Comprehensive Laboratory Animal Monitoring System Research Articles

Regulation of the Novel MSTN-GAL3-NOX1 Axis Reverses Obesity-Induced Skeletal Muscle Quality Decline Resulting in Improved Glycemic Indices and Restored Angiogenesis Following Ischemia

Objective: To test the hypothesis that restored muscle quality achieved through myostatin KO improves angiogenesis in obese mice through the resolution of GAL3-NOX1-mediated endothelial dysfunction.Methods: db/db mice, a well-described model of obesity, were crossed with mice lacking myostatin (MSTN KO), a myokine that negatively regulates muscle differentiation and growth, galectin-3 (GAL3 KO), a pro-inflammatory/atherogenic RAGE (receptor for advanced glycation endproducts), and NADPH oxidase-1 (NOX1 KO), a TGF-b-stimulated primary producer of the reactive oxygen species (ROS) O2−, to generate db+/−-MSTN+/−, db+/−-GAL3+/−, and db+/−-NOX1+/− mice. We utilized nuclear magnetic resonance (NMR) spectroscopy to assess skeletal muscle (SKM) lean/fat content in db+/−-MSTN+/− mice. Muscle and plasma lipid content were analyzed via mass spectrometry. Mice were placed in metabolic cages and monitored with CLAMS (Comprehensive Lab Animal Monitoring System) in order to determine their whole-body metabolism. Insulin metabolism indices were determined via appropriate blood and plasma testing kits. Morphology of SKM was determined via microscopy of H&E and Mason’s Trichrome-stained gastrocnemius cross sections. Femoral artery ligation was used to stimulate in vivo angiogenesis in response to hind limb ischemia (HLI); whereas 1mm aortic rings cultured in ECBM (2% FBS) were used to study ex vivo angiogenesis. Gene expression was assessed by RNA-Seq and RT-qPCR. Results: MSTN deletion results in significantly increased SKM mass and restored SKM quality (i.e. morphology and metabolic function) without altering whole-body mass, fat percentage, or activity in obese (db−) mice. We observed that obesity inhibits and MSTN KO restores angiogenesis in both HLI and aortic ring assay models. Previous studies in our lab revealed that MSTN deletion was suffcient to restore endothelial function in db/db mice. Additionally, our lab has shown that GAL3 or NOX1 KO in db/db mice phenocopied the vascular improvements seen in db−-MSTN− mice without restoring SKM size or quality. RNA-Seq and RT-qPCR analysis of SKM and endothelial cells (ECs) revealed significant upregulation of GAL3 and NOX1 expression in db−-MSTN+ mice but not in db−-MSTN− and db−-GAL3− mice. Finally, either GAL3 or NOX1 deletion results in amended angiogenesis in obese animals. Conclusion: In summary, restoration of muscle quality in obese individuals provides potent protection of vascular and metabolic health. A correlate of this improvement is the attenuation of a novel MSTN-GAL3-NOX1 axis, which we have shown to mediate vascular inflammation/redox signaling in obesity. These data suggest that pharmacological targeting of upstream MSTN, downstream GAL3 and/or NOX1 could be effective in treating cardiovascular disease in the context of obesity and metabolic syndrome. A. C. Speese is supported by NIH 1F31HL165858-01A1. D.W. Stepp and D. J. Fulton are supported by NIH 1R01HL147159. 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.

Effects of Hydrocodone Overdose and Ceftriaxone on Astrocytic Glutamate Transporters and Glutamate Receptors, and Associated Signaling in Nucleus Accumbens as well as Locomotor Activity in C57/BL Mice.

Chronic opioid treatments dysregulate the glutamatergic system, inducing a hyperglutamatergic state in mesocorticolimbic brain regions. This study investigated the effects of exposure to hydrocodone overdose on locomotor activity, expression of target proteins related to the glutamatergic system, signaling kinases, and neuroinflammatory factors in the nucleus accumbens. The locomotor activity of mice was measured using the Comprehensive Laboratory Animal Monitoring System (CLAMS). CLAMS data showed that exposure to hydrocodone overdose increased locomotion activity in mice. This study tested ceftriaxone, known to upregulate major glutamate transporter 1 (GLT-1), in mice exposed to an overdose of hydrocodone. Thus, ceftriaxone normalized hydrocodone-induced hyperlocomotion activity in mice. Furthermore, exposure to hydrocodone overdose downregulated GLT-1, cystine/glutamate antiporter (xCT), and extracellular signal-regulated kinase activity (p-ERK/ERK) expression in the nucleus accumbens. However, exposure to an overdose of hydrocodone increased metabotropic glutamate receptor 5 (mGluR5), neuronal nitric oxide synthase activity (p-nNOS/nNOS), and receptor for advanced glycation end products (RAGE) expression in the nucleus accumbens. Importantly, ceftriaxone treatment attenuated hydrocodone-induced upregulation of mGluR5, p-nNOS/nNOS, and RAGE, as well as hydrocodone-induced downregulation of GLT-1, xCT, and p-ERK/ERK expression. These data demonstrated that exposure to hydrocodone overdose can cause dysregulation of the glutamatergic system, neuroinflammation, hyperlocomotion activity, and the potential therapeutic role of ceftriaxone in attenuating these effects.

