Objective: Parental nutrition and lifestyle impact the metabolic phenotype of the offspring. We have reported that grandpaternal chronic high-fat diet (HFD) transgenerationally impairs glucose metabolism in subsequent generations. Here we determined whether grandpaternal diet transgenerationally impacts the transcriptome and lipidome in skeletal muscle. Our aim was to identify tissue-specific pathways involved in transgenerational inheritance of environmental-induced phenotypes.
Methods: F0 male Sprague–Dawley rats were fed a HFD or chow for 12 weeks before breeding with chow-fed females to generate the F1 generation. F2 offspring were generated by mating F1 males fed a chow diet with an independent line of chow-fed females. F1 and F2 offspring were fed chow or HFD for 12 weeks. Transcriptomic and LC-MS lipidomic analyses were performed in extensor digitorum longus muscle from F2-females rats. Gene set enrichment analysis (GSEA) was performed to determine pathways reprogrammed by grandpaternal diet.
Results: GSEA revealed an enrichment of the unfolded protein response pathway in skeletal muscle of grand-offspring from HFD-fed grandfathers compared to grand-offspring of chow-fed males. Activation of the stress sensor (ATF6α), may be a pivotal point whereby this pathway is activated. Interestingly, skeletal muscle from F1-offspring was not affected in a similar manner. No major changes were observed in the skeletal muscle lipidome profile due to grandpaternal diet.
Conclusions: Grandpaternal HFD-induced obesity transgenerationally affected the skeletal muscle transcriptome. This finding further highlights the impact of parental exposure to environmental factors on offspring's development and health.[Hide abstract]
Objective: A significant portion of the heritable risk for complex metabolic disorders cannot be attributed to classic Mendelian genetic factors. At least some of this missing heritability is thought to be due to the epigenetic influence of parental and grandparental metabolic state on offspring health. Previous work suggests that this transgenerational phenomenon is evolutionarily conserved in Drosophila. These studies, however, have all depended on dietary paradigms to alter parental metabolic state, which can have inconsistent heritable effects on the metabolism of offspring.
Methods: Here we use AKHR null alleles to induce obesity in the parental generation and then score both metabolic parameters and genome-wide transcriptional responses in AKHR heterozygote F1 progeny and genetically wild-type F2 progeny.
Results: Unexpectedly, we observe elevated glycogen levels and changes in gene expression in AKHR heterozygotes due to haploinsufficiency at this locus. We also show that genetic manipulation of parental metabolism using AKHR mutations results in significant physiological changes in F2 wild-type offspring of the grandpaternal/maternal lineage.
Conclusions: Our results demonstrate that genetic manipulation of parental metabolism in Drosophila can have an effect on the health of F2 progeny, providing a non-dietary paradigm to better understand the mechanisms behind the transgenerational inheritance of metabolic state.[Hide abstract]
Objective: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can generate any given cell type in the human body. One challenge for cell-replacement therapy is the efficient differentiation and expansion of large quantities of progenitor cells from pluripotent stem cells produced under good manufacturing practice (GMP). FOXA2 and SOX17 double positive definitive endoderm (DE) progenitor cells can give rise to all endoderm-derived cell types in the thymus, thyroid, lung, pancreas, liver, and gastrointestinal tract. FOXA2 is a pioneer transcription factor in DE differentiation that is also expressed and functionally required during pancreas development and islet cell homeostasis. Current differentiation protocols can successfully generate endoderm; however, generation of mature glucose-sensitive and insulin-secreting β-cells is still a challenge. As a result, it is of utmost importance to screen for small molecules that can improve DE and islet cell differentiation for cell-replacement therapy for diabetic patients.
Methods: The aim of this study was to identify and validate small molecules that can induce DE differentiation and further enhance pancreatic progenitor differentiation. Therefore, we developed a large scale, high-content screen for testing a chemical library of 23,406 small molecules to identify compounds that induce FoxA2 in mouse embryonic stem cells (mESCs).
