bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of the OVLT in the Development of DOCA‐salt Hypertension
Authors: Collister John P , Nahey David B , Banek Christopher T , Osborn John W
Published: November 2023

Lesions of the anteroventral third ventricle (AV3V region) are known to prevent many forms of experimental hypertension, including mineralocorticoid (DOCA‐salt) hypertension in the rat. However, AV3V lesions include the organum vasculosum of the lamina terminalis (OVLT) and portions of the median preoptic nucleus, as well as efferent fibers from the subfornical organ (SFO), thereby limiting the ability to define the individual contribution of these structures to the prevention of experimental hypertension. Having previously reported that the SFO does not play a significant role in the development of DOCA‐salt hypertension, the present study was designed to test the hypothesis that the OVLT is necessary for DOCA‐salt hypertension in the rat. In uninephrectomized OVLT lesioned (OVLTx; n = 4) and SHAM (n = 3) Sprague‐Dawley rats consuming a 2% NaCl diet and 0.9% NaCl drinking solution, 24‐hour mean arterial pressure (MAP) was recorded telemetrically 5 days before and 21 days after DOCA implantation (100 mg/rat; SQ). No differences in control MAP were observed between groups. The chronic pressor response to DOCA was attenuated in OVLTx rats such that MAP increased to 132±4 mmHg in SHAM rats by Day 21 of DOCA compared to 123±4 mmHg in OVLTx rats. These results support the hypothesis that the OVLT is an important brain site of action for the pathogenesis of DOCA‐salt hypertension in the rat.

Support or Funding Information

University of Minnesota Academic Health Center Grant 15.05
Compartment‐Specific Effects of Alveolar Heparan Suflate Shedding after Intratracheal Lipopolysaccharide
Authors: Haeger Sarah , Yang Yimu , Ford Joshay , Zhang Fuming , Suflita Matt , Dedent Alison , Zemans Rachel , Tuder Rubin , Schmidt Eric
Published: November 2023

Acute lung injury can occur as a consequence of direct (pulmonary) insults that target the alveolar epithelium or indirect (extrapulmonary) insults that injure the alveolar endothelium. During indirect lung injury, the endothelial glycocalyx is degraded, releasing highly‐sulfated heparan sulfate (HS) octasaccharides into the vasculature. In contrast to indirect lung injury, it is unknown whether direct lung injury induces release of HS from the apical surface of the damaged alveolar epithelium or the luminal endothelial glycocalyx. We hypothesized that during direct lung injury HS would be shed from injured epithelium, and aimed to determine the function of the shed HS.

To determine if HS was shed from the alveolar epithelium into the airspace or from the luminal endothelium into the vasculature, we intratracheally (IT) instilled mice with lipopolysaccharide (LPS) and performed mass spectrometry for HS on bronchoalveolar lavage fluid (BALF) and plasma collected from these mice. We measured increased BALF HS in mice treated with IT LPS vs PBS on day 2 (24.5 vs 6.5ng/ml) and day 4 (18.8 vs 7.5ng/ml) after injury. There was also a trend of increased plasma HS in mice treated with IT LPS vs PBS 4 days after injury (106.8 vs 63.9ng/ml, p = 0.10). We next aimed to determine the function of the shed HS within both the airspace and the vasculature by supplementing mice with exogenous IT or IV heparin octasaccharides (a heavily sulfated HS fragment) on days 2 and 4 after IT LPS injury. Mice supplemented with IV heparin octasaccharides vs PBS on day 2 after injury were protected from injury, exhibiting decreased lung permeability (BALF protein: 0.34 vs 0.40mg/ml) and inflammation (BALF total cells: 3.14 vs 5.63×106). In contrast, animals treated with IT heparin octasaccharides on day 4 after injury exhibited a trend of increased lung permeability (BALF protein: 0.23 vs 0.20mg/ml, p=0.07). Our data indicate that during direct lung injury HS is largely shed from the damaged epithelium, but may also be shed from the endothelium. While intravascular HS is pro‐reparative and decreases injury, airway HS surprisingly appears to exacerbate injury and reduce repair. We are currently investigating the mechanisms by which IT and IV heparin affect lung injury.

Support or Funding Information

NHLBI RO1 HL125371

NHLBI F30 HL132424
Examination of the Physiological Effects of Ethylene Glycol Exposure on Muscle and Liver Tissue of the Leopard Frog, Rana pipiens
Authors: Funk Alexander N , Stabenau Erich K
Published: November 2023

Newly developed and utilized industrial processes use anthropogenic agents that may adversely affect ecosystem biodiversity. Hydraulic fracturing technology involves the use of a fluid, composed of 98–99% water and 1–2% chemical additives, to break up shale reservoirs for access to natural gas and oil reserves. One of the chemical additives in fracturing fluid is ethylene glycol. To date, there is no information available of the effects of ethylene glycol exposure on aquatic organisms at concentrations used by the hydraulic fracturing industry (i.e., 0.14–15.8 mM). Therefore, the present study examined the physiological effects of ethylene glycol exposure on Leopard frogs, Rana pipiens. Frogs were exposed to control water or to water containing 5.7 mM ethylene glycol for five days. Ethylene glycol‐exposure significantly decreased the swimming time to exhaustion from 284.5±22.8 sec in control animals to 194.8±26.3 sec (P≤0.05, n=6 in each group). Moreover, contraction of isolated gastrocnemius muscles was significantly less in ethylene glycol‐exposed frogs at 3, 6, 12 and 24 V when compared to control animals. Mitochondrial membrane potential was also significantly decreased in ethylene glycol‐exposed frogs when compared to control animals, decreasing 40.1% and 42.4% in liver and muscle tissue, respectively. Culture of isolated satellite cells from skeletal muscle resulted in a dose‐dependent, ethylene glycol‐induced increase in cell proliferation and differentiation. Taken together, these data indicate that 5.7 mM ethylene glycol significantly reduced muscle and liver performance. Moreover, the activation and proliferation of the satellite cells when exposed to ethylene glycol suggests that the hydraulic fracturing agent is myotoxic to frog muscles. We believe that the agent is altering mitochondrial function by reducing the available ATP for cellular or organ system processes like cell volume regulation and muscle contraction.

