United States

Background: The adipocyte-derived hormone leptin is a key mediator in homeostatic appetite regulation and satiety via the arcuate nucleus (ARC) of the hypothalamus. Interestingly, serotonin (5-HT) produced in the dorsal raphe nucleus (DRN) modulates anorectic effects of its own in the ARC, yet the underlying mechanism through which 5-HT acts in tandem with leptin remains inconclusive. Our lab has identified leptin-sensitive serotonergic neurons in the DRN that project to the ARC, and has demonstrated that activation of this circuit significantly decreases food intake. Through the unique co-expression of leptin receptors and 5-HT within these neurons, the current study aimed to elucidate the role of 5-HT in leptin’s regulation of food intake between the DRN and ARC.

Methods: To examine the role of 5-HT on leptin’s action in the DRN, adult male Sprague Dawley rats underwent stereotaxic surgery for guide cannula implantation in the DRN. After recovery, rats received 100 μg of intra-raphe p-chlorophenylalanine (PCPA), an inhibitor of 5-HT synthesis, each day for four days. On the fourth day, an additional injection of leptin was administered in the DRN (5 μg/rat) and food intake was measured over a 24-hour time course. To examine the role of 5-HT on leptin’s hypothalamic action, a subsequent experiment was conducted by implanting an additional cannula into the ARC for the administration of leptin on the fourth day of treatment. Post-mortem, DRN tissue sections were immunolabeled to confirm 5-HT depletion and visualize leptin-sensitive serotonergic neurons.

Results: 24-hour food intake analyses across both experiments revealed only vehicle-treated rats administered leptin exhibited a significant decrease in food intake compared to controls, an effect not observed in PCPA-treated rats. These results suggest depletion of 5-HT within the DRN attenuated leptin’s ability to decrease food intake in both the DRN and ARC. 5-HT immunolabeling confirmed PCPA significantly decreased 5-HT in the DRN of all treated rats and displayed co-localization of 5-HT and phosphorylated-STAT3 (a marker for leptin receptor activation) in DRN neurons.

Conclusion: Depleting DRN-derived 5-HT diminishes leptin’s action on hypothalamic and DRN leptin receptors, suggesting 5-HT is a regulator of leptin-mediated food intake control within this novel neuronal circuit.

AMA Research Challenge 2022

Endocrinology - Serotonin, Leptin, and a Novel Neuronal Circuit: Rewiring Central Appetite Regulation As We Know It - Endocrinology

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TITLE: EFFECTS OF ADENOSINE ON LIPID ACCUMULATION IN A HUMAN HEPATOMA CELL CULTURE MODEL WHEN CHALLENGED WITH EXCESS FATTY ACID


Authors: Michael Brown B.S., School of Medicine, New York Medical College MS2
Dr. Robin DaSilva, PhD, Department of Food Science and Human Nutrition, University of Florida
Background:

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent liver disorder in western populations and is rapidly becoming one of the most common indications for orthotopic liver transplantation in the world. Fully understanding lipid metabolism in the liver is necessary for further understanding the progression of NAFLD to non-alcoholic steatohepatitis (NASH) and in finding an alternative treatment to weight loss for those diagnosed with NAFLD.

Design/Methods:

HepG2 cells treated with oleic acid (OA) were used as a model of liver cell lipid accumulation.
Methionine, choline and purines are important metabolites and nutrients that are dysregulated in
NAFLD, so we used methionine and choline deficiency (MCD) and excess purines in cultured
HepG2 cells to assess the influence of these conditions.

Cell Culture
Quantitative real-time polymerase chain reaction
Western Blot
TG Assay

Statistics:
Analysis for statistics and all figures were made using the software Prism (GraphPad). A one
way-ANOVA was used to test for statistical significance. A P-value less than 0.05 was taken as
a significant difference.