Absence of Slc39a14/Zip14 in mouse pancreatic beta cells results in hyperinsulinemia.

Zinc is an essential component of the insulin protein complex synthesized in β cells. The intracellular compartmentalization and distribution of zinc are controlled by 24 transmembrane zinc transporters belonging to the ZnT or Zrt/Irt-like protein (ZIP) family. Downregulation of SLC39A14/ZIP14 has been reported in pancreatic islets of patients with type 2 diabetes (T2D) as well as mouse models of high-fat diet (HFD)- or db/db-induced obesity. Our previous studies observed mild hyperinsulinemia in mice with whole body knockout of Slc39a14 (Zip14 KO). Based on our current secondary data analysis from an integrative single-cell RNA-seq dataset of human whole pancreatic tissue, SLC39A14 (coding ZIP14) is the only other zinc transporter expressed abundantly in human β cells besides well-known zinc transporter SLC30A8 (coding ZnT8). In the present work, using pancreatic β cell-specific knockout of Slc39a14 (β-Zip14 KO), we investigated the role of SLC39A14/ZIP14-mediated intracellular zinc trafficking in glucose-stimulated insulin secretion and subsequent metabolic responses. Glucose-stimulated insulin secretion, zinc concentrations, and cellular localization of ZIP14 were assessed using in vivo, ex vivo, and in vitro assays using β-Zip14 KO, isolated islets, and murine cell line MIN6. Metabolic evaluations were done on both chow- and HFD-fed mice using time-domain nuclear magnetic resonance and a comprehensive laboratory animal monitoring system. ZIP14 localizes on the endoplasmic reticulum regulating intracellular zinc trafficking in β cells and serves as a negative regulator of glucose-stimulated insulin secretion. Deletion of Zip14 resulted in greater glucose-stimulated insulin secretion, increased energy expenditure, and shifted energy metabolism toward fatty acid utilization. HFD caused β-Zip14 KO mice to develop greater islet hyperplasia, compensatory hyperinsulinemia, and mild insulin resistance and hyperglycemia. This study provided new insights into the contribution of metal transporter ZIP14-mediated intracellular zinc trafficking in glucose-stimulated insulin secretion and subsequent metabolic responses.NEW & NOTEWORTHY Metal transporter SLC39A14/ZIP14 is downregulated in pancreatic islets of patients with T2D and mouse models of HFD- or db/db-induced obesity. However, the function of ZIP14-mediated intracellular zinc trafficking in β cells is unknown. Our analyses revealed that SLC39A14 is the only Zn transporter expressed abundantly in human β cells besides SLC30A8. Within the β cells, ZIP14 is localized on the endoplasmic reticulum and serves as a negative regulator of insulin secretion, providing a potential therapeutic target for T2D.

Targeting phospholipid remodeling pathway improves insulin resistance in diabetic mouse models.

Previous studies have revealed that membrane phospholipid composition controlled by lysophosphatidylcholine acyltransferase 3 (LPCAT3) is involved in the development of insulin resistance in type 2 diabetes. In this study, we aimed to investigate the therapeutic potential of targeting Lpcat3 in the treatment of insulin resistance in diabetic mouse models. Lpcat3 expression was suppressed in the whole body by antisense oligonucleotides (ASO) injection or in the liver by adeno-associated virus (AAV)-encoded Cre in high-fat diet (HFD)-induced and genetic ob/ob type 2 diabetic mouse models. Glucose tolerance test (GTT), insulin tolerance test (ITT), fasting blood glucose, and insulin levels were used to assess insulin sensitivity. Lipid levels in the liver and serum were measured. The expression of genes involved in de novo lipogenesis was analyzed by real-time RT-PCR. Metabolic rates were measured by indirect calorimetry using the Comprehensive Lab Animal Monitoring System (CLAMS). Our data demonstrate that acute knockout of hepatic Lpcat3 by AAV-Cre improves both hyperglycemia and hypertriglyceridemia in HFD-fed mice. Similarly, whole-body ablation of Lpcat3 by ASO administration improves obesity and insulin resistance in both HFD-fed and ob/ob mice. These findings demonstrate that targeting LPCAT3 could be a novel therapy for insulin resistance.

SAT389 Reproductive And Metabolic Characterization of Mice Overexpressing CYP17 in Ovarian Theca Cells