Support or Funding Information

This research was supported by grants from Beta Beta Beta, the Bradley University Biology Department and the Bjorklund Endowment, and a Caterpillar Master's Fellowship to ANF.
Mouse prostate organoids as a model for prostate cancer
Authors: Foley Justin A , Shah Kalpit , Bradbury Neil A.
Published: November 2023

Prostate cancer affects around 14% of men in their lifetime, and is the second leading cause of cancer deaths among the male population. Prostate cancer arises from the epithelial cells of the prostate, and is associated with unregulated activation of the androgen signaling pathway (androgen receptor and associated co‐factors), leading to enhanced expression of androgen dependent genes (e.g., PSA, VEGF) and unregulated cell proliferation. Initially, androgen depravation therapy slows the rate of cancer growth, but this eventually leads to a so‐called androgen‐independent prostate cancer in which cancers are immune to further androgen depletion. Heretofore, research on prostate cancer has relied on the use of prostate cell lines or cells heterologously transfected with androgen signaling components. As these are cancerous or immortalized cell lines, their recapitulation of the native tissue is not always clear. We have previously shown that loss of the kinase LMTK2 is seen in prostate cancer, and is associated with enhanced expression of androgen dependent genes. We have retrieved primary prostate epithelial cells from wt and LMTK2−/− mice. These cells grow and divide in culture and are responsive to androgens. Moreover under low attachment conditions in a 3‐D matrix, the cells form organoids (prostanoids) that display many of the features of native tissues. Our studies reveal prostanoids to be a viable model for normal and cancerous prostate tissues. Indeed, they provide an excellent model for evaluating the physiology of LMTK2 kinase activity within a physiologically relevant context. In addition they provide a rational model for the evaluation of pharmacological agents aimed at treating individuals with prostate cancer.

Support or Funding Information

NIH R01HL102208
Obesity, Independent of the Type of Diet, Drives Lasting Effects on Intestinal Epithelial Stem Cell Proliferation
Authors: Zhou Weinan , Blackmore Katherine , Feldner Mary Kate , Dailey Megan J.
Published: November 2023

The intestinal epithelium plays an essential role in nutrient absorption, satiety hormone release and immune barrier function. Maintenance of the epithelium is driven by continuous cell renewal by intestinal epithelial stem cells (IESCs) located in the intestinal crypts. Obesity affects this process and results in changes in the size and cellular make‐up of the tissue. In addition, isolated IESCs from obese mice have a reduced growth potential to develop into organoids in culture compared with IESCs from lean mice, indicating that obesity may produce long‐lasting effects on IESC proliferation and differentiation. Because the amount and type of diet contribute to obesity, we wanted to determine whether the amount (in kilocalories) or type of diet differentially alters IESC proliferation. C57BL/6J mice were fed two different diet combinations, a high‐fat diet or a Westernized high‐fat/high‐sugar diet. The high‐fat and control groups were as follows: 1) 60% high‐fat fed, 2) 10% low‐fat fed, and 3) 60% high‐fat pair fed (fed a high‐fat diet, but in equal kcal as that eaten by the low‐fat fed animals to keep them lean). The Westernized high‐fat/high‐sugar and control groups were as follows: 1) 31.8% high‐fat, 25% sugar fed, 2) 10.6% low‐fat, 25% sugar fed, and 3) 31.8% high‐fat, 25% sugar pair fed group (fed a high‐fat high‐sugar diet, but in equal kcal as that eaten by the low‐fat high‐sugar fed animals to keep them lean). After 3 months, IESCs were isolated from each mouse using antibody‐based fluorescent cell sorting and grown in media containing inhibitors of differentiation. Crypt development was tracked for 14 days post‐plating and glucose‐induced crypt cell proliferation was measured on day 14. We found that crypts developed from IESCs isolated from high‐fat fed, obese mice formed a reduced number and size of budding structures compared with low‐fat fed, lean mice and high‐fat pair fed, lean mice, while the number of crypts developed per area measured were the same between the groups. Furthermore, glucose increased the proliferation of crypts developed from IESCs isolated from both low‐fat fed, lean mice and high‐fat pair fed, lean mice, but had no effect on the proliferation of crypts developed from IESCs isolated from high‐fat fed, obese mice. In contrast, the mice in the Westernized diet groups ate equal kcal amounts of each diet, were equal in body weight, and there were no significant differences in crypt cell proliferation between the groups. These results suggest that factors related to obesity and not the type of diet (high‐fat or high‐fat/high‐sugar) are driving lasting effects on IESC proliferation.

Support or Funding Information

USDA Hatch ILLU‐538‐926
Focal Adhesion Kinase forms Inhibitory Complexes with its Endogenous Inhibitor
Authors: Zak Taylor J , Robia Seth L , Samarel Allen M
Published: November 2023

