Evaluation of association between center colorectal neuroendocrine neoplasm volume and survival among patients with colorectal neuroendocrine carcinoma.

BACKGROUND: Although correlation between center volume and survival has been reported for several complex cancers, it remains unknown if this is true for colorectal neuroendocrine carcinomas (CRNECs). We hypothesized that higher center annual volume of colorectal neuroendocrine neoplasm resections would be associated with overall survival (OS) for patients with CRNECs. METHODS: Patients in the National Cancer Database diagnosed with stages I-III CRNEC between 2006 and 2018 and who underwent surgical resection were identified. The mean annual colorectal neuroendocrine neoplasm resection volume threshold associated with significantly worse mortality hazard was determined using restricted cubic splines. Kaplan-Meier (KM) method was used to compare OS, while Cox proportional hazards model was used for multivariable analysis. RESULTS: There were 694 patients with CRNEC who met inclusion criteria across 1229 centers. Based on the cubic spline, centers treating fewer than one colorectal neuroendocrine neoplasm patient every 3 years on average had worse outcomes. Centers below this threshold were classified as low-volume (LV) centers corresponding with 42% of centers and about 15% of the patient cohort. In unadjusted survival analysis, LV patients had a median OS of 14 months (95% confidence interval [CI]: 10-19) while those treated at HV centers had a median OS of 33 months (95% CI: 25-49). In multivariable analysis, resection at a LV center was associated with increased risk of mortality (1.42 [95% CI: 1.01-2.00], p = 0.04). CONCLUSION: CRNEC patients have a dire prognosis; however, treatment at an HV center may be associated with decreased risk of mortality.

Use of Multimodal Long-Wave Infrared Thermography Devices in Clinical Practice.

Background: The current practice of assessing wounds is highly dependent on visual examination and clinical judgment; these methods are highly subjective and leave great room for error. Objective measures of wound severity and healing are necessary tools that have been lacking in clinical practice. Long-wave infrared thermography (LWIT) has diverse applications that can be optimized to help detect and monitor wounds. Methods: This work is a retrospective case series of pertinent patients encountered by the authors in clinical practice. Results: Nine cases were ultimately selected to best represent the multitude of benefits that can be seen with the utilization of LWIT devices. Conclusions: Through this case series, we show the many advantages of LWIT devices. This technology is safe, noninvasive, and user friendly and, most importantly, gives objective, instant, and repeatable measurements.

Identification of Binding Regions of Bilirubin in the Ligand-Binding Pocket of the Peroxisome Proliferator-Activated Receptor-A (PPARalpha).

Recent work has shown that bilirubin has a hormonal function by binding to the peroxisome proliferator-activated receptor-α (PPARα), a nuclear receptor that drives the transcription of genes to control adiposity. Our previous in silico work predicted three potential amino acids that bilirubin may interact with by hydrogen bonding in the PPARα ligand-binding domain (LBD), which could be responsible for the ligand-induced function. To further reveal the amino acids that bilirubin interacts with in the PPARα LBD, we harnessed bilirubin's known fluorescent properties when bound to proteins such as albumin. Our work here revealed that bilirubin interacts with threonine 283 (T283) and alanine 333 (A333) for ligand binding. Mutational analysis of T283 and A333 showed significantly reduced bilirubin binding, reductions of 11.4% and 17.0%, respectively. Fenofibrate competitive binding studies for the PPARα LBD showed that bilirubin and fenofibrate possibly interact with different amino acid residues. Furthermore, bilirubin showed no interaction with PPARγ. This is the first study to reveal the amino acids responsible for bilirubin binding in the ligand-binding pocket of PPARα. Our work offers new insight into the mechanistic actions of a well-known molecule, bilirubin, and new fronts into its mechanisms.

Bilirubin as a metabolic hormone: the physiological relevance of low levels.

Recent research on bilirubin, a historically well-known waste product of heme catabolism, suggests an entirely new function as a metabolic hormone that drives gene transcription by nuclear receptors. Studies are now revealing that low plasma bilirubin levels, defined as "hypobilirubinemia," are a possible new pathology analogous to the other end of the spectrum of extreme hyperbilirubinemia seen in patients with jaundice and liver dysfunction. Hypobilirubinemia is most commonly seen in patients with metabolic dysfunction, which may lead to cardiovascular complications and possibly stroke. We address the clinical significance of low bilirubin levels. A better understanding of bilirubin's hormonal function may explain why hypobilirubinemia might be deleterious. We present mechanisms by which bilirubin may be protective at mildly elevated levels and research directions that could generate treatment possibilities for patients with hypobilirubinemia, such as targeting of pathways that regulate its production or turnover or the newly designed bilirubin nanoparticles. Our review here calls for a shift in the perspective of an old molecule that could benefit millions of patients with hypobilirubinemia.

