The Glucagon-Like Peptide-1 Receptor Agonist Exendin 4 Has a Protective Role in Ischemic Injury of Lean and Steatotic Liver by Inhibiting Cell Death and Stimulating Lipolysis

Nonalcoholic fatty liver disease is an increasingly prevalent spectrum of conditions characterized by excess fat deposition within hepatocytes. Affected hepatocytes are known to be highly susceptible to ischemic insults, responding to injury with increased cell death, and commensurate liver dysfunction. Numerous clinical circumstances lead to hepatic ischemia. Mechanistically, specific means of reducing hepatic vulnerability to ischemia are of increasing clinical importance. In this study, we demonstrate that the glucagon-like peptide-1 receptor agonist Exendin 4 (Ex4) protects hepatocytes from ischemia reperfusion injury by mitigating necrosis and apoptosis. Importantly, this effect is more pronounced in steatotic livers, with significantly reducing cell death and facilitating the initiation of lipolysis. Ex4 treatment leads to increased lipid droplet fission, and phosphorylation of perilipin and hormone sensitive lipase – all hallmarks of lipolysis. Importantly, the protective effects of Ex4 are seen after a short course of perioperative treatment, potentially making this clinically relevant. Thus, we conclude that Ex4 has a role in protecting lean and fatty livers from ischemic injury. The rapidity of the effect and the clinical availability of Ex4 make this an attractive new therapeutic approach for treating fatty livers at the time of an ischemic insult.The incidence of obesity and fatty liver disease is increasing worldwide. Non alcoholic fatty liver disease (NAFLD) includes a spectrum of liver abnormalities ranging from simple steatosis with preserved synthetic function to end-stage liver disease requiring transplantation.^{1, 2} The cause of hepatic dysfunction related to steatosis remains incompletely defined.³ However, it is known that a steatotic liver has increased susceptibility to ischemic insults, such as those induced during liver resections and liver surgery,^{4, 5, 6} heart failure,⁷ and shock.⁸ In addition, steatotic livers are known to weather the ischemic insult of transplantation poorly,⁹ resulting in increased rates of primary nonfunction and initial graft dysfunction.^{10, 11} As such, fatty livers are routinely turned down for transplantation and this impacts transplant wait list morbidity and mortality.¹² Thus, liver steatosis contributes to the public health burden and methods to mollify the adverse effects of liver steatosis are relevant across a large spectrum of hepatic diseases.The inability of a steatotic liver to withstand ischemic insult is directly related to increased post ischemic cell death, which can occur through necrosis and apoptosis. The fundamental connection between intracellular fat and poor hepatic cell survival¹³ is incompletely understood. However, it has been suggested that methods that decrease intracellular fat reverse this susceptibility and the use of glucagon-like peptide-1 (GLP-1) analogues is one such approach. GLP-1 is secreted from the L cells of the small intestine and its cognate receptor (GLP-1R) is present in several organs, such as the pancreas, brain, heart, kidney, and liver. Although it is well known for its incretin action,¹⁴ it also has pleotropic effects.^{15, 16, 17, 18, 19} In the liver we have shown that GLP-1 or its homologue Exendin 4 (Ex4) acts directly on steatotic hepatocytes to decrease their lipid content.^{20, 21} In addition, a cytoprotective action of Ex4 with improvement in cell survival has also been reported.²² Thus, we hypothesize that anti-steatotic effects of Ex4 in hepatocytes and cytoprotective effects in other organs make it a rational target for investigation in steatotic livers undergoing ischemia reperfusion injury (IRI), a common clinical scenario in people with NAFLD. In this study, we explore the role of Ex4 in protecting against necrosis and apoptosis, the two forms of cell death encountered in hepatic IRI, and we provide evidence to show that Ex4 stimulates lipolysis with a short course of treatment. To our knowledge, this is the first study showing a direct and rapid action of Ex4 in acutely reversing the vulnerability of a steatotic liver to ischemic insults, supporting the investigation of Ex4 as a potential therapeutic agent for treatment of people with NAFLD undergoing ischemic injury and at the time of procurement of a fatty liver for transplantation.     