Results:
MCD HepG2 cells had fewer total triglycerides and had higher expression of carnitine palmitoyltransferase 1 a (CPT-1a) and PPARa, genes involved in b-oxidation, as compared to control cells. MCD cells treated with extracellular adenosine or inosine with OA lowered phosphorylated-acetyl-CoA carboxylase (p-ACC). Adenosine was found to affect lipid metabolism through its role in activating AMP-activated protein kinase (AMPK) in the AMPK-ACC-CPT 1 pathway.
Conclusions:
HepG2 cells under a methionine and choline-deficient condition increased fatty acid oxidation and prevented cells from lipid-loading after treatment with exogenous fatty acids. The addition of excess purine molecules exacerbated lipid-loading in HepG2 cells in all conditions by decreasing fatty acid oxidation and potentially by increasing fatty acid synthesis. Impaired adenosine production in the MCD condition may reduce the inhibition of fatty acid synthesis and result in enhanced lipid oxidation.


Effects of Adenosine on Lipid Accumulation in a Human Hepatoma Cell Culture Model When Challenged with Excess Fatty Acid

With the increasing prevalence of type 2 diabetes and non-alcoholic fatty liver disease (NAFLD), there is still an unmet need to better treat hyperglycemia and hyperlipidemia. Isthmin (Ism1) is a recently identified adipokine that promotes glucose uptake in adipocytes while suppressing lipid production in hepatocytes. The mechanism by which Ism1 controls lipid metabolism is still unknown but the data show that Ism1 treatment induces a signaling pathway that counteracts lipid production, which could be directly related to its function in reversing hepatic steatosis. Different mechanisms are known to be responsible for the development of hepatosteatosis, including enhanced de novo lipogenesis, impaired fatty acid oxidation or impaired lipid transport. The hypothesis is that Ism1 signaling affects one or several of these mechanisms to control lipid metabolism and reduce hepatosteatosis. The objective of this research was to identify the different lipid species that are controlled by Ism1 treatment or are altered in livers from mice lacking the Ism1 gene, to better understand which of the lipid pathways are regulated by Ism1. To do so, we used a NAFLD mouse model in which mice were pharmacologically treated with Ism1 or in which Ism1 was genetically knocked out. We quantified the liver lipid content by mass spectrometry. We observed that Ism1 treatment and Ism1 loss of function impacted the quantity of many lipids in NAFLD mice liver including diglycerides, triglycerides, and fatty acids, consistent with a major role for Ism1 in regulating de novo lipogenesis. Although Ism1’s mechanism of action needs to be identified, these results suggest that Ism1 may have a therapeutic potential to treat NAFLD.

Isthmin Suppresses Global Hepatic Lipid Synthesis as Revealed by Untargeted Lipidomics of Murine Livers

Sex Specific Differences in Regulation of Estrogen Receptor 𝛣 Signaling in Ulcerative Colitis using Latent Variable Meta-Modeling

Background
Patients with inflammatory bowel disease (IBD) are more susceptible to mental health problems than the general population. The prevalence of psychiatric diagnoses in patients with IBD is lacking in India.
Methods
We ascertained the presence of anxiety, depression, or perceived stress disorder among Indian patients with IBD [Ulcerative colitis (UC) or Crohn's disease(CD)] using the Hospital Anxiety and Depression Scale and Perceived Stress Scale. Patients with prior anxiety, depression, or post-traumatic stress disorder were excluded to create the study cohort.
Results
A total of 318 patients with IBD (mean age 40.13±12.06 years, 168 (52.82%) males) were asked to fill out the questionnaire. Most of the enrolled patients had UC (n=255, 80.2%). The prevalence of anxiety and depression was 14.15% and 12.57%, respectively. According to the perceived stress scale, 58.2%, 36.8%, and 5% of patients had low, moderate, and high stress, respectively. Upon subgroup analysis, the prevalence of anxiety and depression were 12.94% and 12.15% in patients with UC and 19.04% and 14.28% in patients with CD, respectively. Patients with CD had a higher perception of stress as compared to patients with UC.
Conclusion
The prevalence of anxiety, depression, and perceived stress is high among Indian patients with IBD.

