bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of cell surface receptors for the novel adipokine CTRP3
Authors: Peterson Jonathan M
Published: November 2023

C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects throughout the body. Our initial work shows promise in the development of CTRP3‐induced cellular processes as a means to combat nonalcoholic fatty liver disease (NAFLD). Clinically, NAFLD is defined as the excessive accumulation of fat in the liver, usually due to obesity, and NAFLD effects 1 in 10 Americans. Our previous data show that when high fat fed mice are treated with CTRP3 they are protected from developing NAFLD. However, the mechanism for this effect remains unclear. The purpose of this project was to identify the unknown receptor that mediates the hepatic actions of CTRP3.

METHODS

We used Ligand‐receptor glycocapture technology with TriCEPS™‐based ligand‐receptor capture (LRC‐TriCEPS; Dualsystems Biotech AG). The LRC‐TriCEPS experiment with CTRP3‐FLAG protein as ligand and Insulin as the control ligand was performed on H4IIE rat hepatoma cell line. Additional analysis using siRNA induced knockdown of the identified receptors was used to validate the findings.

RESULTS

Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis demonstrated successful oxidation and crosslinking of CTRP3‐TriCEPS and Insulin‐TriCEPS to the cell surface glycans. In total, an enrichment of glycopeptides of 11% (261 peptides) was observed. Under these conditions, INSR (Insulin receptor) could be identified and quantified in the control dataset. For the ligand sample two unique receptors were identified. Flow cytometry analysis with siRNA induced knockdown of these proteins confirmed that the presence of the protein is needed for CTRP3 binding to occur.

CONCLUSION

The LRC‐TriCEPS methodology was successful in identifying the receptor for CTRP3.

RELEVANCE

The identification of the receptors for CTRP3 and CTRP3‐induced cellular signaling pathways are important prerequisites for the development of small molecule drug candidates that exert effects beneficial to human health.

Support or Funding Information

East Tennessee State University Research Development Committee

National Institute On Alcohol Abuse And Alcoholism of the National Institutes of Health under Award Number R03AA023612
Exercise training differentially impacts 13C4‐octanoic acid oxidation in obese individuals with high and low intrahepatic lipid content‐a pilot study
Authors: Winn Nathan C , Lee Nhan , Kanaley Jill A , Rector R. Scott , Parks Elizabeth J , Ibdah Jamal A
Published: November 2023

The beneficial adaptations to chronic physical activity (PA) reduce the risk of nonalcoholic fatty liver disease (NAFLD) and PA is a first line treatment for patients diagnosed with NAFLD. Accumulating evidence posits that increased utilization of lipid substrates may be one mechanism by which exercise reduces liver fat (LF); nonetheless there is paucity of data regarding the impact of exercise training on fatty acid oxidation (FAO) in individuals with differing levels of hepatic steatosis. Here, we determined whether the level of medium chain FAO is altered in individuals with varying contents of intrahepatic lipid (IHL) in response to exercise training.

Methods

Obese individuals (n=8, body mass index, 38.5±1.6 kg/m2) with 1H‐MRS‐measured hepatic steatosis underwent baseline and post‐training meal tests (3 h) with labeled 13C4‐octanoic acid to assess FAO. Frequent breath samples were taken and analyzed for 13CO2 enrichment (%). Subjects were stratified into Low‐ (IHL<15%; n=4) and High‐LF (IHL≥15%; n=4) and compared to non‐exercising controls (n=2). Exercise training was performed 4x/wk for 4 wks at 55–80% maximal oxygen consumption for a duration equivalent to ~400 kcal energy expenditure (30–70 min/session).

Results

At baseline, IHL was greater in High‐LF (27.0±4.3%) compared to both Low‐LF (10.1±1.4%) and controls (5.0±1.8%), P<0.05. Exercise training decreased IHL in High‐LF (−16.7±5.3%; P<0.05) with no statistically‐significant changes in Low‐LF (−11.3±10.1%) or non‐exercising controls (17.3±14.5%), P>0.05. Body weight, body fat percentage, and fat mass were unchanged with exercise training within groups. Baseline 13CO2 area under the curve (AUC) [Control, 9.7±2.0 AU; Low‐LF, 8.4±0.5 AU; High‐LF, 6.7±1.2 AU] and steady‐state (SS) 13CO2 [Control, 4.2±0.8%; Low‐LF, 3.7±0.2%; High‐LF, 2.9±0.5%] were not different among groups (P>0.05). Neither 13CO2 AUC nor SS 13CO2 were changed with exercise training in High‐LF or non‐exercising controls. By contrast, 13CO2 AUC and SS 13CO2 were reduced in Low‐LF by 26.8±5.4% and 22.1±5.9%, respectively (P<0.05). The change in SS 13CO2 was inversely associated with the change in IHL (r=−0.75, P<0.05).

Conclusions

Exercise‐induced reductions in IHL, without weight loss, were not accompanied by an increase in medium chain FAO in obese individuals with High‐LF, whereas, FAO was decreased in obese individuals with Low‐LF despite no changes in IHL following exercise training. Larger cohort studies are needed to confirm these findings.
eNOS is expressed by primary murine hepatocytes and regulates fatty acid oxidation in vitro
Authors: Sheldon Ryan D. , Morris E. Matthew , Linden Melissa A. , Meers Grace M. , Thyfault John P. , Laughlin M. Harold , Rector R. Scott
Published: November 2023

INTRODUCTION

We previously have shown that loss of hepatic endothelial nitric oxide synthase (eNOS) activity is linked to nonalcoholic steatohepatitis (NASH) development and progression. In addition, whole‐body eNOS−/−mice are susceptible to western diet induced hepatic mitochondrial dysfunction and NASH. However, it is unclear whether eNOS is expressed in hepatocytes and, if so, whether hepatocyte specific eNOS plays a role in regulating mitochondrial function. Here we tested the hypothesis that eNOS is expressed by primary hepatocytes and is necessary for normal fatty acid oxidation.