Abstract Disclosure: S. Joseph: None. V. Ubba: None. M. K DSilva: None. S. Wu: None. Androgens, in physiological levels are important to normal ovarian function. However, excess androgens are detrimental to ovarian function, and is seen in PCOS, Congenital Adrenal Hyperplasia and Transgender Males undergoing testosterone therapy. Current models of androgen excess in females, mostly involve external administration of the causative agent. Hence, we developed an endogenous model of excess testosterone in female mice. To achieve this, a Triple mutant (TM) mouse was developed using the doxycycline inducible tet- on system, which involved RTTA, Cyp17 Cre and teto operator specifically inserted into CYP17 gene (purpose- double secure CYP17 specifically expressed in theca cells). These mice were fed with Doxycycline 625 mg/kg at 2 months of age. The TM mice showed significantly increased testosterone levels and Cyp17 protein expression in the ovaries. Estradiol, progesterone was unchanged. Reproductive characterization of the mice showed impaired estrous cyclicity and reduced fertility (number of pups and litters). Ovarian histology showed increased number of atretic follicles, with no changes in the number of developmental follicles and corpus leuteum. In the pituitary, mRNA expression of LHβ was significantly decreased, while GnRHR and FSHβ was unchanged. GnRH stimulation showed a decrease in LH levels in the TM mice. Immunoblotting showed increased expression of Androgen Receptor (Ar). Although increased androgens in females is associated with metabolic dysfunction, we did not observe any changes in Glucose, pyruvate and insulin tolerance in TM mice. Comprehensive Laboratory Animal Monitoring System (CLAMS) did not show any changes in the Respiratory Exchange Ratio, heat production and ambulatory movement. These studies show a unique strategy to develop an endogenous excess testosterone producing transgenic mice with decreased fertility. Presentation Date: Saturday, June 17, 2023

FRI276 KNDy-Neuron Kisspeptin Is Dispensable For Feeding Behavior, But Critical For Energy Expenditure Leading To Metabolic Dysfunction In Female Mice

Abstract Disclosure: N. Nandankar: None. H. Ganesh: None. A.L. Negron: None. S. Al-Samerria: None. J.E. Levine: None. S. Radovick: None. Hypothalamic kisspeptin (Kiss1) is a critical master regulator of the hypothalamic-pituitary-gonadal axis responsible for reproductive development and function. Kiss1, originating from KNDy neurons in the arcuate nucleus (ARC) of the hypothalamus, is implicated in having a dual role in both reproductive and metabolic functions. Using KNDy neuron-specific Kiss1 KO mice (Pdyn-Cre/Kissfl/fl or KO), we explored the role of Kiss1 from KNDy neurons in peripheral metabolism without dietary intervention. Here, we report that KO females have significantly greater body weight, fat mass, and glucose intolerance vs. control females. Interestingly, these parameters did not differ between KO male mice and controls. To determine whether this phenotype was due to dysregulated feeding behavior (i.e., hyperphagia) in KO mice, we tracked daily food consumption in adult mice for 3 weeks. We found no significant differences in food intake between genotypes of either sex. We used the Comprehensive Laboratory Animal Monitoring System (CLAMS, Columbus Instruments) to analyze energy expenditure in our mouse model. Data from the CLAMS experiments revealed reduced energy expenditure in KO females vs. control females, whereas there was no difference in energy expenditure between KO males and control males. Specifically, the respiratory exchange ratio (RER) was not significantly different in KO females vs. control females, indicating that both groups utilize both carbohydrates and fats as their primary energy source. However, heat expenditure was significantly lower in KO females (20.27 ± 2.29 kcal/kg/hr vs. 14.02 ± 1.18 kcal/kg/hr, WT vs. KO, p < 0.0001, 2-way ANOVA, n = 6-7) during the active dark phase, suggesting a decreased energy expenditure. This data suggests that the obesity and impaired glucose tolerance in females lacking Kiss1 from KNDy neurons is likely due to lower energy expenditure (with the expended energy derived from both carbohydrates and fats), rather than hyperphagic behavior. Presentation: Friday, June 16, 2023

The effect of voluntary exercise on light cycle stress-induced metabolic resistance.

Disruption of circadian genes affects metabolic homeostasis. Regular exercise programs prevent metabolic dysfunction and alter circadian gene expression In this study, we investigated whether exercise affects light stress-induced circadian rhythm derangement and metabolic resistance. A circadian rhythm derangement mouse model was designed by extending the light exposure by two hours (14 L/10 D) for three weeks. Nine-weekold male mice were single-caged and divided into four groups: sedentary groups with or without light stress, and voluntary wheel-trained groups with or without light stress. In addition, differentiated myotubes were cultured in the presence of dexamethasone with or without 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR). The comprehensive laboratory animal monitoring system was used to analyze the metabolic changes in mice. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify the mRNA expression levels of circadian genes in animal and cell culture models. Three weeks of light stress reduced the running distance and increased the weight of mice. In addition, VO2 consumption and heat production were increased during the night cycle under non-stress conditions but not under stress conditions. PCR analysis revealed that exercise and stress altered the expression levels of circadian genes in the hypothalamus and quadriceps muscles. mRNA expression levels of period circadian regulator 1 were downregulated in the quadriceps muscles of the stressed sedentary group compared to that in muscles of the non-stressed sedentary group. Furthermore, differentiated myotube cells cultured in the presence of dexamethasone, with or without AICAR, showed distinct oscillation patterns at various time points. Our study demonstrates that exercise partially prevents metabolic disruption by regulating the circadian gene expression in skeletal muscles.

Recovery from FOLFOX chemotherapy-induced systemic and skeletal muscle metabolic dysfunction in mice.

FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) chemotherapy is used to treat colorectal cancer and can acutely induce metabolic dysfunction. However, the lasting effects on systemic and skeletal muscle metabolism after treatment cessation are poorly understood. Therefore, we investigated the acute and lasting effects of FOLFOX chemotherapy on systemic and skeletal muscle metabolism in mice. Direct effects of FOLFOX in cultured myotubes were also investigated. Male C57BL/6J mice completed four cycles (acute) of FOLFOX or PBS. Subsets were allowed to recover for 4 wk or 10 wk. Comprehensive Laboratory Animal Monitoring System (CLAMS) metabolic measurements were performed for 5 days before study endpoint. C2C12 myotubes were treated with FOLFOX for 24 hr. Acute FOLFOX attenuated body mass and body fat accretion independent of food intake or cage activity. Acute FOLFOX decreased blood glucose, oxygen consumption (V̇o2), carbon dioxide production (V̇co2), energy expenditure, and carbohydrate (CHO) oxidation. Deficits in V̇o2 and energy expenditure remained at 10 wk. CHO oxidation remained disrupted at 4 wk but returned to control levels after 10 wk. Acute FOLFOX reduced muscle COXIV enzyme activity, AMPK(T172), ULK1(S555), and LC3BII protein expression. Muscle LC3BII/I ratio was associated with altered CHO oxidation (r = 0.75, P = 0.03). In vitro, FOLFOX suppressed myotube AMPK(T172), ULK1(S555), and autophagy flux. Recovery for 4 wk normalized skeletal muscle AMPK and ULK1 phosphorylation. Our results provide evidence that FOLFOX disrupts systemic metabolism, which is not readily recoverable after treatment cessation. FOLFOX effects on skeletal muscle metabolic signaling did recover. Further investigations are warranted to prevent and treat FOLFOX-induced metabolic toxicities that negatively impact survival and life quality of patients with cancer.NEW & NOTEWORTHY The present study demonstrates that FOLFOX chemotherapy induces long-lasting deficits in systemic metabolism. Interestingly, FOLFOX modestly suppressed skeletal muscle AMPK and autophagy signaling in vivo and in vitro. The FOLFOX-induced suppression of muscle metabolic signaling recovered after treatment cessation, independent of systemic metabolic dysfunction. Future research should investigate if activating AMPK during treatment can prevent long-term toxicities to improve health and quality of life of patients with cancer and survivors.

Loss of Brain Angiogenesis Inhibitor-3 (BAI3) G-Protein Coupled Receptor in Mice Regulates Adaptive Thermogenesis by Enhancing Energy Expenditure.

Effective energy expenditure is critical for maintaining body weight (BW). However, underlying mechanisms contributing to increased BW remain unknown. We characterized the role of brain angiogenesis inhibitor-3 (BAI3/ADGRB3), an adhesion G-protein coupled receptor (aGPCR), in regulating BW. A CRISPR/Cas9 gene editing approach was utilized to generate a whole-body deletion of the BAI3 gene (BAI3-/-). In both BAI3-/- male and female mice, a significant reduction in BW was observed compared to BAI3+/+ control mice. Quantitative magnetic imaging analysis showed that lean and fat masses were reduced in male and female mice with BAI3 deficiency. Total activity, food intake, energy expenditure (EE), and respiratory exchange ratio (RER) were assessed in mice housed at room temperature using a Comprehensive Lab Animal Monitoring System (CLAMS). While no differences were observed in the activity between the two genotypes in male or female mice, energy expenditure was increased in both sexes with BAI3 deficiency. However, at thermoneutrality (30 °C), no differences in energy expenditure were observed between the two genotypes for either sex, suggesting a role for BAI3 in adaptive thermogenesis. Notably, in male BAI3-/- mice, food intake was reduced, and RER was increased, but these attributes remained unchanged in the female mice upon BAI3 loss. Gene expression analysis showed increased mRNA abundance of thermogenic genes Ucp1, Pgc1α, Prdm16, and Elov3 in brown adipose tissue (BAT). These outcomes suggest that adaptive thermogenesis due to enhanced BAT activity contributes to increased energy expenditure and reduced BW with BAI3 deficiency. Additionally, sex-dependent differences were observed in food intake and RER. These studies identify BAI3 as a novel regulator of BW that can be potentially targeted to improve whole-body energy expenditure.

