bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting Glycolytic Metabolism in Cancer
Authors: Telang Sucheta , Trent John , Chesney Jason , Mojesky Aubrey
Published: June 2021

Tumors exhibit high glucose uptake and glycolysis even in the presence of oxygen (i.e. the Warburg effect) and this metabolic phenotype serves to satisfy their increased requirement for energy and biosynthetic intermediates. Tumor glucose metabolism is stimulated in part by fructose‐2,6‐bisphosphate (F26BP), which is the product of a family of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatases (PFKFB1–4) and activates a key rate‐limiting enzyme in glycolysis, 6‐phosphofructo‐1‐kinase (PFK‐1). We have found that the PFKFB4 family member is over‐expressed in human tumors and highly induced by HIF‐1α and that silencing PFKFB4 expression markedly reduced F26BP, glucose metabolism, cell survival and the growth of xenograft tumors in vivo. Using virtual screening, we recently discovered a novel small molecule inhibitor of PFKFB4, 5MPN, that selectively inhibits recombinant PFKFB4 activity, and decreases glucose metabolism, proliferation and tumor growth and have now identified a 5MPN derivative that is significantly more effective at decreasing glucose metabolism and proliferation in cancer cells and markedly reduces the growth of established tumors in vivo, without systemic toxicity. Since our previous studies have determined the PFKFB3 family member to be a dominant source of F26BP in cancer cells and additionally have demonstrated co‐expression of the PFKFB4 and PFKFB3 enzymes in multiple patient‐derived cancers, we compared the effects of silencing PFKFB4 and PFKFB3 in human cancer cell lines in vitro. We unexpectedly found that PFKFB4 expression is increased by PFKFB3 silencing, suggesting that PFKFB4 may compensate for decreased PFKFB3 expression and activity. Importantly, an inhibitor of PFKFB3 (PFK158) is currently in Phase I trials and compensation by PFKFB4 may limit the clinical efficacy of this and other PFKFB3 inhibitors. We then examined the effects of simultaneous silencing of PFKFB4 and PFKFB3 on cancer cell lines and observed that co‐knockdown led to a significant decrease in cell viability and glycolysis and a near‐complete abrogation of anchorage independent growth in vitro and, additionally, found that simultaneous small molecule inhibition of the PFKFB4 and PFKFB3 enzymes in vitro resulted in a synergistic decrease in cell viability. Taken together, our data indicate that targeting PFKFB4 may be a valid therapeutic option against cancer and strongly support the further development of PFKFB4 small molecule inhibitors as well as the co‐targeting of PFKFB4 and PFKFB3 as potential strategies for the treatment of cancer.

Support or Funding Information

NIH REACH Program Kentucky Lung Cancer Research Program

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of dietary different energy sources on the growth performance, amino acid profile, blood profile, intestinal morphology and digestive enzyme in weaned piglets
Authors: Lv Dinghong , Xiong Xia , Yang Huansheng , Wang Meiwei , Liu Yanhong , Yin Yulong
Published: June 2021

The low feed intake immediately after weaning is responsible for insufficient energy supply in weaned piglets. We investigated the effects of different dietary energy source on growth performance, serum amino acid profiles, biochemical parameters and immunoglobulin level, antioxidant capacity, and intestinal morphology and enzyme activity in weaned piglets. Forty‐two 21‐day‐old weaned piglets were randomly assigned to one of three treatments. They were fed isocaloric diets supplemented with three energy sources (glucose, soy oil, and glutamine) with two phases (14 and 28 d). Compared with glutamine group, piglets in the glucose and soy oil groups had greater (P < 0.05) average daily gain, average daily feed intake and gain:feed ratio from d 0 to 14, and gain:feed ratio for the overall period. Pigs fed with glutamine had greater (P < 0.05) serum glucose and creatinine on d 14 and high‐density lipoprotein on d 28, compared with pigs in the other two groups. The blood urea nitrogen of pigs was lower (P < 0.05) in soy oil group than in glucose and glutamine groups on d 28. Supplementation of glutamine or soy oil increased (P < 0.05) serum IgA on d 28, while supplementation of glutamine increased (P < 0.05) IgG and IgM on d 14 and 28. No differences were observed in serum amino acids among 3 treatments on d 14, except glycine and tyrosine. The serum concentrations of histidine, serine, threonine, proline, and cysteine in glutamine group was highest (P < 0.05), while the content of glycine and lysine in soy oil group was highest (P < 0.05) in three groups on d 28. Using glucose or soy oil as energy source increased (P < 0.05) serum total superoxide dismutase (T‐SOD) on d 14 and 28, but decreased (P < 0.05) serum malondialdehyde on d 28 compared with glutamine. On d 14, T‐SOD concentration and total antioxidant capacity (T‐AOC) in the duodenum and ileum of pigs supplemented with glutamine were higher (P < 0.05) than those in soy oil group; however, the concentration of T‐AOC in jejunum was lowest (P < 0.05) in the glutamine group on d 14 and 28. Results from the present experiment indicated that weaned pigs supplemented with glucose or soy oil have better growth performance likely due to their enhanced feed intake and better systemic antioxidant capacity, whereas glutamine supplements may increase immunity and intestinal oxidative capacity by modulating nutrition metabolism.