Focal adhesion kinase related non‐kinase (FRNK) is an inhibitor of focal adhesion kinase (FAK)‐dependent cell growth, survival, and migration pathways. In vascular smooth muscle cells, both FAK and FRNK are upregulated after vascular injury, suggesting balanced stimulation and inhibition of growth during recovery. Better understanding of the molecular mechanisms of FRNK inhibition of FAK are needed to understand vascular smooth muscle cell response to injury. Here we propose a novel mechanism by which FRNK can inhibit FAK: direct binding of FRNK to FAK forming an inhibitory complex within focal adhesions. This hypothesis is supported by co‐immunoprecipitation of FAK and FRNK. In addition, fluorescence resonance energy transfer (FRET) measurements suggest that FRNK binds directly to FAK within focal adhesions of live vascular smooth muscle cells. We hypothesized that this interaction might be regulated by serine phosphorylation of FRNK S217, a site we found to be basally highly phosphorylated at S217 by extracellular signal–regulated kinase 1/2 (ERK). To determine the significance of S217 phosphorylation, we generated a non‐phosphorylatable mutant FRNK by mutating serine 217 to alanine (S217A). S217A‐FRNK exhibited significantly higher amounts of FRET compared to wild type FRNK, suggesting increased interaction with FAK. Progressive acceptor photobleaching analysis revealed that the FRNK‐FAK complex consisted of FRNK and multiple FAK proteins in a high order hetero‐oligomer. The functional significance of the S217 phosphorylation site was suggested by S217A FRNK's more potent inhibition of FAK Y397 phosphorylation compared to wild type FRNK. FRNK S217A more potently decreased cell growth / survival compared to wild type. Specifically, S217A FRNK was a much more potent inducer of apoptosis in vascular smooth muscle cells compared to wild type FRNK, as determined by annexin staining and flow cytometry. We hypothesize that upregulation of FRNK after vascular injury results in the formation of a FRNK‐FAK inhibitory complex that serves to attenuate injury related growth and that phosphorylation of FRNK at S217 by ERK decreases the inhibitory FRNK‐FAK complex formation, allowing the cell to return to a normal resting state once healing has completed.

Support or Funding Information

NIH: R01‐HL092321 and R01‐HL106189 to S.L.R., P01‐HL62426 to A.M.S., and F32‐HL096143 to Y.E.K.
Glucagon‐like peptide‐1 treatment stimulates Neurogenin3 expression and pancreatic beta‐cell neogenesis in adult mice
Authors: Dadheech Nidheesh , Buteau Jean
Published: November 2023

Glucagon‐Like Peptide 1 (GLP‐1) is an intestinal hormone secreted after a meal. GLP‐1 is considered a key molecule in diabetes treatment because (a) it acts as a growth factor to promote beta‐cell mass expansion and (b) increases insulin secretion. Whereas GLP‐1 action on beta‐cell proliferation and survival has been extensively studied, its potential action on beta‐cell differentiation remains elusive. We herein performed lineage tracing experiments in genetically engineered mice to test the hypothesis that GLP‐1 stimulates the formation of new mature beta‐cells (neogenesis) in the adult pancreas.

We used mice expressing a tamoxifen‐inducible Cre‐recombinase under the Neurog3 promoter to permanently label differentiating beta‐cells with a ZsGreen marker. This is because Neurog3 is transiently expressed in progenitor cells undergoing differentiation and is otherwise absent in the adult pancreas. Neurog3‐CreER/Rosa26‐LSL‐ZsGreen mice were injected with a moderate dose of streptozotocin to induce low levels of beta‐cell damage and regeneration. Then, mice were treated daily with tamoxifen alone or in combination with the long‐acting GLP‐1 analog Exendin4 (Ex‐4). Pancreata were harvested after 2 or 7 days of treatment.

Control pancreata were devoid of ZsGreen staining, consistent with the fact that beta‐cell differentiation does not occur spontaneously in adult mice. Animals treated with Ex‐4, showed high frequency of ZsGreen+ cells in pancreatic islets and ducts, indicating that Ex‐4 induces Neurog3 expression in the adult pancreas. Mice treated with Ex‐4 for 2 days displayed small clusters of ZsGreen+ cells that were polyhormonal, a feature of immature/differentiating pancreatic endocrine cells. After 7 days of treatment, we detected a high number of insulin/ZsGreen dual positive cells, demonstrating that Ex‐4 stimulates the formation of new mature beta‐cells. We characterized the lineage of ZsGreen+ cells by studying the expression of various progenitor cell markers. Finally, we sorted ZsGreen+ cells to define their expression profile and confirm that newly‐differentiated beta‐cells could secrete insulin in response to physiological concentrations of glucose.

Our study shows that GLP‐1, a naturally occurring hormone and an important anti‐diabetes therapeutic target, stimulates beta‐cell neogenesis. We thereby unveiled a new mode of action for the incretin hormone.
High Casein Diet Differentially Alters FGF21 Levels in Plasma and Cardiac Tissue in Rats
Authors: Pettit‐Mee Ryan J , Cunningham Rory P , Sharma Naveen
Published: November 2023

Fibroblast growth factor‐21 (FGF21) is a cytokine that has been demonstrated to regulate metabolism and have cardioprotective effects in response to obesity and other cardiac stressors. High protein diets have been used to reduce body fat and ameliorate metabolic disorders, however the effects of high protein diets on FGF21‐mediated signaling in cardiac tissue are not well known. We hypothesized that high protein diets will increase circulating levels of FGF21, leading to enhanced FGF21‐mediated signaling in cardiac tissue. Male Sprague‐Dawley rats were placed into one of three dietary groups: 1) control diet (20% of kcal from protein; CON; n=8), 2) high soy protein diet (40% of kcal from soy; SOY; n=8), or high casein protein diet (40% of kcal from casein; CAS; n=8). Food consumption and body mass were recorded weekly. After 15 weeks, blood was collected and hearts were excised after an overnight fast. Western immunoblotting was performed on cardiac homogenates and analyzed for: FGF21, FGF receptor‐1 (FGFR1), phospho‐FGFR1Tyr654, phospho‐ERK1/2Thr202/Tyr204, phospho‐AktSer473, phospho‐AMPKThr172, phospho‐p38 MAPKThr180/Tyr182, and 4‐hydroxynonenal (4‐HNE), a marker of oxidative stress. There was no difference in daily food intake or terminal body mass between dietary groups. Plasma concentrations of FGF21 were significantly (P<0.05) greater for CAS (742.1±149.9 pg/ml) compared to CON (213.6±37.7 pg/ml), determined by ELISA. FGF21 protein levels in the heart were significantly (P<0.05) lower for CAS compared to CON and SOY groups. Phospho‐AktSer473 was significantly (P<0.05) greater in SOY compared to CON and CAS groups. There were no other significant differences between dietary groups for any other analyte measured. These data indicate that high protein diets composed of differing protein sources have non‐uniform effects on plasma and cardiac levels of FGF21. Additionally, 15‐weeks of high protein diet consumption does not appear to alter body mass or FGF21‐mediated signaling in the rat heart.

