Metabolic Cages Research Articles

Investigating the effects of thermoneutral housing on skeletal and cardiac muscle function in D2 mdx mice

Since its discovery, the mdx mouse has been the foundation for research on Duchenne muscular dystrophy (DMD) and its potential therapies. Although, nearly all studies using mdx mice as an experimental model have been conducted under “standard” lab conditions using room (22C) instead of thermoneutral (TN, 30C) temperature housing. Room temperature (RT) is a mild cold stress to mice, causing metabolic adaptations that can confound experimental results, limiting their interpretation and translation to humans. This study investigated the effects of housing temperature on whole-body metabolism and skeletal and cardiac muscle function in the mdx mouse. To this end, 20 D2 mdx mice (age 5 weeks, n = 10 per group) were housed at RT and TN for 10 weeks. Metabolic cage analysis demonstrated that TN housed mdx mice displayed significantly decreased VO2 consumption and reduced food intake that was likely due to lowered daily energy expenditure. TN housed mice also had significantly lower respiratory exchange ratio values, indicative of increased fat metabolism. However, there were no differences in body mass or composition (i.e., % lean and fat mass). Treadmill-to-fatigue experiments revealed no differences in exercise performance. Furthermore, ex vivo muscle contractility analysis displayed no differences in extensor digitorum longus or soleus specific force generation between TN and RT housed mice. Analysis of cardiac structure/function demonstrated that TN housed mdx mice exhibited a reduction in cardiac output, which we attributed to diastolic dysfunction. While ejection fraction and fractional shortening were not different among the two groups, TN housing led to a significant reduction in end diastolic volume (EDV) and left ventricle internal diameter (diastole, LVID;d), as well as increased relative wall thickness (diastole, RWT;d) compared to RT housed mice. Though isovolumic relaxation time was prolonged in TN-housed mice, this did not reach statistical significance. Taken together, our results demonstrate that TN housing had minimal effects on skeletal muscle contractility, but did lead to signs of diastolic dysfunction in young mdx mice. Future studies will examine the effects of TN housing in aged mdx mice, when systolic dysfunction occurs, as well as any influence of TN housing on the efficacy of potential muscle wasting therapeutics. Overall, our results support the growing literature that highlights the importance of selecting the appropriate housing temperature when studying metabolism or muscle wasting conditions in mice. NSERC This is the full abstract presented at the American Physiology Summit 2023 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.

Delta-6 desaturase (Fads2) deficiency alters triacylglycerol/fatty acid cycling in murine white adipose tissue

The Δ-6 desaturase (D6D) enzyme is not only critical for the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from α-linolenic acid (ALA), but recent evidence suggests that it also plays a role in adipocyte lipid metabolism and body weight; however, the mechanisms remain largely unexplored. The goal of this study was to investigate if a D6D deficiency would inhibit triacylglycerol storage and alter lipolytic and lipogenic pathways in mouse white adipose tissue (WAT) depots due to a disruption in EPA and DHA production. Male C57BL/6J D6D knockout (KO) and wild-type (WT) mice were fed either a 7% w/w lard or flax (ALA rich) diet for 21 weeks. Energy expenditure, physical activity, and substrate utilization were measured with metabolic caging. Inguinal and epididymal WAT depots were analyzed for changes in tissue weight, fatty acid composition, adipocyte size, and markers of lipogenesis, lipolysis, and insulin signaling. KO mice had lower body weight, higher serum nonesterified fatty acids, smaller WAT depots, and reduced adipocyte size compared to WT mice without altered food intake, energy expenditure, or physical activity, regardless of the diet. Markers of lipogenesis and lipolysis were more highly expressed in KO mice compared to WT mice in both depots, regardless of the diet. These changes were concomitant with lower basal insulin signaling in WAT. Collectively, a D6D deficiency alters triacylglycerol/fatty acid cycling in WAT by promoting lipolysis and reducing fatty acid re-esterification, which may be partially attributed to a reduction in WAT insulin signaling.

The Effect of Salep Aqueous Extract High Doses on Serum Level of Urea Nitrogen, Creatinine, Uric acid and Kidney Histopathological Changes in Adult Male Wistar Rats

Kidneys are critical in the clearance and maintenance of active metabolites. One of the medical properties of Salep is treating bladder and kidney inflammation. Due to the widespread use of Salep in traditional medicine and the food industry, and since the effects of Salep on kidney function have not been studied, the present study aimed to investigate the impact of Salep on kidney function. In this experimental study, 48 male rats were divided randomly into six groups as control, sham, and four experimental groups receiving different doses of Salep intraperitoneally (80, 160, 320, and 640 mg/kg). On day 29, after weighing the animals, blood samples were taken from the heart, and serum blood urea nitrogen (BUN), uric acid, and creatinine were analyzed and compared in different groups. All the animal’s kidneys were exposed after dissection, and tissue sections were prepared for histopathological evaluation. From day 28 to 29, rats were kept in metabolic cages to collect urine samples and measure water intake and urine volume. The serum concentration of BUN and uric acid in the groups receiving Salep at all doses decreased non-significantly compared to the control group. Furthermore, a significant reduction was seen in creatinine serum levels in groups receiving 320 and 640 mg/kg of Salep extract (P<0.05). No evidence of damage to renal tissue was observed in this study. In conclusion, Salep could decrease serum BUN, uric acid, and creatinine levels due to its antioxidant properties and had no devastating effect on kidneys.

