Chronic Kidney Disease Diets for Kidney Failure Prevention: Insights from the IL-11 Paradigm.

Nearly every fifth adult in the United States and many older adults worldwide are affected by chronic kidney disease (CKD), which can progress to kidney failure requiring invasive kidney replacement therapy. In this review, we briefly examine the pathophysiology of CKD and discuss emerging mechanisms involving the physiological resolution of kidney injury by transforming growth factor beta 1 (TGFß1) and interleukin-11 (IL-11), as well as the pathological consequences of IL-11 overproduction, which misguides repair processes, ultimately culminating in CKD. Taking these mechanisms into account, we offer an overview of the efficacy of plant-dominant dietary patterns in preventing and managing CKD, while also addressing their limitations in terms of restoring kidney function or preventing kidney failure. In conclusion, this paper outlines novel regeneration strategies aimed at developing a reno-regenerative diet to inhibit IL-11 and promote repair mechanisms in kidneys affected by CKD.

Loss of Paneth cells dysregulates gut ILC subsets and enhances weight gain response to high fat diet in a mouse model.

Obesity has been associated with dysbiosis, but innate mechanisms linking intestinal epithelial cell subsets and obesity remain poorly understood. Using mice lacking Paneth cells (Sox9 ΔIEC mice), small intestinal epithelial cells specialized in the production of antimicrobial products and cytokines, we show that dysbiosis alone does not induce obesity or metabolic disorders. Loss of Paneth cells reduced ILC3 and increased ILC2 numbers in the intestinal lamina propria. High-fat diet (HFD) induced higher weight gain and more severe metabolic disorders in Sox9 ΔIEC mice. Further, HFD enhances the number of ILC1 in the intestinal lamina propria of Sox9 ΔIEC mice and increases intestinal permeability and the accumulation of immune cells (inflammatory macrophages and T cells, and B cells) in abdominal fat tissues of obese Sox9 ΔIEC . Transplantation of fecal materials from Sox9 ΔIEC mice in germ-free mice before HFD further confirmed the regulatory role of Paneth cells for gut ILC subsets and the development of obesity.

Iron Deficiency Anemia: Efficacy and Limitations of Nutritional and Comprehensive Mitigation Strategies.

Iron deficiency anemia (IDA) has reached epidemic proportions in developing countries and has become a major global public health problem, affecting mainly 0-5-year-old children and young women of childbearing age, especially during pregnancy. Iron deficiency can lead to life-threatening loss of red blood cells, muscle function, and energy production. Therefore, the pathogenic features associated with IDA are weakness and impaired growth, motor, and cognitive performance. IDA affects the well-being of the young generation and the economic advancement of developing countries, such as India. The imbalance between iron intake/absorption/storage and iron utilization/loss culminates into IDA. However, numerous strategic programs aimed to increase iron intake have shown that improvement of iron intake alone has not been sufficient to mitigate IDA. Emerging critical risk factors for IDA include a composition of cultural diets, infections, genetics, inflammatory conditions, metabolic diseases, dysbiosis, and socioeconomic parameters. In this review, we discuss numerous IDA mitigation programs in India and their limitations. The new multifactorial mechanism of IDA pathogenesis opens perspectives for the improvement of mitigation programs and relief of IDA in India and worldwide.

Retinol Binding Protein 7 Promotes Adipogenesis in vitro and Regulates Expression of Genes Involved in Retinol Metabolism.

