Insulin-, glucagon-, and somatostatin-containing cells, identified by immunofluorescent staining, were quantitated morphometrically in sections of pancreas obtained from diabetic and nondiabetic humans and rats. Both the volume density and number of somatostatin-and glucagon-containing cells were The other diabetic pancreas was obtained from a 31-yearold white female with a 29-year history of juvenile-type diabetes who died of renal insufficiency. Grossly, the pancreas was indurated and microscopic examination revealed acute pancreatitis. Islets were sparse and small, consisting largely of A-cells. No B-cells were noted.The four nondiabetic pancreases were obtained from encephalographically dead kidney donors in Geneva and Dallas in accordance with local regulations.Streptozotocin Diabetic and Nondiabetic Rats. Pancreases were obtained from four normal control rats and four rats with 16 months of diabetes produced by a single intravenous injection of 45 mg of streptozotocin per kg of body weight. These rats never required insulin treatment, but all were glycosuric at the time of sacrifice.Immunofluorescent Staining Techniques. The indirect immunofluorescent technique of Coons et al. (25) was employed with use of a rabbit anti-somatostatin serum (a gift of Dr. M. P. Dubois) at a dilution of 1:50, highly specific rabbit anti-glucagon serum 15K at a 1:20 dilution, and a guinea pig anti-insulin serum (a gift of Dr. P. H. Wright) at a 1:50 dilution. Anti-rabbit or anti-guinea pig gamma globulin labeled with fluorescein isothiocyanate (Pasteur Institute, Paris) was employed as the second antibody.Control experiments were performed using the specific antiserum absorbed with the corresponding antigen, 200,g of cyclic somatostatin (a gift of Drs. J. Rivier and R. Guillemin), 50 jig of glucagon, or 2 units of insulin per ml of undiluted antiserum. After removal of the paraffin, sections were rehydrated and incubated for 2 hr with anti-somatostatin, anti-insulin, or anti-glucagon serum. After rinsing in phosphate-buffered saline, the sections were incubated with the fluorescein-labeled antibody for 1

No abstract

Liver regeneration is of major clinical importance in the setting of liver injury, resection, and transplantation. A20, a potent anti-inflammatory and NF-κB inhibitory protein, has established pro-proliferative properties in hepatocytes, in part through decreasing expression of the Cyclin Dependent Kinase Inhibitor, p21. Both C-terminal (7-Zinc fingers; 7Zn) and N-terminal (Nter) domains of A20 were required to decrease p21 and inhibit NF-κB. However, both independently increased hepatocyte proliferation, suggesting that additional mechanisms contributed to the pro-proliferative function of A20 in hepatocytes. We ascribed one of A20’s pro-proliferative mechanisms to increased and sustained IL-6 induced STAT3 phosphorylation, as a result of decreased hepatocyte expression of the negative regulator of IL-6 signaling, SOCS3. This novel A20 function segregates with its 7Zn not Nter domain. Conversely, total and partial loss of A20 in hepatocytes increased SOCS3 expression, hampering IL-6-induced STAT3 phosphorylation. Following liver resection in mice pro-proliferative targets downstream of IL-6/STAT3 signaling were increased by A20 overexpression and decreased by A20 knockdown. In contrast, IL-6/STAT3 pro-inflammatory targets were increased in A20 deficient livers, and decreased or unchanged in A20 overexpressing livers. Upstream of SOCS3, levels of its microRNA regulator miR203 were significantly decreased in A20-deficient livers. Altogether these results demonstrate that A20 enhances IL-6/STAT3 pro-proliferative signals in hepatocytes by down-regulating SOCS3, likely through a miR203-dependent manner. This finding together with A20 reducing the levels of the potent cell cycle brake p21 establishes its pro-proliferative properties in hepatocytes and prompts the pursuit of A20-based therapies to promote liver regeneration and repair.