Difference in endocrine and behavior between short-term single- and paired-housing mice in metabolic cage

Metabolic cage housing which is exposed to a number of environmental stressors is often used in pharmacokinetic studies. In this study, we compared the difference in stress response between single- and paired-housing in metabolic cages by evaluating the alteration of urinary stress hormones and behavior. Mice were randomly divided into single- or paired-housing groups and placed in a metabolic cage with wire mesh. Their urine was collected every 24 h for consecutive 4 days to determine excreted catecholamine and corticosterone. The change in body weight was significantly decreased at 3 and 4 days in the single-housing group compared with that before the experiment, but not paired-housing group. The level of urinary catecholamines, such as noradrenaline, adrenaline, and their metabolite vanillylmandelic acid, was significantly increased in the single-housing compared with paired housing group and urinary corticosterone increased as well. Next, for the two similarly housed groups, we observed spontaneous behavior on the fourth day and conducted an elevated plus-maze test on the fifth day. Spontaneous behavior was not different between experimental groups. In the elevated plus-maze test, the proportion of time spent in the open arms was significantly prolonged in the paired-housing group compared to that of the single-housing group. Short-term social isolation stress loading in metabolic cages was suggested to exhibit endocrinological and behavioral changes in mice. To reduce such interference due to stress exposure, it was suggested to keep two mice in a metabolic cage.

Kir4.1 deletion prevents salt-sensitive hypertension in early streptozotocin-induced diabetic mice via Na + -Cl - cotransporter in the distal convoluted tubule.

Functional impairment of renal sodium handling and blood pressure (BP) homeostasis is an early characteristic manifestation of type 1 diabetes. However, the underlying mechanisms remain unclear. Metabolic cages, radio-telemetry, immunoblotting, and electrophysiology were utilized to examine effects of high salt (8% NaCl, HS) intake on Na + /K + balance, BP, Na + -Cl - cotransporter (NCC) function, and basolateral K + channel activity in the distal convoluted tubule (DCT) under diabetic conditions. Improper Na + balance, hypernatremia, and a mild but significant increase in BP were found in streptozotocin (STZ)-induced diabetic mice in response to HS intake for 7 days. Compared to the vehicle, STZ mice showed increased Kir4.1 expression and activity in the DCT, a more negative membrane potential, higher NCC abundance, and enhanced hydrochlorothiazide-induced natriuretic effect. However, HS had no significant effect on basolateral Kir4.1 expression/activity and DCT membrane potential, or NCC activity under diabetic conditions, despite a downregulation in phosphorylated NCC abundance. In contrast, HS significantly downregulated the expression of Na + -H + exchanger 3 (NHE3) and cleaved epithelial sodium channel-γ in STZ mice, despite an increase in NHE3 abundance after STZ treatment. Kir4.1 deletion largely abolished STZ-induced upregulation of NCC expression and prevented BP elevation during HS intake. Interestingly, HS causes severe hypokalemia in STZ-treated kidney-specific Kir4.1 knockout (Ks-Kir4.1 KO) mice and lead to death within a few days, which could be attributed to a higher circulating aldosterone level. We concluded that Kir4.1 is required for upregulating NCC activity and may be essential for developing salt-sensitive hypertension in early STZ-induced diabetes.

