Active p38α causes macrovesicular fatty liver in mice

One third of the western population suffers from nonalcoholic fatty liver disease (NAFLD), which may ultimately develop into hepatocellular carcinoma (HCC). The molecular event(s) that triggers the disease are not clear. Current understanding, known as the multiple hits model, suggests that NAFLD is a result of diverse events at several tissues (e.g., liver, adipose tissues, and intestine) combined with changes in metabolism and microbiome. In contrast to this prevailing concept, we report that fatty liver could be triggered by a single mutated protein expressed only in the liver. We established a transgenic system that allows temporally controlled activation of the MAP kinase p38α in a tissue-specific manner by induced expression of intrinsically active p38α allele. Here we checked the effect of exclusive activation in the liver. Unexpectedly, induction of p38α alone was sufficient to cause macrovesicular fatty liver. Animals did not become overweight, showing that fatty liver can be imposed solely by a genetic modification in liver per se and can be separated from obesity. Active p38α-induced fatty liver is associated with up-regulation of MUC13, CIDEA, PPARγ, ATF3, and c-jun mRNAs, which are up-regulated in human HCC. Shutting off expression of the p38α mutant resulted in reversal of symptoms. The findings suggest that p38α plays a direct causative role in fatty liver diseases and perhaps in other chronic inflammatory diseases. As p38α activity was induced by point mutations, it could be considered a proto-inflammatory gene (proto-inflammagene).