Retinol is an essential nutrient in animals. Its metabolites, specifically retinoic acid (RA), are crucial for cell differentiation, including adipogenesis. Retinol binding protein 7 (Rbp7) is under the control of PPARγ, the master regulator of adipogenesis. However, the role of RBP7 in adipogenesis is unclear. Our study showed that Rbp7 was abundantly expressed in white and brown mouse adipose tissues and had a higher expression in adipocytes than in stromal vascular fraction. Rbp7 overexpression promoted 3T3-L1 preadipocyte differentiation with increased triglyceride accumulation and up-regulation of Pparγ, Fabp4, C/ebpα, and AdipoQ. Rbp7 deficient adipocytes had opposite effects of the overexpression, which were rescued by RA supplementation. Indirect assessment of relative nuclear RA levels using RAR response element (RARE)-Luc reporter assay demonstrated that Rbp7 overexpression significantly increased RARE-Luc reporter activity. Rbp7 overexpression significantly increased expression of Raldh1, responsible for RA production, and up-regulation of Lrat and Cyp26a1, involved in retinol storage and RA catabolism, respectively, in 3T3-L1 adipocytes. Rbp7 deficient adipocytes had opposite effects of the overexpression of those genes involved in retinol metabolism. These data suggest that RBP7 increases transcriptional activity of RARE that may induce negative feedback responses via regulation of the gene expression for retinol homeostasis. Our data indicate critical RBP7 functions in adipocytes: regulation of transcriptional activity of RARE and adipocytes differentiation, potentially providing a new target for obesity therapy.

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo.

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro, or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis assumes that the metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. This article describes a method for measuring extracellular FD-glucose depletion and validates its correlation with intracellular FD-glucose uptake in cells and tissues ex vivo. Extracellular glucose depletion may be potentially applicable for high-throughput kinetic and dose-dependent studies, as well as identifying compounds with glycemic activity and their tissue-specific effects.

Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity.

The leptin receptor (LepR) acts as a signaling nexus for the regulation of glucose uptake and obesity, among other metabolic responses. The functional role of LepR under leptin-deficient conditions remains unclear. This study reports that epiregulin (EREG) governed glucose uptake in vitro and in vivo in Lepob mice by activating LepR under leptin-deficient conditions. Single and long-term treatment with EREG effectively rescued glucose intolerance in comparative insulin and EREG tolerance tests in Lepob mice. The immunoprecipitation study revealed binding between EREG and LepR in adipose tissue of Lepob mice. EREG/LepR regulated glucose uptake without changes in obesity in Lepob mice via mechanisms, including ERK activation and translocation of GLUT4 to the cell surface. EREG-dependent glucose uptake was abolished in Leprdb mice which supports a key role of LepR in this process. In contrast, inhibition of the canonical epidermal growth factor receptor (EGFR) pathway implicated in other EREG responses, increased glucose uptake. Our data provide a basis for understanding glycemic responses of EREG that are dependent on LepR unlike functions mediated by EGFR, including leptin secretion, thermogenesis, pain, growth, and other responses. The computational analysis identified a conserved amino acid sequence, supporting an evolutionary role of EREG as an alternative LepR ligand.

Paracardial fat remodeling affects systemic metabolism through alcohol dehydrogenase 1.

The relationship between adiposity and metabolic health is well established. However, very little is known about the fat depot, known as paracardial fat (pCF), located superior to and surrounding the heart. Here, we show that pCF remodels with aging and a high-fat diet and that the size and function of this depot are controlled by alcohol dehydrogenase 1 (ADH1), an enzyme that oxidizes retinol into retinaldehyde. Elderly individuals and individuals with obesity have low ADH1 expression in pCF, and in mice, genetic ablation of Adh1 is sufficient to drive pCF accumulation, dysfunction, and global impairments in metabolic flexibility. Metabolomics analysis revealed that pCF controlled the levels of circulating metabolites affecting fatty acid biosynthesis. Also, surgical removal of the pCF depot was sufficient to rescue the impairments in cardiometabolic flexibility and fitness observed in Adh1-deficient mice. Furthermore, treatment with retinaldehyde prevented pCF remodeling in these animals. Mechanistically, we found that the ADH1/retinaldehyde pathway works by driving PGC-1α nuclear translocation and promoting mitochondrial fusion and biogenesis in the pCF depot. Together, these data demonstrate that pCF is a critical regulator of cardiometabolic fitness and that retinaldehyde and its generating enzyme ADH1 act as critical regulators of adipocyte remodeling in the pCF depot.