MP03-19 ROLE OF MICROFIBRILLAR-ASSOCIATED PROTEIN 4 IN PARTIAL BLADDER OUTFLOW OBSTRUCTION-RELATED BLADDER FIBROSIS

You have accessJournal of UrologyCME1 Apr 2023MP03-19 ROLE OF MICROFIBRILLAR-ASSOCIATED PROTEIN 4 IN PARTIAL BLADDER OUTFLOW OBSTRUCTION-RELATED BLADDER FIBROSIS Reine Kanaan, Anders Schlosser, Grith Lykke Sorensen, and Peter Zvara Reine KanaanReine Kanaan More articles by this author , Anders SchlosserAnders Schlosser More articles by this author , Grith Lykke SorensenGrith Lykke Sorensen More articles by this author , and Peter ZvaraPeter Zvara More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003214.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Partial bladder outflow obstruction (pBOO) causes remodeling of the bladder wall, with smooth muscle hypertrophy and an excessive deposition of extracellular matrix (ECM) proteins, leading to bladder fibrosis. Our understanding of the pathophysiology of this process is limited and no effective treatment to prevent or reverse bladder fibrosis exists. Microfibrillar-associated protein 4 (MFAP4) is an ECM glycoprotein, localized to elastic fiber-rich regions. It binds ECM components including elastin and collagen and contributes to the process of elastogenesis in healthy tissues. Emerging data suggest the involvement of MFAP4 in the pathogenesis of disease-induced tissue remodeling, including fibrosis. To determine its value as a potential therapeutic target, we compared MFAP4 expression in sham-operated bladders to bladders 6 weeks following pBOO. MFAP4 levels were then correlated with bladder function. METHODS: Partial BOO, was induced in 9 male Sprague Dawley rats, 5 underwent sham surgery. Six weeks following surgery, rats were placed in a metabolic cage overnight to record 12-hour micturition frequency and volume per void. Gene expression levels of MFAP4, alpha-smooth muscle actin (α-SMA) and collagen I were quantified in the bladder tissue using real-time PCR. RESULTS: The bladder/body ratio was significantly higher in the pBOO group (0.07±0.04) when compared to the SHAM group (0.024±0.003). Obstructed rats with increased MFAP4 expression demonstrated an increase in α-SMA expression. No significant increase in collagen I levels was recorded in the pBOO group. Animals with bladder overactivity as determined by frequent low volume micturitions (≥4 micturitions with <0.1 mL per void/12 hours) had higher MFAP4 expression than animals in the SHAM group, which exhibited normal micturition pattern. CONCLUSIONS: These initial observations show that pBOO lasting 6 weeks results in detrusor hypertrophy. Although bladder wall fibrosis did not develop within the 6-week timeframe, our data suggest that bladder remodeling following pBOO is associated with increased MFAP4 levels and that MFAP4 might be involved in the pathogenesis of pBOO-induced bladder dysfunction. Experiments addressing processes extending beyond 6 weeks post-pBOO and possible involvement of MFAP4 in their pathophysiology are ongoing. Source of Funding: Odense University hospital © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e31 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Reine Kanaan More articles by this author Anders Schlosser More articles by this author Grith Lykke Sorensen More articles by this author Peter Zvara More articles by this author Expand All Advertisement PDF downloadLoading ...

In Vivo Digestibility of &amp;lt;i&amp;gt;Brachiaria deflexa&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;Echinocloa colona&amp;lt;/i&amp;gt; Supplemented with Cowpea Haulms in Sahelian Goats

A study of In vivo digestibility of <i>Brachiaria deflexa</i> and <i>Echinocloa colona </i>fed<i> </i>Sahelian bucks supplemented with 30% cowpea haulms was conducted in the Scientific Garden of the University of Sciences and Technology of Ati in Chad by September 2018. Twelve (12) Sahelian bucks weighing around 28. 62 ±2. 97 kg, aged between 20 and 24 months were used for this feeding trial. Four (4) groups of three (3) animals were housed in individual metabolic cages, where they were treated with Oxytetracycline 20% long-acting (1ml/10 kg) and 1% of Ivermectin against gastrointestinal and pulmonary worms before trials. Four rations were made and distributed hazardly to each group as follows: <i>Brachiaria deflexa</i> without supplement of cowpea haulms (BF₀), <i>Brachiaria deflexa</i> suplemented with 30% cowpea haulms (BF₃₀), <i>Echinocloa colona</i> without suplement (EF₀) and <i>Echinocloa colona</i> suplmented with 30% cowpea haulms (EF₃₀). Each animal received 1000g of feeds twice a day for basal feeds during the adaptation period (14 days). After this period, data were collected for seven (7) days. The result shows that <i>Echinocloa</i> <i>colona</i> supplemented with 30% cowpea haulm induced a significant (p<0.05) intake of DM (697.2g), OM (525.14g), and CF (214.35g) in Sahelian bucks. While, digestive utilization of these elements was comparable (p> 0.05). Though, digestive utilization of nitrogen (58.68g) was significantly (P<0.05) high in bucks. Biochemically, blood concentration with protein elements (protein, albumin, and globulin) was similar in bucks fed <i>Brachiaria deflexa </i>or<i> Echinocloa colona </i>alone or associated with 30% cowpea haulms. As such, bucks could be fed with dried forage without cowpea supplements to minimize production costs.

