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Maternal undernutrition (MU) during fetal development profoundly effects offspring metabolism, resulting in either blunting or premature shifting of the neonatal leptin surge, and can cause metabolic dysfunction in adulthood. We developed a mild MU model in FVB mice, where dams received 20% less caloric intake (underfed) compared to control, ad libitum fed (Fed) mice. As previously reported, underfed progeny exhibited a leptin surge that was 3 days earlier (PND8) than controls (PND11) and PND1 GH levels were 354% higher. However serum IGF-1 was 55% lower (p=0.027) at PND16 in underfed progeny, resulting in delayed puberty and growth deficiencies which persisted in the male offspring. At 3 months, fed and underfed offspring received either a 45% HFD or 10% fat (Control) diet for 16 weeks, resulting in a weight gain (&gt;12 g) in progeny from fed dams. However, underfed females gained only 8.74 g on the HFD, [lower than fed females (p=0.03)]. More surprisingly, underfed males showed no weight gain in response to the HFD. The study was replicated, yielding the same results.
We hypothesize that resistance to HFD induced weight gain by underfed males may be due to altered transcriptome expression. We investigated this unusual response through RNA sequencing of the pituitary, fat and liver of both fed and underfed male progeny. Pituitary and liver transcriptome analysis are ongoing. This presentation focuses on changes in the fat transcriptome.
Fat from Fed HFD males had 592 upregulated and 153 downregulated transcripts when compared to controls. Strikingly, fat of underfed HFD males had only 3 upregulated and 3 downregulated transcripts when compared to underfed controls. To ascertain potential protective influences, transcripts in the fat of Fed HFD males were compared with those of underfed HFD males. Prelid3a, Peg3, and Thbs1 were differentially expressed in underfed HFD males compared to Fed HFD males. Prelid3a, (involved in mitochondrial lipid metabolism and transport) was downregulated 2.9 fold (0.035 adj. P) in underfed HFD fat. Peg3,(involved in fat accumulation when decreased) was increased in underfed HFD fat by 2.4 fold (0.0297 adj. P). Thbs1 was also increased by 2.7 fold (0.0476 adj. P) in underfed HFD fat. Thbs1 contributes to HFD weight gain when increased, but can also improve hepatic steatosis and promote a protective ER stress response. Pde3b, found to be protective against obesity, was reduced 1.4 fold (0.002 adj. P) in Fed HFD males, but unchanged in underfed HFD males.
These data indicate that mild undernutrition causes a premature leptin surge, marked by sex-specific differences in offspring growth and transcriptome regulation in the fat of male offspring that is beneficial to blunting HFD induced weight gain.

ENDO 2022

Mild Maternal Undernutrition Results in Transcript Regulation in the Fat of Male Offspring that Correlates with Resistance to High Fat Diet Induced Weight Gain

