Jaideep Behari

Lipotoxicity Causes Multisystem Organ Failure and Exacerbates Acute Pancreatitis in Obesity

Unsaturated fatty acids cause lipotoxicity and mediate acute adverse outcomes in obese individuals with pancreatitis. The Burden of Adiposity As if diabetes and heart disease were not burden enough, obese people who suffer trauma, burns, or other critical conditions have an increased likelihood of death. During these exacerbated illnesses, multiple organs can fail, a situation that is particularly hard to reverse. How the presence of excess adipose tissue contributes to the severity of these diseases is not clear, but understanding the mechanisms could provide clues for possible treatments. Pancreatitis is a relatively well-defined disease that tends to be worse in the obese and, in its most severe form, is accompanied by multi-organ failure. By using a combination of patient investigation, in vitro cell studies, and an animal model, Navina et al. have assembled evidence that pinpoints the culprits in the obesity-related complications of this disease: unsaturated fatty acids liberated by lipolysis from adipose tissue. The authors carefully examine the pancreases of 24 patients who had died of pancreatitis. The staining patterns indicated that nonesterified fatty acids, derived by lipolysis of excess intrapancreatic fat, contributed to the pancreatic necrosis in these patients. To test this idea, the authors used a cell culture system and showed that it is unsaturated fatty acids that do the damage, impairing acinar cell activities, inhibiting mitochondrial function, releasing calcium, and causing cell death. But what about the failure of other organs? To answer this question, the authors used obese mice with pancreatitis and, by inhibiting lipolysis with the drug orlistat, were able to prevent the pancreatic-associated rise in serum unsaturated fatty acids and, of most importance, to reduce damage to the lung and kidney, as well as mortality. It is not yet clear which lipase is the critical one for multiorgan failure or where it is located. But once revealed, this potential therapeutic target may specify a treatment that enhances the survival of critically ill obese patients. Obesity increases the risk of adverse outcomes during acute critical illnesses such as burns, severe trauma, and acute pancreatitis. Although individuals with more body fat and higher serum cytokines and lipase are more likely to experience problems, the roles that these characteristics play are not clear. We used severe acute pancreatitis as a representative disease to investigate the effects of obesity on local organ function and systemic processes. In obese humans, we found that an increase in the volume of intrapancreatic adipocytes was associated with more extensive pancreatic necrosis during acute pancreatitis and that acute pancreatitis was associated with multisystem organ failure in obese individuals. In vitro studies of pancreatic acinar cells showed that unsaturated fatty acids were proinflammatory, releasing intracellular calcium, inhibiting mitochondrial complexes I and V, and causing necrosis. Saturated fatty acids had no such effects. Inhibition of lipolysis in obese (ob/ob) mice with induced pancreatitis prevented a rise in serum unsaturated fatty acids and prevented renal injury, lung injury, systemic inflammation, hypocalcemia, reduced pancreatic necrosis, and mortality. Thus, therapeutic approaches that target unsaturated fatty acid–mediated lipotoxicity may reduce adverse outcomes in obese patients with critical illnesses such as severe acute pancreatitis.

Outcomes of curative treatment for hepatocellular cancer in nonalcoholic steatohepatitis versus hepatitis C and alcoholic liver disease.

Concomitant increasing incidences of hepatocellular carcinoma (HCC) and nonalcoholic steatohepatitis (NASH) suggest that a substantial proportion of HCC arises as a result of hepatocellular injury from NASH. The aim of this study was to determine differences in severity of liver dysfunction at HCC diagnosis and long‐term survival outcomes between patients undergoing curative therapy for HCC in the background of NASH compared to hepatitis C virus (HCV) and/or alcoholic liver disease (ALD). Patient demographics and comorbidities, clinicopathologic data, and long‐term outcomes among patients who underwent liver transplantation, hepatic resection, or radiofrequency ablation for HCC were reviewed. From 2000 to 2010, 303 patients underwent curative treatment of HCC; 52 (17.2%) and 162 (53.5%) patients had NASH and HCV and/or alcoholic liver disease. At HCC diagnosis, NASH patients were older (median age 65 versus 58 years), were more often female (48.1% versus 16.7%), more often had the metabolic syndrome (45.1% versus 14.8%), and had lower model for end‐stage liver disease scores (median 9 versus 10) (all P < 0.05). NASH patients were less likely to have hepatic bridging fibrosis or cirrhosis (73.1% versus 93.8%; P < 0.001). After a median follow‐up of 50 months after curative treatment, the most frequent cause of death was liver failure. Though there were no differences in recurrence‐free survival after curative therapy (median, 60 versus 56 months; P = 0.303), NASH patients had longer overall survival (OS) (median not reached versus 52 months; P = 0.009) independent of other clinicopathologic factors and type of curative treatment. Conclusion: Patients with HCC in the setting of NASH have less severe liver dysfunction at HCC diagnosis and better OS after curative treatment compared to counterparts with HCV and/or alcoholic liver disease. (HEPATOLOGY 2012;55:1811–1821)