Support or Funding Information

Central Michigan University Herbert H. and Grace A. Dow College of Health Proffesions, and the Office of Research and Graduate Studies Early Career Award.
Liraglutide Promotes Browning of White Adipose Tissue in Mice with Diet Induced Obesity
Authors: Oliveira Fernanda Cerqueira Barroso , Ribeiro Carolina Martins , Christ Camila Greice , Pereira Sidney Alcântara , Beserra Bruna Teles Soares , da Costa Silva Viana Janice , de Assis Rocha Neves Francisco , Coelho Michella Soares , Amato Angélica Amorim
Published: November 2023

Liraglutide is a long‐acting glucagon‐like peptide 1 (GLP‐1) receptor agonist used as an anti‐hyperglycemiant agent in type 2 diabetes and recently approved as an anti‐obesity agent. The mechanisms underlying weight loss include delayed gastric emptying and induction of satiety, and there are data indicating it may also result in increased energy expenditure. Beige adipocytes are an inducible cell type in white adipose tissue (WAT), in a process so‐called browning of WAT. Upon appropriate stimuli, beige adipocytes are fully capable of executing thermogenesis.

Objective

To investigate the effects of liraglutide on browning of WAT in obese mice.

Methods

Male C57Bl/6 mice with obesity induced by high fat diet (HFD) were randomly assigned into four groups and treated with saline (vehicle), the b3‐adrenergic agonist CL316,243, liraglutide or liraglutide and CL316,243. Liraglutide was administered intraperitoneally for 21 days (200 μg/kg, twice daily) and CL316,243 was administered intraperitoneally from day 17 to day 21 (1 mg/kg/d, once daily, for 5 days). Weight gain, energy intake and metabolic efficiency were determined. Two different WAT (inguinal and epidydimal) and the interscapular brown adipose tissue (BAT) depots were excised for determination of adiposity, histological analysis, oxygen consumption rates and relative expression of Ucp‐1 by RT‐qPCR.

Results

(p<0.05, ~ 5 mice/group): Liraglutide treatment reduced HFD‐induced body weight gain, energy intake, metabolic efficiency, visceral and subcutaneous adiposity (epidydimal and inguinal WAT depots), when compared to saline treatment. BAT mass decreased only in response to treatment with CL316,243, either alone or in combination with liraglutide. In all three adipose tissue depots, liraglutide treatment induced the expression of Ucp‐1 when compared to saline, and this effect was additive to that of treatment with CL316,243. Liraglutide significantly increased oxygen consumption rates in epidydimal WAT and interscapular BAT, compared saline treatment. In addition, in epidydimal WAT, inguinal WAT and interscapular BAT, the effect of liraglutide on oxygen consumption was additive to that of CL316,243 stimulation.

Conclusion

Our findings suggest that liraglutide treatment induces browning of WAT and increases the thermogenic capacity of both WAT and BAT. In addition, its effects seem additive to those of b3‐adrenergic signalling, a classical pathway of adaptive thermogenesis activation. This suggests that GLP‐1 receptor agonists may result in increased energy expenditure by activating thermogenic adipocytes, in addition to decreasing food intake, and opens new horizons to treat obesity.

Support or Funding Information

CNPq/CAPES/FAPDF
Prior Exercise Training Alters the Response of White and Brown Adipose Tissue to Cold Stress
Authors: Knuth Carly M , Peppler Willem T , Trottier Sarah K , Townsend Logan K , Wright David C
Published: November 2023

Brown adipose tissue is a thermogenic tissue bed that produces heat in conditions of cold environmental temperatures. While possessing much lower oxidative capacity than brown adipose tissue, recent findings have suggested that exercise training can increase the thermogenic capacity of white adipose tissue. What is not known is if exercise training can alter the response of an animal to cold stress. To address this question, male C57BL/6 mice were subject to either exercise training by voluntary wheel running (EX) or a sedentary (SED) intervention for 12 days. EX mice ran approximately 6km/day, which led to decreased body weight and increased glucose tolerance, while maintaining a similar pattern of food intake to SED. Mice were then further divided into groups that would be kept at room temperature (22°C) or a cold challenge of 4°C for 48‐hrs, thus we examined four groups in this study; (i) SED kept at room temperature (SRT), (ii) exercise trained kept at room temperature (EXRT), (iii) SED kept at 4°C (SC), and (iv) EX kept at 4°C (EXC). Rectal temperatures (Tb) of SC and EXC were lower than that of SRT and EXRT, and the initial drop in Tb of EXC was less than that observed in the SC 2‐hours following the start of the cold, however this difference was not maintained throughout the rest of the cold exposure. 48‐hrs of cold exposure led to reductions in body weight and epididymal and subcutaneous adipose tissue mass in the SED but not EX mice. Conversely, interscapular brown adipose tissue mass was increased following cold exposure in previously EX but not SED animals Taken together, our data demonstrates a unique effect of prior exercise on altering the response of adipose tissue to cold stress.