Results: Based on our high-content screen algorithm, we selected 84 compounds that directed differentiation of mESCs towards the FoxA2 lineage. Strikingly, we identified ROCK inhibition (ROCKi) as a novel mechanism of endoderm induction in mESCs and hESCs. DE induced by the ROCK inhibitor Fasudil efficiently gives rise to PDX1+ pancreatic progenitors from hESCs.
Conclusions: Taken together, DE induction by ROCKi can simplify and improve current endoderm and pancreatic differentiation protocols towards a GMP-grade cell product for β-cell replacement.[Hide abstract]
Objective: Insulin release from pancreatic β-cells is controlled by plasma glucose levels via mitochondrial fuel metabolism. Therefore, insulin secretion is critically dependent on mitochondrial DNA (mtDNA) and the genes it encodes. Mitochondrial transcription factor B2 (TFB2M) controls transcription of mitochondrial-encoded genes. However, its precise role in mitochondrial metabolism in pancreatic β-cells and, consequently, in insulin secretion remains unknown.
Methods: To elucidate the role of TFB2M in mitochondrial function and insulin secretion in vitro and in vivo, mice with a β-cell specific homozygous or heterozygous knockout of Tfb2m and rat clonal insulin-producing cells in which the gene was silenced were examined with an array of metabolic and functional assays.
Results: There was an effect of gene dosage on Tfb2m expression and function. Loss of Tfb2m led to diabetes due to disrupted transcription of mitochondrial DNA (mtDNA) and reduced mtDNA content. The ensuing mitochondrial dysfunction activated compensatory mechanisms aiming to limit cellular dysfunction and damage of β-cells. These processes included the mitochondrial unfolded protein response, mitophagy, and autophagy. Ultimately, however, these cell-protective systems were overridden, leading to mitochondrial dysfunction and activation of mitochondrial-dependent apoptotic pathways. In this way, β-cell function and mass were reduced. Together, these perturbations resulted in impaired insulin secretion, progressive hyperglycemia, and, ultimately, development of diabetes.
Conclusions: Loss of Tfb2m in pancreatic β-cells results in progressive mitochondrial dysfunction. Consequently, insulin secretion in response to metabolic stimuli is impaired and β-cell mass reduced. Our findings indicate that TFB2M plays an important functional role in pancreatic β-cells. Perturbations of its actions may lead to loss of functional β-cell mass, a hallmark of T2D.[Hide abstract]
Objective: Type 1 diabetes is characterized by autoimmune destruction of β-cells leading to severe insulin deficiency. Although many improvements have been made in recent years, exogenous insulin therapy is still imperfect; new therapeutic approaches, focusing on preserving/expanding β-cell mass and/or blocking the autoimmune process that destroys islets, should be developed. The main objective of this work was to test in non-obese diabetic (NOD) mice, which spontaneously develop autoimmune diabetes, the effects of local expression of Insulin-like growth factor 1 (IGF1), a potent mitogenic and pro-survival factor for β-cells with immunomodulatory properties.
Methods: Transgenic NOD mice overexpressing IGF1 specifically in β-cells (NOD-IGF1) were generated and phenotyped. In addition, miRT-containing, IGF1-encoding adeno-associated viruses (AAV) of serotype 8 (AAV8-IGF1-dmiRT) were produced and administered to 4- or 11-week-old non-transgenic NOD females through intraductal delivery. Several histological, immunological, and metabolic parameters were measured to monitor disease over a period of 28–30 weeks.
Results: In transgenic mice, local IGF1 expression led to long-term suppression of diabetes onset and robust protection of β-cell mass from the autoimmune insult. AAV-mediated pancreatic-specific overexpression of IGF1 in adult animals also dramatically reduced diabetes incidence, both when vectors were delivered before pathology onset or once insulitis was established. Transgenic NOD-IGF1 and AAV8-IGF1-dmiRT-treated NOD animals had much less islet infiltration than controls, preserved β-cell mass, and normal insulinemia. Transgenic and AAV-treated islets showed less expression of antigen-presenting molecules, inflammatory cytokines, and chemokines important for tissue-specific homing of effector T cells, suggesting IGF1 modulated islet autoimmunity in NOD mice.
