Proteomic predictors of individualized nutrient-specific insulin secretion in health and disease.

Population level variation and molecular mechanisms behind insulin secretion in response to carbohydrate, protein, and fat remain uncharacterized despite ramifications for personalized nutrition. Here, we define prototypical insulin secretion dynamics in response to the three macronutrients in islets from 140 cadaveric donors, including those diagnosed with type 2 diabetes. While islets from the majority of donors exhibited the expected relative response magnitudes, with glucose being highest, amino acid moderate, and fatty acid small, 9% of islets stimulated with amino acid and 8% of islets stimulated with fatty acids had larger responses compared with high glucose. We leveraged this insulin response heterogeneity and used transcriptomics and proteomics to identify molecular correlates of specific nutrient responsiveness, as well as those proteins and mRNAs altered in type 2 diabetes. We also examine nutrient-responsiveness in stem cell-derived islet clusters and observe that they have dysregulated fuel sensitivity, which is a hallmark of functionally immature cells. Our study now represents the first comparison of dynamic responses to nutrients and multi-omics analysis in human insulin secreting cells. Responses of different people's islets to carbohydrate, protein, and fat lay the groundwork for personalized nutrition. ONE-SENTENCE SUMMARY: Deep phenotyping and multi-omics reveal individualized nutrient-specific insulin secretion propensity.

Hyperinsulinemia acts via acinar insulin receptors to initiate pancreatic cancer by increasing digestive enzyme production and inflammation.

The rising pancreatic cancer incidence due to obesity and type 2 diabetes is closely tied to hyperinsulinemia, an independent cancer risk factor. Previous studies demonstrated reducing insulin production suppressed pancreatic intraepithelial neoplasia (PanIN) pre-cancerous lesions in Kras-mutant mice. However, the pathophysiological and molecular mechanisms remained unknown, and in particular it was unclear whether hyperinsulinemia affected PanIN precursor cells directly or indirectly. Here, we demonstrate that insulin receptors (Insr) in KrasG12D-expressing pancreatic acinar cells are dispensable for glucose homeostasis but necessary for hyperinsulinemia-driven PanIN formation in the context of diet-induced hyperinsulinemia and obesity. Mechanistically, this was attributed to amplified digestive enzyme protein translation, triggering of local inflammation, and PanIN metaplasia in vivo. In vitro, insulin dose-dependently increased acinar-to-ductal metaplasia formation in a trypsin- and Insr-dependent manner. Collectively, our data shed light on the mechanisms connecting obesity-driven hyperinsulinemia and pancreatic cancer development.

Transcriptomics for Clinical and Experimental Biology Research: Hang on a Seq.

Sequencing the human genome empowers translational medicine, facilitating transcriptome-wide molecular diagnosis, pathway biology, and drug repositioning. Initially, microarrays are used to study the bulk transcriptome; but now short-read RNA sequencing (RNA-seq) predominates. Positioned as a superior technology, that makes the discovery of novel transcripts routine, most RNA-seq analyses are in fact modeled on the known transcriptome. Limitations of the RNA-seq methodology have emerged, while the design of, and the analysis strategies applied to, arrays have matured. An equitable comparison between these technologies is provided, highlighting advantages that modern arrays hold over RNA-seq. Array protocols more accurately quantify constitutively expressed protein coding genes across tissue replicates, and are more reliable for studying lower expressed genes. Arrays reveal long noncoding RNAs (lncRNA) are neither sparsely nor lower expressed than protein coding genes. Heterogeneous coverage of constitutively expressed genes observed with RNA-seq, undermines the validity and reproducibility of pathway analyses. The factors driving these observations, many of which are relevant to long-read or single-cell sequencing are discussed. As proposed herein, a reappreciation of bulk transcriptomic methods is required, including wider use of the modern high-density array data-to urgently revise existing anatomical RNA reference atlases and assist with more accurate study of lncRNAs.

Flow-Induced Secretion of Endothelial Heparanase Regulates Cardiac Lipoprotein Lipase and Changes Following Diabetes.

