Pair-fed Mice Research Articles

Benefits of calorie restriction in mice are mediated via energy imbalance, not absolute energy or protein intake

Caloric restriction (CR) results in reduced energy and protein intake, raising questions about protein restriction’s contribution to CR longevity benefits. We kept ad libitum (AL)–fed male C57BL/6J mice at 27°C (AL27) and pair-fed (PF) mice at 22°C (22(PF27)). The 22(PF27) group was fed to match AL27 while restricted for calories due to cold-induced metabolism. The 22(PF27) mice had significantly lower body weight, lean mass, fat mass, leptin, IGF-1, and TNF-α levels than AL27 mice (p<0.001 for all). Manipulations over ~11 weeks resulted in significant differences in body temperature, physical activity, and expression of key genes linked to hunger in the hypothalamus. Survival was significantly greater in 22(PF27) compared to AL27 overall (p<0.001). CR in the context of equivalent energy and protein intake resulted in hormonal, metabolic, and physiological benefits and extended longevity. Hence, energy imbalance, rather than low energy or protein intake per se, mediates the benefits of CR.

Sodium Bicarbonate Decreases Alcohol Consumption in Mice

pH is an important biological factor in the brain as H+ alters the structure and function of many ion channels, transporters and receptors, thus affecting relevant synaptic activities and complex firing patterns. pH may change activities in the neural circuits responsible for motivation and reward, such that drug reward values or modulatory properties are modified to change drinking frequency and amount. In this study, we tested whether alcohol consumption can be affected by systemic pH in mice. C57BL/6J mice were given free access to 0.2 M NaHCO3 for 6 days, which resulted in a 0.2 pH unit increase compared to water-fed control mice ( p &lt; 0.05; n = 6/group). Mice were then subjected to voluntary alcohol consumption in the two-bottle free choice procedure with one bottle containing water and the other containing 5-20% alcohol (5% increment for 4 days each). NaHCO3-fed mice consumed similar amounts of 5-15% alcohol as pair-fed mice but decreased the consumption of 20% alcohol (p &lt; 0.05). Consistent with this result, NaHCO3-fed mice had decreased alcohol preference (i.e., alcohol intake/total fluid intake) at 20%. These changes were observed in both males and females. In separate experiments, mice were subjected to the two-bottle free choice procedure using water and 20% alcohol, with both bottles containing 0.05-0.4 M NaHCO3. The alcohol consumption was significantly decreased in the presence of 0.1 M or higher NaHCO3. The minimum effective dose of NaHCO3 was 0.08 g/kg body weight, calculated from the amount of alcohol mice consumed. Determined by the diarrhea scoring system, mice produced a normal state of feces after receiving 0.4 M NaHCO3 for 2 days, thus indicating negligible abdominal discomfort at this concentration. In other experiments, mice were given NaHCO3 by oral gavage and 1 hour later subjected to 20% alcohol in the drinking in the dark procedure. The alcohol consumption was significantly decreased in NaHCO3-fed mice, compared to pair-fed mice. In conclusion, our study shows that mildly raising blood pH by NaHCO3 decreases alcohol consumption in mice. Given that pH change has been considered as a consequence of alcohol metabolism, our data provide first evidence on the pH as a factor affecting alcohol consumption. This work was supported by the grant from the National Institute of Alcohol Abuse and Alcoholism (R21AA028606). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

C-Jun N-Terminal Kinase-2 (JNK2) Plays a Crucial Role in Alcohol and Corticosterone-Induced Dysbiosis of Gut Microbiota and Tissue Injury at the Gut-Liver Axis