Inflammatory liver diseases form a serious epidemic in the Western world. Nonalcoholic fatty liver disease (NAFLD) has grown from a relatively unknown disease to the most common cause of chronic liver diseases in the world over the last two decades, with a global prevalence of 25% (1). NAFLD is characterized by steatosis of the liver, involving greater than 5% of parenchyma, with no evidence of hepatocyte injury. In nonalcoholic steatohepatitis (NASH), the liver cells become injured in a background of steatosis (2). NAFLD may further develop to NASH and, at a later stage, to liver cirrhosis that could eventually progress to liver cancer (3).Many cases of liver diseases are associated with viral infections (hepatitis B and C and HIV), alcoholism, drugs, or autoimmunity. NAFLD and NASH are commonly associated with obesity, insulin resistance (IR), and type 2 diabetes. In addition, a large number of patients develop fatty liver with no known background (3, 4).Regardless of the background conditions, the molecular basis of the triggering and the etiology of the diseases are unclear (3). As is the case for all other chronic inflammatory diseases, the current understanding of the underlying mechanism of the onset of NAFLD is that it involves some combination of genetic and environmental conditions. The latest theory is the “multiple hit” hypothesis, which suggests that a number of events, occurring in parallel in several tissues and systems, combined with environmental and genetic factors, lead to NAFLD (5–7). Such hits include IR, adipose tissue inflammation, hormones secreted from adipose tissue, nutritional factors, gut microbiota, and genetic and epigenetic factors (5, 8, 9). This explanation for the onset of NAFLD and other chronic inflammatory diseases stands in a sharp contrast to the molecular mechanism underlying the onset of cancer diseases, which are triggered by genetic alterations in proto-oncogenes and in tumor suppressor genes (10, 11). Namely, the “multiple hit” concept for the onset of NAFLD does not consider the possibility of proto-inflammatory genes that may become disease-causing genes (“inflammagenes”) following some genetic alteration. It is shown here, nonetheless, that fatty liver could be induced by a single mutated protein.The lack of deep understanding of the molecular triggers of fatty liver diseases hinders the development of accurate experimental models (12). Current models rely on treatments that indirectly lead to accumulation of lipids in the liver. These models can be broadly categorized as diet induced, genetics, or a combination of more than one intervention. Diet-induced models include mainly the use of high-fat diet (HFD) that causes obesity and in turn NAFLD-like disease in mice. In these models, the development of the disease is slow as it must be preceded by a serious increase in body weight (13). In the genetic models, lipidosis (fatty liver) is enhanced in response to HFD as the animal is engineered to develop obesity faster. The principal genetic animal models of NASH are the ob/ob (leptin-deficient) and db/db (leptin receptor–deficient) mice, the Zucker (fa/fa) rat, foz mice (deficient in the Alstrom syndrome 1 gene) and several transgenic or conditional knockout mice (12). Exposure of the animals to drugs such as the hepatotoxin CCl₄ to amplify injury and fibrosis also appears to closely resemble human NASH (12, 14). In all models, numerous biochemical pathways and systemic responses are induced in various tissues, making it difficult to extract the relevant activities that impose the liver disease. No genetic model exists in which fatty liver is induced solely by activation of a single protein by mutation or gene amplification, analogously to models of cancer diseases. By contrast, this study describes a transgenic system in which induction of a single mutated gene, exclusively in the liver, leads rapidly to the development of fatty liver, with no effect on body weight. The mutated gene encodes the mitogen-activated protein kinase (MAPK) p38α.p38α, a member of the p38 family, which is composed of four isoforms (15), is abnormally active in chronic inflammatory diseases including NAFLD (15–18). Inhibition of p38α resulted in reversal of pathological symptoms in some experimental systems, suggesting that p38α plays a role in the maintenance of the disease and perhaps in its etiology as well (17, 18). Finally, mice knocked out for various MAPK phosphatases are prone to the development of fatty liver. Although these phosphatases affect all members of the MAPK family (i.e., p38s, JNKs, and ERKs) it seems that the effect on the liver is a result of overactive p38 (19–23).In other inflammatory diseases in which p38α is abnormally active, including rheumatoid arthritis, asthma, and inflammatory bowel diseases (24–26), its role in these maladies, if any, is also not clear, similarly to the case of fatty liver diseases (18). We thus sought the establishment of an experimental model that will disclose the exact function of p38α in each tissue and may also point at its relative contribution to disease etiology. Achieving this goal necessitates in vivo activation of p38α per se in a tissue-specific and temporally controlled manner. Accomplishing such a task is far from trivial as, like all MAPKs, p38α manifests no basal catalytic activity when not activated, making an overexpression approach insufficient. Natural activation of p38α is obtained, in most cases, through a complex signaling cascade that culminates in p38α’s dual phosphorylation on a TGY motif located at its activation loop, a reaction catalyzed by the MAPK kinases MKK3 and MKK6 (27, 28). In the absence of this dual phosphorylation, p38α is catalytically impotent (27). Activation of upstream components of the p38 cascade induces the activity of various components in addition to p38α, including all p38 isoforms.In this study, the challenge of activating p38α individually in a tightly controlled manner was met by combining a unique double cassette expression system in transgenic mice, with an intrinsically active mutant of p38α, p38α^D176A+F327S (29, 30). p38α^D176A+F327S was shown to be intrinsically catalytically active in vitro and in cell cultures, confirming its independence from MKK3/6 activation or any upstream induction (31–35). Importantly, apart from gaining intrinsic activity, p38α^D176A+F327S maintained the biochemical, pharmacological, and physiological properties of naturally activated p38α molecules (35).We describe a transgenic model in which p38α could be specifically activated in a reversible manner, in vivo, in any tissue of choice. This model could be applied for studying the role of p38α per se in various inflammatory diseases, and here, we apply it to reveal the role of p38α in the etiology of fatty liver. It was originally expected that activation of p38α in the liver would render the liver prone to stimuli that impose lipidosis, such as HFD, or CCl₄. Unexpectedly, it was observed that expression of p38α^D176A+F327S in liver of mice unprovoked by any treatment was sufficient to cause an increase in serum and hepatic triglycerides and a histological appearance that resembles macrovesicular lipidosis. This observation proposes a perspective on the onset of NAFLD.Looking into the mechanism through which active p38α imposes fatty liver, it was assumed that p38α’s substrates would be strongly phosphorylated. Unexpectedly, the expression level and phosphorylation of the p38α’s substrate MAPKAPK2 (MK2) is down-regulated along with reduced phosphorylation of heat shock protein 27 (Hsp27) in mice expressing p38α^D176A+F327S in the liver. This suggests that constitutive activity of p38α may impose fatty liver by causing down-regulation of downstream components, perhaps via a feedback inhibition mechanism.     

Pre-emptive analgesic effect of tramadol after mandibular third molar extraction: a pilot study.

PURPOSE: We compared the efficacy of tramadol given before or immediately after surgical extraction of an impacted mandibular third molar under local anesthesia. MATERIALS AND METHODS: In this prospective, randomized, controlled, double-blind pilot study, 3 groups of 20 patients each were included: tramadol preoperative, 100 mg intramuscularly (IM) 1 hour before surgery (group A); tramadol postoperative, 100 mg IM immediately after surgery (group B); and saline (group C). We evaluated intensity of pain and analgesic consumption as was requested. RESULTS: The analgesic efficacy measured as complete relief of pain at 24 hours was 86% in the preemptive tramadol compared with 70% and 36% for postoperative tramadol administration and control group. A significant reduction in the consumption of analgesics was seen in preoperative group as compared with the postoperative and control groups. Adverse events were minimal and similar in all groups. CONCLUSIONS: This study suggests the preemptive use of tramadol as an alternative for the acute pain treatment after the removal of an impacted mandibular third molar carried out under local anesthesia.     