Support or Funding Information

D.C.W. is a Tier II Canada Research Chair. This work was funded by an NSERC Discovery Grant to D.C.W.
Effect of Nonylphenol on Adiposity and Glucose Homeostasis in Mice with Diet Induced Obesity
Authors: Ribeiro Carolina Martins , Oliveira Fernanda Cerqueira Barroso , Pereira Sidney Alcântara , de Assis Rocha Neves Francisco , Coelho Michella Soares , Amato Angélica Amorim
Published: November 2023

Nonylphenol (NP) is the predominant environmental biodegradation product of NP ethoxylates and is widely use in detergents, plastic products and paints. It is an endocrine disrupter found to compete with 17β‐estradiol for the binding site of estrogen receptor, due to their structural similarity. More recently, it was also identified as an environmental obesogenic, both in cell culture and in vivo. Acute exposure to NP has been found to induce the expression of adipogenesis and lipogenesis‐related genes in adipose tissue. Additionally, perinatal exposure to different concentrations of NP has resulted in increased body mass and adiposity in male and female offspring. However, the obesogenic potential of long‐term exposure to NP outside the perinatal period has not been investigated.

Objective

To investigate the effects of chronic exposure to NP on body weight, adiposity and glucose homeostasis in female C57BL/6 mice.

Methods

Female C57BL/6 mice were fed a high‐fat diet (HFD, 60% kcal as fat) from the 5th to 20th week of age. The animals were randomly divided into five groups (n=6) and treated with vehicle (drinking water) or NP 0.5 mg/kg/d or 2.5 mg/kg/d for 16 weeks (5th to 20th wk) or for 6 weeks (14th to 20th wk) in drinking water. Weight, weight gain, food and water consumption were measured. Two different white adipose tissue (WAT‐retroperitoneal and perigonadal) and one interscapular brown adipose tissue (BAT) depots were weighed. Glucose homeostasis was assessed by fasting glucose levels, glucose tolerance test (GTT), and insulin tolerance test (ITT).

Results

There was a slight, but nonsignificant trend towards increased body weight and weight gain in response to NP treatment at doses of 0.5 mg/kg/d for 6 or 16 weeks and 2.5 mg/kg/d for 16 weeks when compared to vehicle. On the other hand, mice treated with NP 2.5 mg/kg/d for 6 weeks showed a trend towards decreased body weight. Treatment with NP did not affect energy intake, but increased water consumption when compared to vehicle. Fasting glucose levels at the 20th week were nonsignificantly decreased by NP treatment (0.5 mg/kg/d, 6 wk). However, there were no changes in glucose tolerance or insulin sensitivity. Visceral WAT, subcutaneous WAT and BAT mass remained unchanged in response to treatment with NF.

Conclusions

Our findings suggest long‐term exposure of female mice to environmentally relevant NP concentrations, outside of the developmental period, does not affect glucose homeostasis or adiposity.

Support or Funding Information

CNPq/CAPES/FAPDF
The effects of CTRP3 on lipid oxidation in adult mouse primary hepatocytes
Authors: Li Ying , Wright Gary L , Peterson Jonathan M
Published: November 2023

C1q Tumor Necrosis Factor‐Related Protein (CTRP3) is an adipose tissue‐derived secreted factor, or adipokine, that contributes to the processes of metabolism and inflammation. Our previous work has demonstrated that CTRP3 inhibits hepatic lipid biogenesis, in vivo and in vitro. Additionally, recombinant CTRP3 treatment induced the up‐regulation of lipid oxidation in rat H4IIE hepatoma cell line (a liver‐like cell line). However, CTRP3‐induced increase to lipid utilization in primary hepatocytes has not been examined. Although the H4IIE can provide insight into hepatic metabolism, by design immortalized cell lines function very differently than the tissue from which they were derived. Therefore, we isolated primary hepatocytes from wildtype (WT) and transgenic mice which overexpress CTRP3 (CTRP3 Tg), to test the effects of elevated CTRP3 on hepatocyte metabolism (primary hepatocytes, ex vivo, mirror the physiological metabolism of in vivo hepatocytes). Real‐time oxygen consumption rates (OCR) of hepatocytes were recorded by Extracellular Flux Analyzer. Protein expression was measured by Western Blot. We found that OCR were unchanged between WT and CTRP3 Tg mice under standard conditions, after the addition of 100 μM palmitic acid, however, hepatocytes from CTRP3 Tg mice had a significant ~45% increase in OCR consistent with a shift of respiratory substrate from glucose to fatty acid. Further, primary hepatocytes from CTRP3 Tg mice had a 50% increase in Akt phosphorylation (Ser473) in response to insulin. In conclusion, this study confirmed that CTRP3 increases hepatic lipid oxidation, ex vivo, and has provided more evidence to support that lipid oxidation increased by CTRP3 relies on b‐oxidation of fatty acid in primary hepatocytes.

Support or Funding Information

This research was supported in part National Institute On Alcohol Abuse And Alcoholism of the National Institutes of Health under Award Number R03AA023612 and East Tennessee State University Research Development Committee (E82262). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Cold induced Ucp1 expression in brown and white adipose tissue of WFS1‐deficient mice
Authors: Ehrlich Marite , Ivask Marilin , Kõks Sulev
Published: November 2023

Ucp1 expression in brown adipose tissue (BAT) is induced by cold temperatures and is fully consistent with temperature adaptation and thermogenesis. Transcription of the Ucp1 gene is increased within minutes of cold exposure. Beige adipocytes are mainly in the subcutaneous white adipose tissues (WAT) and they arise postnatally in response to chronic cold exposure. This process is often referred as the browning of WAT. The aim of this study was to compare the cold induced metabolic and Ucp1 expression differences between WFS1‐deficient mice and their wild‐type littermates.

Experiments were performed with 2–3 months old male and female F2 hybrids (129S6/SvEvTac × 129S6/SvEvTac). Five to twelve WFS1‐deficient mice and wild‐type mice were used in the study. Mice were kept at 8–12°C up to 7 days and metabolic effect was measured with calorimetric cages (TSE Phenomaster). Ucp1 expression was measured in BAT and WAT with qPCR (Applied Biosystems).