Introduction: About 15% of heavy drinkers develop liver disease, suggesting the “Multiple hit hypothesis” of Alcohol-Associated Liver Diseases (AALD). Chronic stress is a potential second hit in AALD pathogenesis. Our recent study showed that alcohol-induced gut and liver injury is exacerbated by chronic restraint stress or corticosterone. JNK2 plays an essential role in stress-induced cell injury. In this study, we investigated the role of JNK2 in ethanol (EtOH) and corticosterone-induced gut and liver injury and microbiota dysbiosis in mice. Methods: Wildtype (WT) and JNK2 knockout (JNK2-KO) mice (male and female; 8-10 wks) were fed a Lieber-DiCarli liquid diet with or without EtOH (0% 2d, 1% 2d, 2% 2d, 4% 1wk, 5% 1wk, &amp; 6% 1wk). Control groups were pair-fed isocaloric EtOH-free diet. One group of pair-fed and EtOH-fed WT and JNK2-KO mice were administered corticosterone (CORT, 25 mg/kg/day; s.c.). Intestinal permeability was measured by vascular-to-luminal flux of FITC-inulin in vivo, tight junction (TJ) and adherens junction (AJ) integrity was assessed by confocal microscopy for occludin, ZO-1, E-cadherin, and β-catenin, and mucosal inflammation by RT-PCR for cytokines/chemokines. Plasma lipopolysaccharide (LPS) and cytokines were measured to evaluate endotoxemia and systemic inflammation. Liver damage was assessed by measuring plasma AST/ALT, liver triglyceride, and cytokine/chemokine mRNA. Gut microbiota was analyzed by 16S rRNA sequencing of colonic flushing followed by bioinformatics analyses. Results and conclusion: In WT mice, EtOH feeding increased intestinal permeability, disrupted epithelial TJ and AJ, and elevated mucosal cytokine expression, indicating epithelial barrier dysfunction and mucosal inflammation. EtOH increased plasma LPS and cytokine/chemokine levels, indicating endotoxemia and systemic inflammation. Plasma AST/ALT and liver triglyceride levels were increased, indicating EtOH-induced liver damage. CORT significantly elevated EtOH-induced intestinal permeability, TJ/AJ disruption, endotoxemia, systemic inflammation, and liver damage in WT mice. These effects of EtOH and CORT were absent in JNK2-KO mice. Microbiota α-diversity was higher in JNK2-KO mice than in WT mice, and β-diversity analysis showed that the microbiota composition in WT mice differed from that of KO mice. Microbiota α- and β-diversities were altered by EtOH and CORT in WT mice but not in JNK2-KO mice. The relative abundance of Nocardiaceae and Verrucomicrobiaceae was high, and Ruminococcaceae and Bacillaceae were low in KO mice. CORT decreased the abundance of Caulobacteriaceae and Ruminococcaceae and increased Verrucomicrobiaceae and Lactobacillaceae in WT but not KO mice. EtOH increased Ruminococcaceae and Bacillaceae and decreased Lactobacillaceae in WT mice but not KO mice. CORT-induced modulation of EtOH-induced microbiota dysbiosis was observed in WT mice but not KO mice. In conclusion, these data demonstrate that JNK2 regulates gut microbiota in mice, and JNK2 deficiency confers resistance to alcohol and corticosterone-induced tissue injury and microbiota dysbiosis. Acknowledgements: NIH/NIAAA - R01AA012307, AA029270; VA - IO1BX003014. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Spatial Proteomics Reveals Alcohol-Induced Damages to the Crypts and Villi of the Mouse Small Intestine.

Alcohol consumption perturbs the gut immune barrier and ultimately results in alcoholic liver diseases, but little is known about how immune-related cells in the gut are perturbed in this process. In this study, we employed laser capture microdissection and a label-free proteomics approach to investigate the consequences of alcohol exposure to the proteomes of crypts and villi in the proximal small intestine. Intestinal tissues from alcohol-fed and pair-fed mice were microdissected to selectively capture cells in the crypts and villi regions, followed by one-pot protein digestion and data-independent LC-MS/MS analysis. We successfully identified over 3000 proteins from each of the crypt or villi regions equivalent to ∼3000 cells. Analysis of alcohol-treated tissues indicated an enhanced alcohol metabolism and reduced levels of α-defensins in crypts, alongside increased lipid metabolism and apoptosis in villi. Immunofluorescence imaging further corroborated the proteomic findings. Our work provides a detailed profiling of the proteomic changes in the compartments of the mouse small intestine and aids in molecular-level understanding of alcohol-induced tissue damage.

Sex-Specific Effect of Ethanol on Colon Content Lipidome in a Mice Model Using Nontargeted LC/MS.

Consumption of alcohol has widespread effects on the human body. The organs that are most significantly impacted are the liver and digestive system. When alcohol is consumed, it is absorbed in the intestines and processed by the liver. However, excessive alcohol use may affect gut epithelial integrity, microbiome composition, and lipid metabolism. Despite past studies investigating the effect of ethanol on hepatic lipid metabolism, the focus on colonic lipid metabolism has not been well explored. In this study, we investigated the sex-specific effect of ethanol on the colonic content lipidome in a mouse model using nontargeted liquid chromatography-mass spectrometry. Comprehensive lipidome analysis of colonic flush samples was performed using ethanol-fed (EF) and pair-fed (PF) mice of each sex. Partial least-squares discriminant analysis revealed that ethanol altered colonic lipid composition largely in male mice compared with female mice. A significant increase in free fatty acids, ceramides, and hexosylceramides and decreased phosphatidylglycerols (PG) was observed in the EF group compared to the PF group in male mice. Phosphatidylethanolamine (PE) levels were increased significantly in the EF group of both sexes compared to the PF group. The volcanic plot shows that PG (O-15:1/15:0) and PE (O-18:2/15:0) are common markers that are increased in both sexes of the EF group. In addition, decreased fatty acid esters of hydroxy fatty acids (FAHFA) were observed specifically in the EF group of female mice. Overall, a significant variation in the mice colonic content lipidome between the EF and PF groups was observed. Target pathways, such as sphingolipid metabolism in males, FAHFA in females, and PE metabolism in both sexes, were suggested. This study provides new insight into the sex-dependent lipid change associated with alcohol-induced gut-microbiota dysfunction and its potential health impacts.