METHODS

Primary murine hepatocytes from wild‐type and eNOS−/− mice were isolated using the two‐step collagenase perfusion and digestion method. For siRNA experiments, WT cells were plated with media containing either scrambled or eNOS siRNA and maintained in culture for 48 hours. In a separate set of experiments, primary hepatocytes from WT or eNOS−/− mice were maintained in a collagen matrix for 5 days. On day 4 in culture, cells were placed in media with or without 500 μM free fatty acids (FFA; 250 μM palmitate + 250 μM oleate) for 21 hours and subsequently placed in 1‐14C palmitate (200μM) containing media for 3‐hours and 14CO2 production was measured to indicate complete palmitate oxidation.

RESULTS

Complete 1‐14C palmitate oxidation was reduced by 30% (p<0.05) in primary hepatocytes from eNOS−/− versus WT mice, and prior exposure to FFA further reduced 1‐14C palmitate oxidation by 19% and 15% in WT and eNOS−/− hepatocytes, respectively. eNOS mRNA and protein were expressed in isolated primary hepatocytes from WT but not eNOS−/−mice and eNOS siRNA knocked down eNOS mRNA expression in WT cells by ~50%. Knockdown of eNOS reduced PGC1α, NQO1, and BNIP3 mRNA expression by 40–50%, suggesting that hepatocellular eNOS is important for normal mitochondrial biogenesis, antioxidative capacity and mitophagy, respectively.

CONCLUSIONS

These findings strongly support a hepatocellular specific role for eNOS in the regulation of mitochondrial content and function in vitro. These data suggest that loss of hepatocellular eNOS function in vivo may represent a novel disease mechanism leading to mitochondrial impairments in NASH.

Support or Funding Information

Support: MU Life Sciences Pre‐doctoral Fellowship (RDS), AHA 14POST20110034 (EMM), NIH R01DK088940 (JPT), NIH HL‐36088 (MHL), VHA‐CDA2 IK2BX001299‐01 (RSR).
Modulation of Cerebral Activity Induced by α‐casozepine, a Benzodiazepine‐like Peptide Derived from Bovine Casein
Authors: Benoit Simon , Chaumontet Catherine , Cakir‐Kiefer Céline , Tomé Daniel , Miclo Laurent
Published: November 2023

The tryptic hydrolysate of bovine αs1‐casein (CH) displays anxiolytic properties highlighted in several animal species and in humans. Unlike benzodiazepines (BZD), the most prescribed anxiolytic drugs, CH shows neither addiction, dependence, sedation nor toxicity. The search for a carrier bioactive molecule within CH led to the α‐casozepine (α‐CZP), a decapeptide which anxiolytic properties were confirmed in rats. Its affinity for the benzodiazepine site of the GABAA receptor has helped getting the α‐CZP closer to the BZD family, despite a much lower affinity than the BZD reference diazepam. The aim of this study was to characterise the changes in the activity of the brain areas involved in the reduction of anxiety after intraperitoneal administration of α‐CZP by labelling neuronal activity in mice brain, in order to characterise its mechanism of action.

Swiss mice were fed with a soy‐protein based diet containing no caseins, in order to prevent the presence or potential release of bioactive peptides from these milk proteins. Animals (8 per group) were placed in an anxiety‐producing situation (light‐dark box) 30 minutes after an intraperitoneal injection of α‐CZP (1 mg/kg), diazepam (1 mg/kg) or of the vehicle used to solubilize the molecules. The molecules were then perfused with formalin 1h30 after this stimulus. Brain expression of c‐Fos (a marker of neuronal activity) was measured by automatic counting with immunofluorescence on sagittal and coronal brains sections.

The anxiolytic effects of α‐CZP on mice were confirmed using the light‐dark box (significant augmentation of the time spent in the aversive lit box). Immunofluorescence analysis showed a significant lower expression of c‐Fos in the prefrontal cortex (−60%), hippocampus (−40%), nucleus accumbens (−50%) and hypothalamus (−60%) after administration of the α‐CZP compared to the vehicle. The same profiles were observed after diazepam injection. A significant increase in the expression of c‐Fos in the amygdala (+300%), observed only after the administration of α‐CZP, indicates a different mechanism of action compared to diazepam. Results were confirmed on coronal sections.

In conclusion, this study showed that an intraperitoneal administration of α‐CZP, a bioactive peptide resulting of a food protein hydrolysis, allows a modulation of neuronal activity in different brain regions involved in the regulation of anxiety and thereby can partly explain the anxiolytic activity of the peptide. A binding of α‐CZP on BZD receptors could explain the diminution of neuronal activity in different brain regions associated with the anxiolytic effects of the peptide. Moreover, a different mechanism of action of that of diazepam could also account for the absence of side effects observed with CH and this could be explained by the differences observed in the amygdala activation.
The effect of wheat peptides on ethanol‐induced gastric mucosal damage in rats
Authors: Yang Xian , Sun Guiju , Wang Shaokang
Published: November 2023

Objecive

Excessive alcohol consumption can lead to gastric mucosal damage and the present work was aimed to examine the protective effect of wheat peptides in the model rats with gastric mucosal damage induced by ethanol.

Methods

Wheat peptides (83, 167 or 333 mg/kg), cimetidine (65 mg/kg), or distilled water were administered daily by gavage for 30 days before the rats were treated with anhydrous ethanol (1mL) and then animals were euthanized 1 h after ethanol ingestion. Histological observations was performed, so were antioxidation effect, anti‐inflammatory action and caspase‐3.