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Maternal undernutrition (MU) during fetal development profoundly effects offspring metabolism, resulting in either blunting or premature shifting of the neonatal leptin surge, and can cause metabolic dysfunction in adulthood. We developed a mild MU model in FVB mice, where dams received 20% less caloric intake (underfed) compared to control, ad libitum fed (Fed) mice. As previously reported, underfed progeny exhibited a leptin surge that was 3 days earlier (PND8) than controls (PND11) and PND1 GH levels were 354% higher. However serum IGF-1 was 55% lower (p=0.027) at PND16 in underfed progeny, resulting in delayed puberty and growth deficiencies which persisted in the male offspring. At 3 months, fed and underfed offspring received either a 45% HFD or 10% fat (Control) diet for 16 weeks, resulting in a weight gain (>12 g) in progeny from fed dams. However, underfed females gained only 8.74 g on the HFD, [lower than fed females (p=0.03)]. More surprisingly, underfed males showed no weight gain in response to the HFD. The study was replicated, yielding the same results.
We hypothesize that resistance to HFD induced weight gain by underfed males may be due to altered transcriptome expression. We investigated this unusual response through RNA sequencing of the pituitary, fat and liver of both fed and underfed male progeny. Pituitary and liver transcriptome analysis are ongoing. This presentation focuses on changes in the fat transcriptome.
Fat from Fed HFD males had 592 upregulated and 153 downregulated transcripts when compared to controls. Strikingly, fat of underfed HFD males had only 3 upregulated and 3 downregulated transcripts when compared to underfed controls. To ascertain potential protective influences, transcripts in the fat of Fed HFD males were compared with those of underfed HFD males. Prelid3a, Peg3, and Thbs1 were differentially expressed in underfed HFD males compared to Fed HFD males. Prelid3a, (involved in mitochondrial lipid metabolism and transport) was downregulated 2.9 fold (0.035 adj. P) in underfed HFD fat. Peg3,(involved in fat accumulation when decreased) was increased in underfed HFD fat by 2.4 fold (0.0297 adj. P). Thbs1 was also increased by 2.7 fold (0.0476 adj. P) in underfed HFD fat. Thbs1 contributes to HFD weight gain when increased, but can also improve hepatic steatosis and promote a protective ER stress response. Pde3b, found to be protective against obesity, was reduced 1.4 fold (0.002 adj. P) in Fed HFD males, but unchanged in underfed HFD males.
These data indicate that mild undernutrition causes a premature leptin surge, marked by sex-specific differences in offspring growth and transcriptome regulation in the fat of male offspring that is beneficial to blunting HFD induced weight gain.

technical paper

Neurons that co-express the orexigenic agouti-related protein (AgRP) and neuropeptide Y (NPY) are indispensable for normal feeding behavior. Firing activities of AgRP/NPY neurons dynamically fluctuate with energy status and coordinate appropriate feeding behavior to meet nutritional demands. We previously demonstrated that asprosin, a recently discovered fasting-induced glucogenic and orexigenic hormone, crosses the blood-brain barrier and directly activates the orexigenic AgRP/NPY neurons via a cyclic cAMP-dependent pathway. However, intrinsic mechanisms on how asprosin regulates AgRP/NPY neural activities during the fed-to-fasted transition are not fully understood. In the satiated mice with low circulating levels of asprosin, we found that AgRP/NPY neurons expressed high levels of the small conductance calcium-activated potassium channel 3 (SK3) and were inhibited by SK3-mediated potassium currents. Conversely, in 24 hour-fasting mice with high levels of asprosin, AgRP/NPY neurons expressed low levels of SK3 and were activated via inhibition of SK3-mediated potassium currents. Notably, the stimulatory effects of asprosin on AgRP/NPY neurons were blunted in mice with SK3 selectively deleted in the AgRP/NPY neurons, further supporting a mediating role of SK3.
Moreover, we also identified protein tyrosine phosphatase receptor δ (Ptprd), a membrane-bound phosphatase receptor, as the orexigenic asprosin-receptor. Deletion of Ptprd in AgRP/NPY neurons abolished asprosin's stimulatory effect on SK3-mediated currents and protected mice from diet-induced obesity.
Lastly, increased circulating levels of asprosin promoted hyperphagia, body weight gain, and glucose intolerance. On the other hand, anti-asprosin antibody treatment decreased food intake and body weight by inhibiting AgRP/NPY and other orexigenic hypothalamic neurons via SK currents stimulation.
In summary, our data support a model that asprosin acts on Ptprd expressed by AgRP/NPY neurons to inhibit SK3 current, further activating AgRP/NPY neurons to increase food intake and body weight. Our findings also provided preclinical evidence that asprosin-neutralizing antibodies could be used to treat obesity.