Support or Funding Information

The present study was jointly supported by the National Natural Science Foundation of China (31572420; 31330075)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of Omega‐3 Supplementation on the Lipid Profile and Adipose Indices in Hispanics with Type 2 Diabetes Mellitus
Authors: Jehi Toni , Mota Christy , Beeson Lawrence , Firek Anthony , Cordero‐MacIntyre Zaida , De Leon Marino
Published: June 2021

Background

Diabetes mellitus (DM) is considered a serious affliction that highly augments the risk for serious health complications. Evidence is suggesting a role of n‐3 polyunsaturated fatty acids (PUFAs) on improving the lipid profile and adipose indices in patients with type 2 DM (DM2). However, this association hasn't been probed much in underserved populations. Thus, the objective of this study was to investigate the effect of the diabetes education program, En Balance Plus, on lipid profiles and adipose indices among Hispanic adults with DM2, using supplemental fish oil.

Methods

Subjects (n=40) consumed a daily intake of 2000 milligrams of omega 3 fish oil in capsule form for the whole 3‐month study. At baseline and after the intervention, comprehensive assessments were conducted to evaluate the health status, dietary habits and total and regional body compositions of the subjects. During analysis, non‐parametric tests were utilized to conduct the comparisons.

Results

There was a significant decrease in median glucose levels (−4.5 mg/dL, p=0.021), hemoglobin A1c (−0.1%; p = 0.001), trunk fat (−1%; p = 0.0134), and total fat (−0.50%; p = 0.0452). As for the changes in cholesterol levels, the only significant differences were observed in LDL(reduced by 5 mg/dL; p= 0.011)

Conclusion

The omega‐3 supplementation for Hispanic patients in the En Balance Plus program contributed to significant attenuation in LDL cholesterol, an improvement in glycemic control, and a decrease in trunk and total fat. Further investigations should be conducted to assess the long term effects of this intervention.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of FSH‐SP on inhibition winkle formation, melaningenesis, and promoting skin hydration in UVB‐irradiated HRM‐2 hairless mice
Authors: Lee Dasom , Lee Minhee , Park Soo‐Jeng , Yun Jeong‐Moon , Kim Dakyung , Lee Minjae , Woo Young Ho , Lee Jeongmin
Published: June 2021

Skin is important organ to protect against dangerous external factors including bacteria, chemicals, and UV irradiation. UVB is directly induced destruction in skin epidermis barrier by damaging DNA. Expose to UVB can lead to low skin hydration, winkle formation and melanogenesis. The aim of this study was to investigate the effect of FSH‐SP to protect skin barrier in HRM‐2 hairless mice induced UVB irradiation 150 mJ/cm2 three times a week for 6 weeks. The groups divided PL (FSH‐SP 100 mg/kg b.w.) and PH (FSH‐SP 200 mg/kg b.w.). We examined histopathological experiment about skin damage including changes in skin thickness, melanin pigmentation and winkle formation. Also, we experimented SOD, catalase in plasma and expression of mRNA about procollagens, HAS2 (hyaluronan synthesis 2), MMPs (matrix metalloproteinase) and MITF. The serum SOD and catalase activity were significantly increased PH. The result of procollagen, HAS2 and elastin expression was significantly increased in PH. The gene expression of MMPs (1 and 9) and MITF which is a key factor to synthesis melanin were significantly reduced in PH. The skin hydration exposed to back skin was increased in PH. The winkles, skin thickness and melanin pigment looked like decreased in PL and PH. In conclusion, FSH‐SP decreased the expression of MMPs and MITF increasing the activity of antioxidant enzymes, thereby protecting the skin moisturizing factors and inhibiting melanogenesis. It is believed that effect of the FSH‐SP was reduced to suppress the wrinkle formation, whitening effect and moisturizing.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acetaminophen‐Induced Hepatotoxicity in Wistar Rats Treated with Annona Muricata Aqueous Stem Extract
Authors: Okpara Ifeoma Janet , Nosiri Chidi Ijeoma , chisom madukwe
Published: June 2021

The central role of the liver in biochemical homeostasis cannot be overemphasized and its toxicity is owed greatly to its great involvement in xenobiotic metabolism. This study therefore evaluated the hepatoprotective effect of aqueous stem extract of Anonna muricata S. on Acetaminophen‐induced liver damage. Animals were divided into four groups of five rats. Group1‐ normal saline p.o, Groups 2–4 pretreated with 400mg/kg p.o Acetaminophen, then Groups 3 and 4 treated for 7days with 400mg/kg and 800mg/kg extract p.o respectively. The result revealed an elevated level of serum liver enzymes (ALT 32±1.00, AST 42±2.00) to the untreated Group II rats that received only 400 mg/kg acetaminophen, which at concurrent examination showed a significant decrease in these liver enzymes in Groups III (ALT 29±1.00 and AST 34.67±1.53) and IV (ALT 29±3.00 and AST 34±2.00). The increase seen in the treated groups were not dose dependent but were higher than the control. Group II when compared with the control (ALT 17±1.00 and AST 26±1.04) showed a significant (p≤0.05) increase. The ALP, total protein and bilirubin showed little or no effect in both treated and untreated. Acetaminophen overdose induces hepatotoxicity through its intermediate metabolite N‐acetyl‐p‐benzoquinone imine (NAPQI) which reacts with the natural antioxidant Glutathione reducing its concentration and generating more reactive oxygen species that damage the hepatic cells. These reductions in serum liver enzymes by the extract revealed that the aqueous stem extract of Anonna muricata S. has no toxic effect on the liver and could be hepatoprotective at the tested doses.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Wild yeast strains ferment galactose in whey permeate
Authors: Loughrin Molly R. , Nold Stephen , Delshadi Rana
Published: June 2021