Nutrient utilization and ingestive behavior by lambs fed a partial mixed ration consisting of different carbohydrate sources combined with fresh alfalfa

The objective of the current study was to evaluate the nutrient utilization by lambs fed partial mixed ration (PMR) containing either cereal grain or fibrous by-products as an energy source complemented with fresh alfalfa. Eighteen male Corriedale × Ile-de France lambs (32.2 ± 1.82 kg body weight and 4 months old) fitted with permanent rumen catheters were individually housed in metabolism cages and randomly assigned to the treatments (diets): FF, fresh alfalfa ad libitum; PMR-C, PMR containing cereal grains plus fresh alfalfa or PMR-F, PMR containing fibrous by-products plus fresh alfalfa. The amount of PMR offered corresponded to 75% of the potential total dry matter intake of lambs, in two equal meals daily (i.e. morning and afternoon) whereas fresh alfalfa was offered ad libitum from 10:00 to 18:00 h The inclusion of PMR, independently of the energy source, increased the nutrient intake and digestibility of lambs and reduced daily eating time. The ruminal fluid of lambs fed either PMR showed lower pH, higher propionate and butyrate concentration, and lower NH3 concentration compared to lambs fed only alfalfa (P < 0.01). Lambs fed PMR-F showed lower digestibility of N compounds and ruminal ammonia concentration compared to lambs fed PMR-C (P < 0.01). We concluded that offering PMR combined with fresh alfalfa to lambs had positive effects on nutrient intake, regardless of whether the energy source in PMR was starch or fibers.

ARBM101 (Methanobactin SB2) Drains Excess Liver Copper via Biliary Excretion in Wilson’s Disease Rats

Excess copper causes hepatocyte death in hereditary Wilson's disease (WD). Current WD treatments by copper-binding chelators may gradually reduce copper overload; they fail, however, to bring hepatic copper close to normal physiological levels. Consequently, lifelong daily dose regimens are required to hinder disease progression. This may result in severe issues due to nonadherence or unwanted adverse drug reactions and also due to drug switching and ultimate treatment failures. This study comparatively tested bacteria-derived copper binding agents-methanobactins (MBs)-for efficient liver copper depletion in WD rats as well as their safety and effect duration. Copper chelators were tested invitro and invivo in WD rats. Metabolic cage housing allowed the accurate assessment of animal copper balances and long-term experiments related to the determination of minimal treatment phases. We found that copper-binding ARBM101 (previously known as MB-SB2) depletes WD rat liver copper dose dependently via fecal excretion down to normal physiological levels within 8 days, superseding the need for continuous treatment. Consequently, we developed a new treatment consisting of repetitive cycles, each of ∼1 week of ARBM101 applications, followed by months of in-between treatment pauses to ensure a healthy long-term survival in WD rats. ARBM101 safely and efficiently depletes excess liver copper from WD rats, thus allowing for short treatment periods as well as prolonged in-between rest periods.

Evaluation of Nutrient Digestibility and Metabolizable Energy of Yeast-Fermented Acacia mangium Leaf by Japanese Quails

The nutrient digestibility and the metabolizable energy (AME) of Acacia mangium leaf (AM) and yeast-fermented AM (YFAM) by Japanese quails were investigated. Fifty-four of 4-weeks-old Japanese quails were divided into 3 groups with six replications of three quails. Each quail was randomly fed with an experimental diet composed of dextrose (protein-free diet for determining endogenous excretion) 40 % AM and 40% YFAM. The quails were raised individually in a metabolic cage, where feed and water were provided ad-libitum. Both feed intake and feces weight were recorded. Experimental diets and excreta were sampled and subjected to proximate analysis for gross energy. The results of nutrient composition indicated that the fermentation AM with yeast highly significantly increased (P&lt;0.01) dry matter (DM), ash, and nitrogen-free extractives (NFE) content but decreased (P&lt;0.01) crude protein (CP), crude fiber (CF), and gross energy (GE) content. In addition, the birds fed dietary YFAM compared with AM showed significantly increased (P≤0.01) DM, organic matter (OM), CP, and GE digestibility but decreased (P≤0.01) EE and CF digestibility. The protein utilization of birds fed dietary YFAM showed significantly greater (P≤0.01) FI, protein retained, protein intake, net protein utilization (NPU), and AME than those fed dietary AM. In conclusion, the fermentation of AMLM with yeast improved nutrient composition and enhanced the digestibility of nutrients, protein utilization, and AME of AMLM in Japanese quail.

Exploring the Long-Term Tissue Accumulation and Excretion of 3 nm Cerium Oxide Nanoparticles after Single Dose Administration.