Results

Histological observations showed that ethanol‐induced gastric mucosal damage was attenuated by wheat peptides pretreatment. Wheat peptides remarkably increased the superoxide dismutase (SOD) activity, epidermal growth factor (EGF) and prostaglandinE‐2 (PGE2) levels in serum. The treatment of wheat peptides dramatically decreased the malondialdehyde (MDA), platelet‐activating factor (PAF) and interleukin‐8 (IL‐8) levels in serum. The expression level of caspase‐3 in stomach was significantly decreased in wheat peptides‐treated rats comparing with the model group, however, the expression level of EGFR was markedly increased.

Conclusion

Ethanol‐induced gastric mucosal damage in rats can be prevented by wheat peptides. And the potential mechanism may be associated with enhancement of antioxidation, anti‐inflammatory action and anti‐apoptosis function.

Support or Funding Information

This work was supported by the cooperation project with china national research institute of food & fermentation industries.
Metformin improves ileal epithelial barrier function in interleukin‐10 deficient mice
Authors: Xue Yansong , Zhang Hanying , Yang Guan , Zhu Mei‐Jun
Published: November 2023

Background

Accumulating evidence shows that gut epithelial integrity and barrier function is a central predisposing factor in the pathogenesis of inflammatory bowel disease. The proper intestinal epithelial proliferation and differentiation is crucial for maintaining gut homeostasis and intestinal integrity.

Objective

To evaluate the protective role of metformin in ileal epithelial barrier function using interleukin‐10 deficient (IL10KO) mice.

Results

Metformin supplementation had no effect body weight gain and feed intake, but decreased intestinal permeability in IL10KO mice associated with reduced expression of pore forming tight junction protein, claudin 2 and enhanced expression of barrier forming tight junction protein, claudin 3. Histological analysis showed that IL‐10 depletion elongated crypt depth and increased mucosal thickness, resulting in decreased Villi/Crypt ratio, which was restored by metformin supplementation. Consistent with increased crypt depth, proliferation of epithelial cells in IL10KO mice with or without metformin supplementation was enhanced compared to that of WT mice, while metformin supplementation decreased the number of proliferative cells per crypt in IL10KO mice as assayed by Ki67 staining and in vivo BrdU labeling. The density of Paneth cells was increased in metformin treated mice regardless of genotypes, but the Paneth cell density was decreased dramatically in IL10KO mice when compared to those of WT mice regardless of metformin supplementation. Consistently, the density of goblet cells was much lower in the intestinal epithelium of IL10KO mice compared to that of the WT mice, while metformin supplementation increased the goblet cell density in both WT and IL10KO mice.

Conclusion

Our data demonstrate that metformin supplementation inhibits cell proliferation, promotes secretory cell lineage differentiation and further improves epithelial barrier function in IL10KO mice.

Support or Funding Information

R15HD073864
Mast cells promote biliary proliferation and hepatic fibrosis in normal and HDC−/− mice by interacting with cholangiocytes and hepatic stellate cells via TGF‐β1 signaling
Authors: Francis Heather
Published: November 2023

Background

In normal, homeostatic conditions, cholangiocytes are mitotically dormant, hepatic stellate cells (HSCs) are quiescent and mast cell (MC) numbers are relatively low. Upon injury resulting in hepatic fibrosis, there is an activation of HSCs and the release of pro‐inflammatory factors like TGF‐β1 from proliferating cholangiocytes. MCs infiltrate the liver following injury and promote biliary hyperplasia by releasing factors like histamine. In mice lacking the histidine decarboxylase gene (HDC−/− mice), MCs are morphologically altered and also low in number. We tested the hypothesis that MCs promote fibrosis in normal and HDC−/−mice by increasing HSC activation, cholangiocyte proliferation and TGF‐β1 release.

Methods

Normal, wild‐type (WT) mice and HDC−/− mice were injected with 1xPBS or cultured MCs derived from fetal mouse liver (5 × 106) via tail vein delivery. After 6 days, mice were sacrificed and liver blocks, isolated cholangiocytes and serum was obtained. Biliary mass and proliferation was evaluated by immunohistochemistry for CK‐19 and PCNA, respectively, and hepatic fibrosis and collagen deposition was measured by Masson's Trichrome and Fast Green/Sirius Red staining. Real‐time PCR was performed in total liver and isolated cholangiocytes for fibrotic markers, α‐SMA, collagen‐type 1a and fibronectin. HSC activation was measured by immunofluorescence and real‐time PCR in total liver for synaptophysin‐9 (SYP‐9). TGF‐β1 secretion was measured by EIA in serum from all mice and by real‐time PCR in total liver and isolated cholangiocytes. In vitro, cultured HSCs and cholangiocytes were treated with cultured MCs and fibrosis markers and TGF‐β1 expression/secretion was measured.

Results

MC injection increased biliary mass, proliferation and hepatic fibrosis in both WT and HDC−/− mice compared to controls. HSC activation was also enhanced in mice injected with MC compared to controls. TGF‐β1 secretion in serum and gene expression was increased following MC injections in both WT and HDC−/− mice compared to controls. In vitro, co‐culture with MCs increased cholangiocyte and HSC proliferation, activation and fibrotic marker gene expression.

Conclusion

In normal conditions, MCs are low in number, however; when the liver is damaged, MCs infiltrate, release profibrotic factors and induce further damage. Our results demonstrate that MCs promote biliary proliferation, HSC activation and hepatic fibrosis in normal and HDC−/−mice via increased TGF‐β1 signaling. MCs are a pro‐proliferative and pro‐fibrogenic component of hepatic fibrosis. Targeting MCs may be a key therapeutic in fibrotic‐related liver pathologies when MC numbers are increased.