Hunger hormone asprosin activates orexigenic neurons via SK currents

Free fatty acid receptor 2 and free fatty acid receptor 3 (FFA2/3) are two highly similar G protein-coupled receptors belonging to the free fatty acid receptor family. Their ligands are short-chain fatty acids (SCFAs), which are key nutrients that play a diverse role in physiological function, including the regulation of metabolic homeostasis and glycemic control. FFA2/3 are broadly expressed in a multitude of tissues including the intestine, pancreas, adipose and central nervous system, where they contribute to metabolic homeostasis via a summation of tissue-specific effects. Consequently, FFA2/3 have been identified as a potential drug target for metabolic diseases including obesity and type-2 diabetes.
Both FFA2 and FFA3 are highly expressed within the intestinal epithelium – the major site of SCFA generation – and have been identified in hormone-secreting enteroendocrine cells as well as intestinal epithelial cells. However, due conflicting data, the respective roles of FFA2/3 within the intestine and their effects on physiology and metabolism are still largely unclear. Previous in vivo studies involving this receptor have largely relied on global knockout mouse models, making it difficult to isolate their effects in the intestine. To overcome this challenge, we generated a novel intestine-specific knockout mouse model for FFA2 and FFA3 individually, utilizing Cre-mediated recombination under the expression of the villin promoter. Here, we report the first in vivo characterization of FFA2/3 in the intestine and reveal novel insights into receptor function.
Following model validation, we conducted a general metabolic assessment of male Villin-Cre-FFA2 (Vil-FFA2) and Villin-Cre-FFA3 (Vil-FFA3) mice on standard chow and observed no major congenital or time-dependent defects. Because dietary changes are known to alter gut microbial composition, and thereby SCFA production, a pilot study was performed on male Vil-FFA2 and Vil-FFA3 mice and their littermate controls to probe for a phenotype on a high-fat, high-sugar "western diet." Mice were placed on either a low-fat control diet (CD) or western diet (WD) at 10 weeks of age and metabolically profiled for 25 weeks. We found that both Vil-FFA2 and Vil-FFA3 mouse strains were largely protected from diet-induced obesity and had significantly lower fat mass as well as adipose hypertrophy. Additionally, both mouse strains had reduced intestinal inflammation and improved glucose homeostasis. These differences were driven by lower food intake in the Vil-FFA2 strain only. Our findings suggest a novel role of FFA2/3 in mediating the metabolic consequences of a western diet – a state of high inflammation, dysbiosis and metabolic stress. Moreover, these data support an intestine-specific role of FFA2/3 in whole-body metabolic homeostasis and in the development of adiposity and hyperglycemia.

Intestinal FFA2 and FFA3 Mediate Obesogenic Effects in Mice on a Western Diet

Adipogenesis is a complex and tightly regulated process that plays a vital role in multiple aspects of human health.  The molecular mechanisms regulating adipogenesis are incompletely understood, although key facets of the signaling and regulatory pathways have been defined.  ADP-ribosylation – the process whereby ADP-ribose moieties are covalently transferred from NAD+ to substrate proteins – has been shown to control multiple components of the adipogenic regulatory machinery, including the transcription factor C/EBPb. In order to explore the role of mono(ADP-ribosyl)transferases (MARTs) during adipogenesis, we used an siRNA-mediated knockdown screen in mouse 3T3-L1 preadipocytes to determine which MARTs are required for the differentiation of the preadipocytes into mature adipocytes. This screen identified PARP7 the primary candidate. Using a variety of cell-based and biochemical assays, we have identified key aspects of PARP7's involvement throughout adipogenesis. We observed dynamic localization of PARP7 during adipogenesis, moving from the nucleus in early adipogenesis to the cytosol during mid to late adipogenesis. The requirement for PARP7, as determined by knockdown, was most evident in early stages of adipogenesis.  Knockdown of PARP7 was rescued by a PPAR g agonist, suggesting that PARP7 acts before the onset of the later stages of adipogenesis leading to mature adipocytes. Together, these findings led us to hypothesize that PARP7 plays a critical role in the nucleus during early adipogenesis. Preliminary data have revealed that PARP7 binds to C/EBPb, a key "first wave" transcription factor that drives adipogenesis, during a time when C/EBPb is poly(ADP-ribosyl)ated by PARP1 in the nucleus. Ongoing investigations are exploring: (1) the mechanisms by which PARP7 binds to C/EBPb; (2) the impact of PARP7 binding on C/EBPb stability; and (3) the biological outcomes of PARP7-C/EBPb interactions. Collectively, these studies provide an avenue to better understand the role of PARP7 in the regulation of adipogenesis.
This work is supported by a grant from the NIH/NIDDK (R01 DK069710) and funds from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment to W.L.K., and a predoctoral fellowship from the American Heart Association to M.S.S.