The Prevalence of Anxiety, Depression, and Stress in a Cohort of Indian patients With Inflammatory Bowel

A Glycan-based Approach to Cell Characterization & Purification

Introduction
Achyrocline bogotensis is a member of the Asteraceae family of flowering plants. While the Asteraceae family is well known for food crops (lettuce), ornamentals (chrysanthemum), or common weeds (dandelion); A. bogotensis, itself, is know as ‘vira vira’ (the plant of life). Found in the Andean region of South America, A. bogotensis has been used ethnomedically for thousands of years. Ground into a poultice or steeped to make tea, A. bogotensis has been said to help heal wounds, fight infections, reduce inflammation, and even possess anti-neoplastic qualities. Previous studies from our lab have shown that one compound isolated from A. bogotensis, 3,5 dihydroxy-7-methoxyflavone (Achyrocline B, Flavone B, FB), is cytotoxic to colorectal and pancreatic cancer cell lines1,2. 
Surgically unresectable colorectal and pancreatic carcinomas have a high rate of mortality as current treatment options are limited. One common chemotherapeutic used to treat these cancers is 5-flurouracil (5-Fu); however, treatment typically only serves to slow the progression of the disease and comes with many side effects due to 5-Fu’s intrinsic toxicity3-6. Thus, strategies to decrease the amount of 5-Fu used in treatment as well as reduce its off-target toxicity are paramount not only to better treatment of colorectal and pancreatic carcinomas, but also to improve patient quality of life. 

Methods
Viability after treatment was measured in various colorectal and pancreatic cancer cell lines by MTT. Synergy analysis was performed using CompuSyn software. Results were supported by analysis of cellular lysates for apoptotic proteins by western. Xenograft implantation was done using HCT116 into nude mice. Mice were treated 3/week for six weeks with either saline, Achyrocline B + 5-Fu, or 5-Fu alone. Tissue was either flash frozen for western or paraformaldehyde fixed, paraffin embedded, and then analyzed using H&E. All statistical analyses was performed using GraphPad Prism.

Results
Synergy analysis revealed that cotreatment with Achyrocline B and 5-Fu was synergistic and as efficacious as a dose of 5-Fu four-fold higher in multiple colonic and pancreatic cell lines. These results were supported by western analysis of apoptotic proteins. Off target toxicity models showed an opposite profile in which less loss of viability and apoptosis was seen. In our xenograft model, Achyrocline B was shown to increase apoptosis in tumors leading to degeneration of the outer tumoral boundary. Furthermore, in 5-Fu treated animals it was found that Achyrocline B provided protection on both a histological and cellular level. 

Conclusion
Our studies show that Achyrocline B efficiently allows for 5-Fu dose reduction and has dual actions: 1) It works synergistically with 5-Fu to kill cancer cells in vitro and in vivo and 2) protects tissue in vivo from the toxicity of 5-Fu. These results show promise for a new adjuvant therapy for colorectal and pancreatic carcinomas that not only reduces tumor progression, but more importantly has the potential to improve patient quality of life. 


Achyrocline B (3,5 dihydroxy-7-methoxyflavone) synergizes with 5-fluorouracil (5-Fu) allowing for 5-Fu dose reduction and reduced off-target toxicity in the treatment of colonic and pancreatic cancers

Background
In recent years, glycobiology has garnered increasing interest in relation to oncology research. Glycans are complex carbohydrates on the surface of most eukaryotic cells and have been found to contribute to cancer tumorigenesis and metastasis. Patterns of aberrant glycan expression, such as increased sialylation and fucosylation, have been proposed as potential novel biomarkers of cancer. However, to our knowledge, there is little research examining the differences in glycosylation between malignant and surrounding benign tissues that can be applied in a clinical setting. This study examines the N-linked glycan expression and composition in samples of cancerous and surrounding healthy tissue of breast cancer patients, with the aim of establishing signature glycosylation patterns between the two tissue types. 

Methods
Thirty-nine samples of malignant and surrounding benign tissue from breast cancer patients were collected from the Prisma Health Biorepository. The samples included specimens from breast cancer staged I through IV at the time of the patients’ diagnosis. The samples were analyzed using MALDI-TOF mass spectroscopy for the presence of glycans by Emory Glycomics and Molecular Interactions Core. The known masses of sixty-one unique glycans were then compared to the spectra of the samples analyzed to determine the type of glycans present. The relative amount of each glycan present was determined using the area under the curve in the spectral data.