Amino Acid Nanofibers Improve Glycemia and Confer Cognitive Therapeutic Efficacy to Bound Insulin.

Diabetes poses a high risk for debilitating complications in neural tissues, regulating glucose uptake through insulin-dependent and predominantly insulin-independent pathways. Supramolecular nanostructures provide a flexible strategy for combinatorial regulation of glycemia. Here, we compare the effects of free insulin to insulin bound to positively charged nanofibers comprised of self-assembling amino acid compounds (AACs) with an antioxidant-modified side chain moiety (AAC2) in both in vitro and in vivo models of type 1 diabetes. Free AAC2, free human insulin (hINS) and AAC2-bound-human insulin (AAC2-hINS) were tested in streptozotocin (STZ)-induced mouse model of type 1 diabetes. AAC2-hINS acted as a complex and exhibited different properties compared to free AAC2 or hINS. Mice treated with the AAC2-hINS complex were devoid of hypoglycemic episodes, had improved levels of insulin in circulation and in the brain, and increased expression of neurotransmitter taurine transporter, Slc6a6. Consequently, treatment with AAC2-hINS markedly advanced both physical and cognitive performance in mice with STZ-induced and genetic type 1 diabetes compared to treatments with free AAC2 or hINS. This study demonstrates that the flexible nanofiber AAC2 can serve as a therapeutic platform for the combinatorial treatment of diabetes and its complications.

Osmotic Adaptation by Na+-Dependent Transporters and ACE2: Correlation with Hemostatic Crisis in COVID-19.

COVID-19 symptoms, including hypokalemia, hypoalbuminemia, ageusia, neurological dysfunctions, D-dimer production, and multi-organ microthrombosis reach beyond effects attributed to impaired angiotensin-converting enzyme 2 (ACE2) signaling and elevated concentrations of angiotensin II (Ang II). Although both SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus) and SARS-CoV-2 utilize ACE2 for host entry, distinct COVID-19 pathogenesis coincides with the acquisition of a new sequence, which is homologous to the furin cleavage site of the human epithelial Na+ channel (ENaC). This review provides a comprehensive summary of the role of ACE2 in the assembly of Na+-dependent transporters of glucose, imino and neutral amino acids, as well as the functions of ENaC. Data support an osmotic adaptation mechanism in which osmotic and hemostatic instability induced by Ang II-activated ENaC is counterbalanced by an influx of organic osmolytes and Na+ through the ACE2 complex. We propose a paradigm for the two-site attack of SARS-CoV-2 leading to ENaC hyperactivation and inactivation of the ACE2 complex, which collapses cell osmolality and leads to rupture and/or necrotic death of swollen pulmonary, endothelial, and cardiac cells, thrombosis in infected and non-infected tissues, and aberrant sensory and neurological perception in COVID-19 patients. This dual mechanism employed by SARS-CoV-2 calls for combinatorial treatment strategies to address and prevent severe complications of COVID-19.

Antibiotics in Food Chain: The Consequences for Antibiotic Resistance.

Antibiotics have been used as essential therapeutics for nearly 100 years and, increasingly, as a preventive agent in the agricultural and animal industry. Continuous use and misuse of antibiotics have provoked the development of antibiotic resistant bacteria that progressively increased mortality from multidrug-resistant bacterial infections, thereby posing a tremendous threat to public health. The goal of our review is to advance the understanding of mechanisms of dissemination and the development of antibiotic resistance genes in the context of nutrition and related clinical, agricultural, veterinary, and environmental settings. We conclude with an overview of alternative strategies, including probiotics, essential oils, vaccines, and antibodies, as primary or adjunct preventive antimicrobial measures or therapies against multidrug-resistant bacterial infections. The solution for antibiotic resistance will require comprehensive and incessant efforts of policymakers in agriculture along with the development of alternative therapeutics by experts in diverse fields of microbiology, biochemistry, clinical research, genetic, and computational engineering.

Amino acid-based compound activates atypical PKC and leptin receptor pathways to improve glycemia and anxiety like behavior in diabetic mice.