Rats Genetically Selected for High Aerobic Exercise Capacity Have Elevated Plasma Bilirubin by Upregulation of Hepatic Biliverdin Reductase-A (BVRA) and Suppression of UGT1A1.

Exercise in humans and animals increases plasma bilirubin levels, but the mechanism by which this occurs is unknown. In the present study, we utilized rats genetically selected for high capacity running (HCR) and low capacity running (LCR) to determine pathways in the liver that aerobic exercise modifies to control plasma bilirubin. The HCR rats, compared to the LCR, exhibited significantly higher levels of plasma bilirubin and the hepatic enzyme that produces it, biliverdin reductase-A (BVRA). The HCR also had reduced expression of the glucuronyl hepatic enzyme UGT1A1, which lowers plasma bilirubin. Recently, bilirubin has been shown to activate the peroxisome proliferator-activated receptor-α (PPARα), a ligand-induced transcription factor, and the higher bilirubin HCR rats had significantly increased PPARα-target genes Fgf21, Abcd3, and Gys2. These are known to promote liver function and glycogen storage, which we found by Periodic acid-Schiff (PAS) staining that hepatic glycogen content was higher in the HCR versus the LCR. Our results demonstrate that exercise stimulates pathways that raise plasma bilirubin through alterations in hepatic enzymes involved in bilirubin synthesis and metabolism, improving liver function, and glycogen content. These mechanisms may explain the beneficial effects of exercise on plasma bilirubin levels and health in humans.

Chronic Ethanol Consumption Alters Glucocorticoid Receptor Isoform Expression in Stress Neurocircuits and Mesocorticolimbic Brain Regions of Alcohol-Preferring Rats.

Evidence suggests the hypothalamic-pituitary-adrenal (HPA) axis is involved in Alcohol Use Disorders (AUDs), which might be mediated by an imbalance of glucocorticoid receptor (GR), GRα and GRß, activity. GRß antagonizes the GRα isoform to cause glucocorticoid (GC) resistance. In the present study, we aimed to investigate the effects of chronic continuous free-choice access to ethanol on GR isoform expression in subregions of the mesocorticolimbic reward circuit. Adult male alcohol-preferring (P) rats had concurrent access to 15% and 30% ethanol solutions, with ad lib access to lab chow and water, for six weeks. Quantitative Real-time PCR (RT-PCR) analysis showed that chronic ethanol consumption reduced GRα expression in the nucleus accumbens shell (NAcsh) and hippocampus, whereas ethanol drinking reduced GRß in the nucleus accumbens core (NAcc), prefrontal cortex (PFC), and hippocampus. An inhibitor of GRα, microRNA-124-3p (miR124-3p) was significantly higher in the NAcsh, and GC-induced gene, GILZ, as a measure of GC-responsiveness, was significantly lower. These were not changed in the NAcc. Likewise, genes associated with HPA axis activity were not significantly changed by ethanol drinking [i.e., corticotrophin-releasing hormone (Crh), adrenocorticotrophic hormone (Acth), and proopiomelanocortin (Pomc)] in these brain regions. Serum corticosterone levels were not changed by ethanol drinking. These data indicate that the expression of GRα and GRß isoforms are differentially affected by ethanol drinking despite HPA-associated peptides remaining unchanged, at least at the time of tissue harvesting. Moreover, the results suggest that GR changes may stem from ethanol-induced GC-resistance in the NAcsh. These findings confirm a role for stress in high ethanol drinking, with GRα and GRß implicated as targets for the treatment of AUDs.

Bilirubin remodels murine white adipose tissue by reshaping mitochondrial activity and the coregulator profile of peroxisome proliferator-activated receptor α.

Activation of lipid-burning pathways in the fat-storing white adipose tissue (WAT) is a promising strategy to improve metabolic health and reduce obesity, insulin resistance, and type II diabetes. For unknown reasons, bilirubin levels are negatively associated with obesity and diabetes. Here, using mice and an array of approaches, including MRI to assess body composition, biochemical assays to measure bilirubin and fatty acids, MitoTracker-based mitochondrial analysis, immunofluorescence, and high-throughput coregulator analysis, we show that bilirubin functions as a molecular switch for the nuclear receptor transcription factor peroxisome proliferator-activated receptor α (PPARα). Bilirubin exerted its effects by recruiting and dissociating specific coregulators in WAT, driving the expression of PPARα target genes such as uncoupling protein 1 (Ucp1) and adrenoreceptor ß 3 (Adrb3). We also found that bilirubin is a selective ligand for PPARα and does not affect the activities of the related proteins PPARγ and PPARδ. We further found that diet-induced obese mice with mild hyperbilirubinemia have reduced WAT size and an increased number of mitochondria, associated with a restructuring of PPARα-binding coregulators. We conclude that bilirubin strongly affects organismal body weight by reshaping the PPARα coregulator profile, remodeling WAT to improve metabolic function, and reducing fat accumulation.