Nanoparticle (NP) pharmacokinetics significantly differ from traditional small molecule principles. From this emerges the need to create new tools and concepts to harness their full potential and avoid unnecessary risks. Nanoparticle pharmacokinetics strongly depend on size, shape, surface functionalisation, and aggregation state, influencing their biodistribution, accumulation, transformations, and excretion profile, and hence their efficacy and safety. Today, while NP biodistribution and nanoceria biodistribution have been studied often at short times, their long-term accumulation and excretion have rarely been studied. In this work, 3 nm nanoceria at 5.7 mg/kg of body weight was intravenously administrated in a single dose to healthy mice. Biodistribution was measured in the liver, spleen, kidney, lung, brain, lymph nodes, ovary, bone marrow, urine, and faeces at different time points (1, 9, 30, and 100 days). Biodistribution and urinary and faecal excretion were also studied in rats placed in metabolic cages at shorter times. The similarity of results of different NPs in different models is shown as the heterogeneous nanoceria distribution in organs. After the expectable accumulation in the liver and spleen, the concentration of cerium decays exponentially, accounting for about a 50% excretion of cerium from the body in 100 days. Cerium ions, coming from NP dissolution, are most likely excreted via the urinary tract, and ceria nanoparticles accumulated in the liver are most likely excreted via the hepatobiliary route. In addition, nanoceria looks safe and does not damage the target organs. No weight loss or apathy was observed during the course of the experiments.

A moderate-fat diet containing soy protein does not differentially impact energy balance in male and female mice compared to dairy protein

Dairy and nondairy plant-based alternative proteins are reported to differentially influence body weight; however, most research has compared plant-based alternatives with isolated dairy proteins rather than a complete milk protein (containing casein and whey). This is notable given that people do not generally consume isolated dairy proteins. Therefore, the present study aimed to investigate the impact of a soy protein isolate (SPI) on factors influencing body weight gain in male and female mice in comparison to skim milk powder (SMP). Based on current knowledge in rodents, we hypothesized that SPI would promote body weight gain compared with SMP. Mice (n = 8 per sex per diet) consumed a moderate-fat diet (35% kcal from fat) containing either SPI or SMP for 8 weeks. Body weight and food intake were measured weekly. Energy expenditure, physical activity, and substrate use were measured using metabolic cages. Fecal energy content was measured with bomb calorimetry. Body weight gain and food intake during the 8-week feeding study was not different in mice consuming either SPI or SMP; however, males had a higher body weight, adiposity, and feed efficiency compared with females (all P < .05). Fecal energy content was approximately 7% higher in both male and female mice fed the SPI diet compared with the SMP diet. Neither protein source affected substrate utilization, physical activity, or energy expenditure. Physical activity in the dark phase trended higher in females compared with males (P = .0732). The present study suggests that the consumption of SPI in the context of a moderate-fat diet has little impact on numerous factors influencing body weight regulation in male and female mice compared with a complete milk protein.

Mitotic CDK1 and 4E-BP1 II: A single phosphomimetic mutation in 4E-BP1 induces glucose intolerance in mice.

Cyclin-dependent kinase 1 (CDK1)/cyclin B1 phosphorylates many of the same substrates as mTORC1 (a key regulator of glucose metabolism), including the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Only mitotic CDK1 phosphorylates 4E-BP1 at residue S82 in mice (S83 in humans), in addition to the common 4E-BP1 phospho-acceptor sites phosphorylated by both CDK1 and mTORC1. We examined glucose metabolism in mice having a single aspartate phosphomimetic amino acid knock in substitution at the 4E-BP1 serine 82 (4E-BP1S82D) mimicking constitutive CDK1 phosphorylation. Knock-in homozygous 4E-BP1S82D and 4E-BP1S82A C57Bl/6N mice were assessed for glucose tolerance testing (GTT) and metabolic cage analysis on regular and on high-fat chow diets. Gastrocnemius tissues from 4E-BP1S82D and WT mice were subject to Reverse Phase Protein Array analysis. Since the bone marrow is one of the few tissues typically having cycling cells that transit mitosis, reciprocal bone-marrow transplants were performed between male 4E-BP1S82D and WT mice, followed by metabolic assessment, to determine the role of actively cycling cells on glucose homeostasis. Homozygous knock-in 4E-BP1S82D mice showed glucose intolerance that was markedly accentuated with a diabetogenic high-fat diet (p = 0.004). In contrast, homozygous mice with the unphosphorylatable alanine substitution (4E-BP1S82A) had normal glucose tolerance. Protein profiling of lean muscle tissues, largely arrested in G0, did not show protein expression or signaling changes that could account for these results. Reciprocal bone-marrow transplantation between 4E-BP1S82D and wild-type littermates revealed a trend for wild-type mice with 4E-BP1S82D marrow engraftment on high-fat diets to become hyperglycemic after glucose challenge. 4E-BP1S82D is a single amino acid substitution that induces glucose intolerance in mice. These findings indicate that glucose metabolism may be regulated by CDK1 4E-BP1 phosphorylation independent from mTOR and point towards an unexpected role for cycling cells that transit mitosis in diabetic glucose control.

