Accelerated onset of diabetes in non-obese diabetic mice fed a refined high-fat diet.

AIM: Type 1 diabetes results from autoimmune events influenced by environmental variables, including changes in diet. This study investigated how feeding refined versus unrefined (aka 'chow') diets affects the onset and progression of hyperglycaemia in non-obese diabetic (NOD) mice. METHODS: Female NOD mice were fed either unrefined diets or matched refined low- and high-fat diets. The onset of hyperglycaemia, glucose tolerance, food intake, energy expenditure, circulating insulin, liver gene expression and microbiome changes were measured for each dietary group. RESULTS: NOD mice consuming unrefined (chow) diets developed hyperglycaemia at similar frequencies. By contrast, mice consuming the defined high-fat diet had an accelerated onset of hyperglycaemia compared to the matched low-fat diet. There was no change in food intake, energy expenditure, or physical activity within each respective dietary group. Microbiome changes were driven by diet type, with chow diets clustering similarly, while refined low- and high-fat bacterial diversity also grouped closely. In the defined dietary cohort, liver gene expression changes in high-fat-fed mice were consistent with a greater frequency of hyperglycaemia and impaired glucose tolerance. CONCLUSION: Glucose intolerance is associated with an enhanced frequency of hyperglycaemia in female NOD mice fed a defined high-fat diet. Using an appropriate matched control diet is an essential experimental variable when studying changes in microbiome composition and diet as a modifier of disease risk.

Untargeted Pixel-by-Pixel Imaging of Metabolite Ratio Pairs as a Novel Tool for Biomedical Discovery in Mass Spectrometry Imaging.

Mass spectrometry imaging (MSI) is a powerful technology used to define the spatial distribution and relative abundance of structurally identified and yet-undefined metabolites across tissue cryosections. While numerous software packages enable pixel-by-pixel imaging of individual metabolites, the research community lacks a discovery tool that images all metabolite abundance ratio pairs. Importantly, recognition of correlated metabolite pairs informs discovery of unanticipated molecules contributing to shared metabolic pathways, uncovers hidden metabolic heterogeneity across cells and tissue subregions, and indicates single-timepoint flux through pathways of interest. Here, we describe the development and implementation of an untargeted R package workflow for pixel-by-pixel ratio imaging of all metabolites detected in an MSI experiment. Considering untargeted MSI studies of murine brain and embryogenesis, we demonstrate that ratio imaging minimizes systematic data variation introduced by sample handling and instrument drift, markedly enhances spatial image resolution, and reveals previously unrecognized metabotype-distinct tissue regions. Furthermore, ratio imaging facilitates identification of novel regional biomarkers and provides anatomical information regarding spatial distribution of metabolite-linked biochemical pathways. The algorithm described herein is applicable to any MSI dataset containing spatial information for metabolites, peptides or proteins, offering a potent tool to enhance knowledge obtained from current spatial metabolite profiling technologies.

Differential responses to maternal diabetes in embryo and visceral yolk sac.

Introduction: Maternal diabetes during pregnancy is well known to be associated with a higher risk for structural birth defects in the offspring. Recent searches for underlying mechanisms have largely focused on aberrant processes in the embryo itself, although prior research in rodent models implicated dysfunction also of the visceral yolk sac. The objective of our research was to investigate both tissues within the conceptus simultaneously. Methods: We conducted unbiased transcriptome profiling by RNA sequencing on pairs of individual yolk sacs and their cognate embryos, using the non-obese diabetic (NOD) mouse model. The analysis was performed at gestational day 8.5 on morphologically normal specimen to circumvent confounding by defective development. Results: Even with large sample numbers (n = 33 in each group), we observed considerable variability of gene expression, primarily driven by exposure to maternal diabetes, and secondarily by developmental stage of the embryo. Only a moderate number of genes changed expression in the yolk sac, while in the embryo, the exposure distinctly influenced the relationship of gene expression levels to developmental progression, revealing a possible role for altered cell cycle regulation in the response. Also affected in embryos under diabetic conditions were genes involved in cholesterol biosynthesis and NAD metabolism pathways. Discussion: Exposure to maternal diabetes during gastrulation changes transcriptomic profiles in embryos to a substantially greater effect than in the corresponding yolk sacs, indicating that despite yolk sac being of embryonic origin, different mechanisms control transcriptional activity in these tissues. The effects of maternal diabetes on expression of many genes that are correlated with developmental progression (i.e. somite stage) highlight the importance of considering developmental maturity in the interpretation of transcriptomic data. Our analyses identified cholesterol biosynthesis and NAD metabolism as novel pathways not previously implicated in diabetic pregnancies. Both NAD and cholesterol availability affect a wide variety of cellular signaling processes, and can be modulated by diet, implying that prevention of adverse outcomes from diabetic pregnancies may require broad interventions, particularly in the early stages of pregnancy.