Exploring the Role of PARP7 in Adipogenesis

Background: Skeletal muscle is one of to the most dynamic and plastic tissues. The capacity to regenerate arises from adult muscle stem cells termed myogenic progenitors (MPs) that are activated in response to injury and differentiate to replenish damaged muscle fibers. The age-related loss of muscle mass is reflected in the reduced regenerative potential of MPs. The underlying mechanisms are still unclear and insights into alterations in adult muscle stem cells during ageing are urgently needed. We have previously identified a novel muscle-specific microRNA, miR-501-3p, that is enriched in activated MPs. Pharmacological inhibition of miR-501 during muscle regeneration promoted small-diameter neofibers. Here, we provide evidence for the regulation of miR-501 in skeletal muscle during aging and its effect on newly formed myofibers using genetic deletion in mice.Methods: We analysed the regulation and upstream signalling pathways of miR-501 in primary myoblasts from mice and humans and in skeletal muscle from young and aged mice. We generated a novel Cre-loxP mouse model, which allowed for global and MP-specific deletion of miR-501. Skeletal muscle regeneration was induced using cardiotoxin (CTX) and assessed using immunofluorescence and transcriptomic analysis.Results: miR-501 was upregulated in muscle cells by the paracrine growth factors platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) through the Janus tyrosine kinase (JAK)-signal transducer and activator of transcription-3 (STAT3) signalling axis. Expression levels of miR-501 as well as its upstream regulators Pdgf and Vegf were significantly reduced in skeletal muscle from aged mice (22 months) compared to young mice (3 months) by 67.8% (p<0.001), 40.0% (p<0.01) and 36.3% (p<0.05), respectively. Genetic deletion of miR-501 in mice resulted in muscle fibers with decreased size in adult skeletal muscle and in newly formed myofibers during muscle regeneration. RNA-seq of regenerating muscle annotated a critical role for miR-501 in the organisation of muscle filaments. We identified a miR-501-dependent sarcomeric gene signature that involves genes such as myosin heavy chains, titin and tubulin polymerization-promoting protein. This was confirmed in vitro in myotubes from mice and humans in which miR-501 was deleted either genetically or pharmacologically using antagomirs. Altered sarcomeric gene expression correlated with an increased sarcomere length in myotubes lacking miR-501 (2.49 ± 0.08μm vs 3.22 ± 0.15μm, p<0.01).Conclusion: Our data indicate that paracrine growth factor signalling contributes to muscle fiber formation via miR-501 in myogenic progenitor cells and its effect on the sarcomere. Restoring the Pdgf+Vegf/miR-501 axis might have the potential to improve muscle formation during aging.

Paracrine Growth Factor Signaling Regulates Muscle Regeneration in Aged Mice via microRNA 501