The results of measurements showed that WFS1‐deficient mice have significantly higher energy metabolism and higher Ucp1 expression in BAT and WAT after cold exposure.

In conclusion, our results show that WFS1‐deficient mice have a stronger response to cold exposure and upregulation of Ucp1 in BAT and WAT. Thus suggesting that WFS1‐deficient mice have changes in regulation of thermogenesis that should be studied further.

Support or Funding Information

This work was supported by Institutional Research Funding IUT20‐46 of the Estonian Ministry of Education.
Long‐term Western Diet Feeding Causes Severe NASH and Cecal Dysbiosis in Juvenile Ossabaw Swine
Authors: Panasevich Matthew R , Meers Grace M , Linden Melissa A , Ibdah Jamal A , Booth Frank W , Perfield James W , Shankar Kartik , Rector R Scott
Published: November 2023

Pediatric obesity and nonalcoholic fatty liver disease (NAFLD) are on the rise in industrialized countries, yet our ability to mechanistically examine this relationship is limited by the lack of a suitable higher animal model. Here we examined the effects of continued western diet (WD)‐feeding on NAFLD and cecal dysbiosis in juvenile Ossabaw swine. Female Ossabaw swine (5 weeks old) were fed WD (43.0% fat; 17.8% high fructose corn syrup; 2% cholesterol) or low‐fat control diet (CON; 10.5% fat) for 16 or 36 wks. We observed progressive obesity, lower physical activity, dyslipidemia, and systemic insulin resistance through 36 wks of WD feeding (P<0.05). In addition, WD‐fed pigs developed a progressive form of severe nonalcoholic steatohepatitis (NASH), with significant hepatic steatosis, hepatocyte ballooning, inflammatory cell infiltration, fibrosis, and elevated liver enzymes. WD‐fed pigs also had elevated hepatic expression of inflammatory genes TNFα, TGFβ, IL‐1β, TLR4, TLR9, and F4/80 (P<0.05), as well as increased (P<0.05) hepatic protein content of fibrosis marker α‐SMA and JNK1 at 16 and 36 wks. WD feeding also significantly reduced hepatic OXPHOS proteins in complexes I, III and V at 16 and 36 weeks. Assessment of the microbiome in WD‐fed animals for 36 wks revealed dysbiosis showing higher (P<0.05) cecal relative abundance of Proteobacteria, and families and genera within (i.e. Enterobacteriaceae, Succinivibrionaceae, Desulfovrobrionaceae, Succinvibrio, and Desulfovbrio). Furthermore, LPS‐producing Desulfovbrionaceae and Desulfovbrio, were positively correlated with hepatic TLR4 expression (r=0.7997 and r=0.9130, P=0.01, respectively). Consistent with human high fat diet feeding and obesity and suggestive of increased energy harvest, long term WD‐feeding also resulted in an increased (P<0.05) Firmicutes to Bacteroidetes ratio and exhibited a tendency (r=0.6503, P=0.08) to be positively correlated with biochemical hepatic TAG concentrations. The changes observed in the microbiome are consistent with LPS‐induced transcriptional activation of pro‐inflammatory status in the liver and resemble the human NAFLD/NASH phenotype. Overall, WD‐fed Ossabaw swine developed cecal dysbiosis, a pro‐inflammatory signature, and NAFLD progressing to NASH similar to the pediatric/adolescent and young adult populations.

Support or Funding Information

This work was supported by grants from Mizzou Advantage, The Allen Foundation, and partially supported by VA Grant VHA‐CDA2 IK2BX001299.
CTRP3 Alters Lipid Profile in Response to Ethanol Feeding
Authors: DeGroat Ashley R. , Clark William Andrew , Hagood Kendra L. , Peterson Jonathan M.
Published: November 2023

The overall goal of this study is to evaluate the role of a novel adipose‐tissue derived cytokine, C1q TNF Related Protein 3 (CTRP3), in preventing Alcoholic Fatty Liver Disease (AFLD) and related complications. We have recently identified CTRP3 as a potential therapeutic treatment for Non‐Alcoholic Fatty Liver Disease (NAFLD). AFLD contributes to nearly half of all liver‐related mortalities, yet the effects of CTRP3 on AFLD have not been explored. To determine the impact of CTRP3 on AFLD we fed wildtype (WT) and CTRP3 transgenic (Tg) mice an ethanol diet ad libitum for 10 days followed by a gavage of a single dose of ethanol (5 g kg−1), also known as the chronic plus binge model or “The NIAAA model”. This model mimics the hepatic steatosis and liver injury seen with chronic alcohol consumption. Tg and WT littermates placed on isocaloric control diet (without the addition of ethanol) were used as experimental controls.

Results

Ethanol feeding resulted in a significant increase in triglyceride accumulation in both WT and Tg mice, with no difference between WT and Tg mice fed the same diet. However, when the lipid content was examined control fed Tg mice had a higher percentage of linoleic acid (18:2Δ9,12) and a decrease in arachidonic acid (20:4Δ5,8,11,14) compared to WT. Whereas on the ethanol diet Tg mice had a ~45% increase in oleic acid (18:1Δ9) compared to ethanol‐fed WT. There was no difference in oleic acid concentration between control‐fed WT and Tg mice.

Conclusion

Although CTRP3 failed to reduce hepatic triglyceride levels in response to the NIAAA model of AFLD, CTRP3 significantly increased the concentration of the putative anti‐inflammatory and beneficial fatty acid (oleic acid). These findings indicate that although CTRP3 may not prevent alcohol‐induced hepatic steatosis, it may prevent secondary complications associated with AFLD.