Conclusions: Local expression of Igf1 by AAV-mediated gene transfer counteracts progression to diabetes in NOD mice. This study suggests a therapeutic strategy for autoimmune diabetes in humans.[Hide abstract]
Objective: The prevalence of obesity and related co-morbidities is reaching pandemic proportions. Today, the most effective obesity treatments are glucagon-like peptide 1 (GLP-1) analogs and bariatric surgery. Interestingly, both intervention paradigms have been associated with adaptive growth responses in the gut; however, intestinotrophic mechanisms associated with or secondary to medical or surgical obesity therapies are poorly understood. Therefore, the objective of this study was to assess the local basal endogenous and pharmacological intestinotrophic effects of glucagon-like peptides and bariatric surgery in mice.
Methods: We used in situ hybridization to provide a detailed and comparative anatomical map of the local distribution of GLP-1 receptor (Glp1r), GLP-2 receptor (Glp2r), and preproglucagon (Gcg) mRNA expression throughout the mouse gastrointestinal tract. Gut development in GLP-1R-, GLP-2R-, or GCG-deficient mice was compared to their corresponding wild-type controls, and intestinotrophic effects of GLP-1 and GLP-2 analogs were assessed in wild-type mice. Lastly, gut volume was determined in a mouse model of vertical sleeve gastrectomy (VSG).
Results: Comparison of Glp1r, Glp2r, and Gcg mRNA expression indicated a widespread, but distinct, distribution of these three transcripts throughout all compartments of the mouse gastrointestinal tract. While mice null for Glp1r or Gcg showed normal intestinal morphology, Glp2r−/− mice exhibited a slight reduction in small intestinal mucosa volume. Pharmacological treatment with GLP-1 and GLP-2 analogs significantly increased gut volume. In contrast, VSG surgery had no effect on intestinal morphology.
Conclusions: The present study indicates that the endogenous preproglucagon system, exemplified by the entire GCG gene and the receptors for GLP-1 and GLP-2, does not play a major role in normal gut development in the mouse. Furthermore, elevation in local intestinal and circulating levels of GLP-1 and GLP-2 achieved after VSG has limited impact on intestinal morphometry. Hence, although exogenous treatment with GLP-1 and GLP-2 analogs enhances gut growth, the contributions of endogenously-secreted GLP-1 and GLP-2 to gut growth may be more modest and highly context-dependent.[Hide abstract]
Objective: To identify, purify, and characterize the proteins responsible for glutenase activity in the feces of healthy subjects and patients with celiac disease (CD).
Methods: Sixteen subjects were included in this study; 8 were healthy with no known food intolerances, and 8 were treated CD patients on a gluten-free diet. Fecal samples were homogenized, and precipitated proteins were purified by chromatography. Glutenase activity was evaluated by bioassays, zymography, and high-performance liquid chromatography with immunogenic 33-mer, 19-mer, and 13-mer gliadin peptides.
Results: The gastrointestinal elastase 3B (CEL3B), elastase 2A (CEL2A), and carboxypeptidase A1 (CBPA1) enzymes degraded human gluten. These proteins fully hydrolyzed 13-mer and 19-mer gliadin peptides that trigger immune-mediated enteropathy in individuals genetically predisposed to CD and partially digested a 33-mer. Feces from patients with CD showed more glutenase activity than feces from individuals without CD (171–466% higher). Peptidase activity against the gliadin peptides also increased in patients with CD.