Sarcolipin ablation decreases whole body VO2 in young female nice

The sarco(endo)plasmic reticulum calcium (Ca 2+ ) ATPase (SERCA) pump is a major regulator of cytosolic Ca 2+ in striated muscle. Sarcolipin (SLN) reduces SERCA-mediated Ca 2+ pumping efficiency and is a key regulator of muscle metabolism and contractile function. With SERCA activity comprising approximately 12% of whole-body metabolic rate, the inefficient SERCA Ca 2+ cycling promoted by SLN would be predicted to increase metabolic rate. Although SLN is involved in adaptive diet-induced thermogenesis and overexpression of SLN increases metabolic rate and protects against diet-induced obesity, we found previously that SLN ablation had no effect on whole body metabolic rate or body composition in male mice that were fed a standard chow diet. However, unpublished work from our lab indicates that SLN expression in mouse soleus is significantly higher in females compared with males. Therefore, the purpose of this study was to investigate the effects of SLN ablation on body composition and metabolic rate of female mice. Adult (4-6mo) female wild type (WT) and SLN knockout (KO) mice (n=4 per group) were used to collect anthropometric measures (adiposity and body weight) and were housed in a comprehensive lab animal monitoring system (CLAMS) over a 48-hour period to assess daily whole body VO2 and total cage activity. With SLN ablation, total whole body VO 2 (ml/kg/hr) was lower (p<0.05) in the KO females (3534 ± 95.1) compared to WT females (5080 ± 98.8), with no differences (p>0.05) found for total cage activity (counts) (WT, 14935.42 ± 1799.6 vs. KO, 11860.83 ± 679.48), body weight (grams) (WT, 20.97 ± 0.76 vs. KO, 22.18 ± 0.80), or adiposity (arbitrary units) (WT, 5.18 ± 1.71 vs. KO, 2.12 ± 0.36). These results showing that high levels of endogenous muscle SLN expression in female mice influence whole-body metabolism even under chow-fed conditions, demonstrate a potential important sex difference in whole body physiology and metabolism. Natural Sciences and Engineering Research Council of Canada (NSERC) [grant number 311922-05 (to A.R.T.)] 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.

Differential Responses to Sigma-1 or Sigma-2 Receptor Ablation in Adiposity, Fat Oxidation, and Sexual Dimorphism.

Obesity is increasing at epidemic rates across the US and worldwide, as are its co-morbidities, including type-2 diabetes and cardiovascular disease. Thus, targeted interventions to reduce the prevalence of obesity are of the utmost importance. The sigma-1 receptor (S1R) and sigma-2 receptor (S2R; encoded by Tmem97) belong to the same class of drug-binding sites, yet they are genetically distinct. There are multiple ongoing clinical trials focused on sigma receptors, targeting diseases ranging from Alzheimer’s disease through chronic pain to COVID-19. However, little is known regarding their gene-specific role in obesity. In this study, we measured body composition, used a comprehensive laboratory-animal monitoring system, and determined the glucose and insulin tolerance in mice fed a high-fat diet. Compared to Sigmar1+/+ mice of the same sex, the male and female Sigmar1−/− mice had lower fat mass (17% and 12% lower, respectively), and elevated lean mass (16% and 10% higher, respectively), but S1R ablation had no effect on their metabolism. The male Tmem97−/− mice exhibited 7% lower fat mass, 8% higher lean mass, increased volumes of O2 and CO2, a decreased respiratory exchange ratio indicating elevated fatty-acid oxidation, and improved insulin tolerance, compared to the male Tmem97+/+ mice. There were no changes in any of these parameters in the female Tmem97−/− mice. Together, these data indicate that the S1R ablation in male and female mice or the S2R ablation in male mice protects against diet-induced adiposity, and that S2R ablation, but not S1R deletion, improves insulin tolerance and enhances fatty-acid oxidation in male mice. Further mechanistic investigations may lead to translational strategies to target differential S1R/S2R regulations and sexual dimorphism for precision treatments of obesity.

Bile acid conjugation deficiency causes hypercholanemia, hyperphagia, islet dysfunction, and gut dysbiosis in mice.

Bile acid‐CoA: amino acid N‐acyltransferase (BAAT) catalyzes bile acid conjugation, the last step in bile acid synthesis. BAAT gene mutation in humans results in hypercholanemia, growth retardation, and fat‐soluble vitamin insufficiency. The current study investigated the physiological function of BAAT in bile acid and lipid metabolism using Baat −/− mice. The bile acid composition and hepatic gene expression were analyzed in 10‐week‐old Baat −/− mice. They were also challenged with a westernized diet (WD) for additional 15 weeks to assess the role of BAAT in bile acid, lipid, and glucose metabolism. Comprehensive lab animal monitoring system and cecal 16S ribosomal RNA gene sequencing were used to evaluate the energy metabolism and microbiome structure of the mice, respectively. In Baat −/− mice, hepatic bile acids were mostly unconjugated and their levels were significantly increased compared with wild‐type mice. Bile acid polyhydroxylation was markedly up‐regulated to detoxify unconjugated bile acid accumulated in Baat −/− mice. Although the level of serum marker of bile acid synthesis, 7α‐hydroxy‐4‐cholesten‐3‐one, was higher in Baat −/− mice, their bile acid pool size was smaller. When fed a WD, the Baat −/− mice showed a compromised body weight gain and impaired insulin secretion. The gut microbiome of Baat −/− mice showed a low level of sulfidogenic bacteria Bilophila. Conclusion: Mouse BAAT is the major taurine‐conjugating enzyme. Its deletion protected the animals from diet‐induced obesity, but caused glucose intolerance. The gut microbiome of the Baat −/− mice was altered to accommodate the unconjugated bile acid pool.