Differences in glucose uptake in peripheral and neural tissues account for the reduced efficacy of insulin in nervous tissues. Herein, we report the design of short peptides, referred as amino acid compounds (AAC) with and without a modified side chain moiety. At nanomolar concentrations, a candidate therapeutic molecule, AAC2, containing a 7-(diethylamino) coumarin-3-carboxamide side-chain improved glucose control in human peripheral adipocytes and the endothelial brain barrier cells by activation of insulin-insensitive glucose transporter 1 (GLUT1). AAC2 interacted specifically with the leptin receptor (LepR) and activated atypical protein kinase C zeta (PKCς) to increase glucose uptake. The effects induced by AAC2 were absent in leptin receptor-deficient predipocytes and in Leprdb mice. In contrast, AAC2 established glycemic control altering food intake in leptin-deficient Lepob mice. Therefore, AAC2 activated the LepR and acted in a cytokine-like manner distinct from leptin. In a monogenic Ins2Akita mouse model for the phenotypes associated with type 1 diabetes, AAC2 rescued systemic glucose uptake in these mice without an increase in insulin levels and adiposity, as seen in insulin-treated Ins2Akita mice. In contrast to insulin, AAC2 treatment increased brain mass and reduced anxiety-related behavior in Ins2Akita mice. Our data suggests that the unique mechanism of action for AAC2, activating LepR/PKCς/GLUT1 axis, offers an effective strategy to broaden glycemic control for the prevention of diabetic complications of the nervous system and, possibly, other insulin insensitive or resistant tissues.

Thermogenic crosstalk occurs between adipocytes from different species.

Visceral obesity increases risks for all-cause mortality worldwide. A small population of thermogenic adipocytes expressing uncoupling protein-1 (Ucp1) regulates energy dissipation in white adipose tissue (WAT) depots. Thermogenic adipocytes subsets decrease obesity in mice, but their efficacy has not been tested in obese large animals. Here we enclosed murine subcutaneous adipocytes with and without engineered thermogenic response in biocompatible microcapsules and implanted them into the left and right side of the visceral falciform depot in six obese dogs. After 28 days of treatment, dogs have markedly reduced waist circumference, body weight, and fat mass. Ucp1 expression in canine WAT was increased at sites implanted with thermogenic vs. wild type murine adipocytes. This site-specific thermogenic remodeling of canine tissue by thermogenic murine adipocytes suggests evolutionary conserved paracrine regulation of energy dissipation across species. These findings have translational potential aimed to reduce deleterious WAT depots in humans and pets.

Assessment of adipogenic, antioxidant, and anti-inflammatory properties of whole and whey bovine colostrum.

Bovine colostrum (BC) has been used for nutraceutical purposes for animals and humans. Bovine colostrum is a complex heterogeneous product and its antimicrobial activity, antioxidant potential, and growth factors can vary depending on age and species of the cow as well as their environment. Bovine colostrum preparation in skimmed or whey fractions can also alter properties of BC. Our goal was to compare cumulative anti-inflammatory, antioxidant, and adipogenic properties of natural (whole) versus whey BC. We compared properties of whole and whey BC in 3T3-L1 preadipocytes permanently transfected with reporters responding to changes in inflammatory (NfκbRE/green fluorescent protein), anti-inflammatory (Nrf2/YFP), and adipogenic (Fabp4/cyan fluorescent protein) status in cells. Interleukin-6 secretion in these cells was measured by ELISA. Whole and whey BC induce IL-6 secretion from 3T3-L1 fibroblasts; however, whey preparation stimulated less IL-6 secretion. Cumulative inflammatory nuclear factor (NF)κB activation in the presence of lipopolysaccharide was reduced by both whole (-27%) and whey BC (-22%) compared with lipopolysaccharide-treated cells (100%). Treatment with whole BC was more effective in the reduction of NFκB activation compared with whey BC and occurred in a dose-dependent manner. In consonance with decreased NFκB activation, the Nrf2 promoter activity was also reduced in response to whole (-27%) and whey (-13%) treatments compared with nontreated cells (100%). Whole and whey BC suppressed adipogenesis, measured as induction of Fabp4, by -27 and -13%, respectively, compared with nontreated 3T3-L1 fibroblasts (100%). Our results showed distinct differences in properties of whey and whole BC that could be used to attain reduced adipogenic or cumulative inflammatory responses.