Conclusions: The digestive tracts of patients with CD and healthy subjects have enzymatic machinery needed for gluten degradation. Patients with CD showed more gluten hydrolysis than did healthy individuals, although, in both cases, a fraction of 33-mer peptide remained intact. Gliadin peptides derived from gastrointestinal digestion, especially the 33-mer, can potentially be used by commensal microbiota from both CD-positive and CD-negative individuals, and differences in bacterial hydrolysis can modify its immunogenic capacity.[Hide abstract]
Objective: The loss of skeletal muscle mass and strength are a central feature of traumatic injury and degenerative myopathies. Unfortunately, pharmacological interventions typically fail to stem the long-term decline in quality of life. Reduced Rev-Erb-mediated gene suppression in cultured C2C12 myoblasts has been shown to stimulate myoblast differentiation. Yet the mechanisms that allow Rev-Erb to pleiotropically inhibit muscle differentiation are not well understood. In this study, we sought to elucidate the role of Rev-Erb in the regulation of muscle differentiation and regeneration in vivo.
Methods: Using Rev-Erbα/β shRNAs, pharmacological ligands, and Rev-Erbα null and heterozygous mice, we probed the mechanism of Rev-Erbα/β regulation of muscle differentiation and muscle regeneration.
Results: ChIP seq analysis of Rev-Erb in differentiating myoblasts showed that Rev-Erbα did not transcriptionally regulate muscle differentiation through cognate Rev-Erb/ROR-response elements but through possible interaction with the cell fate regulator NF-Y at CCAAT-motifs. Muscle differentiation is stimulated by Rev-Erb release from CCAAT-motifs at promoter and enhancer elements of a number of myogenesis proteins. Partial loss of Rev-Erb expression in mice heterozygous for Rev-Erbα accelerated muscle repair in vivo whereas Rev-Erb knockout mice showed deficiencies in regenerative repair compared to wild type mice. These phenotypic differences between heterozygous and knockout mice were not apparently dependent on MRF induction in response to injury. Similarly, pharmacological disruption of Rev-Erb suppressive activity in injured muscle accelerated regenerative repair in response to acute injury.
Conclusions: Disrupting Rev-Erb activity in injured muscle accelerates regenerative muscle repair/differentiation through transcriptional de-repression of myogenic programs. Rev-Erb, therefore, may be a potent therapeutic target for a myriad of muscular disorders.[Hide abstract]
Objective: A potential strategy to treat obesity – and the associated metabolic consequences – is to increase energy expenditure. This could be achieved by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or the induction of browning of white adipose tissue (WAT). Over the last years, it has become clear that several metalloproteinases play an important role in adipocyte biology. Here, we investigated the potential role of ADAMTS5.
Methods: Mice deficient in ADAMTS5 (Adamts5−/−) and wild-type (Adamts5+/+) littermates were kept on a standard of Western-type diet for 15 weeks. Energy expenditure and heat production was followed by indirect calorimetry. To activate thermogenesis, mice were treated with the β3-adrenergic receptor (β3-AR) agonist CL-316,243 or alternatively, exposed to cold for 2 weeks.
Results: Compared to Adamts5+/+ mice, Adamts5−/− mice have significantly more interscapular BAT and marked browning of their subcutaneous (SC) WAT. Thermogenic pathway analysis indicated, in the absence of ADAMTS5, enhanced β3-AR signaling via activation of the cAMP response element-binding protein (CREB). Additional β3-AR stimulation with CL-316,243 promoted browning of WAT in Adamts5+/+ mice but had no additive effect in Adamts5−/− mice. However, cold exposure induced more pronounced browning of WAT in Adamts5−/− mice.
Conclusions: These data indicate that ADAMTS5 plays a functional role in development of BAT and browning of WAT. Hence, selective targeting of ADAMTS5 could provide a novel therapeutic strategy for treatment/prevention of obesity and metabolic diseases.[Hide abstract]
Objective: Obesity variably disrupts human health, but molecular-based patients' health-risk stratification is limited. Adipose tissue (AT) stresses may link obesity with metabolic dysfunction, but how they signal in humans remains poorly-characterized. We hypothesized that a transcriptional AT stress-signaling cascade involving E2F1 and ASK1 (MAP3K5) molecularly defines high-risk obese subtype.