Metal Transporter SLC39A14/ZIP14-Mediated Zinc Transport Modulates the Gut Microbiome and Host Metabolism

ObjectivesIntestinal permeability, gut dysbiosis, and Zn dyshomeostasis are emerging signatures of inflammatory bowel diseases and metabolic disorders such as type-2-diabetes and obesity. Zn deficiency is a common clinical finding among these diseases. Zinc (Zn) plays a critical role in maintaining intestinal homeostasis by regulating intestinal epithelial cells, host immune cells, and gut microbiome community composition. Zn transporters maintain Zn homeostasis by facilitating the cellular and subcellular distribution of Zn to and within tissues. Our previous studies revealed that deletion of metal transporter Slc39a14/Zip14 caused spontaneous intestinal permeability with low-grade chronic inflammation, mild hyperinsulinemia, and greater body fat with insulin resistance in adipose, suggesting a role for ZIP14-mediated intestinal metal transport in regulating both intestinal homeostasis and systemic metabolism. Here we present our novel findings on the function of ZIP14-mediated Zn transport in intestinal tissue, microbial profile, and how ZIP14-linked changes in gut microbiome and metabolites modulate host metabolism.MethodsTissue metal concentrations were measured by microwave plasma atomic emission spectrometers. Gut microbiome community composition Saccharomyces cerevisiae levels were determined using 16S rRNA gene sequencing and < qPCR, respectively. To identify the host and microbial-originated metabolites in WT and KO mice serum, we conducted untargeted metabolomics using Gas chromatography coupled to time-of-flight mass spectrometry. Lastly, we conducted metabolic phenotyping by using a Comprehensive Laboratory Animal Monitoring System.ResultsDeletion of Zip14 generated Zn-deficient epithelial cells and luminal content in the entire intestinal tract; reduced bacterial diversity and Saccharomyces cerevisiae (S. cerevisiae) overgrowth; altered host metabolome; and shifted host energy metabolism toward glucose utilization.ConclusionsThis work provided evidence for the regulation of gut microbiome composition, host metabolome, and energy metabolism by metal transporter ZIP14.Funding SourcesCornell University Division of Nutritional Sciences Funds.

Metabolic trade‐off and adipose tissue role in Neonatal Sepsis

BackgroundNeonatal sepsis contributes substantially to neonatal morbidity and mortality. The activation of an inflammatory response by the host triggers a significant metabolic challenge in which mobilization of stored energy is redistributed. Metabolic costs of immunity are high and physiological trade‐offs that neonates suffering from sepsis undergoes can determine the disease tolerance. Adipose tissue (AT) is a highly dynamic endocrine organ that partakes inter‐organs communication and regulates metabolism. In this study, we investigated whether immunity activation due to distinct TLRs ligands in neonatal sepsis promoted an energetic trade‐off and if the AT depots promoted tissue tolerance.MethodsPostnatal day 5 (PD5) CD1 mice were I.P. treated either with saline (Control), LPS, or Pam3CSK4 (PAM). Body weight and temperature were accessed prior treatments and after 3 hours. Oxygen and CO2 consumption was assessed through Comprehensive Lab Animal Monitoring System (CLAMS). Following euthanasia, blood was collected and subcutaneous white AT (WAT) and brown AT (BAT) depots were dissected out and submitted to microarray analysis utilizing Clariom™ S Assay mouse and the Affymetrix GeneChip Mouse Gene ST 2.0 arrays. Blood glucose was assessed using a glucometer.ResultsThe activation of inflammation promoted either by LPS or PAM resulted in significantly reduced total body weight due to the treatment only after 3 hours. We asked if this effect was due to the growth or nutritional status, we found the control pups kept with the mom increased body weight, whereas the pups fasting did not, as expected. However, these changes were not observed in the TLR4 and TLR1/2 ligands treated pups, in which, the bodyweight was decreased independently of them being fasted or not. The cutaneous temperature remained similar in the control group, while LPS induced a loss of ‐1.5°C degrees post‐treatment, and only ‐0.5°C variation was observed in the PAM treated pups. A reduction in VO2 and VCO2 consumption was stronger in LPS than in PAM treated pups, in which they started to increase these levels after 90 minutes, indicating that TLR4 induced energy conservation also in neonates. LPS and PAM induced diminished blood glucose levels compared to control in fasting pups, and only in LPS in fed pups. Next, we evaluated the contribution of AT to lower metabolic rate and promote energy conservation. Microarray analysis showed LPS induced a total of 103 up‐regulated genes, and 238 due to PAM in BAT. Enrichment analysis of this depot was related to Collagen‐containing extracellular matrix, and Inflammatory response in LPS treated pups, while mRNA metabolic process was highlighted in pups treated with PAM. Moreover, in the WAT, LPS promoted enrichment of inflammatory responses along with lipid storage, fatty acid‐binding and lipid metabolism, whereas PAM induced several pathways related to lipid storage, catabolism, and temperature homeostasis.ConclusionsCollectively, these data highlight the physiological trade‐offs occurring between immunity and energy suggesting that the hypometabolic‐hypothermic state is inducing energy conservation. AT depots are promoting fatty acid oxidation and perhaps mediating this energetic trade‐off. Thus, these physiological outcomes demonstrate the importance of AT regulation over the course of neonatal sepsis.