Correspondence of Yolk Sac and Embryonic Genotypes in F0 Mouse CRISPants.

CRISPR-mediated genome editing in vivo can be accompanied by prolonged stability of the Cas9 protein in mouse embryos. Then, genome edited variant alleles will be induced as long as Cas9 protein is active, and unmodified wildtype target loci are available. The corollary is that CRISPR-modified alleles that arise after the first zygotic cell division potentially could be distributed asymmetrically to the cell lineages that are specified early during morula and blastocyst development. This has practical implications for the investigation of F0 generation individuals, as cells in embryonic and extraembryonic tissues, such as the visceral yolk sac, might end up inheriting different genotypes. We here investigated the hypothetically possible scenarios by genotyping individual F0 CRISPants and their associated visceral yolk sacs in parallel. In all cases, we found that embryonic genotype was accurately reflected by yolk sac genotyping, with the two tissues indicating genetic congruence, even when the conceptus was a mosaic of cells with distinct allele configurations. Nevertheless, low abundance of a variant allele may represent a private mutation occurring only in the yolk sac, and in those rare cases, additional genotyping to determine the mutational status of the embryo proper is warranted.

Aberrant lipid accumulation in the mouse visceral yolk sac resulting from maternal diabetes and obesity.

Maternal diabetes and obesity in pregnancy are well-known risk factors for structural birth defects, including neural tube defects and congenital heart defects. Progeny from affected pregnancies are also predisposed to developing cardiometabolic disease in later life. Based upon in vitro embryo cultures of rat embryos, it was postulated that nutrient uptake by the yolk sac is deficient in diabetic pregnancies. In contrast, using two independent mouse models of maternal diabetes, and a high-fat diet-feeding model of maternal obesity, we observed excessive lipid accumulation at 8.5 days in the yolk sac. The numbers as well as sizes of intracellular lipid droplets were increased in yolk sacs of embryos from diabetic and obese pregnancies. Maternal metabolic disease did not affect expression of lipid transporter proteins, including ApoA1, ApoB and SR-B1, consistent with our earlier report that expression of glucose and fatty acid transporter genes was also unchanged in diabetic pregnancy-derived yolk sacs. Colocalization of lipid droplets with lysosomes was significantly reduced in the yolk sacs from diabetic and obese pregnancies compared to yolk sacs from normal pregnancies. We therefore conclude that processing of lipids is defective in pregnancies affected by maternal metabolic disease, which may lead to reduced availability of lipids to the developing embryo. The possible implications of insufficient supply of lipids -and potentially of other nutrients-to the embryos experiencing adverse pregnancy conditions are discussed.

Nutrient Transporter Gene Expression in the Early Conceptus-Implications From Two Mouse Models of Diabetic Pregnancy.

Maternal diabetes in early pregnancy increases the risk for birth defects in the offspring, particularly heart, and neural tube defects. While elevated glucose levels are characteristic for diabetic pregnancies, these are also accompanied by hyperlipidemia, indicating altered nutrient availability. We therefore investigated whether changes in the expression of nutrient transporters at the conception site or in the early post-implantation embryo could account for increased birth defect incidence at later developmental stages. Focusing on glucose and fatty acid transporters, we measured their expression by RT-PCR in the spontaneously diabetic non-obese mouse strain NOD, and in pregnant FVB/N mouse strain dams with Streptozotocin-induced diabetes. Sites of expression in the deciduum, extra-embryonic, and embryonic tissues were determined by RNAscope in situ hybridization. While maternal diabetes had no apparent effects on levels or cellular profiles of expression, we detected striking cell-type specificity of particular nutrient transporters. For examples, Slc2a2/Glut2 expression was restricted to the endodermal cells of the visceral yolk sac, while Slc2a1/Glut1 expression was limited to the mesodermal compartment; Slc27a4/Fatp4 and Slc27a3/Fatp3 also exhibited reciprocally exclusive expression in the endodermal and mesodermal compartments of the yolk sac, respectively. These findings not only highlight the significance of nutrient transporters in the intrauterine environment, but also raise important implications for the etiology of birth defects in diabetic pregnancies, and for strategies aimed at reducing birth defects risk by nutrient supplementation.