Support or Funding Information

VA Career Development Award
Cecal Microbiota Dysbiosis and Hepatic Transcriptomic Alterations in Western Diet‐Induced NASH in Juvenile Ossabaw Swine
Authors: Panasevich Matthew R , Linden M A , Meers G M , Kanosky K M , Reubel M , Perfield J W , Booth F W , Ibdah J A , Oerther D B , Rector R S
Published: November 2023

Pediatric nonalcoholic steatohepatitis (NASH) rates are on the rise in industrialized nations, yet the mechanisms that contribute to disease development remain poorly defined. Here we sought to test the hypothesis that western diet (WD)‐induced NASH in obese juvenile Ossabaw swine is linked to cecal dysbiosis and altered markers of intestinal permeability, as well as to determine RNA transcriptomic changes within the liver in this model of pediatric NASH. Sixteen weeks of WD (high fat, fructose, cholesterol) feeding caused obesity and a severe NASH phenotype, including hepatocellular ballooning, inflammation, and fibrosis compared with lean pigs fed low fat chow. WD feeding induced marked cecal dysbiosis, including decreases in the relative abundance of Firmicutes (70% vs 50%, P<0.05) and a 6‐fold increase in Proteobacteria (23% vs 4%, p<0.001) vs chow fed pigs. Specifically, WD‐feeding increased relative abundances of classes Gammaproteobacteria and Deltaproteobacteria (gram‐negative genera Eschericia coli and Shigella) which are linked to endotoxemia, inflammation and intestinal permeability. The observed dysbiosis was associated with down regulation of jejunal mRNA expression of tight junction proteins ZO‐1 and ZO‐2, and increased mucosal cell expression of inflammatory markers CD‐14, TLR2, TLR4, and IL‐1β in obese WD‐fed pigs. RNA‐sequencing analyses revealed 2168 up‐ and 67 down‐regulated mRNA transcripts (≥ 2.0 fold, FDR <0.10) in liver between WD and chow fed swine. The top regulated canonical pathways identified by Ingenuity Pathway Analysis were “Interferon Signaling”, “Hepatic Fibrosis/Hepatic Stellate Cell Activation”, and “Antigen Presentation Pathway”. Collectively, these data demonstrate that WD feeding in juvenile Ossabaw swine induces cecal dysbiosis, alters markers of intestinal permeability, and causes robust transcriptomic changes in the liver that may contribute to NASH.

Support or Funding Information

Mizzou Advantage, Allen Foundation, and VA‐CDA2 IK2BX001299 (RSR)
Blockades of neuronal or endothelial synthases of nitric oxide have opposite effects on gastric bicarbonate secretion induced by mild irritation of the mucosa
Authors: Zolotarev Vasiliy A. , Andreeva Julia V. , Khropycheva Raisa P.
Published: November 2023

In the gastric mucosa (GM), under normal physiological circumstances, an increase of endogenous nitric oxide (NO) level due to activity of constitutive synthases of NO is considered an important factor of gastric protection. However, the role of endogenous NO in regulation of gastric bicarbonate secretion, the main process of gastric protection, still is not fully understood. In particular, effects of endothelial (eNOS) and neuronal (nNOS) synthases of nitric oxide which are present almost in all types of GM cells were not compared.

We examined roles of nNOS or eNOS in control of bicarbonate production induced by transient irritation of GM at normal conditions under anesthesia or after suppression of gastric acid secretion by omeprasol.

In rats, concentration of HCO3− in stomach luminal perfusate was calculated basing on measurements of pH and PCO2. GM irritation for 20 min with acidic and hyperosmotic solution (1 M NaCl, pH 2.0) caused a marked and omeprazole‐independent increase of HCO3− secretion. The selective blocker of nNOS in vivo, 7‐nitroindazole (7‐NI), or the nonselective blocker of nNOS and eNOS, NG‐nitro‐L‐arginine (L‐NNA), was applied to the splanchnic circulation via retrograde injection into the splenic artery (1 mg/kg). At the normo‐acid state, irritation‐induced output of HCO3− was inhibited by 7‐NI but was not affected by L‐NNA. After administration of omeprazole, both 7‐NI and L‐NNA equally inhibited HCO3− production. Bicarbonate output was substantially reduced by the blocker of soluble guanylate cyclase (GC), methylene blue. Additionally, the effect of 7‐NI (but not L‐NNA) was abolished by a cyclooxygenase (COX) inhibitor, indomethacin. Infusion of NaHCO3 (10 mmol*kg−1*h−1, i.v.) did not change secretion of bicarbonates induced by irritation at the normo‐acid state but markedly enhanced HCO3− output after treatment with omeprazole. The last was inhibited by 7‐NI but was not affected by L‐NNA.

We conclude that GM secretion of HCO3− caused by gastric intraluminal application of mild irritants is largely mediated by intramural nNOS and depends on GC‐COX interaction. Activity of nNOS promotes gastric transepithelial flux of HCO3− induced by irritation. Assuming that impacts of eNOS and nNOS on gastric bicarbonate secretion were additive, we determined a selective influence of eNOS indirectly by subtracting an effect of 7‐NI on HCO3− output from the effect of L‐NNA. It allows to speculate that eNOS activity causes COX‐independent reduction of HCO3− output in the normo‐acid stomach which was abolished by omeprazole. eNOS activity reduces transport of HCO3− from circulation to the surface of GM.
Gallbladder Motility And The Sex of The Guinea Pig
Authors: Kline Loren William , Karpinski Edward
Published: November 2023