Background and purpose of the study
The overall goal of this study is to develop precision-targeted therapies for type 1 diabetes (T1D) by blocking the presentation of pancreatic antigens to T cells using an innovative translational approach. There is currently no curative therapy for T1D and the only available treatment is insulin replacement, which – although being life-saving – remains problematic and can be associated with often fatal high or low glucose levels. We hypothesize that the presentation of pathogenic pancreatic peptides to T cells within the HLA-DQ8 pocket is a key trigger in T1D, and that blocking the peptide binding to this pocket can be harnessed to treat/prevent the autoimmune response targeting beta cells in T1D. We propose to block antigen presentation to treat/prevent autoimmunity in T1D using retro-inverso D-amino acid peptides (RIPs). To test our hypothesis, we used the following tools: recombinant human HLA-DQ8, human B cells homozygous for HLA-DQ8, humanized mice expressing human HLA-DQ8, NOD mice, and peripheral blood mononuclear cells (PBMCs) isolated from new onset HLA-DQ8 T1D patients. All of them represent exquisite translational instruments to evaluate in vitro, ex vivo, and in vivo the functional role of our RIPs in preventing beta cell destruction.
Summary of results
We have identified a RIP (RI-CT) that inhibits InsB: 9-23 binding to recombinant HLA-DQ8 molecule, as well as its binding to HLA-DQ8 expressed on a human B-cell line. Specifically, RI-CT averted T-cell activation in a mixed lymphocyte reaction containing human DQ8 cells loaded with InsB: 9-23 peptide and murine T-cells expressing a human TCR specific for the InsB: 9-23–DQ8 complex. These results were also confirmed in humanized transgenic B6-DQ8 mice (KO for murine MHC-II) both ex vivo and in vivo, as shown by decreased production of pro-inflammatory cytokines (including IL-2 and IFN-γ) and reduced lymphocyte proliferation. Moreover, RI-CT prevents autoimmune diabetes in NOD mice as demonstrated by a significant decrease in pancreatic immune cell infiltration in treated mice versus controls. Importantly, at 25 weeks of age 70% of RI-CT treated mice were protected from the development of diabetes, while as expected 80% of control NOD mice had developed the disease. Interestingly, RI-CT injection also altered T-cell subpopulations in NOD mice significantly decreasing CD8 expression and significantly increasing Foxp3 levels in treated mice. Of note, RI-CT significantly inhibits InsB: 9-23-mediated lymphocyte activation in peripheral blood mononuclear cells isolated from new onset DQ8-T1D patients.
Conclusions
In summary, we discovered a RIP that blocks InsB: 9-23 binding to HLA-DQ8 and its presentation to T-cells averting beta cells destruction, and that delays T1D in NOD mice. These data set the stage for using our approach of blocking antigen presentation by RIP as a novel therapeutic approach for autoimmune diseases in general.

Cutting-edge translational strategies in type 1 diabetes

The pathogenesis of type 1 diabetes (T1D) involves the interaction of the immune system with pancreatic islets, featured by inflammation in both macrophages and β cells. The enzyme 12-lipoxygenase (12-LOX) is expressed in macrophages and β-cells, produces the eicosanoid 12(S)-HETE and promotes inflammation. GPR31 has recently been identified as a 12(S)-HETE receptor. GPR31 protein levels increase in human islets after cytokine-induced inflammation. To elucidate the role of GPR31 in the 12-LOX pathway and during diabetic inflammation, we generated Gpr31b-/- mice on the C57BL/6J background. Gpr31-/- mice are viable, with normal body weight, glucose tolerance, and β-cell mass. Upon low dose streptozotocin treatment to induce β-cell inflammation, Gpr31b-/- mice remained normoglycemic unlike wildtype littermates and maintained near normal β-cell mass. To examine GPR31 in islet inflammation, RNA sequencing was performed on isolated mouse islets treated with a cytokine cocktail (IL-1β, IFN-γ, TNF-α). Both wildtype and Gpr31b-/- islets showed expected increases in Nos2 and Il1b after cytokine treatment. By contrast, Gpr31b-/- islets, showed changes in Gene Ontology pathways related to ER stress, oxidative stress, protein targeting, and MAPK activity compared to wildtype islets after cytokine treatment. To test a role for GPR31 in macrophage function, we first interrogated its role in the generation of proinflammatory macrophages upon polarization in vitro to the M1-like state using LPS and IFN-γ; under these conditions, we found no differences between Gpr31b-/- and wildtype macrophages, as assessed by flow cytometric analysis, suggesting that GPR31 does not play a role in macrophage polarization. Next, we tested if GPR31 is required for the ability of macrophages to migrate. To do this, bone marrow derived macrophages were isolated from Gpr31b-/- and wildtype littermates and in vitro migration assays were performed using Transwell chambers. We observed a significant decrease in migration of the Gpr31b-/- macrophages compared to wildtype macrophages. Taken together, our data support the concept that GPR31 promotes proinflammatory responses in both macrophages and pancreatic islets.