Cross-Omics Analysis of Fenugreek Supplementation Reveals Beneficial Effects Are Caused by Gut Microbiome Changes Not Mammalian Host Physiology.

Herbal remedies are increasing in popularity as treatments for metabolic conditions such as obesity and Type 2 Diabetes. One potential therapeutic option is fenugreek seeds (Trigonella foenum-graecum), which have been used for treating high cholesterol and Type 2 diabetes. A proposed mechanism for these benefits is through alterations in the microbiome, which impact mammalian host metabolic function. This study used untargeted metabolomics to investigate the fenugreek-induced alterations in the intestinal, liver, and serum profiles of mice fed either a 60% high-fat or low-fat control diet each with or without fenugreek supplementation (2% w/w) for 14 weeks. Metagenomic analyses of intestinal contents found significant alterations in the relative composition of the gut microbiome resulting from fenugreek supplementation. Specifically, Verrucomicrobia, a phylum containing beneficial bacteria which are correlated with health benefits, increased in relative abundance with fenugreek. Metabolomics partial least squares discriminant analysis revealed substantial fenugreek-induced changes in the large intestines. However, it was observed that while the magnitude of changes was less, significant modifications were present in the liver tissues resulting from fenugreek supplementation. Further analyses revealed metabolic processes affected by fenugreek and showed broad ranging impacts in multiple pathways, including carnitine biosynthesis, cholesterol and bile acid metabolism, and arginine biosynthesis. These pathways may play important roles in the beneficial effects of fenugreek.

Effects of Maternal Diabetes and Diet on Gene Expression in the Murine Placenta.

Adverse exposures during pregnancy have been shown to contribute to susceptibility for chronic diseases in offspring. Maternal diabetes during pregnancy is associated with higher risk of pregnancy complications, structural birth defects, and cardiometabolic health impairments later in life. We showed previously in a mouse model that the placenta is smaller in diabetic pregnancies, with reduced size of the junctional zone and labyrinth. In addition, cell migration is impaired, resulting in ectopic accumulation of spongiotrophoblasts within the labyrinth. The present study had the goal to identify the mechanisms underlying the growth defects and trophoblast migration abnormalities. Based upon gene expression assays of 47 candidate genes, we were able to attribute the reduced growth of diabetic placenta to alterations in the Insulin growth factor and Serotonin signaling pathways, and provide evidence for Prostaglandin signaling deficiencies as the possible cause for abnormal trophoblast migration. Furthermore, our results reinforce the notion that the exposure to maternal diabetes has particularly pronounced effects on gene expression at midgestation time points. An implication of these findings is that mechanisms underlying developmental programming act early in pregnancy, during placenta morphogenesis, and before the conceptus switches from histiotrophic to hemotrophic nutrition.

Broad spectrum of CRISPR-induced edits in an embryonic lethal gene.

Mendelian genetics poses practical limitations on the number of mutant genes that can be investigated simultaneously for their roles in embryonic development in the mouse. While CRISPR-based gene editing of multiple genes at once offers an attractive alternative strategy, subsequent breeding or establishment of permanent mouse lines will rapidly segregate the different mutant loci again. Direct phenotypic analysis of genomic edits in an embryonic lethal gene in F0 generation mice, or F0 mouse embryos, circumvents the need for breeding or establishment of mutant mouse lines. In the course of genotyping a large cohort of F0 CRISPants, where the embryonic lethal gene T/brachyury was targeted, we noted the presence of multiple CRISPR-induced modifications in individual embryos. Using long-read single-molecule Nanopore sequencing, we identified a wide variety of deletions, ranging up to 3 kb, that would not have been detected or scored as wildtype with commonly used genotyping methods that rely on subcloning and short-read or Sanger sequencing. Long-read sequencing results were crucial for accurate genotype-phenotype correlation in our F0 CRISPants. We thus demonstrate feasibility of screening manipulated F0 embryos for mid-gestation phenotypic consequences of CRISPR-induced mutations without requiring derivation of permanent mouse lines.