Localization of microcystin-LR in medaka fish tissues after cyanotoxin gavage

Microcystins (MCs) are toxic monocyclic heptapeptides produced by many cyanobacteria. Over 70 MCs have been successfully isolated and identified, of which MC-LR is the most commonly occurring toxin. Microcystins, especially MC-LR, cause toxic effects in mammals, birds and fish and are a recognized potent cause of environmental stress and pose a potential health hazard in aquatic ecosystems when heavy blooms of cyanobacteria appear. They also constitute a public health threat to people via drinking water and food chains. The concentrations of MC-LR can be very low, even in fish displaying severely disrupted tissues, which makes it essential to devise selective and sensitive histochemical methods for identifying and localizing MC-LR in target organs, such as liver and intestine. The aim of the study reported here was to analyze the presence of MC-LR in contaminated fish tissues using immunohistochemical methods. The present experiment involving subacute exposure confirmed our initial hypothesis that subacute and acute exposure to microcystin contamination can exacerbate physiological stress, induce sustained pathological damage, and affect the immune response in exposed medaka fish.     

Pharmacokinetics of DTPA entrapped in conventional and long-circulating liposomes of different size for plutonium decorporation.

The aim of the present study was to develop an efficient DTPA liposome formulation designed for plutonium decorporation. DTPA was encapsulated in conventional (CL) and polyethylene glycol-coated stealth liposomes (SL) prepared by extrusion followed by the freeze-thawing method and sizing from around 100 to 800 nm. DTPA encapsulation percentages were approximately 30% in CL of any size but dropped from 48% to 7% as the diameter of SL was reduced. The pharmacokinetics of [(14)C]-DTPA encapsulated in large and small vesicles was evaluated in rats after a single intravenous administration. Both liposomal composition and size reduction had a significant impact on pharmacokinetic parameters, inducing a marked increased in exposure of the body to DTPA and its delayed excretion. DTPA distribution was moderate in liver but enhanced in spleen and bone and was dose-dependent, especially when SL of 100 nm were given. In conclusion, small and stealth(R) vesicles have interesting properties in delivering DTPA to contaminated tissues.     

Incorporating HIV education and counseling into routine prenatal care: a program model

This article reports how a prenatal clinic in a major urban teaching hospital has developed and integrated an HIV education and counseling program into routine prenatal care. The patient population served are predominantly minority women living in an inner-city community that has been disproportionately affected by the AIDS epidemic. Implementation of the patient program has required training and support for all professional staff. Staff training served as a foundation for this comprehensive patient program, which has reached all prenatal patients regardless of risk behavior. The program has succeeded in involving a large population of women in an educational program, has identified HIV-1 seropositive pregnant women through voluntary testing, and has provided them with the necessary medical and social work services. Principles of program development are identified for use in other settings.     

Endoscopic hemostasis. An effective therapy for bleeding peptic ulcers

We performed a meta-analysis of 25 randomized control trials that compared endoscopic hemostasis with standard therapy for bleeding peptic ulcer. For recurrent or continued bleeding, the mean rate in control patients was 0.39, and the pooled rate difference, or reduction due to therapy, was 0.27 +/- 0.15 (95% confidence interval) (69% relative reduction). For emergency surgery, the mean rate in control patients was 0.26, and the pooled rate difference was 0.16 +/- 0.05 (62% relative reduction). Most important, for overall mortality, the mean rate in control patients was 0.10, and the pooled rate difference was 0.03 +/- 0.02 (30% relative reduction). The effects were greatest in patients with spurting or visible blood vessels and equivocal when the ulcer showed only signs of recent bleeding. We conclude that endoscopic hemostasis is clearly effective but that data were insufficient for direct comparisons between modalities. Randomized control trials to compare the different modes of endoscopic therapy should continue.     

Pteridines. 41. Synthesis and dihydrofolate reductase inhibitory activity of some cycloalka[g]pteridines.

A number of homologous 2,4-diaminocycloalka[g]pteridines varying in ring size from 5 to 15 were prepared by (a) condensation of aminomalononitrile tosylate with alpha-oximinocycloalkanones, deoxygenation of the resulting 2-amino-3-cyanocycloalka[b]pyrazine 1-oxides, and guanidine cyclization; (b) guanidine cyclization of the above pyrazine 1-oxides to give 2,4-diaminocycloalka[g]pteridine 8-oxides, followed by deoxygenation; or (c) condensation of 2,4,5,6-tetraaminopyrimidine with a cycloalka-1,2-dione (for the cyclohepta- and cycloocta[g]pteridines only). These compounds were examined for their activity as dihydrofolate reductase inhibitors against Lactobacillus casei, rat liver, L1210, and Trypanosoma cruzi. Activity was found to depend upon ring size, with the greatest activity exhibited by the cyclododeca derivatives 31.