Treatment of Sleep Disordered Breathing with Leptin Loaded Extracellular Vesicles

IntroductionObstructive sleep apnea (OSA) is the most common type of sleep disordered breathing (SDB), characterized by recurrent periods of upper airway obstruction during sleep leading to increase in morbidity and mortality. The prevalence of OSA exceeds 50% in individuals with obesity and 10‐20% also develop hypercapnia and hypoventilation during sleep which characterizes obesity hypoventilation syndrome (OHS). There is no effective pharmacotherapy for SDB. Leptin, an adipocyte derived hormone, can stimulate breathing and is a potential therapeutic candidate. However, diet‐induced obesity is associated with impairment of leptin transport across the blood‐brain barrier. Previous studies have shown that extracellular vesicles (EVs) naturally derived from macrophages can penetrate the BBB and be used for drug delivery. The objective of this study was to determine if EVs overcome the BBB and treat SDB in DIO mice.MethodsTo examine the ability of EVs to cross the BBB, lean (n=5) and DIO (n=5) C57BL/6J mice were injected with fluorescent EVs or saline into the lateral tail vein. After 4h EV biodistribution was evaluated by Bioimaging and Infrared Spectroscopy (IVIS). Sleep studies were performed in a plethysmography chamber in a separate subgroup of male, DIO (n=10) and lean (n=10) mice. Mice received saline, empty exosomes, free leptin, or leptin‐loaded exosomes in a crossover manner. Similar design was used to determine energy expenditure in a Comprehensive Laboratory Animal Monitoring System (CLAMS).ResultsWe demonstrated that EVs accumulated in the brain and the transport across the BBB was 2‐times more efficient in DIO when compared to lean mice (p&lt;0.005). In DIO mice, leptin EVs induced 1.7‐2.2‐fold increases in minute ventilation and 1.5‐2.0‐fold increases in maximal inspiratory flow during flow‐limited and non‐flow limited breaths (p&lt;0.05). In contrast, free leptin had no effect. There was no effect of leptin EVs on metabolism. These findings suggest that leptin EVs mitigated upper airway obstruction and improved control of breathing in DIO mice. Lean mice did not present significant sleep disordered breathing and no differences were observed between groups.ConclusionWe demonstrated that EVs overcome the BBB and that leptin loaded EVs treat SDB in DIO mice.

Early-Onset Physical Inactivity and Metabolic Dysfunction in Tumor-bearing Mice Is Associated with Accelerated Cachexia.

Male C57BL/6J mice (12 wk of age) were injected with 1 × 106 LLC cells or phosphate-buffered saline (PBS) subcutaneously in the right flank, and tissue was collected 26-28 d after cell injection. Tumor volume was measured every 5 d throughout the study to calculate the tumor growth rate. Fifteen days after tumor inoculation, a subset of PBS (n = 11) and LLC (n = 16) mice were individually housed in metabolic Comprehensive Laboratory Animal Monitoring System cages for 5 d. LLC mice exhibited greater body weight loss (-5.1%), decreased muscle mass (-7%), decreased fat mass (-22%), and increased plasma interleukin-6 (212%) compared with PBS mice. Before the onset of cachexia, total cage activity was decreased in tumor-bearing mice. Cage activity was negatively associated with tumor mass and positively associated with hindlimb muscle mass. In addition, LLC mice had greater lipid oxidation than PBS mice. LLC mice exhibit early-onset physical inactivity and altered systemic lipid oxidation, which are associated with the eventual development of cachexia.

Effects of Micronutrient Supplementation on Glucose and Hepatic Lipid Metabolism in a Rat Model of Diet Induced Obesity.

Obesity increases the risk of metabolic disorders, partly through increased oxidative stress. Here, we examined the effects of a dietary micronutrient supplement (consisting of folate, vitamin B6, choline, betaine, and zinc) with antioxidant and methyl donor activities. Male Sprague Dawley rats (3 weeks old, 17/group) were weaned onto control (C) or high-fat diet (HFD) or same diets with added micronutrient supplement (CS; HS). At 14.5 weeks of age, body composition was measured by magnetic resonance imaging. At 21 weeks of age, respiratory quotient and energy expenditure was measured using Comprehensive Lab Animal Monitoring System. At 22 weeks of age, an oral glucose tolerance test (OGTT) was performed, and using fasting glucose and insulin values, Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) was calculated as a surrogate measure of insulin resistance. At 30.5 weeks of age, blood and liver tissues were harvested. Liver antioxidant capacity, lipids and expression of genes involved in lipid metabolism (Cd36, Fabp1, Acaca, Fasn, Cpt1a, Srebf1) were measured. HFD increased adiposity (p < 0.001) and body weight (p < 0.001), both of which did not occur in the HS group. The animals fed HFD developed impaired fasting glucose, impaired glucose tolerance, and fasting hyperinsulinemia compared to control fed animals. Interestingly, HS animals demonstrated an improvement in fasting glucose and fasting insulin. Based on insulin release during OGTT and HOMA-IR, the supplement appeared to reduce the insulin resistance developed by HFD feeding. Supplementation increased hepatic glutathione content (p < 0.05) and reduced hepatic triglyceride accumulation (p < 0.001) regardless of diet; this was accompanied by altered gene expression (particularly of CPT-1). Our findings show that dietary micronutrient supplementation can reduce weight gain and adiposity, improve glucose metabolism, and improve hepatic antioxidant capacity and lipid metabolism in response to HFD intake.