Biliverdin Reductase A (BVRA) Knockout in Adipocytes Induces Hypertrophy and Reduces Mitochondria in White Fat of Obese Mice.

Biliverdin reductase (BVR) is an enzymatic and signaling protein that has multifaceted roles in physiological systems. Despite the wealth of knowledge about BVR, no data exist regarding its actions in adipocytes. Here, we generated an adipose-specific deletion of biliverdin reductase-A (BVRA) (BlvraFatKO) in mice to determine the function of BVRA in adipocytes and how it may impact adipose tissue expansion. The BlvraFatKO and littermate control (BlvraFlox) mice were placed on a high-fat diet (HFD) for 12 weeks. Body weights were measured weekly and body composition, fasting blood glucose and insulin levels were quantitated at the end of the 12 weeks. The data showed that the percent body fat and body weights did not differ between the groups; however, BlvraFatKO mice had significantly higher visceral fat as compared to the BlvraFlox. The loss of adipocyte BVRA decreased the mitochondrial number in white adipose tissue (WAT), and increased inflammation and adipocyte size, but this was not observed in brown adipose tissue (BAT). There were genes significantly reduced in WAT that induce the browning effect such as Ppara and Adrb3, indicating that BVRA improves mitochondria function and beige-type white adipocytes. The BlvraFatKO mice also had significantly higher fasting blood glucose levels and no changes in plasma insulin levels, which is indicative of decreased insulin signaling in WAT, as evidenced by reduced levels of phosphorylated AKT (pAKT) and Glut4 mRNA. These results demonstrate the essential role of BVRA in WAT in insulin signaling and adipocyte hypertrophy.

Bilirubin Nanoparticles Reduce Diet-Induced Hepatic Steatosis, Improve Fat Utilization, and Increase Plasma ß-Hydroxybutyrate.

The inverse relationship of plasma bilirubin levels with liver fat accumulation has prompted the possibility of bilirubin as a therapeutic for non-alcoholic fatty liver disease. Here, we used diet-induced obese mice with non-alcoholic fatty liver disease treated with pegylated bilirubin (bilirubin nanoparticles) or vehicle control to determine the impact on hepatic lipid accumulation. The bilirubin nanoparticles significantly reduced hepatic fat, triglyceride accumulation, de novo lipogenesis, and serum levels of liver dysfunction marker aspartate transaminase and ApoB100 containing very-low-density lipoprotein. The bilirubin nanoparticles improved liver function and activated the hepatic ß-oxidation pathway by increasing PPARα and acyl-coenzyme A oxidase 1. The bilirubin nanoparticles also significantly elevated plasma levels of the ketone ß-hydroxybutyrate and lowered liver fat accumulation. This study demonstrates that bilirubin nanoparticles induce hepatic fat utilization, raise plasma ketones, and reduce hepatic steatosis, opening new therapeutic avenues for NAFLD.

Loss of hepatic PPARα promotes inflammation and serum hyperlipidemia in diet-induced obesity.

Agonists for PPARα are used clinically to reduce triglycerides and improve high-density lipoprotein (HDL) cholesterol levels in patients with hyperlipidemia. Whether the mechanism of PPARα activation to lower serum lipids occurs in the liver or other tissues is unknown. To determine the function of hepatic PPARα on lipid profiles in diet-induced obese mice, we placed hepatocyte-specific peroxisome proliferator-activated receptor-α (PPARα) knockout (PparaHepKO) and wild-type (Pparafl/fl) mice on high-fat diet (HFD) or normal fat diet (NFD) for 12 wk. There was no significant difference in weight gain, percent body fat mass, or percent body lean mass between the groups of mice in response to HFD or NFD. Interestingly, the PparaHepKO mice on HFD had worsened hepatic inflammation and a significant shift in the proinflammatory M1 macrophage population. These changes were associated with higher hepatic fat mass and decreased hepatic lean mass in the PparαHepKO on HFD but not in NFD as measured by Oil Red O and noninvasive EchoMRI analysis (31.1 ± 2.8 vs. 20.2 ± 1.5, 66.6 ± 2.5 vs. 76.4 ± 1.5%, P < 0.05). We did find that this was related to significantly reduced peroxisomal gene function and lower plasma ß-hydroxybutyrate in the PparaHepKO on HFD, indicative of reduced metabolism of fats in the liver. Together, these provoked higher plasma triglyceride and apolipoprotein B100 levels in the PparaHepKO mice compared with Pparafl/fl on HFD. These data indicate that hepatic PPARα functions to control inflammation and liver triglyceride accumulation that prevent hyperlipidemia.