Methods: ASK1 expression in human AT biopsies was determined by real-time PCR analysis, and chromatin immunoprecipitation (ChIP) adopted to AT explants was used to evaluate the binding of E2F1 to the ASK1 promoter. Dual luciferase assay was used to measure ASK1 promoter activity in HEK293 cells. Effects of E2F1 knockout/knockdown in adipocytes was assessed utilizing mouse-embryonal-fibroblasts (MEF)-derived adipocyte-like cells from WT and E2F1−/− mice and by siRNA, respectively. ASK1 depletion in adipocytes was studied in MEF-derived adipocyte-like cells from WT and adipose tissue-specific ASK1 knockout mice (ASK1-ATKO).
Results: Human visceral-AT ASK1 mRNA (N = 436) was associated with parameters of obesity-related cardio-metabolic morbidity. Adjustment for E2F1 expression attenuated the association of ASK1 with fasting glucose, insulin resistance, circulating IL-6, and lipids (triglycerides, HDL-cholesterol), even after adjusting for BMI. Chromatin-immunoprecipitation in human-AT explants revealed BMI-associated increased occupancy of the ASK1 promoter by E2F1 (r2 = 0.847, p < 0.01). In adipocytes, siRNA-mediated E2F1-knockdown, and MEF-derived adipocytes of E2F1-knockout mice, demonstrated decreased ASK1 expression and signaling to JNK. Mutation/truncation of an E2F1 binding site in hASK1 promoter decreased E2F1-induced ASK1 promoter activity, whereas E2F1-mediated sensitization of ASK1 promoter to further activation by TNFα was inhibited by JNK-inhibitor. Finally, MEF-derived adipocytes from adipocyte-specific ASK1-knockout mice exhibited lower leptin and higher adiponectin expression and secretion, and resistance to the effects of TNFα.
Conclusions: AT E2F1 –ASK1 molecularly defines a metabolically-detrimental obese sub-phenotype. Functionally, it may negatively affect AT endocrine function, linking AT stress to whole-body metabolic dysfunction.[Hide abstract]
Objective: Metformin, the first line drug for treatment of type 2 diabetes, suppresses hepatic gluconeogenesis and reduces body weight in patients, the latter by an unknown mechanism.
Methods: Mice on a high fat diet were continuously fed metformin in a therapeutically relevant dose, mimicking a retarded formulation.
Results: Feeding metformin in pharmacologically relevant doses to mice on a high fat diet normalized HbA1c levels and ameliorated glucose tolerance, as expected, but also considerably slowed down weight gain. This was due to increased energy expenditure, since food intake was unchanged and locomotor activity was even decreased. Metformin caused lactate accumulation in the intestinal wall and in portal venous blood but not in peripheral blood or the liver. Increased conversion of glucose-1-13C to glucose-1,6-13C under metformin strongly supports a futile cycle of lactic acid production in the intestinal wall, and usage of the produced lactate for gluconeogenesis in liver.
Conclusions: The reported glucose–lactate–glucose cycle is a highly energy consuming process, explaining the beneficial effects of metformin given continuously on the development of a type 2 diabetic-like state in our mice.[Hide abstract]
Objective: Adult obesity risk is influenced by alterations to fetal and neonatal environments. Modifying neonatal gut or neurohormone signaling pathways can have negative metabolic consequences in adulthood. Here we characterize the effect of neonatal activation of glucagon like peptide-1 (GLP-1) receptor (GLP1R) signaling on adult adiposity and metabolism.
Methods: Wild type C57BL/6 mice were injected with 1 nmol/kg Exendin-4 (Ex-4), a GLP1R agonist, for 6 consecutive days after birth. Growth, body composition, serum analysis, energy expenditure, food intake, and brain and fat pad histology and gene expression were assessed at multiple time points through 42 weeks. Similar analyses were conducted in a Glp1r conditional allele crossed with a Sim1Cre deleter strain to produce Sim1Cre;Glp1rloxP/loxP mice and control littermates.