Hepatic follistatin increases basal metabolic rate and attenuates diet-induced obesity during hepatic insulin resistance

ObjectiveBody weight change and obesity follow the variance of excess energy input balanced against tightly controlled EE (energy expenditure). Since insulin resistance can reduce energy storage, we investigated whether genetic disruption of hepatic insulin signaling reduced adipose mass with increased EE. MethodsInsulin signaling was disrupted by genetic inactivation of Irs1 (Insulin receptor substrate 1) and Irs2 in hepatocytes of LDKO mice (Irs1L/L·Irs2L/L·CreAlb), creating a state of complete hepatic insulin resistance. We inactivated FoxO1 or the FoxO1-regulated hepatokine Fst (Follistatin) in the liver of LDKO mice by intercrossing LDKO mice with FoxO1L/L or FstL/L mice. We used DEXA (dual-energy X-ray absorptiometry) to assess total lean mass, fat mass and fat percentage, and metabolic cages to measure EE (energy expenditure) and estimate basal metabolic rate (BMR). High-fat diet was used to induce obesity. ResultsHepatic disruption of Irs1 and Irs2 (LDKO mice) attenuated HFD (high-fat diet)-induced obesity and increased whole-body EE in a FoxO1-dependent manner. Hepatic disruption of the FoxO1-regulated hepatokine Fst normalized EE in LDKO mice and restored adipose mass during HFD consumption; moreover, hepatic Fst disruption alone increased fat mass accumulation, whereas hepatic overexpression of Fst reduced HFD-induced obesity. Excess circulating Fst in overexpressing mice neutralized Mstn (Myostatin), activating mTORC1-promoted pathways of nutrient uptake and EE in skeletal muscle. Similar to Fst overexpression, direct activation of muscle mTORC1 also reduced adipose mass. ConclusionsThus, complete hepatic insulin resistance in LDKO mice fed a HFD revealed Fst-mediated communication between the liver and muscle, which might go unnoticed during ordinary hepatic insulin resistance as a mechanism to increase muscle EE and constrain obesity.

A5 A MOUSE MODEL TO UNRAVEL THE PATHOPHYSIOLOGICAL LINK BETWEEN CROHN’S DISEASE AND TYPE-2 DIABETES-ASSOCIATED METABOLIC DISORDERS

Abstract Background Crohn’s disease (CD), an idiopathic inflammatory bowel disease (IBD), has been recently shown to increase the risk of developing type 2 diabetes (T2D). Moreover, treatment with anti-diabetic drugs has a protective role in preventing the severity and course of CD progression. However, the pathophysiological basis of T2D development in CD remains unclear. Findings have highlighted the contribution of adipose tissue (AT) to the development of chronic inflammatory diseases and have identified parallels between T2D and CD that may provide hints to common mechanisms of disease pathogenesis. Typically, microbial dysbiosis, hyperpermeable intestinal barrier, and intra-abdominal AT accumulation are the common features of both diseases, yet how the interplay of these factors contribute to pathogenesis is not known. Therefore, common pathogenic paradigms underlying both T2D and CD have led us to hypothesize that chronic intestinal inflammation serves as an initiator of AT dysfunction in CD, predisposing individuals to T2D. Further, the lack of appropriate animal models of CD with chronic intestinal inflammation that manifests accumulation of intra-abdominal AT, and extra-intestinal metabolic disorder as observed in CD and T2D patients has been a limitation. Purpose To develop a genetic mouse model to investigate if gut inflammation-mediated microbial dysbiosis and metabolic dysregulation of AT are at the nexus that cause T2D in CD. Method We developed a CD-mouse model, where we challenged Nod2-deficient mice (NOD2 being the strongest genetic risk factor contributing to CD) with a chronic inflammatory insult regime, using dextran sulfate sodium (cDSS) for 3 cycles. Subsequently, intraperitoneal insulin and oral glucose tolerance tests, metabolic caging, and MRI imaging of mice were performed. Changes in AT metabolism and microbial infiltration into AT were analyzed by quantitative real-time PCR (qRT-PCR) and/or immunohistochemistry (IHC). Result(s) Our new CD-mouse model revealed increased gut inflammation (TNF and type-I IFN) in Nod2-deficient mice compared to wild-type control mice post-cDSS. Surprisingly, Nod2-deficient mice gained body weight, which was at least in part accounted for by an increased intra-abdominal AT accumulation along with decreased AT fatty-acid metabolism (Cpt1a, Fabp4 expression) and AT browning (Ucp1, Cidea expression, and UCP-1 staining), reduced intestinal goblet cell numbers, increased gut bacterial infiltration within the fat, more insulin resistance and energy expenditure. Conclusion(s) This experimental mouse model mimicking CD-associated T2D will provide insights into how the microbiome-AT axis fuel chronic inflammation-mediated extra-intestinal metabolic disorder and immune dysregulation. Understanding these connections will be transformative, as it will help us devise novel therapeutic strategies to prevent T2D development in progressive CD patients. Disclosure of Interest None Declared

Effect of feeding pomegranate seed pulp on Awassi lambs' nutrient digestibility, growth performance, and carcass quality.