A paracrine role for white thermogenic adipocytes in innervation: an evidence-based hypothesis.

White adipose tissue (WAT) stores energy and also plays an important endocrine role in producing adipokines for communication with the peripheral and central nervous system. WAT consists of the major lipogenic unilocular adipocytes and the minor populations of beige and brite multilocular adipocytes. These multilocular adipocytes express thermogenic genes and have phenotypic similarity with thermogenic brown adipose tissue. According to a current paradigm, multilocular adipocytes have a thermogenic function in WAT. In this mini review, we discuss data revealing heterogeneity among multilocular cell subsets in WAT and their functions beyond thermogenesis. We propose a hypothetical neuroendocrine role for multilocular adipocytes subsets in the formation of adaptive sensory-sympathetic circuits between the central nervous system and adipose tissue, which activate lipolysis and thermogenesis in WAT in high energy demand situations.

Epiregulin induces leptin secretion and energy expenditure in high-fat diet-fed mice.

Adipokine leptin regulates neuroendocrine circuits that control energy expenditure, thermogenesis and weight loss. However, canonic regulators of leptin secretion, such as insulin and malonyl CoA, do not support these processes. We hypothesize that epiregulin (EREG), a growth factor that is secreted from fibroblasts under thermogenic and cachexia conditions, induces leptin secretion associated with energy dissipation. The effects of EREG on leptin secretion were studied ex vivo, in the intra-abdominal white adipose tissue (iAb WAT) explants, as well as in vivo, in WT mice with diet-induced obesity (DIO) and in ob/ob mice. These mice were pair fed a high-fat diet and treated with intraperitoneal injections of EREG. EREG increased leptin production and secretion in a dose-dependent manner in iAb fat explants via the EGFR/MAPK pathway. After 2 weeks, the plasma leptin concentration was increased by 215% in the EREG-treated group compared to the control DIO group. EREG-treated DIO mice had an increased metabolic rate and core temperature during the active dark cycle and displayed cold-induced thermogenesis. EREG treatment reduced iAb fat mass, the major site of leptin protein production and secretion, but did not reduce the mass of the other fat depots. In the iAb fat, expression of genes supporting mitochondrial oxidation and thermogenesis was increased in EREG-treated mice vs control DIO mice. All metabolic and gene regulation effects of EREG treatment were abolished in leptin-deficient ob/ob mice. Our data revealed a new role of EREG in induction of leptin secretion leading to the energy expenditure state. EREG could be a potential target protein to regulate hypo- and hyperleptinemia, underlying metabolic and immune diseases.

Bipolar disorder in youth is associated with increased levels of vitamin D-binding protein.

Genetic, dietary, and inflammatory factors contribute to the etiology of major mood disorders (MMD), thus impeding the identification of specific biomarkers to assist in diagnosis and treatment. We tested association of vitamin D and inflammatory markers in 36 adolescents with bipolar disorder (BD) and major depressive disorder (MDD) forms of MMD and without MMD (non-mood control). We also assessed the overall level of inflammation using a cell-based reporter assay for nuclear factor kappa-B (NFκB) activation and measuring antibodies to oxidized LDL. We found that these factors were similar between non-mood and MMD youth. To identify potential biomarkers, we developed a screening immunoprecipitation-sequencing approach based on inflammatory brain glia maturation factor beta (GMFß). We discovered that a homolog of GMFß in human plasma is vitamin D-binding protein (DBP) and validated this finding using immunoprecipitation with anti-DBP antibodies and mass spectrometry/sequencing analysis. We quantified DBP levels in participants by western blot. DBP levels in BD participants were significantly higher (136%) than in participants without MMD (100%). The increase in DBP levels in MDD participants (121.1%) was not statistically different from these groups. The DBP responds early to cellular damage by binding of structural proteins and activating inflammatory cells. A product of enzymatic cleavage of DBP has been described as macrophage-activating factor. Circulating DBP is comprised of heterogenous high and low molecular fractions that are only partially recognized by mono- and polyclonal ELISA and are not suitable for the quantitative comparison of DBP in non-mood and MDD participants. Our data suggest DBP as a marker candidate of BD warranting its validation in a larger cohort of adolescent and adult MMD patients.