Fenugreek Counters the Effects of High Fat Diet on Gut Microbiota in Mice: Links to Metabolic Benefit.

Fenugreek (Trigonella foenum-graecum) is an annual herbaceous plant and a staple of traditional health remedies for metabolic conditions including high cholesterol and diabetes. While the mechanisms of the beneficial actions of fenugreek remain unknown, a role for intestinal microbiota in metabolic homeostasis is likely. To determine if fenugreek utilizes intestinal bacteria to offset the adverse effects of high fat diets, C57BL/6J mice were fed control/low fat (CD) or high fat (HFD) diets each supplemented with or without 2% (w/w) fenugreek for 16 weeks. The effects of fenugreek and HFD on gut microbiota were comprehensively mapped and then statistically assessed in relation to effects on metrics of body weight, hyperlipidemia, and glucose tolerance. 16S metagenomic analyses revealed robust and significant effects of fenugreek on gut microbiota, with alterations in both alpha and beta diversity as well as taxonomic redistribution under both CD and HFD conditions. As previously reported, fenugreek attenuated HFD-induced hyperlipidemia and stabilized glucose tolerance without affecting body weight. Finally, fenugreek specifically reversed the dysbiotic effects of HFD on numerous taxa in a manner tightly correlated with overall metabolic function. Collectively, these data reinforce the essential link between gut microbiota and metabolic syndrome and suggest that the preservation of healthy populations of gut microbiota participates in the beneficial properties of fenugreek in the context of modern Western-style diets.

Maternal diet modulates placental nutrient transporter gene expression in a mouse model of diabetic pregnancy.

Diabetes in the mother during pregnancy is a risk factor for birth defects and perinatal complications and can affect long-term health of the offspring through developmental programming of susceptibility to metabolic disease. We previously showed that Streptozotocin-induced maternal diabetes in mice is associated with altered cell differentiation and with smaller size of the placenta. Placental size and fetal size were affected by maternal diet in this model, and maternal diet also modulated the risk for neural tube defects. In the present study, we sought to determine the extent to which these effects might be mediated through altered expression of nutrient transporters, specifically glucose and fatty acid transporters in the placenta. Our results demonstrate that expression of several transporters is modulated by both maternal diet and maternal diabetes. Diet was revealed as the more prominent determinant of nutrient transporter expression levels, even in pregnancies with uncontrolled diabetes, consistent with the role of diet in placental and fetal growth. Notably, the largest changes in nutrient transporter expression levels were detected around midgestation time points when the placenta is being formed. These findings place the critical time period for susceptibility to diet exposures earlier than previously appreciated, implying that mechanisms underlying developmental programming can act on placenta formation.

Extensive metabolic remodeling after limiting mitochondrial lipid burden is consistent with an improved metabolic health profile.

Mitochondrial lipid overload in skeletal muscle contributes to insulin resistance, and strategies limiting this lipid pressure improve glucose homeostasis; however, comprehensive cellular adaptations that occur in response to such an intervention have not been reported. Herein, mice with skeletal muscle-specific deletion of carnitine palmitoyltransferase 1b (Cpt1bM-/-), which limits mitochondrial lipid entry, were fed a moderate fat (25%) diet, and samples were subjected to a multimodal analysis merging transcriptomics, proteomics, and nontargeted metabolomics to characterize the coordinated multilevel cellular responses that occur when mitochondrial lipid burden is mitigated. Limiting mitochondrial fat entry predictably improves glucose homeostasis; however, remodeling of glucose metabolism pathways pales compared with adaptations in amino acid and lipid metabolism pathways, shifts in nucleotide metabolites, and biogenesis of mitochondria and peroxisomes. Despite impaired fat utilization, Cpt1bM-/- mice have increased acetyl-CoA (14-fold) and NADH (2-fold), indicating metabolic shifts yield sufficient precursors to meet energy demand; however, this does not translate to enhance energy status as Cpt1bM-/- mice have low ATP and high AMP levels, signifying energy deficit. Comparative analysis of transcriptomic data with disease-associated gene-sets not only predicted reduced risk of glucose metabolism disorders but was also consistent with lower risk for hepatic steatosis, cardiac hypertrophy, and premature death. Collectively, these results suggest induction of metabolic inefficiency under conditions of energy surfeit likely contributes to improvements in metabolic health when mitochondrial lipid burden is mitigated. Moreover, the breadth of disease states to which mechanisms induced by muscle-specific Cpt1b inhibition may mediate health benefits could be more extensive than previously predicted.