The sex hormones 17 β‐estradiol (E2), progesterone (P), and dihydrotestosterone (DHT) have all been shown to affect the motility of the gallbladder. A decrease in the motility of the gallbladder has been observed during pregnancy. This decrease in motility was attributed to elevated P levels. Gallstones are also more common in women than men. One suggested reason was that in gallbladders taken from women and men, women have more estrogen receptors than men, as well as, more P receptors than men. Both P and E2 had an inhibitory effect upon gallbladder contractility in men and premenopausal and postmenopausal women. Similar findings have been reported in gallbladder strips taken from male and female guinea pigs. In the present study, using pharmacologic techniques, there was no significant difference in the amount of E2‐, P‐, or DHT‐induced relaxation of CCK‐induced tension when the responses in gallbladder strips from male and female guinea pigs were compared at each concentration used. In addition, 3 metabolites of P were used‐17‐hydroxyprogesterone (17‐P), 20α ‐hydroxyprogesterone (20‐P), and 21‐hydroxyprogesterone (21‐P). There was no significant difference in the responses from strips from male and female guinea pigs at each concentration used. In order to determine if the effects of E2 and P were additive, strips from male animals were exposed to either E2 or P and the amount of relaxation recorded. After recovery the strips were exposed to E2 or P in reverse order to ensure the order of treatment had no effect. After recovery, the strips were treated with both E2 and P simultaneously and the relaxation recorded. This procedure was repeated with strips from female guinea pigs. The effect of E2 and P was found to be additive; suggesting that each hormone was acting through different messenger systems; however, the response of the strips from each sex were not significantly different. It is concluded that the sex of the guinea pig has no significant effect on the response to the sex hormones used.

Support or Funding Information

Internal Grant
Sex Differences in GABAergic Neurotransmission to Gastric‐Projecting DMV Neurons
Authors: Jiang Yanyan , D'Angelo Michael Paul , Anselmi Laura , Travagli R. Alberto
Published: November 2023

Functional gastrointestinal disorders, including delayed gastric emptying and decreased gastric motility, are more prevalent in women than men. It is suggested that the abundance of circulating estrogen in women may account for this difference. Gastric motility is finely tuned by the inputs arising from the dorsal motor nucleus of the vagus (DMV), which is itself controlled by a tonic, inhibitory GABAergic input. Estrogen has been shown to increase GABA expression in various CNS area, however the effect of estrogen to modulate GABAergic inputs onto DMV neurons, has not been investigated.

The aims of the present study were to test the hypotheses that: i) in female rats the GABAergic tone controlling gastric motility is higher than in male rats; ii) estrogen levels in female rats influence the gastric motility response; and, iii) the response to exogenous application of estrogen in the DMV varies between male and female rats.

In vivo anesthetized preparations of Sprague‐Dawley rats were used to compare gastric tone and motility in age‐matched males and virgin females; female rats were further subdivided according to the high estrogen (pro‐estrus and estrus) or low‐estrogen (meta‐ and di‐estrus) period of their cycle. To record gastric tone and motility, miniature strain gauges were sutured onto both the anterior corpus and antrum portions of the stomach. Following exposure of the dorsal brainstem, microinjections of the GABA‐A selective antagonist bicuculline methiodide (BIC, 0.1–5 pmoles/60nL) increased gastric tone and motility in a dose‐dependent manner; the increase was significantly higher in high estrogen (BIC 0.1pmoles increased gastric tone by 192±51 mg and motility by 158±53%; N=5) than in low estrogen females (100±29 mg and 175±112%; N=4) or males (56±17 mg and 111±19%; N=6). Conversely, microinjections of 17b‐estradiol (10pmoles/60nl) decreased gastric motility and tone by −141±26 mg and 145±33% (N=9) in males and by −111±54 mg and 153±40% (N=4) and −205±64 mg and 84±15% (N=5) in low and high‐estrogen females, respectively. The tone and motility effects of 17b‐estradiol were antagonized by pretreatment with the GPER receptor antagonist ICI182,720 (3nmol).

The data suggests that i) in female rats the GABAergic tone controlling gastric motility is higher than in male rats; ii) estrogen levels in female rats influence the gastric motility response; and, iii) the response to exogenous application of estrogen in the DMV varies between male and female rats. Taken together, the data supports the hypothesis that estrogen mediates a greater inhibitory input to the DMV through increased expression GABAergic inputs.

Support or Funding Information

Supported by ANMS and by AHA grants
Salmonella Enteritidis effector protein AvrA stabilizes tight junctions via the JNK pathway in epithelial cells
Authors: Lin Zhijie , Zhang Yong‐Guo , Xu Xiulong , Jiao Xinan , Sun Jun
Published: November 2023