Functional regulation of human neutrophil Fc γ receptors

Interaction between Fc receptors expressed on phagocytic cells and antibodies play a critical role in innate immune response. Interestingly, immune cells such as neutrophils, monocytes, and dendritic cells (DCs) express multiple Fc receptors for IgG (FγR) with overlapping ligand specificity. These receptors compete for the same ligand on the target and are known to transduce positive and negative signals to the same cell, depending on presence of type of signaling motif in their cytoplasmic domain. Neutrophils, the first line of defense against bacterial infection and the major phagocytic cell in the blood, express two types of FcγRs depending on the species. In humans, the neutrophils co-express immunoreceptor tyrosine-based activation motif (ITAM) containing CD32A and glycosylphosphatidyl inositol (GPI)-anchored CD16B, which is in contrast to co-expression of ITAM containing CD16A and ITIM containing CD32B in mouse neutrophils. Recent studies in gene knockout mice have demonstrated that the negative signaling by CD32B plays a critical role in preventing immune complex (IC)-mediated autoimmune diseases by regulating the activation signal delivered by CD16A. However, it is not known how the function of ITAM signaling CD32A is regulated in human neutrophils. Recent observations from our laboratory suggest that in human neutrophils, the CD32A receptor is regulated at the ligand-binding stage. Using a CD16B-deficient donor, we found that the CD32A expressed on resting neutrophils is unable to bind ligand; however, once neutrophils are activated with fMLP, a bacterial chemotactic peptide, the CD32A is functionally active in binding ligand. We also observed that this regulation is neutrophil-specific phenomenon. These observations suggest that FcγR can be regulated by distinct mechanisms and factors such as membrane-anchoring, cell-specific signaling, and avidity modulation that may be coordinately involved in regulating the function of human FγR. Because neutrophils may be activated during infectious and inflammatory diseases, the knowledge of functional regulation of Fc γR will be useful in designing therapies for many autoimmune diseases.     

The pharmacokinetics of letrozole in brain and brain tumor in rats with orthotopically implanted C6 glioma, assessed using intracerebral microdialysis

Purpose

Emerging evidence suggests that primary and metastatic brain tumors may be sensitive to hormonal manipulations. However, the pharmacokinetics of compounds against such targets in the brain and, more importantly, in the brain tumor are not well characterized. Here, we investigated the pharmacokinetics of letrozole, a third-generation aromatase inhibitor, in the normal brain and in orthotopically implanted C6 glioma in Sprague–Dawley rats.

Methods

Intracerebral microdialysis was employed to determine the concentrations of unbound letrozole in the brain extracellular fluid (ECF) while simultaneously collecting blood samples (via jugular vein) to assess plasma levels of letrozole. Letrozole was administered intravenously at doses of 4, 6, 8 and 12 mg/kg, and ECF and blood samples were collected over 8 h. For assessing normal versus tumoral brain pharmacokinetics, letrozole (4 or 8 mg/Kg; i.v.) was administered 10 days after implantation of C6 glioma in the brain. Dual-probe intracerebral microdialysis was employed for assessing ECF samples from tumor-free and tumor-bearing regions of the brain.

Results

Normal brain ECF and plasma C _max and AUC_0–8h increased linearly with letrozole doses up to 8 mg/kg dose, but at 12 mg/kg, the pharmacokinetics were nonlinear. The relative brain distribution coefficients, AUC_ECF/AUC_{plasma (ub)}, were 0.3–0.98. The tumoral uptake of letrozole was 1.5- to 2-fold higher relative to tumor-free region.

Conclusions

Thus, letrozole permeability across the blood brain barrier is high, and the exposure to the brain is dose dependent. Furthermore, the brain tumoral letrozole levels are markedly higher than those in the tumor-free regions, which underscore potential selectivity of its activity against tumor cells.     

TLR5 or NLRC4 is necessary and sufficient for promotion of humoral immunity by flagellin

The fact that some TLR-based vaccine adjuvants maintain function in TLR-deficient hosts highlights that their mechanism of function remains incompletely understood. Thus, we examined the ability of flagellin to induce cytokines and elicit/promote murine antibody responses upon deletion of the flagellin receptors TLR5 and/or NLRC4 (also referred to as IPAF) using a prime/boost regimen. In TLR5-KO mice, flagellin failed to induce NF-κB-regulated cytokines such as keratinocyte-derived chemokine (CXCL1) but induced WT levels of the inflammasome cytokine IL-18 (IL-1F4). Conversely, in NLRC4-KO mice, flagellin induced keratinocyte-derived chemokine, but not IL-18, whereas TLR5/NLRC4-DKO lacked induction of all cytokines measured. Flagellin/ovalbumin treatment resulted in high-antibody titers to both flagellin and ovalbumin in WT, TLR5-KO and DKO mice but did not elicit antibodies to either in TLR5/NLRC4-DKO mice. Thus, flagellin's ability to elicit/promote humoral immunity requires a germ-line-encoded receptor capable of recognizing this molecule. Such promotion of adaptive immunity can be effectively driven by either TLR5-mediated activation of NF-κB or NLRC4-mediated activation of the inflammasome.     