Whey permeate is a by‐product of cheese production that is rich in lactose but currently has little commercial value. Lactose in whey can act as a carbon source for ethanol production by yeast such as Kluyveromyces marxianus, but ethanol yield is typically low due to the inability of most strains to ferment galactose. In this study, we cultivated and characterized wild yeast strains that ferment galactose. Samples were collected from dairy operations and inoculated into enrichment cultures containing whey amended with galactose, then were streaked for isolation on plates to obtain isolates. Wild strains and K. marxianus were tested for their ethanol production efficiency and their ability to utilize different carbon sources. Of the 56 strains we obtained, 22 strains produced as much or more ethanol than K. marxianus and 37 strains fermented galactose as the sole carbon source. We are currently characterizing the optimal growth conditions and suitability for industrial ethanol production of these strains compared to a commercially available yeast. These findings may be useful in our efforts to convert a common dairy waste into ethanol, a useful fermentation product.

Support or Funding Information

University of Wisconsin ‐ Stout

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Group A Streptococcus Prevents Lysosomal Acidification in THP‐1 Macrophages By Inhibiting V‐ATPase Complex Fformation
Authors: Nishioka Scott , Snipper Joshua , Franscioni Henry , Yuen Isabelle , Gao Yu , Delahunty Claire , Yates John , Okumura Cheryl
Published: June 2021

Group A Streptococcus (GAS) is a Gram‐positive bacterial pathogen responsible for approximately 750 million human‐specific infections worldwide every year, ranging in severity from mild pharyngitis to necrotizing fasciitis. To defend against invasive pathogens such as GAS, the innate immune system deploys macrophages to phagocytose and eradicate the bacterial pathogen via engulfment into the phagosome, fusion with lysosomes, phagosomal acidification, and activation of hydrolytic enzymes. We have demonstrated that GAS persists within macrophages despite trafficking normally through the phagocytic pathway. Because GAS is not capable of surviving highly acidic conditions, the data suggest that GAS instead prevents lysosomal acidification. Vacuolar‐ATPase (V‐ATPase), the proton pump on lysosomes responsible for generation of the proton gradient, is a complex of several proteins that must be properly assembled. Here, we investigated disruption of the formation of the V‐ATPase as a possible mechanism employed by GAS to promote intracellular survival. Immunofluorescence data suggest that membrane subunits fundamental to V‐ATPase assembly and function do not localize to the phagolysosomal membrane in GAS‐infected macrophages. Preliminary cell fractionation and mass spectrometry data also indicate that cytosolic components of the V‐ATPase are present in lower quantities in GAS‐infected phagosomes. We are in the process of immunoprecipitating the V‐ATPase complex from GAS‐infected macrophages to further confirm complex formation. The information gained through our investigations provide important insights on the specific mechanisms GAS has evolved to evade the innate immune response, which may contribute toward the development of more effective therapies.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cocaine Enhances the Discriminative Stimulus Effects of Mephedrone
Authors: Erwin Laura , Jursic Branko , Winsauer Peter
Published: June 2021

Mephedrone (4‐methylmethcathinone) is one of the major constituents of “bath salts.” It is a drug with discriminative effects similar to CNS stimulants and it can produce auditory and visual hallucinations, as well as problematic cardiovascular effects. This study compared the discriminative stimulus effects of mephedrone (0.32–10 mg/kg) with other prototypical drugs with CNS effects, such as ketamine (1.8–18 mg/kg), bupropion (5.6–56 mg/kg), and 3,4‐methylenedioxyamphetamine (MDA) (0.32–5.6 mg/kg). In addition, mephedrone was administered in combination with cocaine (5.6–32 mg/kg), which inhibits the reuptake of catecholamines and serotonin. Rats (n=6) were trained to discriminate an intraperitoneal injection of 3.2 mg/kg mephedrone from saline under a fixed‐ratio 20 schedule. Following training, increasing cumulative doses of mephedrone produced dose‐dependent increases in mephedrone‐lever responding, with full substitution considered to be greater than 80% responding and partial substitution to be greater than 50% responding. During substitution tests, ketamine never produced more than 17% drug‐lever responding up to doses that significantly decreased overall response rate. In addition, bupropion and MDA produced 36% and 77% drug‐lever responding, respectively, without significantly decreasing response rate. When 3.2 mg/kg of cocaine was administered prior to increasing doses of mephedrone, there was no substitution for cocaine alone; however, this dose produced a rightward shift in the dose‐effect curve for mephedrone‐lever responding. Unlike this dose, 5.6 mg/kg of cocaine alone did not substitute for mephedrone or shift the mephedrone dose‐effect curve. When the dose of cocaine was increased to 10 mg/kg, there was still no mephedrone substitution, but this dose increased the mephedrone‐lever responding of the low doses of mephedrone. Finally, 18 and 32 mg/kg of cocaine produced 38.1% and 77.7% substitution, respectively, and dose‐dependently increased the substitution of the low doses of mephedrone (0.32 – 1.8 mg/kg). This data suggest that mephedrone may be similar to cocaine in having a higher affinity for the serotonin transorter (SERT) than the dopamine transporter (DAT), but more efficacy at DAT than SERT. Certainly, due to the capacity of cocaine to only enhance the substitution of the low mephedrone doses, the discriminative stimulus effects of the training dose of mephedrone are mediated predominately by dopamine rather than serotonin.