Host‐bacterial interactions play sentential roles in pathogenesis of various human diseases. Bacteria possesses a range of effector proteins that are translocated into the host cells via a type III secretion system (T3SS). These bacterial effector proteins display a large repertoire of biochemical activities and modulate the function of host regulatory molecules, including tight junction proteins and their up‐stream regulators. Bacterial effector proteins paralyze or reprogram the eukaryotic cell to the benefit of the pathogens. AvrA is a Salmonella effector protein that possesses acetyltransferase activity and inhibits JNK/AP‐1 signaling pathways. While most of the Salmonella pathogenesis studies to date have focused on Salmonella typhimurium, the pathogenesis of a major serotype of Salmonella Enteritidis is poorly understood. Earlier studies on AvrA were based on S. typhimurium in vitro and in mice, However, bacterial effectors in Salmonella Enteritidis still need to be explored. We hypothesize that Salmonella Enteritidis protein AvrA stabilizes intestinal epithelial tight junctions (TJs) and decrease bacterial invasion. In the current studies, we established deletion mutant S.E‐AvrA‐ and plasmid‐mediated complementary strain S.E‐AvrA‐/AvrA+ (S.E‐AvrA+) in Salmonella Enteritidis. Using in vitro and in vivo Salmonella Enteritidis infection models, we showed that AvrA stabilizes epithelial TJ proteins, such as ZO‐1, in human intestinal epithelial cells. Trans‐epithelial electric resistance is significantly higher in cells infected with AvrA expressing strain, compared to cells infected with AvrA‐ strain. Inhibition of the JNK pathway suppresses the disassembly of TJ proteins. We further found that AvrA inhibited the activation of JNK activity in cells with wild‐type and AvrA+ strain infection. Therefore, AvrA‐induced ZO‐1 stability is via suppression of the JNK pathway. Furthermore, we found that S.E‐AvrA‐ strain led to more bacterial invasion, compared to wild‐type and AvrA+, in both human epithelial Caco‐2 BBE cells and mouse colon in vivo. Taken together, our data reveal a novel role of AvrA in Salmonella Enteritidis that stabilizes TJs and decreases bacteria invasion in intestinal epithelial cells. Our findings indicate an important role of bacterial protein that influences host cellular functions and bacterial invasion during infection and inflammation. Manipulation of JNK activity and TJs in microbial‐epithelial interactions could be a novel therapeutic approach to treat infectious diseases.
NHE8 is required for goblet cell function
Authors: Xu Hua , Li Jing , Ghishan Fayez
Published: November 2023

We reported previously that NHE8 plays an important role in mucosal protection. We found that mice lacking NHE8 are susceptible to DSS treatment and that loss of NHE8 expression in mice reduced muc2 expression and the number of PAS‐positive goblet cells. To increase our understanding of the impact and mechanism of these cellular changes, we studied cell proliferation using BrdU labeling. Our data showed that the rate of intestinal cell proliferation was enhanced in NHE8KO mice compared with their wild‐type littermates. We also analyzed additional genomic alterations by microarray and real‐time PCR. At the gene expression level, the intestinal expression of E2F2, a transcriptional factor involved in cell cycle regulation, Lgr5, an adult stem cell marker, and Wnt3, a secreted signaling protein, were elevated in NHE8KO. Therefore, the reduced mucosal protection in NHE8KO mice is most likely due to the disruption of normal goblet cell function.

Support or Funding Information

This study is supported by NIH grant R01DK073638.
Impact of Dietary Fat on Murine Gut Microbiota and Fatty Liver
Authors: Shang Yue , Derakhshani Hooman , Sarna Lindsei K , Siow Yaw L , Khafipour Ehsan , Karmin O
Published: November 2023

Overconsumption of fat‐rich diets is a potential risk for obesity and metabolic disease such as non‐alcoholic fatty liver disease (NAFLD). Gut microbiota is an important factor that plays a role in regulating energy homeostasis and body weight control. An obesity‐associated gut microbiota can be induced by a high‐fat diet (HFD). Dietary intervention is one of the approaches for obesity management. The aim of our study was to investigate the impact of dietary fat intervention on the composition of gut microbiota. Male C57BL/6J mice were fed a control (10% kcal fat) or a HFD (60% kcal fat) for 7 weeks. A third group of mice was fed a HFD for 5 weeks followed by a control diet for 2 weeks (HFD + Control). DNA was extracted from cecal and colonic mucosa samples for amplification of V4 region of bacterial 16S rRNA genes and subjected to Illumina sequencing. Bioinformatics analyses were performed using QIIME. Statistical difference was determined by one‐way ANOVA and Permutational MANOVA at P < 0.05. At the end of 7 week trial, body weight gain of the control or the HFD+Control group was significantly lower than that of the HFD. Significant differences were observed on richness, abundance, diversity and functional properties of the gut microbiota in cecal and colonic mucosa between the control and the HFD group. The HFD+Control diet partially shifted microbiota profile to the control and reduced hepatic lipid accumulation. In conclusion, our results suggested that 5‐week HFD induced obesity‐associated microbiota. Withdrawal of HFD could prevent excess body weight gain and attenuated fatty liver while only partially restored the composition, diversity and profile of gut microbiota.

Support or Funding Information

The study was supported, in part, by NSERC, CIHR and St. Boniface Hospital Research Centre.
A Systematic Examination of the Association between Parental and Child Obesity: Systematic Review and Meta‐Analysis
Authors: Wang Youfa , Min Jungwon , Khuri Jacob , Li Miao
Published: November 2023

Objectives

Childhood obesity has become a global epidemic. Many factors contribute to childhood obesity. Parents and family influence children's energy‐balanced related behaviors and thus likely affect their weight status. However, the reported intergenerational association in obesity varies and no systematic examination has been reported. This study systematically examined the association between parental and child obesity/overweight and its variation by demographic characteristics and countries.

Methods

A systematic literature search was conducted using PubMed for relevant studies published worldwide between Jan 1, 2000 and July 31, 2015. Strength of overall parent‐child (P‐C) obesity (including overweight, body mass index) associations was evaluated and compared across demographic factors and country economic development level including by meta‐analysis of meta‐regression models with random effects.

Results

In total, 32 studies from 21 countries met inclusion criteria, and 27 of them reported odds ratios (ORs) as the key measure of P‐C obesity association and were included in meta‐analysis. About 2/3 of the 32 studies reported moderate or strong P‐C association in obesity. Meta‐analysis showed overall P‐C weight status association was significant (pooled OR=2.22 (2.09–2.36), ie, children with overweight/obese parents were twice more likely to be overweight/obese than those with normal weight parents), and the association varied by type of P‐C pairs, child age, parent/child weight status, and country economic development level. The association with both parents examined was more likely to be strong (63.0%) than those with either father (24.5%) or mother (30.2%) examined separately. The proportion of studies reporting strong P‐C association in high‐ and middle‐income countries were 40.2% and 16.1%, respectively. Meta‐analysis shows a stronger association (ie, higher ORs) was more likely to be reported for older children (β (SE)= 0.02 (0.01)), parental obesity (0.26 (0.10)) and child obesity (0.28 (0.12)) than if younger children and parental and child overweight were examined.