Liver-Specific β-Catenin Knockout Mice Exhibit Defective Bile Acid and Cholesterol Homeostasis and Increased Susceptibility to Diet-Induced Steatohepatitis

Although the role of Wnt/beta-catenin signaling in liver growth and development is well established, its contribution in non-neoplastic hepatic pathologies has not been investigated. Here, we examine the role of beta-catenin in a murine model of diet-induced liver injury. Mice with hepatocyte-specific beta-catenin deletion (KO) and littermate controls were fed the steatogenic methionine and choline-deficient (MCD) diet or the corresponding control diet for 2 weeks and characterized for histological, biochemical, and molecular changes. KO mice developed significantly higher steatohepatitis and fibrosis on the MCD diet compared with wild-type mice. Both wild-type and KO livers accumulated triglyceride on the MCD diet but, unexpectedly, higher hepatic cholesterol levels were observed in KO livers on both control and MCD diets. Gene expression analysis showed that hepatic cholesterol accumulation in KO livers was not attributable to increased synthesis or uptake. KO mice had lower expression of bile acid synthetic enzymes but exhibited higher hepatic bile acid and serum bilirubin levels, suggesting defects in bile export. Therefore, loss of beta-catenin in the liver leads to defective cholesterol and bile acid metabolism in the liver and increased susceptibility to developing steatohepatitis in the face of metabolic stress.

The Wnt/β-catenin signaling pathway in liver biology and disease

The Wnt signaling pathway is an evolutionarily conserved, highly complex signaling pathway that is critical for development, differentiation and cellular homeostasis. The protein β-catenin is the central player in one major arm of the Wnt pathway called the canonical Wnt pathway. As in other organs, the Wnt/β-catenin pathway is critical for liver development. However, recent research suggests that the pathway is also important in liver regeneration, liver metabolism and maintenance of normal function in the adult liver. Aberrant activation of β-catenin has also been implicated in the pathogenesis of hepatobiliary neoplasia, ranging from benign lesions to liver cancer. The explosion of research into the many roles of the Wnt/β-catenin pathway promises to change our fundamental understanding of normal liver biology and the aberrations that lead to disease and cancer.

Liver-Specific β-Catenin Knockout Mice Have Bile Canalicular Abnormalities, Bile Secretory Defect, and Intrahepatic Cholestasis

Beta‐catenin plays important roles in liver physiology and hepatocarcinogenesis. While studying the role of β‐catenin in diet‐induced steatohepatitis, we recently found that liver‐specific β‐catenin knockout (KO) mice exhibit intrahepatic cholestasis. This study was undertaken to further characterize the role of β‐catenin in biliary physiology. KO mice and wild‐type (WT) littermates were fed standard chow or a diet supplemented with 0.5% cholic acid for 2 weeks. Chow‐fed KO mice had higher serum and hepatic total bile acid levels and lower bile flow rate than WT mice. Expression levels of bile acid biosynthetic genes were lower and levels of major bile acid exporters were similar, which therefore could not explain the KO phenotype. Despite loss of the tight junction protein claudin‐2, KO mice had preserved functional integrity of tight junctions. KO mice had bile canalicular morphologic abnormalities as evidenced by staining for F‐actin and zona occludens 1. Electron microscopy revealed dilated and tortuous bile canaliculi in KO livers along with decreased canalicular and sinusoidal microvilli. KO mice on a cholic acid diet had higher hepatic and serum bile acid levels, bile ductular reaction, increased pericellular fibrosis, and dilated, misshapen bile canaliculi. Compensatory changes in expression levels of several bile acid transporters and regulatory genes were found in KO livers. Conclusion: Liver‐specific loss of β‐catenin leads to defective bile canalicular morphology, bile secretory defect, and intrahepatic cholestasis. Thus, our results establish a critical role for β‐catenin in biliary physiology. (HEPATOLOGY 2010)

Hepatocyte γ-catenin compensates for conditionally deleted β-catenin at adherens junctions