Support or Funding Information

This research was supported by the Louisiana Board of Regents Fellowship.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of Porphyromonas gingivalis Lipopolysaccharide (LPS) on Rat Brain Microglia Classical and Alternative Activation in vitro: MMP‐9 generation
Authors: Czerwonka Evan , Memedovski Zylfi , Han Jin , Mayer Joshua , Klemm Lucas C. , Hall Mary L. , Mayer Alejandro M.
Published: June 2021

Background

The putative association between periodontal disease (PD), a chronic inflammatory disease affecting 64% of adults 65 years and older, and neuroinflammation, a condition which involves microglia, a cell of the brain innate immune system, remains relatively unexplored. We recently reported that lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg), a pathogenic bacteria implicated in periodontitis activates both classical (M1‐type) and alternative (M2‐type) activation of neonatal rat microglia (BMG) and concomitant release of superoxide anion, thromboxane B2, cytokines, and chemokines (Toxicological Sciences Supplement, 150 (1), Abstract # 2974, 2017). We hypothesized that classical activated microglia might also release the pro‐inflammatory matrix metalloproteinase‐9 (MMP‐9).

Methods

P. gingivalis LPS (Pg LPS) was purchased from InvivoGen (San Diego, CA). E. coli LPS (Ec LPS) 026:B6 (from Difco Lab, Detroit, MI) was used as a positive control. BMG were isolated from neonatal rats, and treated in vitro with either Pg LPS or Ec LPS, in a concentration‐dependent manner, for 18 hours at 35.9°C. BMG were identified by confocal microscopy using the surface marker CD11b/c. Matrix metalloproteinase‐9 was determined with a rat total MMP‐9 DuoSet ELISA kit (R&D Systems).

Results

Statistically significant and concentration‐dependent release of pro‐inflammatory MMP‐9 was observed at > than 1ng/mL Ec LPS and 105 ng/mL Pg LPS.

Conclusions

Our results provide support for our working hypothesis, because Pg LPS stimulated BMGs to release pro‐inflammatory MMP‐9 in a time‐ and concentration‐dependent manner. Thus, our current observations extend our studies on the effect of Pg LPS on rat brain microglia activation and contribute to further characterize both periodontitis, and the role of BMG in neuroinflammation and potential neurodegeneration.

Support or Funding Information

Support by College of Dental Medicine, Midwestern University is gratefully acknowledged.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Manganese Exposure Activates NLRP3 Inflammasome Signaling and Propagates Exosomal Release of ASC in Microglial Cells
Authors: Malovic Emir , Sarkar Souvarish , Rokad Dharmin , Luo Jie , Harischandra Dilshan , Jin Huajun , Anantharam Vellareddy , Huang Xuemei , Lewis Mechelle , Kanthasamy Arthi , Kanthasamy Anumantha
Published: June 2021

Abnormal metal homeostasis is a key hallmark of multiple neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Toxicological exposure to manganese (Mn) has been linked to PD‐like symptoms. Sustained neuroinflammation is now recognized as a key pathophysiological process of neurodegenerative conditions, but the inflammatory signaling mechanism underlying Mn neurotoxicity remains equivocal. Recent evidence suggests that the NLRP3 inflammasome, a multiprotein complex, can be activated by various exogenous and endogenous stimuli, leading to a chronic inflammatory response. In this study, we examined whether Mn exerts its neurotoxic effect by activating NLRP3 inflammasome signaling. Exposing LPS‐primed microglial cells to Mn significantly increased NLRP3 expression, caspase‐1 cleavage, and IL‐1β maturation, indicating that Mn can induce the NLRP3 inflammasome activation cascade. Mn exposure compromised the mitochondrial dynamics of microglial cells, as measured via Seahorse Mito Stress assay, and downregulated mitochondrial fusion. Furthermore, Mn reduced the expression of the retromer pathway protein VPS35. CRISPR/Cas9‐based knockdown of VPS35 also downregulated the mitochondrial fusion protein Mfn2 by increasing the mitochondrial ubiquitin ligase Mul1, suggesting a probable role of VPS35 in maintaining mitochondrial health. Given that the inflammasome component ASC can propagate inflammasome activation in a prionic manner, we explored whether Mn exposure induces cell‐to‐cell transfer of ASC via exosomes. Exosomes isolated from Mn‐treated, LPS‐primed microglial cells contained higher amounts of ASC, suggesting exosomal release of ASC. Supporting the role of exosomal ASC in inflammasome propagation, we found that exosomes isolated from microglial cells exposed to Mn were able to induce pro‐IL‐1β and NLRP3 expression. Importantly, we confirmed a significantly higher ASC content in exosomes collected from Mn‐exposed welders when compared to exosomes from matched control subjects. Collectively, these results demonstrate that Mn activates NLRP3 inflammasome proinflammatory signaling and exosomal ASC release from microglia to induce neurotoxicity.