Conclusions

Available research from multiple countries and with various study design show association between parent‐child obesity, but the association strength varied according to child age, types of parent‐child pair, their weight statuses and country‐level economic development. Families and parents are a key target for obesity prevention.

Support or Funding Information

Funding: NIH U54 center grant HD070725.
Berberine Ameliorates Hepatic Steatosis and Suppresses Liver and Adipose Tissue Inflammation in Obesity Mice Independent of AMPK
Authors: Woo Shih‐Lung , Guo Ting , Guo Xin , Li Honggui , Zheng Juan , Botchlett Rachel , Cai Yuli , Li Xiaodong , Li Qifu , Xiao Xiaoqiu , Huo Yuqing , Wu Chaodong
Published: November 2023

Much evidence has demonstrated the beneficial effects of berberine (BBR) on obesity‐associated non‐alcoholic fatty liver disease (NAFLD). However, the mechanisms underlying BBR actions remain to be explored. In the present study, we provided evidence to support an AMP‐activated protein kinase (AMPK)‐independent mechanism through which BBR suppresses obesity‐associated inflammation and improves hepatic steatosis. To induce obesity and obesity‐associated NAFLD, C57BL/6J mice were fed a high‐fat diet (HFD) for 12 weeks. During the last 4 weeks of the feeding regimen, the mice also were treated with BBR or vehicle control. Compared with the control, treatment with BBR decreased inflammation in both the liver and adipose tissue as indicated by reduction of the phosphorylation state of JNK1 and the mRNA levels of proinflammatory cytokines. Treatment of HFD‐fed mice with BBR also decreased hepatic steatosis, as well as the expression of lipogenesis‐related enzymes such as acetyl‐CoA carboxylase and fatty acid synthase. Interestingly, treatment of HFD‐fed mice with BBR did not significantly alter the phosphorylation state of AMPK in both the liver and adipose tissue. Consistently, BBR treatment significantly decreased the phosphorylation state of JNK1 in both hepatoma H4IIE cells and mouse primary hepatocytes in dose‐dependent and time‐dependent manners. BBR treatment also caused a decrease in palmitate‐induced fat deposition in primary mouse hepatocytes. However, BBR treatment did not alter the phosphorylation state of AMPK in H4IIE cells. Although increasing AMPK phosphorylation in primary mouse hepatocytes, BBR treatment suppressed the inflammatory responses in a manner irrelevant to AMPK. Taken together, these results suggest that BBR improves aspects of NAFLD mainly by suppressing inflammation in an AMPK‐independent manner.

Support or Funding Information

This study was support in part by NIH grants.
Deficiency of Vascular TLR4 Protects Against Lipopolysaccharide‐Induced Endothelial Dysfunction and NADPH oxidase‐derived Superoxide
Authors: Didion Sean
Published: November 2023

The goal of this study was to test the hypothesis that homozygous toll‐like receptor 4 (TLR4) deficiency protects against lipopolysaccharide (LPS)‐induced endothelial dysfunction. Carotid arteries from wild‐type (TLR4+/+) and TLR4−/− mice were incubated in DMEM with either vehicle (saline) or LPS (0.05–0.50 μg/ml) for 22 hrs. LPS produced concentration‐dependent impairment (P<0.05) of endothelium‐dependent relaxation to acetylcholine in wild‐type mice. Both basal and NADPH‐stimulated superoxide levels were selectively increased in arteries from wild‐type mice treated with LPS. Tempol, a superoxide scavenger, improved endothelial responses and lowered superoxide levels in vessels from wild‐type mice treated with LPS. In contrast, LPS had no effect (P>0.05) on responses to acetylcholine or on basal or NADPH‐stimulated superoxide levels in TLR4−/− mice. The effects of LPS were selective for endothelium as responses to nitroprusside were unaltered (P>0.05) in either LPS‐treated wild‐type or TLR4−/− mice. Taken together, these genetic and pharmacological findings demonstrate that TLR4 expressed within the vascular wall contributes to LPS‐induced impairment of endothelial function, which is mediated in large part by increases in NADPH oxidase‐derived superoxide.

Support or Funding Information

Supported by NIH HL‐089884 and HL‐107632.
Molecular Targets for Nitric Oxide Deficient Priapism
Authors: La Favor Justin D , Fu Zongming , Bivalacqua Trinity J , Van Eyk Jennifer E , Burnett Arthur L
Published: November 2023

Priapism is a disorder in which prolonged penile erection persists uncontrollably, potentially leading to tissue damage. Priapism commonly afflicts patient populations with severe nitric oxide (NO) deficiency. As NO is a primary mediator of erection, the molecular mechanisms involved in the pathophysiology of NO‐deficient priapism are not well understood. The objective of this study was to identify novel molecular targets and signaling pathways in a mouse model of NO deficiency. Double NO synthase (NOS) knockout mutant mice (dNOS−/−), in which endothelial NOS and the α‐splice variant of neuronal NOS are deficient, were used as a model of NO‐deficiency. Intracavernosal pressure (ICP) was measured in response to low level electrical stimulation of the cavernous nerve in male dNOS−/− and age‐matched wild type (WT) mice (n=7/group). Priapic activity was demonstrated in the dNOS−/− mice, as the duration of erection during 4 minutes immediately following electrical stimulation was significantly elevated (64 ± 23 sec vs. 2.6 ± 2.6 sec; P = 0.02) relative to WT. Penile tissue was harvested, and samples processed and analyzed by reversed‐phase liquid chromatography tandem mass spectrometry. Proteins corresponding to differentially (P < 0.05) detected spectral counts were imported into Ingenuity Pathway Analysis (IPA) software for analysis of divergent protein networks and signaling pathways. 1518 total proteins were identified, 53 of which were significantly downregulated and 107 of which were significantly upregulated in dNOS−/− vs. WT. IPA revealed 11 protein networks, 98 transcription factors, and 81 signaling pathways predicted to be significantly dysregulated. Increased myosin light chain phosphatase protein content, protein kinase A signaling, and decreased adenosine catabolism in dNOS−/− penis all represent potential compensatory mechanisms to NO‐deficiency that may lead to priapism. In conclusion, these results identify novel candidate proteins that may play a role in priapism secondary to NO‐deficiency.