Porphyromonas gingivalis Strain W83 Infection Induces Liver Injury in Experimental Alcohol-Associated Liver Disease (ALD) in Mice

The liver plays a vital role in the defense against infections. Porphyromonas gingivalis (P. gingivalis), a dominant etiologic oral bacterium implicated in periodontal disease (PD), has been associated with various systemic diseases. This study aimed to investigate the influence of P. gingivalis on alcohol-associated liver diseases (ALD). Mice were fed a Lieber–DeCarli liquid diet containing 5% ethanol for 10 days after an initial adaptation period on a diet with lower ethanol content for 7 days. Two days before tissue sample collection, the mice were administered P. gingivalis strain W83 (Pg) through intraperitoneal injection (IP). Pair-fed mice with Pg infection (PF+Pg) exhibited an activated immune response to combat infections. However, alcohol-fed mice with Pg infection (AF+Pg) showed liver injury with noticeable abscess lesions and elevated serum alanine aminotransferase (ALT) levels. Additionally, these mice displayed liver infiltration of inflammatory monocytes and significant downregulation of proinflammatory cytokine gene expression levels; and AF+Pg mice also demonstrated increased intrahepatic neutrophil infiltration, as confirmed by chloroacetate esterase (CAE) staining, along with elevated gene expression levels of neutrophil cytosol factor 1 (Ncf1), neutrophilic inflammation driver lipocalin 2 (Lcn2), and complement component C5a receptor 1 (C5ar1), which are associated with neutrophilic inflammation. Interestingly, compared to PF+Pg mice, the livers of AF+Pg mice exhibited downregulation of gene expression levels of NADPH oxidase 2 (Cybb), the leukocyte adhesion molecule Cd18, and the Toll-like receptor adaptor Myd88. Consequently, impaired clearance of P. gingivalis and other bacteria in the liver, increased susceptibility to infections, and inflammation-associated hepatic necrotic cell death were observed in AF+Pg mice, which is likely to have facilitated immune cell infiltration and contributed to liver injury. Furthermore, in addition to the Srebf1/Fasn pathway induced by alcohol feeding, Pg infection also activated carbohydrate response element-binding protein (ChREBP) in AF+Pg mice. In summary, this study demonstrates that P. gingivalis infection, acting as a “second hit”, induces dysfunction of immune response and impairs the clearance of bacteria and infections in alcohol-sensitized livers. This process drives the development of liver injury.

Cytarabine induces cachexia with lipid malabsorption via zippering the junctions of lacteal in murine small intestine

Chemotherapy-induced cachexia causes severe metabolic abnormalities independently of cancer and reduces the therapeutic efficacy of chemotherapy. The underlying mechanism of chemotherapy-induced cachexia remains unclear. Here we investigated the cytarabine (CYT)-induced alteration in energy balance and its underlying mechanisms in mice. We compared energy balance-associated parameters among the three groups of mice: CON, CYT, and PF (pair-fed mice with the CYT group) that were intravenously administered vehicle or CYT. Weight gain, fat mass, skeletal muscle mass, grip strength, and nocturnal energy expenditure were significantly lowered in the CYT group than in the CON and PF groups. The CYT group demonstrated less energy intake than the CON group and higher respiratory quotient than the PF group, indicating that CYT induced cachexia independently from the anorexia-induced weight loss. Serum triglyceride was significantly lower in the CYT group than in the CON group, whereas the intestinal mucosal triglyceride levels and the lipid content within the small intestine enterocyte were higher after lipid loading in the CYT group than in the CON and PF groups, suggesting that CYT inhibited lipid uptake in the intestine. This was not associated with obvious intestinal damage. The CYT group showed increased zipper-like junctions of lymphatic endothelial vessel in duodenal villi compared to that in the CON and CYT groups, suggesting their imperative role in the CYT-induced inhibition of lipid uptake. CYT worsens cachexia independently of anorexia by inhibiting the intestinal lipid uptake, via the increased zipper-like junctions of lymphatic endothelial vessel.