Results: Neonatal administration of Ex-4 reduced adult body weight and fat mass, increased energy expenditure, and conferred protection from diet-induced obesity in female mice. This was associated with induction of brown adipose genes and increased noradrenergic fiber density in parametrial white adipose tissue (WAT). We further observed durable alterations in orexigenic and anorexigenic projections to the paraventricular hypothalamic nucleus (PVH). Genetic deletion of Glp1r in the PVH by Sim1-Cre abrogated the impact of neonatal Ex-4 on adult body weight, WAT browning, and hypothalamic architecture.
Conclusions: These observations suggest that the acute activation of GLP1R in neonates durably alters hypothalamic architecture to limit adult weight gain and adiposity, identifying GLP1R as a therapeutic target for obesity prevention.[Hide abstract]
Objective: Upregulation of uncoupling protein 2 (UCP2) is associated with impaired glucose-stimulated insulin secretion (GSIS), which is thought to be an important contributor to pathological β cell failure in obesity and type 2 diabetes (T2D); however, the physiological function of UCP2 in the β cell remains undefined. It has been suggested, but not yet tested, that UCP2 plays a physiological role in β cells by coordinating insulin secretion capacity with anticipated fluctuating nutrient supply, such that upregulation of UCP2 in the inactive/fasted state inhibits GSIS as a mechanism to prevent hypoglycemia. Therefore, we hypothesized that daily cycles of GSIS capacity are dependent on rhythmic and predictable patterns of Ucp2 gene expression such that low Ucp2 in the active/fed phase promotes maximal GSIS capacity, whereas elevated Ucp2 expression in the inactive/fasted phase supresses GSIS capacity. We further hypothesized that rhythmic Ucp2 expression is required for the maintenance of glucose tolerance over the 24 h cycle.
Methods: We used synchronized MIN6 clonal β cells and isolated mouse islets from wild type (C57BL6) and mice with β cell knockout of Ucp2 (Ucp2-βKO; and respective Ins2-cre controls) to determine the endogenous expression pattern of Ucp2 over 24 h and its impact on GSIS capacity and glucose tolerance over 24 h.
Results: A dynamic pattern of Ucp2 mRNA expression was observed in synchronized MIN6 cells, which showed a reciprocal relationship with GSIS capacity in a time-of-day-specific manner. GSIS capacity was suppressed in islets isolated from wild type and control mice during the light/inactive phase of the daily cycle; a suppression that was dependent on Ucp2 in the β cell and was lost in islets isolated from Ucp2-βKO mice or wild type islets treated with a UCP2 inhibitor. Finally, suppression of GSIS capacity by UCP2 in the light phase was required for the maintenance of normal patterns of glucose tolerance.
Conclusions: Our study suggests that Ucp2/UCP2 in the β cell is part of an important, endogenous, metabolic regulator that controls the temporal capacity of GSIS over the course of the day/night cycle, which, in turn, regulates time-of-day glucose tolerance. Targeting Ucp2/UCP2 as a therapeutic in type 2 diabetes or any other metabolic condition must take into account the rhythmic nature of its expression and its impact on glucose tolerance over 24 h, specifically during the inactive/fasted phase.[Hide abstract]
Objective: MicroRNAs (miRNAs) are increasingly recognized as fine-tuning regulators of metabolism, and are dysregulated in several disease conditions. With their capacity to rapidly change gene expression, miRNAs are also important regulators of development and cell differentiation. In the current study, we describe an impaired myogenic capacity of muscle stem cells isolated from humans with type 2 diabetes (T2DM) and assess whether this phenotype is regulated by miRNAs.
Methods: We measured global miRNA expression during in vitro differentiation of muscle stem cells derived from T2DM patients and healthy controls.
Results: The mir-23b/27b cluster was downregulated in the cells of the patients, and a pro-myogenic effect of these miRNAs was mediated through the p53 pathway, which was concordantly dysregulated in the muscle cells derived from humans with T2DM.
Conclusions: Our results indicate that we have identified a novel pathway for coordination of myogenesis, the miR-23b/27b-p53 axis that, when dysregulated, potentially contributes to a sustained muscular dysfunction in T2DM.[Hide abstract]