Support or Funding Information

Supported by a mini grant from the National Kidney Foundation of Maryland and National Institutes of Health grant 1F32DK100082.
Deficiency in the Anti‐Aging Gene Klotho Promotes Aortic Valve Fibrosis through AMPKα‐mediated Activation of RUNX2
Authors: Chen Jianglei , Lin Yi , Sun Zhongjie
Published: November 2023

Background

Fibrotic aortic valve disease (FAVD) is an important cause of aortic stenosis, yet currently there is no effective treatment for FAVD due to its unknown etiology. The purpose of this study is to investigate whether deficiency in the anti‐aging Klotho gene (KL) promotes high‐fat‐diet‐induced FAVD and to explore the underlying molecular mechanism.

Methods & Results

Heterozygous Klotho‐deficient (KL+/−) mice and WT littermates were fed with a high‐fat diet (HFD) or normal diet for 13 weeks, followed by treatment with the AMPKa activator (AICAR) for an additional 2 weeks. A HFD caused a greater increase in collagen levels in the aortic valves of KL+/− mice than of WT mice, indicating that Klotho deficiency promotes HFD‐induced aortic valve fibrosis (AVF). AMPKa activity (pAMPKα) was decreased while protein expression of collagen I and RUNX2 was increased in the aortic valves of KL+/− mice fed with a HFD. Treatment with AICAR markedly attenuated HFD‐induced AVF in KL+/− mice. AICAR not only abolished the downregulation of pAMPKα but also eliminated the upregulation of collagen I and RUNX2 in the aortic valves of KL+/− mice fed with HFD. In cultured porcine aortic valve interstitial cells, Klotho‐deficient serum plus cholesterol increased RUNX2 and collagen I protein expression, which were attenuated by activation of AMPKa by AICAR. Interestingly, silencing of RUNX2 abolished the stimulatory effect of Klotho deficiency on cholesterol‐induced upregulation of matrix proteins, including collagen I and osteocalcin.

Conclusion

Klotho gene deficiency promotes HFD‐induced fibrosis in aortic valves, likely through the AMPKa–RUNX2 pathway.

Support or Funding Information

supported by NIH R01 HL118558, DK093403, and HL105302.
Lethal H5N1 Virus Invades Medullary Respiratory‐related Neurons Responsible for Respiratory Chemoreflexes and Rhythm/Pattern Generation
Authors: Zhuang Jianguo , Gao Xiuping , Ye Chunyan , Xu Fadi
Published: November 2023

Highly pathogenic H5N1 avian influenza virus is neurotropic and its infection is lethal mainly due to the respiratory failure. Studies have shown that lethal H5N1 HK483 (A/HK/483/97) virus invades the central nervous system, blunts ventilatory responses to hypoxia and hypercapnia, and induces apneas, leading to death at 6–8 days postinfection (dpi) in mice (Gao et al, Journal of virology 1999, Zhuang et al., ATS 2013, EB 2015). However, the relevant mechanisms remain unclear. We asked if HK483 H5N1 virus: 1) invaded neurons and/or astrocytes in the medullary respiratory‐related nuclei; 2) infected those respiratory neurons expressing neurokinin 1 receptor (NK1R), tyrosine hydroxylase (TH, a marker of catecholaminergic neuron), or tryptophan hydroxylase (TPH, a marker of serotonergic neuron); and 3) altered the these neural expressions of NK1R, TH, or TPH. Female Balb/c mice were intranasally inoculated with vehicle (Ctrl) or HK483 virus (100 PFU) and euthanized at 6–7 dpi. After fixation, the medullary sections were immunohistochemically processed to detect the nucleoprotein of H5N1 influenza A (NP) in neurons and/or astrocytes marked by glial fibrillary acidic protein (GFAP) and its co‐labeling with immunoreactivity (IR) of NK1R, TH, or TPH. We found that: 1) HK483 virus mainly invaded the respiratory‐related nuclei including the nucleus of solitary tract (NTS), ambiguus nucleus (Amb), pre‐Botzinger complex (PBC), Botzinger complex (BC), retrotrapzoid nucleus (RTN) and their neighboring areas with the raphe less infected and the invasion in astrocytes was limited; 2) approximately 20–50% of NK1R neurons (the NTS, PBC, and RTN), 10–30% of TH‐IR neurons (C1/2, A1 areas, the NTS, and locus ceruleus), and < 10% TPH (the raphe) were co‐labeled with NP; and 3) significantly reduced the density of NK1R‐IR in the corresponding nuclei with less effect on reduction of the raphe neural population expressing TPH and no effect on TH‐IR. We conclude that HK483 is capable of invading the medullary respiratory nuclei, particularly those neurons expressing NK1R or TPH to depress NK1R or TPH expression, which likely contributes to depression of the ventilatory responses to hypoxia and hypercapnia and introduction of apneas/respiratory failure, leading to the death.

Support or Funding Information

Supported by HL119683 and HL107462