Support or Funding Information

ES026892, NS088206, ES027245, and Endowed Lloyd Chair

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EFFECT OF VESSILEN®, A NEW FORMULATION OF ADELMIDROL AND SODIUM HYALURONATE, ON OVERACTIVE URINARY BLADDER SYNDROME
Authors: Cuzzocrea Salvatore , Peritore Alessio , Crupi Rosalia , Campolo Michela , Siracusa Rosalba , Cordaro Marika , Filippone Alessia , Esposito Emanuela
Published: June 2021

The disruption of coordinated control between the brain, spinal cord and peripheral nervous system caused by spinal cord injury leads to several secondary pathological conditions, including lower urinary tract dysfunctions. In fact, a disability after SCI is urinary dysfunction, which could be attributable to lack of neuroregeneration of supraspinal pathways that control the bladder function. As a consequence, individuals develop overactive urinary bladder (OAB). The aim of the current study was to explore the effects of vessilen®, a new formulation of 2% adelmidrol + 0.1% sodium hyaluronate, in OAB generated after SCI in mice. Mice were treated with vessilen® (100ml/mouse, intravesical) daily for 48 h and 7 days after SCI. Vessilen® reduced significantly mast cells degranulation and down regulated NFκB pathway in the bladder after SCI both at 48h and 7 days. Moreover vessilen® was able to reduce NGF expression suggesting an association between neurotrophins and bladder pressure. Moreover, 7 days after SCI, bladder was characterized by a considerable bacterial infection and proteinuria, surprisingly vessilen® reduced significantly both parameters. These data show the protective roles of vessilen® in OAB following SCI, highlighting a potential therapeutic target for controlling OAB, making a significant impact on improving overall quality of life.

Support or Funding Information

no funding

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hyperlocomotion induced by inhalation of nicotine and a low dose of Δ9‐Tetrahydrocannabinol in female rats
Authors: Paydar Mehrak Javadi , Vandewater Sophia A. , Taffe Michael Javadi
Published: June 2021

Backgrounds

Growing availability of e‐cigarette devices permits users to prepare their own cocktail of drugs for inhalation, including polysubstance inhalation. Some individuals use nicotine and (−)‐Δ9‐tetrahydrocannabinol (THC) in combination by smoking tobacco and cannabis together or in sequence; this practice may extend to electronic cigarette users. THC exerts biphasic effects on locomotion in rats, with high doses (e.g., 5 mg kg−1, i.p.) decreasing, and lower doses (e.g., 0.03 mg kg−1, i.p.) increasing, locomotor activity. Nicotine (NIC) also increases locomotor activity in rodents. This study was designed to determine if interactive effects of THC and NIC result from vapor inhalation in rats using a novel e‐cigarette based procedure.

Methods

Female Sprague‐Dawley rats were exposed to vapor produced by the propylene glycol vehicle (PG), NIC (1 mg/mL in PG), THC (12.5 mg/ml in PG) or a combination of NIC with THC for a duration of 15 minutes. Body temperature and locomotor responses were evaluated post‐inhalation using a radiotelemetry system.

Results

Locomotor activity was increased in the rats for the first 60 min after NIC (1 mg/ml) exposure and this effect was blocked by pretreatment with mecamylamine. Activity was also increased 15 min after initiation of THC vapor compared with PG. Female rats did not show any further hyperlocomotion following exposure to the combination of NIC+THC vapor compared with either NIC or THC vapor inhalation, alone. The body temperature was decreased following the THC (12.5 mg/ml) or a combination of THC (12.5 mg/ml) +NIC (1 mg/ml) inhalation with significant effects starting 120 min post‐exposure, while NIC (1 mg/ml) inhalation did not alter the body temperature.

Discussion

Vapor inhalation of NIC or low dose THC stimulates locomotor activity in female rats. The co‐administration of a lower dose of THC with NIC produced no additive locomotor stimulation in female rats compared to either THC or NIC alone. The observed data provided behavioral evidence to suggest that the effect of nicotine on locomotion might not be modified by the physiological action of cannabinoids.

Support or Funding Information

Funding support provided by USPHS grants AA007456, DA035482 and DA041967

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization of ML321: A Novel and Selective D2 Dopamine Receptor Antagonist with Predicted Atypical Antipsychotic Properties
Authors: Free R. Benjamin , Xiao Jingbo , Ferrer Marc , Southall Noel , Mach Robert , Bertz Jeremiah , Shi Lei , Woods James H. , Marugan Juan J. , Wetsel William , Sibley David R.
Published: June 2021

Dopamine receptors (DARs) belong to the G protein‐coupled receptor (GPCR) superfamily, whose members play critical roles in cellular signaling, especially modulating the transfer of information within the nervous system. Amongst DARs, the D2 dopamine receptor (D2R) is one of the most validated drug targets in neuropsychiatry and there is a strong correlation between the clinical doses of antipsychotics and their potencies for blocking D2Rs. However, current FDA‐approved antipsychotic drugs typically cross‐react with other GPCRs, leading to many serious off‐target side‐effects. A potent and highly selective D2R antagonist would thus be extremely valuable for the treatment of psychosis and related CNS disorders. Using high throughput screening techniques, we have identified, optimized, and characterized a lead D2R antagonist with high selectivity – ML321. In a functional profiling screen of 168 different GPCRs, ML321 showed relatively little reactivity beyond inhibition of the D2R, and to a lesser extent the D3R, demonstrating exceptional GPCR selectivity. Interestingly, we found that the binding of ML321 to the D2R is entirely dependent on Na+. PET imaging studies in non‐human primates demonstrated that ML321 can penetrate the CNS and occupy the D2R in a dose‐dependent manner. Preliminary in vitro ADME studies showed that ML321 is metabolically stable and did not block hERG channels, whereas pharmacokinetic studies in mice demonstrated elevated plasma and brain levels of ML321 for up to 8 hrs. Behavioral paradigms in rats demonstrated that that ML321 can selectivity antagonize a D2R‐mediated response (hypothermia) while not affecting a D3R‐mediated response (yawning) using the same dose of drug, thus demonstrating good in vivo selectivity. We also investigated the effects of ML321 in animal models that are predictive of antipsychotic efficacy in humans. We found that ML321 can attenuate both PCP and amphetamine‐induced locomotor activity and pre‐pulse inhibition (PPI) in a dose‐dependent manner. Importantly, using doses that were maximally effective in the locomotor and PPI studies, ML321 promoted little‐no catalepsy compared with the nonselective antipsychotic haloperidol. This latter observation suggests that ML321 may produce fewer extrapyramidal (motor) side‐effects, a common problem with FDA‐approved antipsychotics. Overall, these results suggest that the ML321 scaffold could serve as a lead compound for the development of an improved therapeutic with greatly reduced side‐effects for treating schizophrenia and other psychotic syndromes.