BACKGROUND & AIMS Wnt/β-catenin signaling is important in liver physiology. Moreover, β-catenin is also pivotal in adherens junctions (AJ). Here, we investigate hepatocyte-specific β-catenin conditional null mice (KO) for any alterations in AJ and related tight junctions (TJ). METHODS Using gene array, PCR, Western blot, immunohistochemistry, immunofluorescence, and co-immunoprecipitation, we compare and contrast the composition of AJ and TJ in KO and littermate wild-type (WT) control livers. RESULTS We show association of E-cadherin with β-catenin in epithelial cells of WT livers, which is lost in the KOs. While total levels of α-catenin, E-cadherin, and F-actin were modestly decreased, KO livers show increased γ-catenin/plakoglobin. By co-immunoprecipitation, E-cadherin/β-catenin/F-actin association was observed in WT livers, while the association of E-cadherin/γ-catenin/F-actin was evident in KO livers. γ-Catenin was localized at the hepatocyte membrane at baseline in the KO liver. While γ-catenin gene expression remained unaltered, an increase in serine- and threonine-phosphorylated, but not tyrosine-phosphorylated γ-catenin was observed in KO livers. A continued presence of γ-catenin at the hepatocyte membrane, without any nuclear localization, was observed in liver regeneration after partial hepatectomy at 40 and 72 h, in both KO and WT. Analysis of TJ revealed lack of claudin-2 and increased levels of JAM-A and claudin-1 in KO livers. CONCLUSIONS β-Catenin adequately maintains AJ in the absence of β-catenin in hepatocytes; however, it lacks nuclear localization. Moreover, β-catenin/claudin-2 may be an important mechanism of crosstalk between the AJ and TJ.

Transcriptional regulation of CXC-ELR chemokines KC and MIP-2 in mouse pancreatic acini

Neutrophils and their chemoattractants, the CXC-ELR chemokines keratinocyte cytokine (KC) and macrophage inflammatory protein-2 (MIP-2), play a critical role in pancreatitis. While acute pancreatitis is initiated in acinar cells, it is unclear if these are a source of CXC-ELR chemokines. KC and MIP-2 have NF-κB, activator protein-1 (AP-1) sites in their promoter regions. However, previous studies have shown increased basal and reduced caerulein-induced AP-1 activation in harvested pancreatic tissue in vitro, which limits interpreting the caerulein-induced response. Moreover, recent studies suggest that NF-κB silencing in acinar cells alone may not be sufficient to reduce inflammation in acute pancreatitis. Thus the aim of this study was to determine whether acinar cells are a source of KC and MIP-2 and to understand their transcriptional regulation. Primary overnight-cultured murine pancreatic acini were used after confirming their ability to replicate physiological and pathological acinar cell responses. Upstream signaling resulting in KC, MIP-2 upregulation was studied along with activation of the transcription factors NF-κB and AP-1. Cultured acini replicated critical responses to physiological and pathological caerulein concentrations. KC and MIP-2 mRNA levels increased in response to supramaximal but not to physiological caerulein doses. This upregulation was calcium and protein kinase C (PKC), but not cAMP, dependent. NF-κB inhibition completely prevented upregulation of KC but not MIP-2. Complete suppression of MIP-2 upregulation required dual inhibition of NF-κB and AP-1. Acinar cells are a likely source of KC and MIP-2 upregulation during pancreatitis. This upregulation is dependent on calcium and PKC. MIP-2 upregulation requires both NF-κB and AP-1 in these cells. Thus dual inhibition of NF-κB and AP-1 may be a more successful strategy to reduce inflammation in pancreatitis than targeting NF-κB alone.

β-Catenin is Essential for Ethanol Metabolism and Protection Against Alcohol-mediated Liver Steatosis in Mice