Clostridium difficile Infection and Patient-Specific Antimicrobial Resistance Testing Reveals a High Metronidazole Resistance Rate

Digestive Diseases and Sciences - Clostridium difficile (CD) infection (CDI) causes marked morbidity and mortality, accounting for large healthcare expenditures annually. Current CDI treatment...     

Retroviral Rem protein requires processing by signal peptidase and retrotranslocation for nuclear function

Mouse mammary tumor virus (MMTV) is a complex murine retrovirus that encodes an HIV Rev-like export protein, Rem, from a doubly spliced version of envelope (Env) mRNA. Previously, the N-terminal 98-amino acid sequence of Rem, which is identical to Env signal peptide (SP), and full-length Rem were shown to be functional in a reporter assay that measures a postexport function. Here we show that MMTV-infected cells or cells transfected with rem or env cDNAs express SP, which is the active component in the reporter assay. Uncleaved Rem was partially glycosylated, but mutations in both glycosylation sites within the C terminus prevented Rem function. Mutations that reduced Rem or Env cleavage by signal peptidase greatly reduced SP levels and functional activity in the reporter assay and allowed accumulation of the uncleaved protein. Fluorescence microscopy revealed that GFP-tagged cleavage-site mutants are unstable and lack fluorescence compared with wild-type Rem, suggesting improper folding. Proteasome inhibitors allowed accumulation of uncleaved Rem relative to SP and increased reporter activity, consistent with SP retrotranslocation and proteasome escape before nuclear entry. Expression of a dominant-negative p97 ATPase did not alter levels of unprocessed Rem and SP but decreased reporter activity, suggesting p97-facilitated retrotranslocation of SP. Our results provide an example of a SP that is processed by signal peptidase and retrotranslocated to allow nuclear localization and function.     

Goniometric and conoscopic measurements of angular display contrast for one-, three-, five-, and nine-million-pixel medical liquid crystal displays

Active-matrix liquid crystal displays (AMLCDs) have become the preferred choice for displaying digital diagnostic images because of their low cost of ownership, high contrast, and small footprint. However, the contrast and luminance of AMLCDs changes significantly with viewing direction. In this paper, we use a goniometric and a conoscopic method to measure angular contrast and luminance in one-, three-, five-, and nine-million-pixel medical AMLCDs. The viewing angle characteristics of the displays are compared by evaluating the compliance with the desired grayscale and recommended tolerance limits. Using the measured contrast responses, we determined the angle along each of the orthogonal and diagonal axes for which the luminance ratio drops to 0.5, 0.2, and 0.1 of the maximum ratio, seen at perpendicular viewing. The results show a reduction as large as 54% in available JND levels between the perpendicular and off-normal viewing directions. All of the display systems proved to have better angular response in the horizontal and vertical direction as compared to the diagonal directions, and none were able to satisfy a 20% contrast tolerance limit for any viewing direction lying in a cone at an angle of 45 degrees from the normal. Additionally, we show that the measured contrast can be significantly affected by the measurement method used to record the angular change in luminance.     

Toxicity profile of lutein and lutein ester isolated from marigold flowers (Tagetes erecta)

Lutein is a carotenoid with antioxidant properties and is commonly present in many fruits, vegetables, and egg yolk. Lutein affords protection against the development of the two common eye diseases of aging: cataract and macular degeneration. As the dietary lutein concentration is much lower compared to the actual requirement to reduce macular degeneration, supplementation of lutein is under consideration. There are very few data on the toxicity of lutein. In the present study, the authors have evaluated the short-term and long-term toxicity profile of lutein and its esterified form isolated from marigold flowers (Tagetes erecta) in young adult male and female Wistar rats. Lutein and its ester form administered orally at doses of 4, 40, and 400 mg/kg body weight for 4 weeks for short-term toxicity study and 13 weeks for a subchronic toxicity study did not produced any mortality, change in body weight, food consumption pattern, organ weight, and other adverse side reactions. Administration of lutein and ester form did not alter the hepatic and renal function, and did not produce any change in the hematological parameters and in lipid profile. Histopathological analysis of the organs supported the nontoxicity of lutein and its ester form.