Support or Funding Information

NINDS IRP

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fibroblast Growth Factor Binding Protein 3 A Novel Target for Glucose Intolerance and Nonalcoholic Fatty Liver Disease Treatment
Authors: Garman Khalid , Tassi Elena , Schmidt Marcel , Wellstein Anton
Published: June 2021

Endocrine Fibroblast Growth Factor 21 (FGF21) has been shown to improve insulin sensitivity, glucose tolerance, and hepatosteatosis in many species. Previous studies have demonstrated that Fibroblast Growth Factor Biding Proteins (FGFBPs) protects FGFs from proteolytic degradation and enhance their binding to their cognate receptors. In our studies, we report that FGFBP3−/− mice suffer insulin resistance and impaired glucose homeostasis, which was further aggravated by diet‐induced obesity. In FGFBP3−/− mice, we further report on the scant liver fat content in lean regular diet mice and the paradoxical high‐fat diet‐induced hepatosteatosis. We show that endogenous FGFBP3 controls liver fat content through its effect on fatty acid synthesis (Fasn, ACAC, ACLY, AACS, ACAS2, and SCD1), fatty acid uptake (SLC27A5), long‐chain fatty acid activation (ACSL1 and ACSL5), and triglyceride synthesis (Dgat1 and Dgat2). In contrast, we demonstrate that exogenous FGFBP3 administration ameliorates hepatosteatosis in ob/ob mice via promoting peroxisomal fatty acid oxidation (EHHADH) and triglyceride secretion (MTTP). We also describe the high‐fat diet‐induced upregulation of hepatic and white adipose tissue FGF21 and KL® in the FGFBP3−/− mice. Our studies indicate a more prominent FGFBP3 effect on the liver compared to white adipose tissue. Collectively, our studies indicate a possible collaboration between FGFBP3 and FGF21 on blood glucose and liver fat content homeostasis.

Support or Funding Information

Supported by NIH grants P01 HL068686 (CSW, AW), R01 CA71508 (AW), P30 CA51008 (AW)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring the Mechanisms by which Small Molecule Nef Inhibitors Suppress HIV‐1 Infectivity
Authors: Staudt Ryan P. , Emert‐Sedlak Lori , Smithgall Thomas E.
Published: June 2021

Nef is an HIV‐1 accessory factor essential for viral pathogenesis and immune escape of HIV‐infected cells. Recently, our group reported the discovery of small molecule Nef antagonists that reverse Nef‐mediated downregulation of MHC‐I and restore the anti‐HIV cytotoxic T cell response. These compounds also inhibit Nef's ability to enhance the inherent infectivity of HIV‐1 virions, although their mechanism of action in this regard is not known. Recent studies have identified the host cell factor SERINC5 (S5) as a suppressor of HIV‐1 infectivity that is countermanded by Nef. Here we investigated whether our Nef inhibitors suppress viral infectivity through a S5‐dependent mechanism. We first confirmed that expression of S5 within viral producer cells results in Nef‐reversible inhibition of HIV‐1 infectivity. For these experiments, we co‐transfected 293T cells with wild‐type and ΔNef HIV‐1 proviral DNA along with various amounts of a S5 expression plasmid. The infectivity of each resulting virus was then quantified using the luciferase‐based HIV‐1 reporter cell line, TZM‐bl. As expected, co‐expression of S5 resulted in suppression of HIV‐1 infectivity. This S5 effect was reduced by Nef in cells producing wild‐type HIV‐1, but not in cells producing the ΔNef virus. Co‐expression of Nef from a separate expression plasmid during the production of the ΔNef virus completely reversed S5 inhibition of viral infectivity, demonstrating Nef dependence in this system. To determine whether our inhibitors block Nef‐enhanced infectivity in a S5‐specific manner, we treated cells producing wild‐type or ΔNef virus in the presence or absence of S5 with either DMSO or the diphenylpyrazolo Nef inhibitor, B9 (3 μM). This compound inhibited the infectivity of wild‐type but not ΔNef HIV‐1 at the producer cell level, consistent with direct action on Nef. However, the effect of the compound was independent of S5 expression, suggesting that mechanisms in addition to S5 may contribute to inhibition of HIV‐1 infectivity by B9 through Nef.