The use of alternative feeds in feeding livestock as an alternative to traditional feeds has been used for many years, on the one hand, to lower the price of feed and, on the other hand, to raise the profitability of raising livestock. The study aimed to investigate the effect of feeding pomegranate seed pulp (PSP) on the growth performance and carcass characteristics and the health of Awassi lambs. Twenty-four male lambs (16.9 ± 0.42 kg) were assigned randomly to one of two isonitrogenous (160 g/kg crude protein of dietary dry matter [DM]) treatment diets. The diets were the control (CON) and PSP-containing diet (100 g/kg of dietary DM; PSP100). The experimental period was 70 days preceded by 7 days of adaptation to diets. Feed intake was measured and lambs were weighed on day one and then biweekly. On day 49, eight lambs (four lambs per treatment) were chosen randomly and placed in metabolic cages for a digestibility trial. At the end of the trial, lambs were slaughtered to evaluate carcass characteristics and meat quality. The data were analyzed using Proc Mixed procedures of SAS. The results revealed that nutrient intake was greater (p < 0.05) in lambs fed PSP100 than those fed the CON diet. Nitrogen intake and nitrogen retention were greater (p < 0.05) for lambs who consumed the PSP100 diet compared to CON. Final weight, total gain, and average daily gain were greater (p < 0.05) with lambs fed PSP100. Hot and cold carcass weights were higher (p < 0.05) by the PSP100 group than by the CON group. Carcass cut weight increased (p < 0.05) with feeding PSP100 diet. No differences were detected in blood parameters except high-density lipoprotein content, which was greater (p < 0.05) in the PSP100 group compared with the CON group. It could be concluded that adding PSP to lambs' diets improved growth and carcass measurements positively and did not negatively affect lambs' health; therefore, it is recommended to use PSP as an alternative to traditional feeds in lambs formulated rations.

Targeting FABP4 in elderly mice rejuvenates liver metabolism and ameliorates aging-associated metabolic disorders

IntroductionAging is characterized by progressive metabolic dyshomeostasis that increases morbidity and mortality. Solutions for optimizing healthy aging are challenged by lacking appropriate biomarkers. Moreover, druggable targets to rejuvenate the aging-associated metabolic phenotypes remain unavailable. MethodsProteomics analysis was performed in a cohort of young and elderly adults. Circulating levels of insulin-like growth factor 1 (IGF-1) and fatty acid binding protein 4 (FABP4) were evaluated by ELISA. FABP4 was silenced in elderly mice by adeno-associated virus. Metabolic activities were measured by metabolic cages. Cognitive function was evaluated by Morris water maze. Glucose and lipid metabolism were evaluated by biochemistry assays with blood samples. RNA-seq in mouse liver was performed for transcriptome analysis. ResultsAmong 9 aging-sensitive proteins shared by both male and female, FABP4 was identified as a reliable aging biomarker in both human and mouse. Silencing FABP4 in elderly mice significantly rejuvenated the aging-associated decline in metabolic activities. FABP4 knockdown reversed the aging-associated metabolic disorders by promoting degradation of cholesterol and fatty acids, while suppressing gluconeogenesis. Transcriptome analysis revealed a restoration of the pro-aging gene reprogramming towards inflammation and metabolic disorders in the liver after FABP4 knockdown. FABP4 overexpression promoted human LO2 cell senescence. Moreover, administration of an FABP4 inhibitor BMS309403 delivered metabolic benefits in elderly mice. ConclusionOur findings demonstrate FABP4 as a reliable aging biomarker as well as a practicable target to improve healthy aging in the elderly.

Determination of standardised ileal digestibility of amino acids in high-fibre feedstuffs and additivity of apparent and standardised ileal amino acids digestibility of diets containing mixtures of maize, sorghum, and soybean meal