Changes in immunoreactive somatostatin were examined in islets, whole pancreas, stomach, and hypothalamus of streptozotocin-diabetic rats. There was no change in islet somatostatin content at 2 days after the administration of streptozotocin, but thereafter, somatostatin progressively increased in the diabetic animals by 45% at 2 weeks, 230% at 6 weeks, and 500% by 6 months. By contrast, islet glucagon rose acutely and maintained a constant 2-fold elevation irrespective of the duration of the diabetes. Morphometric analysis of the somatostatin- and glucagon-producing cells in the islets revealed an apparent augmentation of both cell types. The concentration of somatostatin per total pancreas was also increased in the diabetic animals, suggesting that the islet increase was part of a true increase in pancreatic somatostatin. Pancreatic glucagon was unchanged despite the islet increase. The increase in pancreatic somatostatin was paralleled by an elevation in gastric somatostatin concentration, implying a common mechanism in response to streptozotocin for the somatostatin cells in these two sites. There was no change in hypothalamic somatostatin concentration. Islet somatostatin was also increased in alloxan-diabetic rats. suggesting that streptozotocin does not stimulate the D cells directly.

BackgroundAccelerated atherosclerosis is the leading cause of morbidity and mortality in diabetic patients. Hyperglycemia is a recognized independent risk factor for heightened atherogenesis in diabetes mellitus (DM). However, our understanding of the mechanisms underlying glucose damage to the vasculature remains incomplete.Methodology/Principal FindingsHigh glucose and hyperglycemia reduced upregulation of the NF-κB inhibitory and atheroprotective protein A20 in human coronary endothelial (EC) and smooth muscle cell (SMC) cultures challenged with Tumor Necrosis Factor alpha (TNF), aortae of diabetic mice following Lipopolysaccharide (LPS) injection used as an inflammatory insult and in failed vein-grafts of diabetic patients. Decreased vascular expression of A20 did not relate to defective transcription, as A20 mRNA levels were similar or even higher in EC/SMC cultured in high glucose, in vessels of diabetic C57BL/6 and FBV/N mice, and in failed vein grafts of diabetic patients, when compared to controls. Rather, decreased A20 expression correlated with post-translational O-Glucosamine-N-Acetylation (O-GlcNAcylation) and ubiquitination of A20, targeting it for proteasomal degradation. Restoring A20 levels by inhibiting O-GlcNAcylation, blocking proteasome activity, or overexpressing A20, blocked upregulation of the receptor for advanced glycation end-products (RAGE) and phosphorylation of PKCβII, two prime atherogenic signals triggered by high glucose in EC/SMC. A20 gene transfer to the aortic arch of diabetic ApoE null mice that develop accelerated atherosclerosis, attenuated vascular expression of RAGE and phospho-PKCβII, significantly reducing atherosclerosis.ConclusionsHigh glucose/hyperglycemia regulate vascular A20 expression via O-GlcNAcylation-dependent ubiquitination and proteasomal degradation. This could be key to the pathogenesis of accelerated atherosclerosis in diabetes.

The value of serial lactate and pH measurements to predict the length of necrotic bowel is very limited. Length of necrotic bowel and lactate values are independent risk factors for mortality.