Support or Funding Information

T32 AI65380 (R.P.S.)R01 AI057083, R42 GM112516 (T.E.S.)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitophagy is Required for Proper Mitochondrial Network Remodeling during Cardiac Progenitor Cell Differentiation
Authors: Lampert Mark , Orogo Amabel , Najor Rita , Gustafsson Åsa
Published: June 2021

Cell based therapies represent a very promising strategy to repair and regenerate the injured heart to prevent progression to heart failure. To date, cell based therapies have had limited success due to a lack of survival and retention of the infused cells. Therefore, it is important to increase our understanding of the biology of these cells and utilize this information to enhance their survival and function in the unfavorable environment of the injured heart. Mitochondria are critical for progenitor cell function and survival. Here we demonstrate the importance of mitochondrial autophagy, or mitophagy, in the differentiation process in adult cardiac progenitor cells (CPCs). We found that mitophagy is rapidly induced upon initiation of differentiation in CPCs. We also found that mitophagy was mediated by the mitophagy receptors pathway, rather than the PINK1/Parkin pathway. Mitophagy receptors Nix and Fundc1, but not Bnip3, were upregulated during differentiation. Mitophagy mediated by Nix and Fundc1 was not involved in regulating progenitor cell fate determination, mitochondrial biogenesis, or reprogramming. Instead, mitophagy facilitated the CPCs to undergo proper mitochondrial network reorganization during differentiation. Abrogating Nix/Fundc1‐mediated mitophagy during differentiation led to mitochondrial fragmentation and failure to form an interconnected mitochondrial network. It also led to increased susceptibility to hydrogen peroxide mediated cell death during differentiation. Finally, aging is associated with accumulation of mtDNA mutations in cells and we found that acquiring mtDNA mutations selectively disrupted the differentiation‐activated mitophagy program in CPCs. These findings demonstrate the importance of Nix/Fundc1‐mediated mitophagy as a critical regulator of mitochondrial network formation in differentiating progenitor cells, as well as the consequences of accumulating mtDNA mutations.

Support or Funding Information

A.B. Gustafsson is supported by an AHA Established Investigator Award, and by NIH R21AG052280, R01HL087023, R01HL132300 and P01HL085577. M.A. Lampert is supported by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology grant T32GM007752. A.M. Orogo is supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences T32GM007752, and National Institutes of Health NRSA Predoctoral Fellowship F31HL123309.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Age Differences in the Stimulation of Murine Hepatic XBP‐1 Splicing in Response to the Anti‐HIV Drug Efavirenz
Authors: Hamlin Allyson Naugle , Heck Carley J.S. , Bumpus Namandje N.
Published: June 2021

Efavirenz is a non‐nucleoside reverse transcriptase inhibitor used in combination with other antiretroviral drugs to treat HIV infection. Efavirenz is one of the most commonly prescribed antiretrovirals and is used in both adult and pediatric populations. Efavirenz treatment has been associated clinically with hepatotoxicity; however, the mechanism underlying this is not fully understood and work in this area has largely been focused on adults. With this in mind, using primary mouse hepatocytes, we probed whether the activation status of transcription factors, such as X‐box binding protein‐1 (XBP‐1), that play a crucial role in regulating cell survival are differentially impacted by efavirenz as a function of age. To do so, the ratio of spliced (activated) to unspliced XBP‐1 mRNA was measured following efavirenz treatment. Primary mouse hepatocytes were isolated from pediatric (3–4 week old) and adult (8–12 week old) male C57Bl/6J mice. XBP‐1 splicing following incubation with efavirenz for 4 hours was measured using reverse transcriptase PCR. In 3–4 week old mice, treatment with 10 μM efavirenz for 4 hours resulted in a 3.4‐fold increase in the ratio of spliced to unspliced XBP‐1 as compared to vehicle controls; however, XBP‐1 splicing was not elevated in response to this same treatment in adult mice. In fact, a 30 μM concentration of efavirenz was required in adult mice to stimulate a comparable (3.9‐fold) increase. Because activation of XBP‐1 splicing can affect cell survival, a cell death assay using ethidium bromide and acridine orange was performed and these experiments were confirmed using a TUNEL assay. In primary hepatocytes isolated from 3–4 week old mice, a 4.8‐fold increase in cell death was observed following treatment with 20 μM efavirenz. Similarly, using hepatocytes from adult mice, cell death was stimulated 4‐fold by treatment with 20 μM efavirenz. In the presence of STF080310, a known inhibitor of XBP‐1 splicing, cell death in response to 20 μM efavirenz was reduced to 2.5‐fold and 1.8‐fold over controls in 3–4 week old and adult mice, respectively. These results were commensurate with data obtained using TUNEL staining. In conclusion, hepatocytes from mice at early stages of development may be more sensitive to activation of XBP‐1 splicing than adult mice. Furthermore, cell death was similar in the 3–4 week old mice and adult mice suggesting that age may not result in differential susceptibility to hepatocyte death in response to efavirenz.

Support or Funding Information

This research is funded by NIH R01 GM103853.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gender Differences on the Effects of Ibuprofen on Proteasome Function in Mice Heart
Authors: Tiwari Shuchita , Gomes Aldrin V.
Published: June 2021

Introduction and objectives

Ibuprofen, a cyclo‐oxygenase inhibitor, is a commonly used non‐steroidal anti‐inflammatory (NSAID) agent used in the treatment of pain, fever and inflammation. The long‐term use of ibuprofen is associated with increased risks for cardiovascular events and stroke. The effect of ibuprofen on cardiac function as well as if gender differences exist with respect to ibuprofen's effect on cardiac function are not well understood. Accordingly, the aim of this study was to explore the effects of ibuprofen on protein homeostasis in mice cardiac tissue and to determine if ibuprofen affects male and female cardiac proteasome activity in a similar manner.

Method/Results

Aged matched male and female C57BL/6J mice were randomly assigned to two groups: (A) control (B) group treated with ibuprofen. Mice were treated with ibuprofen (100 mg/kg) dissolved in drinking water for 6 weeks.

