneficial not only for the prevention, but also for the therapy of acute pancreatitis.

Topics: Acute Disease; Animals; Body Weight; Cholecystokinin; Cysteine Proteinase Inhibitors; Cytokines; HSP72 Heat-Shock Proteins; Leupeptins; Male; NF-kappa B; Organ Size; Oxidative Stress; Pancreas; Pancreatitis; Peroxidase; Proteasome Inhibitors; Rats; Rats, Wistar

The mechanisms of the increased serum leptin in response to feeding are poorly understood. Therefore, we used metabolic labeling to directly assess leptin biosynthesis, secretion, and turnover in adipose tissue from 14 h-starved compared with fed 12-14 week old rats. Starvation decreased serum leptin (-47 +/- 7%), adipose tissue leptin content (-32 +/- 5%), and leptin secretion during 3 h of incubation (-65 +/- 12%). Starvation did not affect leptin mRNA levels but decreased rates of leptin biosynthesis by tissue fragments, as determined by [(35)S]methionine/cysteine incorporation into immunoprecipitable leptin. Insulin in vitro did not acutely increase leptin biosynthesis or rates of (125)I-leptin degradation. Pulse-chase studies showed that in adipose tissue from fed but not starved rats, insulin accelerated the secretion of [(35)S]leptin by approximately 2-fold after 30 and 60 min of chase. Degradation of newly synthesized leptin was slower in adipose tissue of starved than fed rats (half-lives of 50 and 150 min, respectively). Inhibitor experiments showed that both lysosomes and proteosomes contributed to leptin degradation. In conclusion, feeding compared with starvation influences leptin production at multiple posttranscriptional levels: synthesis, tissue storage, turnover, and secretion. The insulin-stimulated release of leptin from a preformed intracellular leptin pool may contribute to increases in serum leptin levels after meals.

Topics: Adipocytes; Adipose Tissue; Animals; Blotting, Northern; Body Weight; Chloroquine; Cycloheximide; Insulin; Iodine Isotopes; Leptin; Leupeptins; Lysosomes; Male; Rats; Rats, Wistar; RNA, Messenger; Starvation; Sulfur Isotopes

Non-small cell lung cancer frequently presents as a locally advanced disease. In this setting, radiation has a prominent role in cancer therapy. However, tumor adaptation to oxidative stress may lessen the efficacy of radiation therapy. Recent studies demonstrate that proteasome inhibitors increase the efficacy of radiation against a range of tumors. Although proteasome inhibition impacts on NF-kappaB translocation, the precise mechanism through which proteasome inhibitors induce tumor cell death and promote radiation efficacy remains unclear. The purpose of this study is to evaluate the potential of the proteasome inhibitor, MG-132, to improve the efficacy of radiation therapy and to determine whether its effect is linked to the suppression of the antioxidant enzyme, manganese superoxide dismutase (MnSOD). Human NSCLC (A549) cells were utilized both in vivo and in vitro to evaluate proteasome inhibition on radiation response. In vivo, mice that received combined treatments of 2.5 microg/g body weight MG-132 and 30 Gy demonstrated a delay in tumor regrowth in comparison to the 30 Gy control group. In vitro, clonegenic survival assays confirmed a dose-dependent enhancement of radiation sensitivity in combination with MG-132 and a significant interaction between the two. The levels of IkappaB-alpha, a NF-kappaB target gene and also an inhibitor of NF-kappaB nuclear translocation, decreased in a time-dependent manner following administration of MG-132 confirming the inhibition of the 26S proteasome. The MnSOD protein level was increased consistent with lower levels of IkappaB-alpha, confirming a NF-kappaB-mediated effect. Cells treated with radiation demonstrated an induction of MnSOD; however, the administration of MG-132 suppressed this induction These results support the hypothesis that proteasome inhibitors such as MG-132 can increase the efficacy of radiation therapy, in part, by suppression of cytoprotective NF-kappaB-mediated MnSOD expression.

Topics: Active Transport, Cell Nucleus; Animals; Antioxidants; Blotting, Western; Body Weight; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Combined Modality Therapy; Dose Fractionation, Radiation; Dose-Response Relationship, Drug; Female; Humans; I-kappa B Proteins; Leupeptins; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; NF-kappa B; NF-KappaB Inhibitor alpha; Oxidative Stress; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Transport; Radiation Tolerance; Reactive Oxygen Species; Superoxide Dismutase; Time Factors