Iron Status Alters Murine Systemic Lupus Erythematosus

The effect of Fe status on murine systemic lupus erythematosus was investigated. Weanling female MRL/MPJ-lpr/lpr mice (systemic lupus erythematosus strain) were fed diets with the following levels (mg Fe/kg diet): 3 (severely deficient), 10 (moderately deficient), 35 (control) and 250 (supplemented). A fifth group was pair fed the control diet in the amounts consumed by the severely deficient group. C3H/Hej mice fed the same diets were used as nonlupus controls. Anemia was more severe in severely deficient mice than in all other MRL groups and C3H severely deficient mice. Incidence of skin lesions was highest in MRL severely and moderately deficient mice compared with pair-fed, control and supplemented mice. By 22 wk of age, mortality was higher in supplemented and severely deficient mice than in moderately deficient, pair-fed and control MRL mice. Anti-dsDNA activity in serum was not altered by Fe. In a second experiment, kidney function was examined in mice fed severely deficient, control, supplemented and pair-fed diets. Urine protein concentration was highest in supplemented mice at 14 wk of age. Serum urea nitrogen was significantly higher in MRL severely deficient mice than in pair-fed and control mice at 18 wk of age. Glomerular filtration rate, measured by creatinine clearance, was significantly lower in MRL severely deficient mice than in pair-fed and Fe supplemented mice at 16 wk of age and pair-fed and control mice at 18 wk of age. Renal histopathology was more severe in Fe supplemented mice than in pair-fed and control mice, and more severe in severely deficient and pair-fed mice than in control mice. Fluorescent staining of kidneys with anti-Ig G and anti-C3 fluorescein-conjugated antibodies was most intense in severely deficient mice, and the concentration of circulating immune complexes in serum was significantly higher in severely deficient mice than in all other groups. These data demonstrate that systemic lupus erythematosus in MRL/MPJ-lpr/lpr mice is altered by dietary iron.

Skeletal Muscle Myokine Expression in Critical Illness, Association With Outcome and Impact of Therapeutic Interventions.

Muscle expresses and secretes several myokines that bring about benefits in distant organs. We investigated the impact of critical illness on muscular expression of irisin, kynurenine aminotransferases, and amylase; association with clinical outcome; and impact of interventions that attenuate muscle wasting/weakness. We studied critically ill patients who participated in 2 randomized controlled trials (EPaNIC/NESCI) and documented time profiles in critically ill mice. Included in the study were 174 intensive care unit (ICU) patients (day 8 ± 1) vs 19 matched controls, and 60 mice subjected to surgery/sepsis vs 60 pair-fed healthy mice. Interventions studied included 7-day neuromuscular electrical stimulation (NMES), and withholding parenteral nutrition (PN) in the first ICU week (late PN) vs early PN. The main outcome measures were FNDC5 (irisin- precursor), KYAT1, KYAT3, and amylase mRNA expression in skeletal muscle. Critically ill patients showed 34% to 80% lower mRNA expression of FNDC5, KYAT1, and amylases than controls (P < .0001). Critically ill mice showed time-dependent reductions in all mRNAs compared with healthy mice (P ≤ .04). The lower FNDC5 expression in patients was independently associated with a higher ICU mortality (P = .015) and ICU-acquired weakness (P = .012), whereas the lower amylase expression in ICU survivors was independently associated with a longer ICU stay (P = .0060). Lower amylase expression was independently associated with a lower risk of death (P = .048), and lower KYAT1 expression with a lower risk of weakness (P = .022). NMES increased FNDC5 expression compared with unstimulated muscle (P = .016), and late PN patients had a higher KYAT1 expression than early PN patients (P = .022). Expression of the studied myokines was affected by critical illness and associated with clinical outcomes, with limited effects of interventions that attenuate muscle wasting or weakness.

Reversal of maternal obesity attenuates hypoxia and improves placental development in the preeclamptic-like BPH/5 mouse model.

Women with obesity have higher risk of adverse pregnancy outcomes, including preeclampsia (PE). Late-gestational hypertension, aberrant fetoplacental development, and fetal growth restriction (FGR), hallmarks of PE, are observed spontaneously in BPH/5 mice. Similar to obese preeclamptic women, BPH/5 mice have higher visceral white adipose tissue (WAT) and circulating leptin. We hypothesized that attenuation of maternal obesity and serum leptin in pregnant BPH/5 mice will improve fetoplacental development by decreasing hypoxia markers and leptin expression at the maternal-fetal interface. To test this hypothesis, BPH/5 mice were fed ad libitum (lib) and pair-fed (PF) to C57 ad lib controls beginning at embryonic day (e) 0.5. Hypoxia-related genes, hypoxia inducible factor (Hif) 1α, stem cell factor (Scf), heme oxygenase-1 (Ho-1), leptin (Lep), and leptin receptor (LepR) were assessed in e7.5 implantation sites. BPH/5 ad lib had 1.5 to 2-fold increase in Hif1α, Scf, and Ho-1 mRNA and a greater than 3-fold increase in leptin mRNA vs. C57 that was attenuated with PF. Exogenous leptin promoted Hif1α and Ho-1 mRNA expression in e7.5 decidua in vitro. While hypoxic conditions in vitro did not change decidual leptin mRNA. Furthermore, BPH/5 PF mice demonstrated improved fetal and placental outcomes later in gestation, with greater placental vascular area by e18.5 and attenuation of FGR. In conclusion, pair-feeding BPH/5 mice beginning at conception may improve placental vasculature formation via decreased leptin and hypoxia-associated markers in this model. Future investigations are needed to better determine the effect of hypoxia and leptin on pregnancy outcomes in obese pregnant women.