The liver plays a central role in ethanol metabolism, and oxidative stress is implicated in alcohol‐mediated liver injury. β‐Catenin regulates hepatic metabolic zonation and adaptive response to oxidative stress. We hypothesized that β‐catenin regulates the hepatic response to ethanol ingestion. Female liver‐specific β‐catenin knockout (KO) mice and wild‐type (WT) littermates were fed the Lieber‐Decarli liquid diet (5% ethanol) in a pairwise fashion. Liver histology, biochemistry, and gene‐expression studies were performed. Plasma alcohol and ammonia levels were measured using standard assays. Ethanol‐fed (EtOH) KO mice exhibited systemic toxicity and early mortality. KO mice exhibited severe macrovesicular steatosis and 5 to 6‐fold higher serum alanine aminotransferase and aspartate aminotransferase levels. KO mice had a modest increase in hepatic oxidative stress, lower expression of mitochondrial superoxide dismutase (SOD2), and lower citrate synthase activity, the first step in the tricarboxylic acid cycle. N‐Acetylcysteine did not prevent ethanol‐induced mortality in KO mice. In WT livers, β‐catenin was found to coprecipitate with forkhead box O3, the upstream regulator of SOD2. Hepatic alcohol dehydrogenase and aldehyde dehydrogenase activities and expression were lower in KO mice. Hepatic cytochrome P450 2E1 protein levels were up‐regulated in EtOH WT mice, but were nearly undetectable in KO mice. These changes in ethanol‐metabolizing enzymes were associated with 30‐fold higher blood alcohol levels in KO mice. Conclusion: β‐Catenin is essential for hepatic ethanol metabolism and plays a protective role in alcohol‐mediated liver steatosis. Our results strongly suggest that integration of these functions by β‐catenin is critical for adaptation to ethanol ingestion in vivo. (HEPATOLOGY 2012;)

Weight Loss in Nonalcoholic Fatty Liver Disease Patients in an Ambulatory Care Setting Is Largely Unsuccessful but Correlates with Frequency of Clinic Visits

Background and Aims Nonalcoholic fatty liver disease (NALFD) is a leading cause of liver disease. Weight loss improves clinical features of NAFLD; however, maintenance of weight loss outside of investigational protocols is poor. The goals of this study were to characterize patterns and clinical predictors of long-term weight loss in ambulatory patients with NAFLD. Methods We retrospectively reviewed 924 non-cirrhotic patients with NAFLD presenting to a liver clinic from May 1st 2007 to April 30th 2013. Overweight and obese patients were counseled on lifestyle modifications for weight loss as per USPSTF guidelines. The primary outcome was percent weight change between the first and last recorded visits: % weight change  =  (weightinitial – weightfinal)/(weightinitial). Baseline BMI and percent BMI change were secondary measures. Predictors of weight loss were determined using logistic regression. Results The mean baseline BMI was 33.3±6.6 kg/m2, and the mean follow-up duration was 17.3±17.6 months. Most patients with NAFLD were in either overweight (26.1%) or class I obesity (30.5%) categories at baseline, while the prevalence of underweight and class III obesity was lower (0.2% and 15.4%, respectively). Overall, there was no change in mean weight or BMI during the follow-up period, and only 183 patients (19.8%) lost at least 5% body weight during the follow up period. Independent predictors of weight loss included number of clinic visits and baseline BMI, and patients with higher baseline BMI required more clinic visits to lose weight. Conclusions Weight loss is largely unsuccessful in NAFLD patients in the ambulatory care setting. Frequent clinical encounters are associated with weight reduction, especially among individuals with high baseline BMI. Future studies are required to define effective weight loss strategies in NAFLD patients.

Risk factors for frequent readmissions and barriers to transplantation in patients with cirrhosis.

Background Hospital readmission rate is receiving increasing regulatory scrutiny. Patients with cirrhosis have high hospital readmissions rates but the relationship between frequent readmissions and barriers to transplantation remains unexplored. The goal of this study was to determine risk factors for frequent readmissions among patients with cirrhosis and identify barriers to transplantation in this population. Methods We retrospectively reviewed medical records of 587 patients with a confirmed diagnosis of cirrhosis admitted to a large tertiary care center between May 1, 2008 and May 1, 2009. Demographics, clinical factors, and outcomes were recorded. Multivariate logistic regression was performed to identify risk factors for high readmission rates. Transplant-related factors were assessed for patients in the high readmission group. Results The 587 patients included in the study had 1557 admissions during the study period. A subset of 87 (15%) patients with 5 or more admissions accounted for 672 (43%) admissions. The factors associated with frequent admissions were non-white race (OR = 2.45, p = 0.01), diabetes (OR = 2.04, p = 0.01), higher Model for End-Stage Liver Disease (MELD) score (OR = 35.10, p<0.0001 for MELD>30) and younger age (OR = 0.98, p = 0.02). Among the 87 patients with ≥5 admissions, only 14 (16%) underwent liver transplantation during the study period. Substance abuse, medical co-morbidities, and low (<15) MELD scores were barriers to transplantation in this group. Conclusions A small group of patients with cirrhosis account for a disproportionately high number of hospital admissions. Interventions targeting this high-risk group may decrease frequent hospital readmissions and increase access to transplantation.