The cell-permeant MG132 tripeptide (Z-Leu-Leu-Leu-aldehyde) is a peptide aldehyde proteasome inhibitor that also inhibits other proteases, including calpains and cathepsins. By blocking the proteasome, this tripeptide has been shown to induce the expression of cell-protective heat shock proteins (HSPs) in vitro. Effects of MG132 were studied in an in vivo model of acute pancreatitis. Pancreatitis was induced in male Wistar rats by injecting 2 x 100 microug/kg cholecystokinin octapeptide intraperitoneally (ip) at an interval of 1 h. Pretreating the animals with 10 mg/kg MG132 ip before the induction of pancreatitis significantly inhibited IkappaB degradation and subsequent activation of nuclear factor-kappaB (NF-kappaB). MG132 also increased HSP72 expression. Induction of HSP72 and inhibition of NF-kappaB improved parameters of acute pancreatitis. Thus MG132 significantly decreased serum amylase, pancreatic weight/body weight ratio, pancreatic myeloperoxidase activity, proinflammatory cytokine concentrations, and the expression of pancreatitis-associated protein. Parameters of oxidative stress (GSH, MDA, SOD, etc.) were improved in both the serum and the pancreas. Histopathological examinations revealed that pancreatic specimens of animals pretreated with the peptide demonstrated milder edema, cellular damage, and inflammatory activity. Our findings show that simultaneous inhibition of calpains, cathepsins, and the proteasome with MG132 prevents the onset of acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Body Weight; Cysteine Proteinase Inhibitors; Cytokines; HSP72 Heat-Shock Proteins; Leupeptins; Lung; Male; NF-kappa B; Oxidative Stress; Pancreas; Pancreatitis; Pancreatitis-Associated Proteins; Proteasome Inhibitors; Rats; Rats, Wistar; Sincalide

Marked loss of body weight and profound waste of both skeletal muscle and white adipose tissue occur in rats into which the ascites hepatoma Yoshida AH-130 has been transplanted, associated with marked perturbations in the hormonal homoeostasis and the presence of circulating tumour necrosis factor and high plasma levels of prostaglandin E2 [Tessitore, Costelli and Baccino (1993) Br. J. Cancer 67, 15-23]. On the basis of previous findings, the present study examined whether the development of cachexia in this model system could be significantly affected by adrenalectomy or by pharmacological treatments that may interfere with proximal or distal mediators of tissue hypercatabolism. In no instance was tumour growth modified. Medroxyprogesterone acetate, an anabolic-hormone-like drug, was completely ineffective. In adrenalectomized animals, although changes such as the elevation of plasma triacylglycerols and corticosterone were corrected, the general course of cachexia was not modified. A partial prevention of muscle waste was observed with acetylsalicylic acid, a non-steroidal anti-inflammatory drug, or with leupeptin, a proteinase inhibitor. Insulin afforded the most significant preservation of muscle protein and adipose-tissue mass, which were maintained close to control values even 10 days after transplantation. The effects of insulin on gastrocnemius muscle and liver protein content were exerted by slowing down protein turnover, mainly enhancing synthesis. Consistently, the total free amino acid concentration in the gastrocnemius of insulin-treated rats 10 days after tumour transplantation was close to that of controls. Although treatment with insulin decreased plasma corticosterone to normal values, it did not modify the circulating level of tumour necrosis factor. On the whole these data show that it seems possible to prevent, at least in part, the tissue waste that characterizes cancer cachexia by purely pharmacological means.

Topics: Adrenalectomy; Animals; Aspirin; Blood Glucose; Body Weight; Cachexia; Cathepsin D; Cholesterol; Endopeptidases; Hormones; Insulin; Leupeptins; Male; Medroxyprogesterone Acetate; Muscle Proteins; Neoplasm Transplantation; Neoplasms, Experimental; Rats; Rats, Wistar; Triglycerides

Previous studies in young normal rats have shown that intracerebral administration of the proteinase inhibitor, leupeptin, caused a rapid accumulation of lipofuscin-like pigment in lysosomes of brain cells (Ivy et al., 1984a). On the other hand, we have recently found that the administration of lovastatin, an inhibitor of HMG-CoA reductase, reduced the ceroid-like pigment and dolichol contents in the crushed epididymal fat pad of rats (Porta et al., 1988). In order to study now the possible modulating effects of these enzyme inhibitors on ceroidogenesis associated with vitamin E deficiency, two main groups of weanling Wistar female rats were respectively fed ad libitum a vitamin E-deficient basal diet, or the same diet supplemented with 16 mg% of dl-alpha-tocopherol acetate. The vitamin E-deficient and -supplemented rats were further subdivided and received for 8 weeks their diets alone or with 2, 1, or 0.5 g of lovastatin/kg of diet. Other subgroups were treated with constant peritoneal infusion of 0.5 mg/day of leupeptin by means of osmotic minipumps (Alzet 2002) consecutively implanted at days 15, 30, and 45. Lovastatin treatment to vitamin E-deficient rats was associated with dose-dependent toxicity, resulting in 100%, 75%, and 50% mortality at concentrations of 2, 1, and 0.5 g/kg diet, respectively. This mortality was mainly due to extensive hepatic necrosis. Food intake and growth rates were reduced, while the relative weights of liver, kidneys, spleen, heart and brain, as well as the serum levels of GPT and GOT were significantly increased over the values of the untreated vitamin E-deficient control rats. The volumetric densities of ceroid pigment and the dolichol contents in liver and kidneys were not significantly modified. Lovastatin toxicity was partially prevented by vitamin E supplementation. However, in these supplemented rats, lovastatin treatment did not modify the volumetric densities of hepatic and renal ceroid, although the contents of hepatic and renal dolichol were significantly increased. No correlations could be found between levels of hepatic or renal ceroid and total dolichol content in vitamin E-deficient and supplemented rats. Leupeptin treatment to vitamin E-deficient rats only slightly reduced food intake and growth rates, and did not significantly modify the relative organ weights or the serum levels of cholesterol, GOT and GPT. Although in both vitamin E-deficient and -supplemented rats the leupeptin treatment consistently s