Embryonic cell migratory capacity is impaired upon exposure to glucose in vivo and in vitro.

BACKGROUND: Impairments in cell migration during vertebrate gastrulation lead to structural birth defects, such as heart defects and neural tube defects. These defects are more frequent in progeny from diabetic pregnancies, and we have recently provided evidence that maternal diabetes leads to impaired migration of embryonic mesodermal cells in a mouse model of diabetic pregnancy. METHODS: We here report the isolation of primary cell lines from normal and diabetes-exposed embryos of the nonobese diabetic mouse strain, and characterization of their energy metabolism and expression of nutrient transporter genes by quantitative real-time PCR. RESULTS: Expression levels of several genes in the glucose transporter and fatty acid transporter gene families were altered in diabetes-exposed cells. Notably, primary cells from embryos with prior in vivo exposure to maternal diabetes exhibited reduced capacity for cell migration in vitro. CONCLUSIONS: Primary cells isolated from diabetes-exposed embryos retained a "memory" of their in vivo exposure, manifesting in cell migration impairment. Thus, we have successfully established an in vitro experimental model for the mesoderm migration defects observed in diabetes-exposed mouse embryos.

Physicochemical differences between malanga (Xanthosoma sagittifolium) and potato (Solanum tuberosum) tubers are associated with differential effects on the gut microbiome.

Malanga (Xanthosoma sagittifolium) is used as a medicinal food for infant development and gastritis. We compared the physicochemical properties and gut microbial effects of malanga versus potato (Solanum tuberosum) using nutritional analysis, rheometry, in vitro TNO Intestinal Model, and C57Bl/6J mouse models. Malanga was characterized by higher starch (70.7% v. 66.3%), lower amylose:amylopectin (0.33 v. 0.59), higher free sugar (5.44% v. 3.23%), lower viscosity (271.0 v. 863.0 mPa.s), and higher bioaccessible and bioavailable sugar (0.89 v. 0.11 g bioaccessible sucrose per 20 g load in vitro; blood glucose levels of 129.1 v. 95.2 and 133.8 v. 104.3 mg/dL after 20 and 60 min in vivo). Gut microbiota of mice fed a high fat diet containing 20% malanga for 14 d exhibited significantly higher α diversity than those fed 20% potato, indicating that minor physicochemical differences between similar tuber crops are associated with significantly different effects on the gut microbiome.

An ethanolic extract of Artemisia scoparia inhibits lipolysis in vivo and has antilipolytic effects on murine adipocytes in vitro.

An ethanolic extract of Artemisia scoparia (SCO) has metabolically favorable effects on adipocyte development and function in vitro and in vivo. In diet-induced obese mice, SCO supplementation significantly reduced fasting glucose and insulin levels. Given the importance of adipocyte lipolysis in metabolic health, we hypothesized that SCO modulates lipolysis in vitro and in vivo. Free fatty acids and glycerol were measured in the sera of mice fed a high-fat diet with or without SCO supplementation. In cultured 3T3-L1 adipocytes, the effects of SCO on lipolysis were assessed by measuring glycerol and free fatty acid release. Microarray analysis, qPCR, and immunoblotting were used to assess gene expression and protein abundance. We found that SCO supplementation of a high-fat diet in mice substantially reduces circulating glycerol and free fatty acid levels, and we observed a cell-autonomous effect of SCO to significantly attenuate tumor necrosis factor-α (TNFα)-induced lipolysis in cultured adipocytes. Although several prolipolytic and antilipolytic genes were identified by microarray analysis of subcutaneous and visceral adipose tissue from SCO-fed mice, regulation of these genes did not consistently correlate with SCO's ability to reduce lipolytic metabolites in sera or cell culture media. However, in the presence of TNFα in cultured adipocytes, SCO induced antilipolytic changes in phosphorylation of hormone-sensitive lipase and perilipin. Together, these data suggest that the antilipolytic effects of SCO on adipose tissue play a role in the ability of this botanical extract to improve whole body metabolic parameters and support its use as a dietary supplement to promote metabolic resiliency.