GPR31 Mediates the Proinflammatory Reponses in the Setting of Diabetic Inflammation

Elevation of glucagon levels and increase in alpha-cell mass are associated with states of hyperglycemia in diabetes. A better understanding of the molecular mechanisms governing glucagon secretion could have major implications in understanding abnormal responses to hypoglycemia in diabetic patients and provide novel avenues for diabetes management. Stimulation of glucagon secretion in hypoglycemia, or by amino acids, induces hepatic glucose production via cellular mechanisms including suppression of glycogenesis and glycolysis and stimulation of glycogenolysis and gluconeogenesis. The close link between amino acids and the alpha-cell is highlighted by the liver–alpha-cell axis. This axis was identified by the major increase in alpha-cell hyperplasia and hyperglucagonemia in models of reduced glucagon action in hepatocytes genetically or pharmacologically by treatment with glucagon receptor antagonists, which was subsequently attributed to the dramatic rise in amino acids. Induction of mTORC1 by constitutive genetic deletion of TSC2 in alpha-cells (aTSC2KO) recapitulated the effects of chronic hyperaminoacidemia with increases in alpha-cell mass and chronic hyperglucagonemia indicating that mTORC1 mediates amino acids signals in alpha-cells. However, the effects of acute versus chronic induction of alpha-cell mTORC1 signaling on these outcomes have not been explored. Using mice with doxycycline induction of Rheb (activator of mTORC1 signaling) (aRhebTg) and inducible Cre-mediated deletion of the mTORC1 inhibitor, TSC2, in alpha-cells (iaTSC2KO), we showed that induction of hyperglucagonemia is associated with two phases: Acute phase characterized by impaired glucose tolerance because of increased gluconeogenesis and glucose output by the liver. And a chronic phase, characterized by normalization of glucose tolerance due to downregulation of the hepatic glucagon receptor (Gcgr), Pepck, and some genes involved in amino acid metabolism and urea production. Importantly, the consequences of chronic hyperglucagonemia were reversible after normalization of glucagon levels by turning off alpha-cell Rheb expression. These studies uncovered the acute versus chronic effects of hyperglucagonemia on glucose homeostasis and further demonstrate that glucagon resistance is a reversible process. These observations provide insight into the impact of glucagon in the pathogenesis of hyperglycemia and the physiological effects of administration of dual GLP1/Glucagon receptor agonists.