BackgroundIn colorectal cancer, CDX2 expression is lost in approximately 20% of cases and associated with poor outcome. Here, we aim to validate the clinical impact of CDX2 and investigate the role of promoter methylation and histone deacetylation in CDX2 repression and restoration.MethodsCDX2 immunohistochemistry was performed on multi-punch tissue microarrays (n = 637 patients). Promoter methylation and protein expression investigated on 11 colorectal cancer cell lines identified two CDX2 low expressors (SW620, COLO205) for treatment with decitabine (DNA methyltransferase inhibitor), trichostatin A (TSA) (general HDAC inhibitor), and LMK-235 (specific HDAC4 and HDAC5 inhibitor). RNA and protein levels were assessed. HDAC5 recruitment to the CDX2 gene promoter region was tested by chromatin immunoprecipitation.ResultsSixty percent of tumors showed focal CDX2 loss; 5% were negative. Reduced CDX2 was associated with lymph node metastasis (p = 0.0167), distant metastasis (p = 0.0123), and unfavorable survival (multivariate analysis: p = 0.0008; HR (95%CI) 0.922 (0.988–0.997)) as well as BRAFV600E, mismatch repair deficiency, and CpG island methylator phenotype. Decitabine treatment alone induced CDX2 RNA and protein with values from 2- to 25-fold. TSA treatment ± decitabine also led to successful restoration of RNA and/or protein. Treatment with LMK-235 alone had marked effects on RNA and protein levels, mainly in COLO205 cells that responded less to decitabine. Lastly, decitabine co-treatment was more effective than LMK-235 alone at restoring CDX2.ConclusionCDX2 loss is an adverse prognostic factor and linked to molecular features of the serrated pathway. RNA/protein expression is restored in CDX2 low-expressing CRC cell lines by demethylation and HDAC inhibition. Importantly, our data underline HDAC4 and HDAC5 as new epigenetic CDX2 regulators that warrant further investigation.Electronic supplementary materialThe online version of this article (10.1186/s13148-018-0548-2) contains supplementary material, which is available to authorized users.

The nuclear factor-B inhibitory protein A20 demonstrates hepatoprotective abilities through combined antiapoptotic, antiinflammatory, and pro-proliferative functions. Accordingly, overexpression of A20 in the liver protects mice from toxic hepatitis and lethal radical hepatectomy, whereas A20 knockout mice die prematurely from unfettered liver inflammation. The effect of A20 on oxidative liver damage, as seen in ischemia/reperfusion injury (IRI), is unknown. In this work, we evaluated the effects of A20 upon IRI using a mouse model of total hepatic ischemia. Hepatic overexpression of A20 was achieved by recombinant adenovirus (rAd.)-mediated gene transfer. Although only 10%-25% of control mice injected with saline or the control rAd.␤ galactosidase survived IRI, the survival rate reached 67% in mice treated with rAd.A20. This significant survival advantage in rAd.A20-treated mice was associated with improved liver function, pathology, and repair potential. A20-treated mice had significantly lower bilirubin and aminotransferase levels, decreased hemorrhagic necrosis and steatosis, and increased hepatocyte proliferation. A20 protected against liver IRI by increasing hepatic expression of peroxisome proliferator-activated receptor alpha (PPAR␣), a regulator of lipid homeostasis and of oxidative damage. A20-mediated protection of hepatocytes from hypoxia/reoxygenation and H 2 O 2 -mediated necrosis was reverted by pretreatment with the PPAR␣ inhibitor MK886.In conclusion, we demonstrate that PPAR␣ is a novel target for A20 in hepatocytes, underscoring its novel protective effect against oxidative necrosis. By combining hepatocyte protection from necrosis and promotion of proliferation, A20-based therapies are well-poised to protect livers from IRI, especially in the context of small-for-size and steatotic liver grafts. Liver Ischemia/reperfusion injury (IRI) of the liver is a major cause of hepatic failure in the context of liver resection surgery, liver trauma, and liver transplantation.1,2 The clinical outcome of IRI is largely dependent on the severity of the insult and on the pre-existing condition of the liver. For instance, steatosis, which affects 25% of the western population, is a critical aggravating factor for IRI. 3The precise sequence of events leading to liver IRI has not been completely elucidated. There is, however, substantial evidence that ischemia depletes ATP levels (energy) and results in the activation of Kü pffer cells, production of proinflammatory cytokines such as tumor necrosis factor (TNF), generation of reactive oxygen species (ROS), and perturbation of the hepatic microcirculation. 4 These events lead to a massive inflammatory Abbreviations: ␤gal, ␤galactosidase; BrdU, 5 bromo-2-deoxyuridine; CDKI, cyclin-dependent kinase inhibitor; FA, fatty acids; HPF, high-power field; IRI, ischemia reperfusion injury; MOI, multiplicity of infection; NF-B, nuclear factor kappa B; PPAR␣, peroxisome proliferator-activated receptor alpha; rAd., recombinant adenovirus; ROS, reactive oxygen species; SEM, ...