Leptin as an Antitorpor Hormone: An Explanation for the Increased Metabolic Efficiency and Cold Sensitivity of ob/ob Mice?

AbstractLeptin is recognized as an anorexigenic hormone. In its absence (e.g., in ob/ob mutant mice), mice become obese, primarily as a result of hyperphagia. A recurrent question is whether, additionally, leptin is thermogenic and thus also an antiobesity hormone in this way. We have earlier reviewed available data and have concluded that most articles implying a thermogenic effect of leptin have based this on a misconstrued division by body weight. Here, we have collected evidence that the remaining observations that imply that leptin is a thermogenic hormone are better understood as implying that leptin is an antitorpor hormone. Leptin levels increase in proportion to the body's energy reserves (i.e., stored lipids in the adipose tissue), and leptin thus serves as an indicator of energy availability. In the absence of leptin, ob/ob mice are exceedingly prone to enter daily torpor, since the absence of leptin causes them to perceive a lack of body energy reserves that, in combination with restricted or no food, induces them to enter the torpid state to save energy. This antitorpor effect of leptin probably explains the following earlier observations. First, ob/ob mice have the ability to gain weight even when pair fed with leptin-treated ob/ob mice. This is understood as follows: In the leptin-treated ob/ob mice, food intake is reduced. Untreated pair-fed mice enter daily torpor, and this markedly lowers total daily energy expenditure; the resulting surplus food energy is then accumulated as fat in these mice. However, ob/ob mice fed ad lib. do not enter torpor, so under normal conditions this mechanism does not contribute to the obesity found in the ob/ob mice. Second, neonatal ob/ob mice have the ability to become obese despite eating the same amount as wild-type mice: this is understood as these mice similarly entering daily torpor. Third, ob/ob mice on the C57BL/6J background have a lower metabolic rate: these mice were examined in the absence of food, and torpor was thus probably induced. Fourth, ob/ob mice have apparent high cold sensitivity: these mice experienced cold in the absence of food and would immediately enter deep torpor. It is suggested that this novel explanation of how the antitorpor effects of leptin affect mouse energy metabolism can open new avenues for leptin research.

Alcohol-dependent downregulation of apolipoprotein H exacerbates fatty liver and gut microbiota dysbiosis in mice

BackgroundAlcohol-related liver disease (ALD) is a major chronic liver ailment caused by alcohol overconsumption and abuse. Apolipoprotein H (APOH) participates in lipid metabolism and might have a potential regulatory role in ALD. Therefore, this study aimed to explore the effects of ApoH on alcohol-induced liver injury and gut microbiota dysbiosis.MethodsApoH−/− mice were generated and the synergic alcoholic steatohepatitis mouse model was constructed, which were used to assess liver function and pathological changes.ResultsApoH−/− mice clearly exhibited spontaneous steatohepatitis. Severe hepatic steatosis was observed in alcohol-fed WT and ApoH−/− mice, in which ApoH expression was reduced post alcohol consumption. Moreover, RNA-seq and KEGG pathway analyses indicated that differential expression genes enriched in lipid metabolism and oxidation–reduction process between in alcohol-fed ApoH−/− mice and pair-fed control mice. Finally, gut microbiota diversity and composition were assessed by 16S rRNA Illumina next-generation sequencing. Alpha diversity of enterobacteria was lower in ApoH−/− mice with ethanol feeding than in ethanol-fed WT mice and all control-fed mice (P < 0.05). Moreover, KEGG enrichment analysis, using PICRUSt software, revealed that metabolic functions were activated in the gut microorganisms of ApoH−/− mice with ethanol feeding (P < 0.05).ConclusionsAlcohol-downregulated ApoH expression, leading to the progress of fatty liver disease and gut microbiota dysbiosis.

Abstract 14292: Binge-on-Chronic Alcohol-Induced Gut Dysbiosis Results in Reduced Nitric Oxide Bioavailability and Endothelial Dysfunction

Introduction: Binge alcohol drinking adversely impacts cardiovascular (CV) homeostasis, leading to increased risk of CV diseases. It is also well-established that chronic alcohol consumption causes gut dysbiosis. However, the effects of alcohol-induced gut dysbiosis on vascular endothelial function remains unknown. Methods: We studied C57BL/6 mice at 10 weeks of age for these studies with n = 8-18 mice per group. Mice underwent a binge-on-chronic alcohol (BoCA) feeding protocol for 20-days and received either vehicle or probiotic treatment. Aortic vascular reactivity (acetylcholine and sodium nitroprusside), plasma nitrite (HPLC) and 8-isoprostane levels (ELISA) were measured. Another group of mice (n = 11-12 per group) received broad-spectrum antibiotic treatments followed by gut microbiotic transplants from either pair-fed or BoCA fed donor mice and plasma nitrite levels were measured to assess vascular function. Results: BoCA resulted in impaired endothelium-dependent vascular relaxation which was accompanied by reduced plasma nitrite levels. Probiotic treatment significantly attenuated vascular dysfunction, increased plasma nitrite and reduced 8-isoprostane levels. Further, mice receiving gut microbiotic transfer from alcohol-treated donor mice exhibited reduced circulating nitrite levels when compared to those who receiving the transfer from pair-fed donor mice. Conclusions: Our data demonstrate that BoCA consumption induced vascular injury resulting in reduced NO bioavailability, impaired vascular relaxation, and elevated oxidative stress which was attenuated by probiotic treatment. Mice receiving gut microbiotic contents from BoCA fed donor mice displayed reduced NO bioavailability, suggesting that gut dysbiosis is involved in the detrimental cardiovascular effects of BoCA. Further studies are underway to define the mechanisms by which alcohol-induced gut dysbiosis promotes cardiovascular disease.