Targeting Hyperglucagonemia Reverses Glucagon Resistance

Background and Aim: One in ten pregnancies suffer from Gestational Diabetes Mellitus (GDM). GDM exposes fetus to high uterine glucose that causes fetal hyperglycemia. At birth Fetal hyperglycemia results in hypoglycemia, macrosomia, and larger than normal infants and these infants are predisposed to a number of metabolic disorders such as obesity, Type 2 Diabetes and non-alcoholic fatty liver disease (NAFLD) in adulthood. Although in-utero glucose exposure provides evidence for fetal lipid dysregulation linked to metabolic disorders in adults, the molecular mechanisms are still unclear. Increase in circulating insulin and glucose are implicated in the enhanced activation of hepatic De novo lipogenesis (DNL) in dyslipidemia, hence, we aim to investigate the potential beneficial effect of the pharmacological inhibition of DNL or glycolysis in our well-characterized zebrafish model of fetal hyperglycemia that mimics the metabolic disorders in human GDM against lipid derangement mediated by excess glucose in embryos in a short term and avoid the long-term outcome of obesity.
Methods: We developed a zebrafish model of fetal hyperglycemia by exposing embryos to high glucose (4.5% w/v) during the last day of embryogenesis from 96 to 120 hours post-fertilization (hpf). Glucose level was detected in embryo homogenates by glucometer. Insulin expression was visualized by whole-mount in-situ Hybridization. Gene expression of glycolytic (Hexokinase) and lipogenic [Acetyl-CoA Carboxylase (acca)] markers by RT-qPCR and malonyl CoA levels (product of DNL) by ELISA were also assessed. Lipid accumulation was detected by Oil-Red-O staining (ORO) and quantified by spectrophotometry. Pharmacological intervention using metformin or ND646 (known inhibitors of acca) or 2-Deoxyglucose, 2DG (Known inhibitor of Hexokinase) with glucose for 24hours were utilized to attenuate fetal hyperglycemia-mediated lipid accumulation in embryos.
Results: Zebrafish embryos exposed to high glucose for 24 hours are transiently hyperglycemic while normoglycemia is restored 24 hours after removal from glucose media. At 120 hpf embryos exposed to high-glucose demonstrated increased insulin, hexokinase, and acca expression compared to untreated controls. We also observed a significant reduction in malonyl CoA levels in ND 646 and metformin cotreated embryos with glucose compared to controls. Interestingly, quantification of ORO staining revealed a significant increase in lipid deposition in glucose treated embryos compared to controls at 120 hpf (0.2±0.01 vrs 0.13±0.005), which was significantly attenuated in embryos co-treated with glucose and 2-DG- (0.15±0.003) or metformin- (0.11±0.007) compared to glucose-treated embryos. Similarly, a moderate reduction in ORO staining was observed in embryos that were co-treated with glucose and ND646 (0.17 ± 0.006).
Conclusion and significance: Together our results show that embryonic glucose exposure leads to increased lipid accumulation which can be rescued by blocking glycolysis or de novo lipogenesis. Intervening glycolysis and de novo lipogenesis could be a promising therapeutic approach for modulating fetal hyperglycemia-related lipid dysregulations.

Zebrafish in utero glucose exposure induces lipid dysregulation that is abrogated by Acetyl CoA carboxylase or glycolysis inhibition

Glycosylation is an important posttranslational modification that modulates protein expression and function. Sialic acid is the most abundant terminal monosaccharide of N-linked glycosylation in mammalian cells, and its biosynthesis is controlled by the rate-limited enzyme UDP-GlcNAc 2-epimerase/ManNac kinase (GNE). There is a positive association between the abundance of terminally sialylated plasma glycoproteins and the risk of type 2 diabetes (T2DM).  The majority of glycosylated plasma proteins are liver-derived, and key hepatic receptors involved in glucose metabolism, such as the insulin receptor (IR) and glucagon receptor (GCGR), are terminally sialylated. Whether and how protein sialylation in the liver influences glucose homeostasis is unknown. To address this question we generated floxed GNE mice and at 4 weeks of age injected males with either AAV8 encoding a liver-specific targeted Cre recombinase or GFP, to yield mice lacking or normally expressing GNE in liver (GNELKO and GNELWT, respectively). Two weeks post AAV8 injection, GNELKO showed a >98% loss of GNE protein selectively in liver and drastic reductions in the sialylation of liver and plasma proteins detected by glycan-specific lectin-based analyses. Whereas body weights/body composition and plasma lipids were unaltered, GNELKO had lower fasting serum glucose and greater glucose tolerance and insulin sensitivity compared to GNELWT; serum insulin or glucose-stimulated insulin secretion were unchanged. In GNELKO mice hyperinsulinemic-euglycemic clamps displayed a loss of insulin suppression of hepatic glucose production, hepatic IRa and IRb subunit expression and Akt and GSK phosphorylation were decreased, and liver glycogen content was lowered >85%.  However, there was also evidence of markedly impaired GCGR function in GNELKO mice, with serum glucagon levels raised 18-fold and attenuation of hepatic cAMP production, CREB phosphorylation and gluconeogenesis in response to glucagon.  Regarding extrahepatic mechanisms, GNELKO mice displayed enhanced peripheral glucose disposal, and this was associated with increased IRb, Akt and GSK phosphorylation in skeletal muscle, and with serum FGF21 levels elevated by 406%. In additional studies, when invoked following 12 weeks of high fat diet feeding, liver GNE silencing caused dramatic improvements in glucose tolerance and insulin sensitivity.  To evaluate the relevance of these findings to humans, liver GNE transcript levels were queried in a publicly available cohort, and they were higher in T2DM patients compared to non-diabetic controls in two independent cohorts (n= 54 and n=9 for Cohort I and n=4 and n=4 for Cohort II, respectively). Collectively these observations reveal that protein sialylation in the liver has dramatic impact on both insulin and glucagon action in the liver, and on peripheral glucose disposal.  Further interrogation of the modulation of glucose control mechanisms by protein sialylation may lead to novel approaches to combatting glucose intolerance in T2DM and other conditions.