Induction of innervation by encapsulated adipocytes with engineered vitamin A metabolism.

Innervation is a fundamental basis for function and survival of tissues. In the peripheral tissues, degenerative diseases create a neurotoxic metabolic milieu that either causes neurodegeneration or fails to sustain regenerative growth and reinnervation of injured/diseased tissues. Encapsulation of cells producing neurotrophic factors can augment axon growth and neuron survival; however, sustained innervation in vivo requires a combination of factors promoting axon growth and guidance pathway that are released in a tissue-specific context. Using novel encapsulation techniques and genetic tools, we manipulated retinoic acid-generating enzyme aldehyde dehydrogenase 1a1 (Aldh1a1) in adipocytes that are capable of promoting growth and innervation of white adipose tissue by sympathetic neurons. Aldh1a1-/- adipocytes secrete molecules that regulate axon guidance and markedly stimulate neurite outgrowth in vitro and in vivo. Based on studies with natural and synthetic RAR agonists and antagonists, gene microarray and nanostring arrays, we concluded that ephrin A5/ephrin A4 is a downstream pathway regulated by Aldh1a1. Encapsulation of Aldh1a1-/- adipocytes into alginate poly-L-lysine microcapsules induced functional innervation of adipose tissue in obese wild-type mice. We propose that encapsulated Aldh1a1-/- adipocytes could provide a therapeutic solution for the reinnervation of damaged tissues.

Aldehyde dehydrogenase 1 a1 regulates energy metabolism in adipocytes from different species.

BACKGROUND: Survival and longevity of xenotransplants depend on immune function and ability to integrate energy metabolism between cells from different species. However, mechanisms for interspecies cross talk in energy metabolism are not well understood. White adipose tissue stores energy and is capable of mobilization and dissipation of energy as heat (thermogenesis) by adipocytes expressing uncoupling protein 1 (Ucp1). Both pathways are under the control of vitamin A metabolizing enzymes. Deficient retinoic acid production in aldehyde dehydrogenase 1 A1 (Aldh1a1) knockout adipocytes (KO) inhibits adipogenesis and increases thermogenesis. Here we test the role Aldh1a1 in regulation of lipid metabolism in xenocultures. METHODS: Murine wide-type (WT) and KO pre-adipocytes were encapsulated into a poly-L-lysine polymer that allows exchange of humoral factors <32kD via nanopores. Encapsulated murine adipocytes were co-incubated with primary differentiated canine adipocytes. Then, expression of adipogenic and thermogenic genes in differentiated canine adipocytes was detected by real-time polymerase chain reaction (PCR). The regulatory factors in WT and KO cells were identified by comparison of secretome using proteomics and in transcriptome by gene microarray. RESULTS: Co-culture of encapsulated mouse KO vs WT adipocytes increased expression of peroxisome proliferator-activated receptor gamma (Pparg), but reduced expression of its target genes fatty acid binding protein 4 (Fabp4), and adipose triglyceride lipase (Atgl) in canine adipocytes, suggesting inhibition of PPARγ activation. Co-culture with KO adipocytes also induced expression of Ucp1 in canine adipocytes compared to expression in WT adipocytes. Cumulatively, murine KO compared to WT adipocytes decreased lipid accumulation in canine adipocytes. Comparative proteomics revealed significantly higher levels of vitamin A carriers, retinol binding protein 4 (RBP4), and lipokalin 2 (LCN2) in KO vs WT adipocytes. CONCLUSIONS: Our data demonstrate the functional exchange of regulatory factors between adipocytes from different species for regulation of energy balance. RBP4 and LCN2 appear to be involved in the transport of retinoids for regulation of lipid accumulation and thermogenesis in xenocultures. While the rarity of thermogenic adipocytes in humans and dogs precludes their use for autologous transplantation, our study demonstrates that xenotransplantation of engineered cells could be a potential solution for the reduction in obesity in dogs and a strategy for translation to patients.