Results
The means of the area under the curve for high-mannose glycans in the malignant and normal tissue samples were 247.18 and 163.6 respectively. The t-test reveals a significant difference in the expression of high-mannose glycans between the two tissue types (p-value, 0.0138).

Conclusion
The results of this data confirm a higher expression of high-mannose glycans to be a potential biomarker for malignant tissue in breast cancer. Future research could include expanding the sample size and comparing the glycan composition of the samples to a library specific for high-mannose type glycans, with the goal of establishing a signature high-mannose expression pattern. This could lead to important clinical applications such as being a helpful distinction in obtaining negative margins during tumor resection, as well as providing a more refined diagnostic method and more informed treatment decisions. 




Analysis of Glycan Expression in Malignant versus Adjacent Benign Tissue in Breast Cancer Patients

In our previous work, we identified clustering of kidney cancers based on differential metabolic pathway activation, and an association between metabolic clustering and survival in renal cell carcinoma. Here we further study clear cell renal carcinoma, papillary cell renal carcinoma and chromophobe renal cell carcinoma by applying machine learning models to pinpoint specific metabolic reactions, and their master regulators, that best explain differences in survival in these cancers. We constructed a cohort of 941 patients in The Cancer Genome Atlas (TCGA) diagnosed with kidney cancer (project codes KIPR [papillary cell RCC], KIRC [clear cell RCC] and KICH [chromophobe RCC]). We used tumor RNA-Seq data to construct reaction activity scores (RAS) using metabolic genes according to the HMRcore rules and taking into account enzymatic subunits and multiple different genes coding for proteins that catalyze the same reaction. We then used a machine learning model to estimate survival as a function of metabolic pathways, age and gender. By using Cox regularization with elastic-net penalty we were able to simultaneously perform variable selection and estimation of survival in presence of a large number of regressors. Using master regulatory analysis, we estimated activity of key transcription factors and then used a multinomial model with elastic net penalty to identify those key transcription factors that have the greatest impact on dysregulation of tumor metabolic pathways in renal cell carcinomas. Using glucose transport as a known hallmark of renal cell carcinoma, we identified 19 metabolic reactions that are better predictors of survival than glucose transport in our machine learning model. The metabolic reactions we identified fit into larger categories that include folate and protein metabolism, as well as beta-oxidation. These are consistent with metabolic changes induced by mTOR, and it is notable that inhibitors of mTOR are already used in the clinic for treatment of renal cell carcinoma. Our research raises the question of whether effectiveness of mTOR inhibitors can be increased by amplifying the effects of mTOR inhibition on oncogenesis through targeting of related master regulators of metabolism.