A map of the PGC-1α- and NT-PGC-1α-regulated transcriptional network in brown adipose tissue.

Transcriptional coactivator PGC-1α and its splice variant NT-PGC-1α play crucial roles in regulating cold-induced thermogenesis in brown adipose tissue (BAT). PGC-1α and NT-PGC-1α are highly induced by cold in BAT and subsequently bind to and coactivate many transcription factors to regulate expression of genes involved in mitochondrial biogenesis, fatty acid oxidation, respiration and thermogenesis. To identify the complete repertoire of PGC-1α and NT-PGC-1α target genes in BAT, we analyzed genome-wide DNA-binding and gene expression profiles. We find that PGC-1α-/NT-PGC-1α binding broadly associates with cold-mediated transcriptional activation. In addition to their known target genes in mitochondrial biogenesis and oxidative metabolism, PGC-1α and NT-PGC-1α additionally target a broad spectrum of genes involved in diverse biological pathways including ubiquitin-dependent protein catabolism, ribonucleoprotein complex biosynthesis, phospholipid biosynthesis, angiogenesis, glycogen metabolism, phosphorylation, and autophagy. Our findings expand the number of genes and biological pathways that may be regulated by PGC-1α and NT-PGC-1α and provide further insight into the transcriptional regulatory network in which PGC-1α and NT-PGC-1α coordinate a comprehensive transcriptional response in BAT in response to cold.

Lipid peroxidation regulates podocyte migration and cytoskeletal structure through redox sensitive RhoA signaling.

Early podocyte loss is characteristic of chronic kidney diseases (CKD) in obesity and diabetes. Since treatments for hyperglycemia and hypertension do not prevent podocyte loss, there must be additional factors causing podocyte depletion. The role of oxidative stress has been implicated in CKD but it is not known how exactly free radicals affect podocyte physiology. To assess this relationship, we investigated the effects of lipid radicals on podocytes, as lipid peroxidation is a major form of oxidative stress in diabetes. We found that lipid radicals govern changes in podocyte homeostasis through redox sensitive RhoA signaling: lipid radicals inhibit migration and cause loss of F-actin fibers. These effects were prevented by mutating the redox sensitive cysteines of RhoA. We therefore suggest that in diseases associated with increased lipid peroxidation, lipid radicals can determine podocyte function with potentially pathogenic consequences for kidney physiology.

A dietary isothiocyanate-enriched moringa (Moringa oleifera) seed extract improves glucose tolerance in a high-fat-diet mouse model and modulates the gut microbiome.

Moringa oleifera (moringa) has been traditionally used for the treatment of diabetes and in water purification. We previously showed that moringa seed extract (MSE), standardized to its primary bioactive isothiocyanate (MIC-1), modulated inflammatory and antioxidant signaling pathways in vitro. To understand the efficacy and mechanisms of action of MSE in vivo, we incorporated MSE into the diets of normal and obese C57Bl/6J male mice fed a standard low-fat diet or a very high-fat diet for 12 wk, respectively. MSE supplementation resulted in reduced body weight, decreased adiposity, improved glucose tolerance, reduced inflammatory gene expression, and increased antioxidant gene expression. 16S rRNA gene sequencing and quantitative PCR of fecal/cecal samples showed major modulation of the gut microbial community and a significantly reduced bacterial load, similar to an antibiotic response. This suggests that MSE improves metabolic health by its intracellular anti-inflammatory and antioxidant activities, and/or its antibiotic-like restructuring of the gut microbiota.