Distinct Basal Metabolism in Three Mouse Models of Neurodevelopmental Disorders.

Prevalence of metabolic disturbances is higher among individuals with neurodevelopmental disorders (NDDs), yet this association has been largely overlooked. Investigation into human disease remains challenging, as a complete pathophysiological understanding relies on accurate modeling and highly controlled variables. Genetically engineered mouse models are widely used to gain insight into the biology of human NDDs, but research focus has been on behavioral and neurophysiological abnormalities. Such models not only allow for evaluating usefulness in reproducing human features, including similarities and discrepancies with rodent phenotypes, but they also represent a unique opportunity to observe and quantify novel anomalies. Here, we present the first characterization and comparison of basal metabolism in three mouse models of NDDs, namely, Down syndrome (DS; Dp(16)Yey/+ mice), 16p11.2 deletion syndrome (16pDel; 16p11.2df/+ mice), and fragile X syndrome [FXS; Fmr1 knock-out (KO) mice] and their wild-type (WT) counterparts. Using the Comprehensive Lab Animal Monitoring System (CLAMS) coupled to EchoMRI, as well as quantification of key plasma metabolites by liquid chromatography mass spectrometry (LC-MS), our in vivo study reveals that each mouse model expresses a unique metabolic signature that is sex-specific, independent of the amount of food consumed and minimally influenced by physical activity. In particular, we identify striking differences in body composition, respiratory exchange ratio (RER), caloric expenditure (CE), and concentrations of circulating plasma metabolites related to mitochondrial function. Providing novel insight into NDD-associated metabolic alterations is an essential prerequisite for future preclinical and clinical interventions.

Brief high fat high sugar diet results in altered energy and fat metabolism during pregnancy in mice

During pregnancy several maternal adaptations occur in order to support the growing fetus which are further exacerbated by gestational diabetes mellitus (GDM). Previously we developed a mouse model of GDM, however we did not evaluate alterations to energy and fat metabolism. We have also shown that alterations in lipid metabolism are mediated by adrenomedullin (ADM) in normal and GDM pregnancies. Our objectives were: (1) evaluate energy and fat homeostasis in our GDM mouse model and (2) determine if ADM may play a role in these changes. Female mice were placed on either control (P-CD) or high fat, high sucrose diet (P-HFHS) 1 week prior to and throughout pregnancy. Mice were placed into comprehensive lab animal monitoring system (CLAMS) chambers throughout pregnancy. Visceral adipose tissue (VAT) was collected at d17.5 of pregnancy for analysis. Energy Expenditure was significantly increased (p < 0.05) in P-HFHS dams compared to all other groups. VAT ex-vivo lipolysis was increased (p < 0.05) in P-HFHS compared to P-CD dams. VAT gene expression of ADM receptors Crlr, Ramp2, and Ramp3 was increased (p < 0.05) in P-HFHS dams. ADM dose dependently increased ex vivo lipolysis. This data further validates our animal model of GDM and is usefulness in investigating the pathophysiology of GDM.

Combination of Pancreastatin inhibitor PSTi8 with metformin inhibits Fetuin-A in type 2 diabetic mice

In the preceding study, we delineated that high-fat diet (HFD) consumption in mice increases the circulatory level of pancreastatin (PST), which additionally enhances the free fatty acid (FFA) concentration in circulation. Consequently, the aggravated FFA activates Fetuin-A, which facilitates hepatic lipid accumulation, insulin resistance (IR), and culminates in type 2 diabetes (T2D). Metformin (Met) is a widely known first-line drug for the treatment of T2D. We previously unveiled PSTi8, an inhibitor of PST, comprising antidiabetic property. Hence, we hypothesized that combination therapy of Met and PSTi8, at reduced therapeutic doses, would mitigate HFD-induced IR by inhibiting hepatic Fetuin-A in mice model of T2D. C57BL/6 mice were fed HFD for 12 weeks, followed by treatment with Met, PSTi8, and its combination for 10 days. Glucose and insulin tolerance tests were conducted. Circulatory levels of PST, Fetuin-A, and lipid markers were determined. Also, the mRNA and protein expression of Fetuin-A was assessed by qPCR, western blotting, and immunofluorescence. Moreover, the energy expenditure was measured by comprehensive laboratory animal monitoring system (CLAMS). Combination therapy displayed improved PST, Fetuin-A, and lipid profile in plasma. We also found reduced hepatic Fetuin-A, which reduced inhibitory phosphorylation of IRS and increased phosphorylation of AKT. Consequently, ameliorated hepatic lipogenesis, gluconeogenesis, and inflammation. Also, combination treatment attenuated Fetuin-A expression, lipid accumulation, and glucose production in palmitate-induced HepG2 cells. Altogether current study promulgates the beneficial effect of combination therapy of Met and PSTi8 (comparable to alone higher therapeutic doses) to ameliorate Fetuin-A activation, hepatic lipid accumulation, insulin resistance, and associated progressive pathophysiological alterations in T2D.