Inhibition of the Renin-Angiotensin System Reduces Gene Expression of Inflammatory Mediators in Adipose Tissue Independent of Energy Balance.

Obesity is a growing health problem worldwide. The renin-angiotensin system (RAS) is present in adipose tissue, and evidence suggests that it is involved in both diet-induced obesity and the inflammation associated with obesity. The present experiments determined the effect of (1) different angiotensin-converting enzyme (ACE) inhibitors (captopril, perindopril, enalapril) and angiotensin receptor blockers (ARBs: telmisartan, losartan) on adiposity of mice fed a high-fat diet for 28 days (2); acute treatment with the ACE-inhibitor captopril on gene expression of inflammatory markers in mice fed a high-fat diet (HFD); and (3) short-term (2 days) and chronic (28 days) treatment of ACE-inhibition on energy expenditure (EE) and energy balance in mice fed HFD ad libitum (AL), as well as receiving HFD limited to the amount of calories eaten by controls (pair-fed (PF) group). Body weight, food intake, adiposity and plasma leptin were lower in ACE inhibitor or ARB-treated groups over 28 days compared with HFD untreated mice. Short-term treatment with captopril led to increased EE relative to the level in the PF group. After 28 days, EE was lower in both captopril-treated and PF mice compared with AL, but the effect was greater in the captopril-treated group. Adiponectin was elevated in captopril-treated mice, but not in PF mice, after both 2 and 28 days. Additionally, acute RAS blockade in HFD-fed mice reduced mRNA expression for MCP-1, IL-6, TLR4, and leptin in adipose tissue relative to values in untreated groups. These data demonstrate that ACE inhibition and angiotensin receptor blockade reduce food intake to produce weight loss and suggest that the anti-inflammatory effects of ACE inhibition may be independent of weight loss.

Increased hepatic JNK activation by ethanol is mediated by transcriptional suppression of mitogen‐activated protein kinase phosphatase 1 (Mkp1): role of cAMP‐specific protein kinase A

Background Increased and sustained JNK activation has been shown to be detrimental for hepatocyte survival and liver injury in various diseases, including alcohol-associated liver disease (ALD). Among three JNK isoforms, JNK1 and 2 are expressed in hepatocytes and both play a role in mouse models of ALD. Mkp1 phosphatase has been identified as a regulator of JNK activation. A previous study showed that Mkp1 degradation plays a critical role in sustained JNK activation and hepatocyte death from acute ethanol exposure. However, the effect of chronic ethanol exposure on hepatocyte Mkp1 expression is not known. Methods Male C57B/6 mice were subjected to intragastric (iG) feeding of ethanol for 8 weeks with weekly ethanol binge (5g/kg) starting in the second week of iG feeding. Pair-fed control mice were fed an isocaloric high-fat liquid diet. Plasma ALT, AST and histologic grading were performed to evaluate steatosis, liver inflammation and injury. Liver immunohistochemistry was performed to evaluate apoptosis (TUNEL staining). Myeloperoxidase (MPO) activity assay and chloroacetate esterase (CAE) staining for neutrophil infiltration were also performed. Liver RNA sequencing was performed on an Illumina NextSeq 500. Data analysis was done using the Tuxedo suite pipeline. Validation studies were carried out by performing real time qPCR and Western blot. Primary mouse hepatocytes were exposed to 50 mM ethanol for 48 hours followed by TNF stimulation. Statistical analysis was performed using ANOVA. Results Histological examination of liver sections confirmed the development of severe alcohol-associated steatohepatitis in ethanol fed mice via significant micro- and macro-vesicular steatosis, polymorphonuclear leukocyte (PMN) infiltration, CAE and TUNEL-positive cells. ALT and AST levels were significantly elevated in ethanol-fed mice. JNK activation was evident by Western blot analysis of phosphorylated JNK levels. RNA Seq analysis showed significant changes in expression of several Mkps; however, only Mkp1 levels were significantly downregulated, which was further validated by real time qPCR. Importantly, ethanol exposure of primary mouse hepatocytes led to a significant decrease in Mkp1 mRNA expression which further declined following TNF stimulation. These changes in Mkp1 were accompanied by sustained JNK activation and cell death. Moreover, ethanol exposure and TNF stimulation markedly increased cAMP specific PDE4 expression resulting in reduction in hepatocyte cAMP levels. Significantly, PDE4 inhibition and PKA specific agonists prevented the suppressive effect of ethanol and TNF on Mkp1 and resultant activation of JNK and cell death. Conclusion Our results strongly implicate ethanol-induced decline in hepatic Mkp1 as a significant mechanism regulating JNK activation and hepatocyte death in ALD. Further, these data strongly suggest that the ethanol induced decrease in cAMP/PKA signaling plays a causal role in regulating the Mkp1/JNK axis in hepatocytes and development of liver injury.