Since impaired proteasome function is associated with cardiovascular disorders, β1 caspase‐like activity, β2 trypsin‐like activity, and β5 chymotrypsin‐like proteasome activities were measured using bortezomib as a specific inhibitor of proteasome in cardiac tissue of both male and female mice. In female mice chronic treatment with ibuprofen showed decreased proteasome function as decreased β1, β2, and β5 activity was observed in ibuprofen treated female animals. However, proteasome activity was not affected in male animals treated with ibuprofen. Excess accumulation of misfolded and oxidized proteins alters proteasome function, therefore levels of oxidized proteins in heart lysates was measured by oxyblot method using dinitrophenylhydrazine (DNPH). The levels of oxidized proteins increased in only female animals treated with ibuprofen. The levels of polyubiquitinated proteins was similar in male and female ibuprofen treated hearts compared to control hearts. An increase in free ubiquitin levels was only observed in the hearts of female animals treated with ibuprofen when compared to control animals and ibuprofen treated male animals.

Conclusion

Our findings suggest, for the first time, that gender differences on proteasome function occur in the heart of ibuprofen treated animals. These data show that chronic treatment of ibuprofen decreased proteasome activities in females as well as increased the levels of oxidized proteins and free ubiquitin levels in only ibuprofen treated female mice hearts.

Support or Funding Information

American Heart Association Award #16GRNT31350040

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CCR5 antagonism as a potential therapeutic approach for cardiac fibrosis
Authors: Liang Wenjing , Gorr Matthew W. , Insel Paul A.
Published: June 2021

Fibrosis of the heart occurs in multiple cardiac disorders and is associated with significant morbidity and mortality. Effective therapy to treat and prevent this pathological process remains a major clinical challenge. G protein‐coupled receptors (GPCRs) play a vital role in altering the function of cardiac cells and are thus major therapeutic targets for cardiovascular disorders. We tested the hypothesis that the chemokine receptor CCR5, which is a co‐receptor for HIV infection of immune cells, may contribute to and thus be a novel therapeutic target in cardiac fibrosis. We found prominent CCR5 expression in cardiac fibroblasts isolated from mice and rats. Expression of CCR5 was significantly increased when fibroblasts were treated with hypoxia to induce a myofibroblast phenotype. Cardiac fibroblasts exposed 72 hours to hypoxia increased CCR5 expression 5.8±1.7‐fold compared to normoxia (p=0.0327) in cells isolated from mice, and 4.2±1.1‐fold in cells isolated from rat (p=0.0178). To assess the role for CCR5 in experimental cardiac fibrosis, we used pharmacological CCR5 activation and inactivation in mouse and rat cardiac fibroblasts. Maraviroc, an FDA‐approved antagonist of CCR5 reduced the expression of fibrotic markers, including α‐smooth muscle actin and collagens I and III in mouse and rat cardiac fibroblasts grown under both normoxic and hypoxic conditions. Treatment with recombinant CCL4, the agonist of CCR5, increased these fibrotic markers in rat cardiac fibroblasts. In addition, Maraviroc reduced cardiac fibroblast proliferation and migration while CCL4 had the opposite effect. We conclude that the CCL4‐CCR5 pathway can regulate activity of cardiac fibroblasts and may be a therapeutic target for cardiac fibrosis.

Support or Funding Information

Supported by R21AG52914 and T32HL007444‐34

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ursodesoxycholic acid Display a Role of Experimental Anti‐hepatic Fibrosis by Inhibiting the Expression of Bnip3L Protein
Authors: Teng Lin‐Hua , Liu Qi‐Lin , Zhang Ji‐Wang , Liu Ze‐Feng , Chen Li , Zhang Guo
Published: June 2021

Backgound & Aims

Previous reports showed that ursodesoxycholic acid (UDCA) might has an therapeutic effect on liver fibrosis, but the detail pharmacological mechanism remains unclear. This study was aimed to study the relationship between the expression change of mitochondrial Bnip3L protein and inhibitory role of UDCA for experimental hepatic fibrosis.

Methods

Hepatic stellate cell (HSC)‐LX2 cell line and CCl4‐induced hepatic fibrotic rats model were used in the in vitro or in vivo regimens with UDCA or vehicle control. Western Blot was then performed to analyze the expression of α‐SMA and Bnip3L in LX2 cells. Real‐time fluorescence quantitative PCR (qRT‐PCR) was performed to compare the differential expression of COL1 mRNA in LX2 cells and COL1 mRNA. Bnip3L mRNA in liver tissues among these different groups. The liver fibrosis of rats was evaluated by Masson staining.

Results

Our research showed that the expression of Bnip3L protein was significantly inhibited by UDCA in LX2 cells which were activated by TGF‐β1, and that was positively correlated with the down‐regulated expression of α‐SMA and COL1 mRNA. UDCA could significantly reduce the production of collagen in liver tissues of CCl4‐induced liver fibrosis. In parallel, the expression of Bnip3L mRNA and COL1 mRNA were simultaneously inhibited by UDCA in these liver tissues.

Conclusions

Our findings demonstrate that UDCA displays a critical role of anti‐hepatic fibrosis by down‐regulating the expression of Bnip3L protein, which may provide a novel pharmacological strategy for liver fibrosis in the future.

Support or Funding Information

Supported by NSFC Grants 81360077, 30630145, 81172260 and GXNSFC Grants 2015GXNSFDA139022 to Guo Zhang

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.