Support or Funding Information

This research was supported in part National Institute On Alcohol Abuse And Alcoholism of the National Institutes of Health under Award Number R03AA023612, East Tennessee State University Research Development Committee (E82262)
Diet‐induced obesity and non‐alcoholic fatty liver disease in TRPV1 null mice
Authors: Connell Pat , Eusebio Luke , Fahmy Joseph N. , DelloStritto Daniel J. , Ferrell Jessica M. , Bratz Ian N
Published: November 2023

The TRPV1 ion channel has been linked to metabolic homeostasis through a variety of proposed mechanisms including; control of appetite and weight, regulation of pancreatic function, thermogenesis, metabolism, and energy balance. Despite this, little information is known regarding its role in liver homeostasis. To better understand the role of TRPV1 in liver metabolism, we explored the effects of either a 24 week chow or high fat/sugar diet (Western, 42.7% carbohydrates, 42% fat, 15.2% protein, 0.2% cholesterol) regimen in male and female wild type (WT) and TRPV1‐null (V1KO) mice. Our data reveals that a loss of TRPV1 channel predisposes male mice to diet‐induced obesity and non‐alcoholic fatty liver disease (NAFLD). V1KO male mice on a western diet displayed numerous phenotypic changes associated with diet‐induced obesity, including increased body mass, adiposity, insulin resistance and glucose intolerance compared to their WT counterparts. Further, western diet fed male V1KO mice exhibited gross changes in liver morphology and size compared to western diet fed WT mice; which were visualized using histological H&E and Oil Red O staining. Moreover, western diet fed male V1KO mice exhibited significantly elevated triglyceride, cholesterol and free fatty acid liver lipid levels compared to western fed WT mice. Gene expression studies revealed decreases in CYP8B1, SREBP2 and HNFα1 gene expression as potential targets for further examination. Of note, western diet fed female V1KO mice were resistant to many of the diet‐induced changes in male counterparts. Taken together, these data demonstrate that loss of TRPV1 promotes fat accumulation, NAFLD development and severe changes in liver lipid profiles, while illustrating that TRPV1 presence is protective against NAFLD development in diet‐induced obesity.
Mechanisms of amelioration of NASH development by deletion of the orphan nuclear receptor SHP: interaction between bile acid and gut microbiota
Authors: Mifflin Ryan , Kim Chunki , Park Jung Eun , Lee Mikang , Shriver Leah , Barton Hazel , Lee Yoon Kwang
Published: November 2023

Whole‐body deletion of small heterodimer partner (SHP, NR0B2), an orphan nuclear receptor, protects mice from diet‐induced obesity and hepatic steatosis upon feeding of a western diet (WD). Additionally, a six‐month WD regimen promoted the progression of nonalcoholic steatohepatitis (NASH) in wild type C57BL/6 mice (WT). Interestingly, SHP deletion was also protective in progression of NASH, demonstrated by lower expression of inflammatory and fibrotic genes, reduced oxidative stress, and less macrophage infiltration in the liver of SHP−/− mice. To clarify its role in hepatic fibrosis, mice were challenged with methionine and choline deficient (MCD) diet for four weeks. While WT mice developed fibrosis after the MCD diet regimen, no fibrotic phenotype was evident in SHP−/− mice when analyzed with qPCR and sirus red staining. Importantly, the observed protection against NASH was recapitulated in liver‐specific SHP‐null mice, suggesting that hepatic SHP plays a role in NASH development. Hepatic SHP is a critical component in bile acid (BA) synthesis through direct regulation of Cyp7a1 gene expression, which encodes the rate‐limiting enzyme in BA synthesis. Since a strong interrelationship between BA and the gut bacteria has been suggested, changes in BA composition possibly alters the composition of the gut microbiome, thereby affecting liver physiology significantly in the SHP−/− mice. Therefore, we performed 16s rDNA pyrosequencing using DNA extracted from fecal samples and ran a principal coordinate analysis (PCoA) with the obtained taxonomic composition. Indeed, mice lacking SHP display a distinctly different microbiome from WT mice, characterized by distinct PCoA clustering and a decreased abundance ratio of the Firmicutes phylum compared to the Bacteroidetes phylum. Using fluorescent staining, we found that the deletion of SHP does not result in loss of fecal bacterial abundance when fed WD. To confirm a potential effect of the gut microbiome on the liver phenotype, WT and SHP−/− mice were cohoused and challenged with WD. This cohousing abolished the protective phenotypes against body weight gain, hepatic inflammation and oxidative stress induced by WD feeding but not hepatic steatosis in the SHP−/− mice. In addition, cohousing also eliminated the distinctiveness in taxonomic composition of gut bacteria. These results suggest a relationship between SHP and the gut microbiome in the development of DIO, inflammation, and nonalcoholic steatohepatitis.

Support or Funding Information

This work had been supported by the National Institutes of Health 344 Grant R01DK093774 (to YKL)
CRISPR Cas9‐mediated deletion of Biliverdin reductase A (BVRA) in mouse liver cells models a broad spectrum of NAFLD Phenotype
Authors: Adeosun Samuel O. , Nwaneri Assumpta C. , Hinds Terry D. , Stec David E.
Published: November 2023

Obesity is the leading cause of Non Alcoholic Fatty Liver Disease (NAFLD) which is also associated with insulin resistance and diabetes. NAFLD include a spectrum of pathologies which starts with fat accumulation (steatosis). The presence of a ‘second hit’ drives the continuum from steatosis to steatohepatitis, fibrosis, cirrhosis and liver failure/cancer. We previously showed that bilirubin treatment protects mice from hepatic steatosis. Biliverdin reductase (BVRA) ‐the key enzyme for bilirubin synthesis, also signals through MAPK/AKT which is an important insulin‐regulated pathway.

The aim of this study was to test the hypothesis that BVRA protects against steatosis via regulation of intracellular fat processing through a combination of direct signaling and bilirubin generation.