Treatment with Fenretinide Diminishes Alcohol Liver Disease

Alcohol liver disease (ALD) is a major cause of liver-related mortality globally, yet there remains an unmet demand for approved ALD drugs. The pathogenesis of ALD involves perturbations to the intestinal barrier and subsequent translocation of bacterial endotoxin that, acting through toll-like receptor 4 (TLR4), promotes hepatic inflammation and progression of ALD. In the present study we investigated the ability of fenretinide (Fen) [N-(4-hydroxyphenyl) retinamide], a synthetic retinoid with known anti-cancer and anti-inflammatory properties, to modulate intestinal permeability and clinical hallmarks of ALD in a mouse model of chronic ethanol (EtOH) exposure. Our results show that EtOH-treated mice had reductions in mRNA and protein expression of intestinal tight junction proteins, including claudin 1 and occludin, and increases in intestinal permeability and endotoxemia compared to pair-fed mice. Also, EtOH-treated mice had marked increases in hepatic steatosis, liver injury, and expression of pro-inflammatory mediators, including TNF-α, and TLR4-positive macrophages, Kupffer cells, and hepatocytes in the intestines and liver, respectively. In contrast, EtOH+Fen-treated mice were resistant to the effects of EtOH on promoting intestinal permeability and had higher intestinal protein levels of claudin 1 and occludin. Also, EtOH+Fen-treated mice had significantly lower plasma levels of endotoxin, and reductions in expression of TNF-α and TLR4 positive macrophages, Kupffer cells, and hepatocytes in the intestine and liver. Lastly, we found that EtOH+Fen-treated mice exhibited major reductions in hepatic triglycerides, steatosis, and liver injury compared to EtOH-treated mice. Our findings are the first to demonstrate that Fen possesses anti-ALD properties, potentially through modulation of the intestinal barrier function, endotoxemia, and TLR4-mediated inflammation. These data warrant further pre-clinical investigations of Fen as a potential anti-ALD drug.

Alcohol-associated intestinal dysbiosis alters mucosal-associated invariant T-cell phenotype and function.

Chronic alcohol consumption is associated with a compromised innate and adaptive immune responses to infectious disease. Mucosa-associated invariant T (MAIT) cells play a critical role in antibacterial host defense. However, whether alcohol-associated deficits in innate and adaptive immune responses are mediated by alterations in MAIT cells remains unclear. To investigate the impact of alcohol on MAIT cells, mice were treated with binge-on-chronic alcohol for 10days and sacrificed at day 11. MAIT cells in the barrier organs (lung, liver, and intestine) were characterized by flow cytometry. Two additional sets of animals were used to examine the involvement of gut microbiota on alcohol-induced MAIT cell changes: (1) Cecal microbiota from alcohol-fed (AF) mice were adoptive transferred into antibiotic-pretreated mice and (2) AF mice were treated with antibiotics during the experiment. MAIT cells in the barrier organs were measured via flow cytometry. Binge-on-chronic alcohol feeding led to a significant reduction in the abundance of MAIT cells in the barrier tissues. However, CD69 expression on tissue-associated MAIT cells was increased in AF mice compared with pair-fed (PF) mice. The expression of Th1 cytokines and the corresponding transcriptional factor was tissue specific, showing downregulation in the intestine and increases in the lung and liver in AF animals. Transplantation of fecal microbiota from AF mice resulted in a MAIT cell profile aligned to that of AF mouse donor. Antibiotic treatment abolished the MAIT cell differences between AF and PF animals. MAIT cells in the intestine, liver, and lung are perturbed by alcohol use and these changes are partially attributable to alcohol-associated dysbiosis. MAIT cell dysfunction may contribute to alcohol-induced innate and adaptive immunity and consequently end-organ pathophysiology.

Fenretinide Improves Intestinal Barrier Function and Mitigates Alcohol Liver Disease.

