Probes for INSULIN

The insulin receptor substrate of 53 kDa, IRSp53, is an adaptor protein that works with activated GTPases, Cdc42 and Rac, to modulate actin dynamics and generate membrane protrusions in response to cell signaling. Adult mice that lack IRSp53 fail to regulate synaptic plasticity and exhibit hippocampus-associated learning deficiencies. Here, we show that 60% of IRSp53 null embryos die at mid to late gestation, indicating a vital IRSp53 function in embryonic development. We find that IRSp53 KO embryos displayed pleiotropic phenotypes such as developmental delay, oligodactyly and subcutaneous edema, and died of severely impaired cardiac and placental development. We further show that double knockout of IRSp53 and its closest family member, IRTKS, resulted in exacerbated placental abnormalities, particularly in spongiotrophoblast differentiation and development, giving rise to complete embryonic lethality. Hence, our findings demonstrate a hitherto under-appreciated IRSp53 function in embryonic development, and further establish an essential genetic interaction between IRSp53 and IRTKS in placental formation.

Diabetic kidney disease (DKD) is the most common cause of severe renal disease, and few treatment options are available today that prevent the progressive loss of renal function. DKD is characterized by altered glomerular filtration and proteinuria. A common observation in DKD is the presence of renal steatosis, but the mechanism(s) underlying this observation and to what extent they contribute to disease progression are unknown. Vascular endothelial growth factor B (VEGF-B) controls muscle lipid accumulation through regulation of endothelial fatty acid transport. Here, we demonstrate in experimental mouse models of DKD that renal VEGF-B expression correlates with the severity of disease. Inhibiting VEGF-B signaling in DKD mouse models reduces renal lipotoxicity, re-sensitizes podocytes to insulin signaling, inhibits the development of DKD-associated pathologies, and prevents renal dysfunction. Further, we show that elevated VEGF-B levels are found in patients with DKD, suggesting that VEGF-B antagonism represents a novel approach to treat DKD.

Acquisition of T cell central tolerance involves distinct pathways of self-antigen presentation to thymocytes. One pathway termed indirect presentation requires a self-antigen transfer step from thymic epithelial cells (TECs) to bone marrow (BM)-derived cells before the self-antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context-dependent, potentially due to variation in self-antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane-bound transgenic self-antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared to thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHCII expression in BM-APCs, insHEL-mediated thymocyte deletion was abolished, whereas thyroHEL-mediated deletion occurred at a later stage of thymocyte development and Foxp3+ T-regulatory cell differentiation increased. Indirect presentation increased the strength of TCR signalling that both self-antigens induced in thymocytes, as assessed by Helios expression. Thus, indirect presentation limits the differentiation of naïve and regulatory T cells by promoting deletion of self-reactive thymocytes.

Pancreatic α-cells proliferate with low rate and little is known about the control of this process. Here we report the characterization of human α-cells by large-scale single cell RNA sequencing coupled with pseudotime ordering. We identified two large subpopulations and a smaller cluster of proliferating α-cells with increased expression of genes involved in cell cycle regulation. The proliferating α-cells were differentiated, had normal levels of GCG expression and showed no signs of cellular stress. Proliferating α-cells were detected in both the G1S and G2M phases of the cell cycle. Human α-cells proliferate with 5-fold higher rate than human β-cells and express lower levels of the cell cycle inhibitors CDKN1A and CDKN1C. Collectively, this study provides the gene signatures of human α-cells and the genes involved in their cell division. The lower expression of two cell cycle inhibitors in human α-cells could account for their higher rate of proliferation compared to their insulin producing counterparts.

Fetal bovine serum (FBS) is used commonly in cell culture. Charcoal-stripped FBS (CS-FBS) is used to study androgen responsiveness and androgen metabolism in cultured CaP cells. Switching CaP cells from FBS to CS-FBS may reduce activity of androgen receptor (AR), inhibit cell proliferation, or modulate intracellular androgen metabolism. Removal of proteins by charcoal stripping may cause changes in biological functions. Proteins in FBS and CS-FBS were profiled using an ion current-based quantitative platform consisting of reproducible surfactant-aided precipitation/on-pellet digestion, long-column nano-liquid chromatography (LC) separation, and ion current-based analysis (ICan). A total of 143 proteins were identified in FBS, among which 14 proteins including insulin-like growth factor 2 (IGF-2) and IGF binding protein (IGFBP)-2 and -6 were reduced in CS-FBS. IGF1 receptor (IGF1R) and insulin receptor (IR) were sensitized to IGFs in CS-FBS. IGF1 and IGF2 stimulation fully compensated for the loss of AR activity to maintain cell growth in CS-FBS. Endogenous production of IGF and IGFBPs was verified in CaP cells and clinical CaP specimens. This study provided the most comprehensive protein profiles of FBS and CS-FBS, and offered an opportunity to identify new protein regulators and signaling pathways that regulate AR activity, androgen metabolism and proliferation of CaP cells.

Mitochondrial oxidative phosphorylation (OXPHOS) and substrate utilization critically regulate the function of hypothalamic proopiomelanocortin (POMC)-expressing neurons. Here, we demonstrate that inactivation of apoptosis-inducing factor (AIF) in POMC neurons mildly impairs mitochondrial respiration and decreases firing of POMC neurons in lean mice. In contrast, under diet-induced obese conditions, POMC-Cre-specific inactivation of AIF prevents obesity-induced silencing of POMC neurons, translating into improved glucose metabolism, improved leptin, and insulin sensitivity, as well as increased energy expenditure in AIFΔPOMC mice. On a cellular level, AIF deficiency improves mitochondrial morphology, facilitates the utilization of fatty acids for mitochondrial respiration, and increases reactive oxygen species (ROS) formation in POMC neurons from obese mice, ultimately leading to restored POMC firing upon HFD feeding. Collectively, partial impairment of mitochondrial function shifts substrate utilization of POMC neurons from glucose to fatty acid metabolism and restores their firing properties, resulting in improved systemic glucose and energy metabolism in obesity.

The composition of the gastrointestinal (GI) microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine (fMLF), are elevated in high fat diet (HFD)-induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon-like peptide-1 (GLP-1). Obese Fpr1-knockout (Fpr1-KO) mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota. Overall, we describe a new mechanism by which the gut microbiota can modulate glucose metabolism, providing a potential approach for treatment of metabolic disease.

Neuropeptide Y (NPY) exerts a powerful orexigenic effect in the hypothalamus. However, extra-hypothalamic nuclei also produce NPY, but its influence on energy homeostasis is unclear. Here we uncover a previously unknown feeding stimulatory pathway that is activated under conditions of stress in combination with calorie-dense food; NPY neurons in the central amygdala are responsible for an exacerbated response to a combined stress and high-fat-diet intervention. Central amygdala NPY neuron-specific Npy overexpression mimics the obese phenotype seen in a combined stress and high-fat-diet model, which is prevented by the selective ablation of Npy. Using food intake and energy expenditure as readouts, we demonstrate that selective activation of central amygdala NPY neurons results in increased food intake and decreased energy expenditure. Mechanistically, it is the diminished insulin signaling capacity on central amygdala NPY neurons under combined stress and high-fat-diet conditions that leads to the exaggerated development of obesity.