Analysis of Metabolic Profile and Survival Data in Renal Cancer

Multiple myeloma (MM) is a plasma cell malignancy that grows in the bone marrow and is characterized by severe bone destruction. The growth of MM cancer cells in the bone marrow increases osteoclasts, bone-resorbing cells, which create debilitating bone lesions. MM cells also decrease osteoblasts, bone-forming cells, which reduces bone formation. Despite recent advances in anti-MM therapy, MM patients frequently relapse and exhibit high fracture incidence even during disease remission. Our lab showed that inhibition of Notch, a pathway regulating cell-to-cell communication in the tumor niche, with a bone-targeted Notch inhibitor (BT-GSI) decreases tumor growth and bone resorption without inducing toxicity. We also found that pharmacologic inhibition of Sclerostin (Scl-Ab), a potent inhibitor of Wnt signaling and osteoblast function, increases bone formation without affecting tumor growth. In this study, we hypothesized that co-administration of BT-GSI and Scl-Ab simultaneously decreases tumor burden and improves bone health by inhibiting bone resorption and increasing bone formation. 6-week-old immunocompetent mice were injected intratibially with 105 murine 5TGM1 myeloma cells. After four weeks, mice with MM were randomized to groups receiving vehicle, Scl-ab (100mg/Kg/1x/wk), BT-GSI (2.5mg/Kg/3x/wk), or Scl-ab+BT-GSI combined for four additional weeks. Longitudinal micro-CT was performed every four weeks to assess bone mass and microarchitecture. Alizarin Red and Calcein fluorescent dyes were injected 5 and 2 days, respectively, before sacrifice. Bone dynamic and static histomorphometry analyzed fluorescent labels and osteoclasts present via TRAP staining using OsteoMeasure. Mice treated with Scl-Ab therapy exhibited similar tumor burden levels than mice receiving vehicle injections. In contrast, mice treated with BT-GSI, either alone or in combination with Scl-ab, exhibited decreased tumor burden compared to vehicle-treated mice. Mice receiving vehicle injections had decreased bone mass compared to control naive mice. All three therapies significantly increased bone mass compared to vehicle mice. There was no organ toxicity with any of the therapies. We performed static and dynamic histomorphometric analysis to determine the mechanism underlying the skeletal protective effects. Vehicle-treated mice had decreased bone formation rate (BFR) compared to naïve mice, although not statistically significant. Scl-ab therapy increased BFR by 28%, bringing it back to naïve levels. BT-GSI therapy did not affect BFR, which remained decreased compared to naïve mice. Co-administration of BT-GSI and Scl-ab also restored BFR to naive mice levels. Vehicle-treated mice had a five-fold increase in osteoclast number corrected by bone surface (Oc.N/BS) compared to naïve mice. Scl-ab and BT-GSI induced a 39% and 75% decrease in Oc.N/BS compared to MM-bearing mice receiving vehicle, respectively. Co-administration of BT-GSI and Scl-ab also resulted in a 75% reduction in Oc.N/BS compared to MM mice, bringing Oc.N/BS back to levels found in naïve mice. BT-GSI + Scl-ab combination therapy decreases tumor burden and improves bone health by decreasing osteoclasts and increasing the bone formation rate. BT-GSI + Scl-ab combination therapy is more effective than these therapies alone in stopping tumors and repairing damaged bone. Because this bone-targeted multimodal therapy lacks toxicity, it provides a new therapeutic avenue to control MM growth and improve bone health in MM patients.

Bone-targeted combination therapy decreases tumor progression and restores bone mass by increasing bone formation in mice with established multiple myeloma

Lung cancer is the leading cause of cancer-related death in both men and women in the United States. Non-small cell lung cancer is the most prevalent and includes adenocarcinoma (AD), and squamous cell lung cancers. The unique cellular biology of these cancers could potentially allow for serological detection and identification via tissue specific protein markers. Galectins are a group of β-galactoside binding proteins which are known to be alternatively expressed in cancers and are being studied for their biomarker potential. Galectin-7 is expressed in epithelial tissues as well as in all layers of the epidermis as it is normally highly expressed by squamous cells. We evaluated the potential for galectins to be used as a serum biomarkers and delineators of two non- small cell lung cancer subtypes.
The serum concentrations of galectin-7 were measured in 85 lung cancer patients using ELISA. Forty-eight samples were from adenocarcinoma patients and 37 were from squamous cell carcinomas patients. The serum galectin-7 concentrations in cancer patients were compared to healthy controls and by their histological subtypes. Oneway analysis was performed using Tukey-Kramer HSD for pairwise comparisons.
Galectin-7 concentrations were significantly elevated in serum samples from squamous cell lung cancer patients (mean, 2.45 ng/mL) relative to healthy controls (N, 26; mean, 1.16 ng/mL; p- value, 0.0010) and relative to adenocarcinoma lung cancer patients (mean, 1.47 ng/mL; p-value, 0.0040).
These results show that serum galectin-7 is increased in patients with squamous cell lung cancer compared to adenocarcinoma lung cancer. Further benchwork would include (1) measuring serum concentrations of galectin-7 by stage of squamous cell lung cancer, (2) identifying concentrations of serum galectin-7 in other lung cancer subtypes, and (3) in other non-pulmonary squamous cell carcinomas such as skin, cervical, and head and neck. Potential clinical applications of this work would include the measurement of galectin-7 in blood samples of cancer patients to aid in cancer screening and tumor type identification.

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Effects of Adenosine on Lipid Accumulation in a Human Hepatoma Cell Culture Model When Challenged with Excess Fatty Acid

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