Alcohol liver disease (ALD) is a major cause of liver-related mortality globally, yet there remains an unmet demand for approved ALD drugs. The pathogenesis of ALD involves perturbations to the intestinal barrier and subsequent translocation of bacterial endotoxin that, acting through toll-like receptor 4 (TLR4), promotes hepatic inflammation and progression of ALD. In the present study we investigated the ability of fenretinide (Fen) [N-(4-hydroxyphenyl) retinamide], a synthetic retinoid with known anti-cancer and anti-inflammatory properties, to modulate intestinal permeability and clinical hallmarks of ALD in a mouse model of chronic ethanol (EtOH) exposure. Our results show that EtOH-treated mice had reductions in mRNA and protein expression of intestinal tight junction proteins, including claudin one and occludin, and increases in intestinal permeability and endotoxemia compared to pair-fed mice. Also, EtOH-treated mice had marked increases in hepatic steatosis, liver injury, and expression of pro-inflammatory mediators, including TNF-α, and TLR4-positive macrophages, Kupffer cells, and hepatocytes in the intestines and liver, respectively. In contrast, EtOH + Fen-treated mice were resistant to the effects of EtOH on promoting intestinal permeability and had higher intestinal protein levels of claudin one and occludin. Also, EtOH + Fen-treated mice had significantly lower plasma levels of endotoxin, and reductions in expression of TNF-α and TLR4 positive macrophages, Kupffer cells, and hepatocytes in the intestine and liver. Lastly, we found that EtOH + Fen-treated mice exhibited major reductions in hepatic triglycerides, steatosis, and liver injury compared to EtOH-treated mice. Our findings are the first to demonstrate that Fen possesses anti-ALD properties, potentially through modulation of the intestinal barrier function, endotoxemia, and TLR4-mediated inflammation. These data warrant further pre-clinical investigations of Fen as a potential anti-ALD drug.

Multiomics-Identified Intervention to Restore Ethanol-Induced Dysregulated Proteostasis and Secondary Sarcopenia in Alcoholic Liver Disease.

Signaling and metabolic perturbations contribute to dysregulated skeletal muscle protein homeostasis and secondary sarcopenia in response to a number of cellular stressors including ethanol exposure. Using an innovative multiomics-based curating of unbiased data, we identified molecular and metabolic therapeutic targets and experimentally validated restoration of protein homeostasis in an ethanol-fed mouse model of liver disease. Studies were performed in ethanol-treated differentiated C2C12 myotubes and physiological relevance established in an ethanol-fed mouse model of alcohol-related liver disease (mALD) or pair-fed control C57BL/6 mice. Transcriptome and proteome from ethanol treated-myotubes and gastrocnemius muscle from mALD and pair-fed mice were analyzed to identify target pathways and molecules. Readouts including signaling responses and autophagy markers by immunoblots, mitochondrial oxidative function and free radical generation, and metabolic studies by gas chromatography-mass spectrometry and sarcopenic phenotype by imaging. Multiomics analyses showed that ethanol impaired skeletal muscle mTORC1 signaling, mitochondrial oxidative pathways, including intermediary metabolite regulatory genes, interleukin-6, and amino acid degradation pathways are β-hydroxymethyl-butyrate targets. Ethanol decreased mTORC1 signaling, increased autophagy flux, impaired mitochondrial oxidative function with decreased tricarboxylic acid cycle intermediary metabolites, ATP synthesis, protein synthesis and myotube diameter that were reversed by HMB. Consistently, skeletal muscle from mALD had decreased mTORC1 signaling, reduced fractional and total muscle protein synthesis rates, increased autophagy markers, lower intermediary metabolite concentrations, and lower muscle mass and fiber diameter that were reversed by β-hydroxymethyl-butyrate treatment. An innovative multiomics approach followed by experimental validation showed that β-hydroxymethyl-butyrate restores muscle protein homeostasis in liver disease.

Regular exercise and branched-chain amino acids prevent ischemic acute kidney injury-related muscle wasting in mice.

Acute kidney injury (AKI) causes glucose and protein metabolism abnormalities that result in muscle wasting, thereby affecting the long‐term prognosis of critical illness survivors. Here, we examined whether early intervention with treadmill exercise and branched‐chain amino acids (BCAA) can prevent AKI‐related muscle wasting and reduced physical performance in mice. Unilateral 15 min ischemia‐reperfusion injury was induced in contralateral nephrectomized mice, and muscle histological and physiological changes were assessed and compared with those of pair‐fed control mice, since AKI causes severe anorexia. Mice exercised for 30 min each day and received oral BCAA for 7 days after AKI insult. By day 7, ischemic AKI significantly decreased wet weight, myofiber cross‐sectional area, and central mitochondrial volume density of the anterior tibialis muscle, and significantly reduced maximal exercise time. Regular exercise and BCAA prevented AKI‐related muscle wasting and low physical performance by suppressing myostatin and atrogin‐1 mRNA upregulation, and restoring reduced phosphorylated Akt and PGC‐1α mRNA expression in the muscle. Ischemic AKI induces muscle wasting by accelerating muscle protein degradation and reducing protein synthesis; however, we found that regular exercise and BCAA prevented AKI‐related muscle wasting without worsening kidney damage, suggesting that early rehabilitation with nutritional support could prevent AKI‐related muscle wasting.