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Body Weight; Ceroid; Cholesterol; Dolichols; Eating; Female; Kidney; Leupeptins; Liver; Lovastatin; Organ Size; Rats; Rats, Inbred Strains; Vitamin E; Vitamin E Deficiency

We investigated the influence of leupeptin (LP) intraperitoneal injection (40 mumol/2 days) on protein and amino-acid metabolism of septic rats (cecal ligation). All septic rats lost weight (-17 +/- 4 g), which was not prevented by LP administration (-24 +/- 1.8 g, n.s.). LP injection evoked weight loss even in normal rats (p less than 0.05 vs controls). Weight loss was accompanied by enhanced urinary nitrogen losses in all three groups. LP reduced food intake for 47% in control rats. Cecal ligation, and also the administration of LP, led to alterations of amino-acid metabolism. The most important changes were found in muscle free amino-acid concentrations with highly decreased levels of free glutamine. A glutamine deficiency is known to be related to a decreased rate of protein synthesis. The proteolytic rate in incubated soleus muscle was increased for 11.5% and even higher in LP-treated septic rats (+22%). It is concluded that the administration of LP cannot reverse protein catabolism in sepsis--possibly because LP does not influence those enzymes or proteases involved in tissue loss, or LP is inactivated by enzymes in rat tissues.

Topics: Amino Acids; Animals; Bacterial Infections; Body Weight; Eating; Leupeptins; Liver; Muscles; Oligopeptides; Proteins; Rats; Rats, Inbred Strains; Tyrosine

Topics: Animals; Bacterial Infections; Body Weight; Creatinine; Leupeptins; Liver; Male; Muscles; Oligopeptides; Rats; Rats, Inbred Strains; Tyrosine

Long bone fracture in the rat is accompanied by enhanced urinary nitrogen loss reflecting changes in protein synthesis and breakdown. The effects of leupeptin, an inhibitor of lysosomal proteases, were assessed on organ weights, RNA, DNA, and protein content after injury in the rat. Two groups of 8-week-old rats were studied: The first group received left femoral fracture. Half of these received leupeptin (25 mumole ip/day), and the remainder received saline. The second group served as uninjured pair-fed controls, with half receiving leupeptin and half receiving saline. On Days 0, 1, 2, 4, and 7 after injury, animals were sacrificed and organs were removed for determination of weight, RNA, DNA, and protein content. All injured rats lost weight, with maximum loss occurring on Day 4. Food intake was also reduced. Pair-fed rats lost the same amount of weight as injured ones, and leupeptin could not prevent whole body weight loss. Expressed as percentage of total body weight, livers from leupeptin-treated injured rats weighed 10% greater than saline-treated ones on Days 2, 4, and 7 after injury (P less than 0.05). No differences occurred in RNA, DNA, or protein contents. Diaphragms similarly weighed 10, 20, and 30% greater on Days 2, 4, and 7 after injury, respectively, in leupeptin-treated rats (P less than 0.05). In addition, the RNA and DNA contents of diaphragms were 96 and 88% greater, respectively, in treated rats than in controls (P less than 0.05) on Day 4. It is concluded that leupeptin causes a relative increase in the weights of livers and diaphragms after injury, and causes a marked increase in the RNA and DNA contents of diaphragms.

Topics: Animals; Body Weight; Diaphragm; DNA; Eating; Femoral Fractures; Leupeptins; Liver; Male; Oligopeptides; Organ Size; Proteins; Rats; Rats, Inbred Strains; RNA

The effect of leupeptin on the induction of lymphoblastic leukemia in ICR/JCL female mice by N-nitrosobutylurea (NBU) was investigated. NBU was given as a 0.02% solution in drinking water for 10 weeks. A 0.1% concentration of leupeptin was given in the diet. Group A was fed on the leupeptin diet from the beginning, Group B received it after the end of NBU treatment, and Group C was fed on a leupeptin-free control diet throughout. The average periods in the appearance of leukemia in groups A, B, and C were 115+/-50, 112+/-43, and 100+/-17 days (mean+/-SD), respectively, and there was a significant difference between groups A and B and Group C at P less than 0.001. In regard to this point, leupeptin might have the effect of retarding the rpocess of leukemogenesis. However, leupeptin showed no effect on the incidence and histopathological finding of leukemia.

Topics: Animals; Body Weight; Female; Leukemia, Experimental; Leukemia, Lymphoid; Leupeptins; Mice; Mice, Inbred ICR; Nitrosourea Compounds; Oligopeptides; Thymus Gland; Thymus Neoplasms