Liver-specific inhibition of sialic acid biosynthesis improves systemic glucose homeostasis in mice

Congenital unilateral defects of male reproductive tracts have been documented in men, suggesting the potential left-right asymmetry in male reproductive tract development. However, classic textbooks of endocrinology have been teaching us that the development of the paired male reproductive tract is governed by the universal action of the androgen receptor (Ar). During sexual differentiation, testis-derived androgens activate the Ar to promote the stabilization of the bilateral Wolffian ducts, the progenitor for the male reproductive tract. The Wolffian ducts then differentiate into the paired male reproductive tract organs, which include the epididymes, vas deferentes, and seminal vesicles. The Ar is expressed both in the epithelium and mesenchyme of the male reproductive tract during fetal development. Landmark tissue recombinant studies and the observation of normal morphology in the epithelium-specific Ar knockout mouse all support the notion that Ar action in the mesenchyme dictates the morphogenesis of the male reproductive tract. However, no genetic study has been performed to investigate the consequence of the loss of mesenchymal Ar on male reproductive tract development. To test the functional significance of the mesenchymal Ar, we designed a mesenchyme-specific Ar knockout mouse model (ARcKO) to ablate Ar specifically in the mesenchyme prior to the initiation of sexual differentiation. We performed immunohistochemistry of AR to confirm that Ar expression was absent in the mesenchyme while epithelial Ar remained intact in this ARcKO model. Based on the widely accepted notion that mesenchymal Ar regulates epithelial morphogenesis of the male reproductive tract, we expected to see abnormal patterning of the male reproductive tract. Indeed, the epididymis lost its characteristic coiling and became cystic. However, 40% of the 23 collected ARcKO males displayed these abnormalities on both horns; 43% and 17% displayed these abnormal phenotype solely on the left and right horn, respectively. This surprising observation might result from asynchronous activities of Cre on the left and right horns. However, when the Cre was crossed with a reporter (tdTomato), we observed comparable reporter expression on both horns at the onset of sexual differentiation of reproductive tracts. These observations suggest that the developmental programs in the morphogenesis of the left and right male reproductive tracts are asymmetrical and there could be a compensation mechanism for the loss of mesenchymal AR. We are currently in the process of comparing the left and the right horns in Ar knockout male mice to determine any biased signaling. Taken together, our study provides a unique model for not only studying congenital defects of male reproductive tracts but also for investigating the potential asymmetry in the masculinization program.

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Mild Maternal Undernutrition Results in Transcript Regulation in the Fat of Male Offspring that Correlates with Resistance to High Fat Diet Induced Weight Gain

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