We used the CRISPR‐Cas9 system to delete/disrupt a 16.5kbp fragment between Exon1 and Exon 5 of the BVRA gene in a mouse liver cell line –Hepa 1c1c7 and studied NAFLD‐related phenotypes. Using a novel protein UnaG which fluoresces only when bound to bilirubin, we show that both intracellular bilirubin and conversion of biliverdin to bilirubin (BVRA activity) by live cells and by cell lysates were markedly reduced in these BVRA‐KO cells. With no treatment, these cells recapitulated the transcriptomic signature of drug‐induced steatosis as FOXA1, HHEX and SREB1F –genes associated with fatty acid processing, were changed in a manner that favors fat accumulation. Consequently, when exposed to free‐fatty acids, Nile‐Red staining coupled with flow cytometry showed that BVRA‐KO cells accumulate 42% more fat (p=0.007) and 100% more triglycerides (p=0.002) compared to wildtype cells. They also showed a blunting of insulin‐stimulated AKT phosphorylation (up to 41% less) compared to wildtype cells. Lastly, untreated BVRA‐KO cells express significantly higher levels (1.7–3.2 folds) of markers of fibrosis versus wildtype cells.

In conclusion, our data shows that BVRA plays a critical role in protecting against lipid accumulation and in the pathophysiology of NAFLD. Studies are on‐going to determine the independent contributions of the signaling and reductase activities of BVRA and thus to identify the best BVRA‐directed therapeutic target. Also, given that the deletion of this important gene required no [additional] second‐hit to precipitate phenotypes like fibrosis which represents a later stage in the NAFLD continuum, these cells may be a novel in‐vitro model for studying a broader spectrum of NAFLD phenotypes.

Support or Funding Information

NIH grant PO1HL 51971 and P20GM‐104357
Western diet feeding downregulates hepatic RECK expression and induces NASH with fibrosis
Authors: Cunningham Rory , Sheldon Ryan , Meers Grace , Kandikattu Hemanth Kumar , Chandrasekar Bysani , Rector Randy Scott
Published: November 2023

Nonalcoholic steatohepatitis (NASH) is characterized by increased accumulation of fat in liver, along with inflammation and fibrosis. During liver fibrosis development, there is a shift in the intrahepatic balance where extracellular matrix accumulation exceeds its degradation, resulting in excessive collagen deposition. Reversion‐inducing‐cysteine‐rich protein with Kazal motifs (RECK) is a novel membrane‐anchored protease inhibitor. While its expression and protective role in myocardial fibrosis are reported, its expression and role in the development and progression of hepatic fibrosis are not known. Here we examined the effects of short‐term (6 weeks) and long‐term (18 weeks) western diet (WD; 44.9% fat; 17% sucrose; 1% cholesterol) feeding on hepatic RECK expression and fibrosis development in male C57/BL6 wild‐type (WT) mice. Six weeks of WD feeding resulted in significant hepatic steatosis and mild inflammatory cell infiltration, but not hepatic fibrosis. This histological presentation occurred in conjunction with significantly elevated hepatic RECK mRNA expression and protein content (p<0.05). However, following 18‐weeks of WD feeding (versus low‐fat control diet; 10% fat), RECK mRNA and protein expression was dramatically reduced by ~50% in the liver, despite no significant changes in hepatic SP‐1, SHP, or FXR mRNA expression. Importantly, reduced RECK expression was associated with (i) significant hepatic steatosis, (ii) histological increases in hepatic immune cell infiltration and hepatic fibrosis, and (iii) elevated expression of key genes involved in hepatic fibrogenesis (Tgfb1 and Col1α1), total macrophage content (Emr1), M1 macrophage markers (Tnfα, Cd11c, Nos2), cellular adhesion (Selectin E, Icam, Ccl2, Ccr2), and extracellular matrix regulators (TIMP‐1, MMP‐13, and MMP‐2). These results demonstrate that the progression of western diet‐induced NASH with fibrosis is associated with suppressed RECK and elevated pro‐fibrotic mediators in the liver. These results suggest that overexpression or induction of RECK may have therapeutic potential in hepatic fibrogenesis. Our future studies will determine this possibility using a transgenic mouse model with liver‐specific overexpression of RECK.

Support or Funding Information

This work was supported by a Veterans Affairs VHA‐CDA2 grant (IK2BX001299).
Palmitate Increases Protein Palmitoylation in Liver Cells
Authors: Estrada Andrea L , Stewart Claire M , Kim Paul Y , Wang Dong , Wei Yuren , Pagliassotti Michael
Published: November 2023

Purpose

Disruption of endoplasmic reticulum (ER) homeostasis, or ER stress, is a characteristic feature of non‐alcoholic fatty liver disease (NAFLD). Elevated fatty acids, in particular saturated fatty acids, induce ER stress in numerous cell types including hepatocytes. However, the specific mechanisms that link fatty acids to ER stress in the liver are poorly understood. The ER is responsible for numerous post‐translational modifications including palmitoylation. Protein palmitoylation plays a crucial role in protein stability, function, and subcellular location. The aim of this study was to assess whether palmitate selectively increases protein palmitoylation in liver cells.

Methods

H4IIE liver cells (n=3) were treated with control media or control media supplemented with palmitate (250μM), a saturated fatty acid that induces ER stress. Treatments were administered in the absence or presence of bromopalmitate (200μM), a potent inhibitor of palmitoylation, for 6 hours. In order to characterize the palmitoylated proteome, we utilized an acyl‐biotin exchange technique to capture palmitoylated proteins. The protein lysate was separated via SDS‐PAGE, and probed for using a fluorophore‐conjugated streptavidin antibody.

Results

Palmitate increased palmitoylation by approximately 20% compared to control. Bromopalmitate restored protein palmitoylation in palmitate treated cells to levels observed in control. Preliminary results suggest that the presence of bromopalmitate reduced palmitate‐induced X‐box binding protein 1 splicing (a marker of ER stress). In conclusion, palmitate increased protein palmitoylation over a time course that coincides with ER stress.

Support or Funding Information

NIH: DIK072017