Leptin Production by Encapsulated Adipocytes Increases Brown Fat, Decreases Resistin, and Improves Glucose Intolerance in Obese Mice.

The neuroendocrine effects of leptin on metabolism hold promise to be translated into a complementary therapy to traditional insulin therapy for diabetes and obesity. However, injections of leptin can provoke inflammation. We tested the effects of leptin, produced in the physiological adipocyte location, on metabolism in mouse models of genetic and dietary obesity. We generated 3T3-L1 adipocytes constitutively secreting leptin and encapsulated them in a poly-L-lysine membrane, which protects the cells from immune rejection. Ob/ob mice (OB) were injected with capsules containing no cells (empty, OB[Emp]), adipocytes (OB[3T3]), or adipocytes overexpressing leptin (OB[Lep]) into both visceral fat depots. Leptin was found in the plasma of OB[Lep], but not OB[Emp] and OB[3T3] mice at the end of treatment (72 days). The OB[Lep] and OB[3T3] mice have transiently suppressed appetite and weight loss compared to OB[Emp]. Only OB[Lep] mice have greater brown fat mass, metabolic rate, and reduced resistin plasma levels compared to OB[Emp]. Glucose tolerance was markedly better in OB[Lep] vs. OB[Emp] and OB[3T3] mice as well as in wild type mice with high-fat diet-induced obesity and insulin resistance treated with encapsulated leptin-producing adipocytes. Our proof-of-principle study provides evidence of long-term improvement of glucose tolerance with encapsulated adipocytes producing leptin.

Graded Maximal Exercise Testing to Assess Mouse Cardio-Metabolic Phenotypes.

Functional assessments of cardiovascular fitness (CVF) are needed to establish animal models of dysfunction, test the effects of novel therapeutics, and establish the cardio-metabolic phenotype of mice. In humans, the graded maximal exercise test (GXT) is a standardized diagnostic for assessing CVF and mortality risk. These tests, which consist of concurrent staged increases in running speed and inclination, provide diagnostic cardio-metabolic parameters, such as, VO2max, anaerobic threshold, and metabolic crossover. Unlike the human-GXT, published mouse treadmill tests have set, not staged, increases in inclination as speed progress until exhaustion (PXT). Additionally, they often lack multiple cardio-metabolic parameters. Here, we developed a mouse-GXT with the intent of improving mouse-exercise testing sensitivity and developing translatable parameters to assess CVF in healthy and dysfunctional mice. The mouse-GXT, like the human-GXT, incorporated staged increases in inclination, speed, and intensity; and, was designed by considering imitations of the PXT and differences between human and mouse physiology. The mouse-GXT and PXTs were both tested in healthy mice (C57BL/6J, FVBN/J) to determine their ability to identify cardio-metabolic parameters (anaerobic threshold, VO2max, metabolic crossover) observed in human-GXTs. Next, theses assays were tested on established diet-induced (obese-C57BL/6J) and genetic (cardiac isoform Casq2-/-) models of cardiovascular dysfunction. Results showed that both tests reported VO2max and provided reproducible data about performance. Only the mouse-GXT reproducibly identified anaerobic threshold, metabolic crossover, and detected impaired CVF in dysfunctional models. Our findings demonstrated that the mouse-GXT is a sensitive, non-invasive, and cost-effective method for assessing CVF in mice. This new test can be used as a functional assessment to determine the cardio-metabolic phenotype of various animal models or the effects of novel therapeutics.