CRISPR Cas9-mediated deletion of biliverdin reductase A (BVRA) in mouse liver cells induces oxidative stress and lipid accumulation.

Obesity is the predominant cause of non-alcoholic fatty liver disease (NAFLD), which is associated with insulin resistance and diabetes. NAFLD includes a spectrum of pathologies that starts with simple steatosis, which can progress to non-alcoholic steatohepatitis (NASH) with the commission of other factors such as the enhancement of reactive oxygen species (ROS). Biliverdin reductase A (BVRA) reduces biliverdin to the antioxidant bilirubin, which may serve to prevent NAFLD, and possibly the progression to NASH. To further understand the role of BVRA in hepatic function, we used CRISPR-Cas9 technology to target the Blvra gene in the murine hepa1c1c7 hepatocyte cell line (BVRA KO). BVRA activity and protein levels were significantly lower in BVRA KO vs. wild-type (WT) hepatocytes. Lipid accumulation under basal and serum-starved conditions was significantly (p < 0.05) higher in BVRA KO vs. WT cells. The loss of BVRA resulted in the reduction of mitochondria number, decreased expression of markers of mitochondrial biogenesis, uncoupling, oxidation, and fusion, which paralleled reduced mitochondrial oxygen consumption. BVRA KO cells exhibited increased levels of ROS generation and decreased levels of superoxide dismutase mRNA expression. In conclusion, our data demonstrate a critical role for BVRA in protecting against lipid accumulation and oxidative stress in hepatocytes, which may serve as a future therapeutic target for NAFLD and its progression to NASH.

RNA sequencing in human HepG2 hepatocytes reveals PPAR-α mediates transcriptome responsiveness of bilirubin.

Bilirubin is a potent antioxidant that reduces inflammation and the accumulation of fat. There have been reports of gene responses to bilirubin, which was mostly attributed to its antioxidant function. Using RNA sequencing, we found that biliverdin, which is rapidly reduced to bilirubin, induced transcriptome responses in human HepG2 hepatocytes in a peroxisome proliferator-activated receptor (PPAR)-α-dependent fashion (398 genes with >2-fold change; false discovery rate P < 0.05). For comparison, a much narrower set of genes demonstrated differential expression when PPAR-α was suppressed via lentiviral shRNA knockdown (23 genes). Gene set enrichment analysis revealed the bilirubin-PPAR-α transcriptome mediates pathways for oxidation-reduction processes, mitochondrial function, response to nutrients, fatty acid oxidation, and lipid homeostasis. Together, these findings suggest that transcriptome responses from the generation of bilirubin are mostly PPAR-α dependent, and its antioxidant function regulates a smaller set of genes.

Loss of biliverdin reductase-A promotes lipid accumulation and lipotoxicity in mouse proximal tubule cells.

Obesity and increased lipid availability have been implicated in the development and progression of chronic kidney disease. One of the major sites of renal lipid accumulation is in the proximal tubule cells of the kidney, suggesting that these cells may be susceptible to lipotoxicity. We previously demonstrated that loss of hepatic biliverdin reductase A (BVRA) causes fat accumulation in livers of mice on a high-fat diet. To determine the role of BVRA in mouse proximal tubule cells, we generated a CRISPR targeting BVRA for a knockout in mouse proximal tubule cells (BVRA KO). The BVRA KO cells had significantly less metabolic potential and mitochondrial respiration, which was exacerbated by treatment with palmitic acid, a saturated fatty acid. The BVRA KO cells also showed increased intracellular triglycerides which were associated with higher fatty acid uptake gene cluster of differentiation 36 as well as increased de novo lipogenesis as measured by higher neutral lipids. Additionally, neutrophil gelatinase-associated lipocalin 1 expression, annexin-V FITC staining, and lactate dehydrogenase assays all demonstrated that BVRA KO cells are more sensitive to palmitic acid-induced lipotoxicity than wild-type cells. Phosphorylation of BAD which plays a role in cell survival pathways, was significantly reduced in palmitic acid-treated BVRA KO cells. These data demonstrate the protective role of BVRA in proximal tubule cells against saturated fatty acid-induced lipotoxicity and suggest that activating BVRA could provide a benefit in protecting from obesity-induced kidney injury.