ABSTRACT 1. Two experiments were conducted to determine the coefficients of standardised ileal amino acid digestibility for selected high-fibre feedstuffs (Expt.1) and to assess the additivity of coefficients of apparent (cAIAAD) and standardised (cSIAAD) ileal amino acid digestibility for diets containing combinations of sorghum, maize and soybean meal. 2. In Expt. 1, broiler chickens (324) were allocated to 54 metabolism cages (nine diets with six replicates). In Expt. 2, broiler chickens (315) were allocated to 63 metabolism cages (seven diets with nine replicates). The diets in Expt. 1 were a nitrogen-free diet plus eight semi-purified diets in which soybean meal (SBM), low-protein SBM (LPSBM), soybean hull, wheat bran, maize bran, rice bran, dried sugarbeet pulp or maize gluten feed were the only sources of protein in the respective diets. In Expt. 2, the seven diets were a nitrogen-free diet, and six semi-purified diets consisting of individual feedstuffs (SBM, maize, or sorghum) or combinations (maize and SBM; sorghum and SBM; or maize, sorghum, and SBM) as the only sources of protein. 3. In both experiments, all the broiler chickens received the same maize-SBM diet formulated to meet nutrient requirements according to the breeder’s recommendations from d 0 to 16. Allocated experimental diets were provided on d 16, and ileal digesta were collected on d 21. 4. In Expt. 1, SBM and LPSBM had greater (P < 0.01) cSIAAD for indispensable and dispensable amino acids than the other feedstuffs. The rice bran had greater (P<0.01) cSIAAD values than wheat bran and maize bran, except for Leu, Cys, and Pro. 5. In Expt. 2, predicted cAIAAD values for maize-SBM and maize-sorghum-SBM were generally lower (P<0.01) than actual values. The actual and predicted cSIAAD values for maize-SBM, sorghum-SBM and maize-sorghum-SBM combinations were not significantly different. 6. It was concluded that the standardised amino acid digestibility of sorghum, when combined with other feedstuffs, was additive and that approximately three-quarters of total amino acids in the assayed high-fibre feedstuffs, except for dried sugarbeet pulp and maize gluten feed, were digestible.

Olive leaves as alternative feed for finishing lambs: evaluation of feed intake, nutrients digestibility, growth performance, and carcase quality

This research aims to evaluate how incorporating sun-dried olive leaves (OL) in the diets of Awassi lambs affects production costs, intake and nutrients digestibility, growth performance, and carcase quality. Thirty-six newly weaned male lambs were randomly allocated to one of three diet groups as dry matter (DM) in diets: 1) the control diet (CON), 2) 50 OL g/kg (OL50), and 3) 100 OL g/kg (OL100). On the 50th day, four lambs were randomly selected from each treatment and transported to metabolic cages to evaluate digestibility and N balance. The DM intake was higher (p = .003) in the OL50 and OL100 diets than those on the CON diet. At the same time, the ether extract and metabolisable energy intakes were the highest (p < .0001) in OL100, followed by OL50 and the CON diet. Intake of neutral detergent fibre and acid detergent fibre for lambs fed the OL50 diet surpassed (p < .0001) the other diets. The growth performance parameters were comparable among diets. However, the cost of gain was lower (p = .03) in diets containing OL. The OL incorporation had no treatment effect (p > .05) on the digestibility, N retention, carcase characteristics and meat quality. In conclusion, while the addition of OL at 50 g/kg and 100 g/kg did not affect the growth performance and characteristics of carcase, it did decrease the cost of production, making it a good candidate to replace part of the wheat straw in diets of growing lambs. Highlights Olive leaves percentage substitution of wheat straw improved the nutrients intake in Awassi lambs fed commercial diets. Olive leaves incorporation decreased both the cost of the diet and the cost per kg of gain in Awassi lambs. Olive leaves inclusion did not have a negative effect on digestibility and N Balance, carcase characteristics and meat quality.

Activation of Kir4.1/Kir5.1 contributes to the cyclosporin A-induced stimulation of the renal NaCl cotransporter and hyperkalemic hypertension.

Cyclosporin A (CsA) is a widely used immunosuppressive drug that causes hypertension and hyperkalemia. Moreover, CsA-induced stimulation of the thiazide-sensitive NaCl cotransporter (NCC) in the kidney has been shown to be responsible for the development of hyperkalemic hypertension. In this study, we tested whether CsA induces the activation of NCC by stimulating the basolateral Kir4.1/Kir5.1 channel in the distal convoluted tubule (DCT). Electrophysiology, immunoblotting, metabolic cages, and radio-telemetry methods were used to examine the effects of CsA on Kir4.1/Kir5.1 activity in the DCT, NCC function, and blood pressure in wild-type (WT) and kidney-specific Kir4.1 knockout (KS-Kir4.1 KO) mice. The single-channel patch clamp experiment demonstrated that CsA stimulated the basolateral 40 pS K+ channel in the DCT. Whole-cell recording showed that short-term CsA administration (2h) not only increased DCT K+ currents but also shifted the K+ current (IK ) reversal potential to the negative range (hyperpolarization). Furthermore, CsA administration increased phosphorylated NCC (pNCC) levels and inhibited renal Na+ and K+ excretions in WT mice but not in KS-Kir4.1 KO mice, suggesting that Kir4.1 is required to mediate CsA effects on NCC function. Finally, long-term CsA infusion (14 days) increased blood pressure, plasma K+ concentration, and total NCC or pNCC abundance in WT mice, but these effects were blunted in KS-Kir4.1 KO mice. We conclude that CsA stimulates basolateral K+ channel activity in the DCT and that Kir4.1 is essential for CsA-induced NCC activation and hyperkalemic hypertension.