Background Lipoprotein lipase (LPL)-derived fatty acid is a major source of energy for cardiac contraction. Synthesized in cardiomyocytes, LPL requires translocation to the vascular lumen for hydrolysis of lipoprotein triglyceride, an action mediated by endothelial cell (EC) release of heparanase. We determined whether flow-mediated biophysical forces can cause ECs to secrete heparanase and thus regulate cardiac metabolism. Methods and Results Isolated hearts were retrogradely perfused. Confluent rat aortic ECs were exposed to laminar flow using an orbital shaker. Cathepsin L activity was determined using gelatin-zymography. Diabetes was induced in rats with streptozotocin. Despite the abundance of enzymatically active heparanase in the heart, it was the enzymatically inactive, latent heparanase that was exceptionally responsive to flow-induced release. EC exposed to orbital rotation exhibited a similar pattern of heparanase secretion, an effect that was reproduced by activation of the mechanosensor, Piezo1. The laminar flow-mediated release of heparanase from EC required activation of both the purinergic receptor and protein kinase D, a kinase that assists in vesicular transport of proteins. Heparanase influenced cardiac metabolism by increasing cardiomyocyte LPL displacement along with subsequent replenishment. The flow-induced heparanase secretion was augmented following diabetes and could explain the increased heparin-releasable pool of LPL at the coronary lumen in these diabetic hearts. Conclusions ECs sense fluid shear-stress and communicate this information to subjacent cardiomyocytes with the help of heparanase. This flow-induced mechanosensing and its dynamic control of cardiac metabolism to generate ATP, using LPL-derived fatty acid, is exquisitely adapted to respond to disease conditions, like diabetes.

Beta-cell specific Insr deletion promotes insulin hypersecretion and improves glucose tolerance prior to global insulin resistance.

Insulin receptor (Insr) protein is present at higher levels in pancreatic ß-cells than in most other tissues, but the consequences of ß-cell insulin resistance remain enigmatic. Here, we use an Ins1cre knock-in allele to delete Insr specifically in ß-cells of both female and male mice. We compare experimental mice to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined ß-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout ß-cells from female, but not male mice, whereas only male ßInsrKO islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female ßInsrKO and ßInsrHET mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr does not alter ß-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include ß-cell insulin resistance, which predicts that ß-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female ßInsrKO and ßInsrHET mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of ß-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1-CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that ß-cell insulin resistance in the form of reduced ß-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts.

Human and mouse muscle transcriptomic analyses identify insulin receptor mRNA downregulation in hyperinsulinemia-associated insulin resistance.

Hyperinsulinemia is commonly viewed as a compensatory response to insulin resistance, yet studies have demonstrated that chronically elevated insulin may also drive insulin resistance. The molecular mechanisms underpinning this potentially cyclic process remain poorly defined, especially on a transcriptome-wide level. Transcriptomic meta-analysis in >450 human samples demonstrated that fasting insulin reliably and negatively correlated with INSR mRNA in skeletal muscle. To establish causality and study the direct effects of prolonged exposure to excess insulin in muscle cells, we incubated C2C12 myotubes with elevated insulin for 16 h, followed by 6 h of serum starvation, and established that acute AKT and ERK signaling were attenuated in this model of in vitro hyperinsulinemia. Global RNA-sequencing of cells both before and after nutrient withdrawal highlighted genes in the insulin receptor (INSR) signaling, FOXO signaling, and glucose metabolism pathways indicative of 'hyperinsulinemia' and 'starvation' programs. Consistently, we observed that hyperinsulinemia led to a substantial reduction in Insr gene expression, and subsequently a reduced surface INSR and total INSR protein, both in vitro and in vivo. Bioinformatic modeling combined with RNAi identified SIN3A as a negative regulator of Insr mRNA (and JUND, MAX, and MXI as positive regulators of Irs2 mRNA). Together, our analysis identifies mechanisms which may explain the cyclic processes underlying hyperinsulinemia-induced insulin resistance in muscle, a process directly relevant to the etiology and disease progression of type 2 diabetes.

Differential Effects of Voclosporin and Tacrolimus on Insulin Secretion From Human Islets.

The incidence of new onset diabetes after transplant (NODAT) has increased over the past decade, likely due to calcineurin inhibitor-based immunosuppressants, including tacrolimus (TAC) and cyclosporin. Voclosporin (VCS), a next-generation calcineurin inhibitor, is reported to cause fewer incidences of NODAT but the reason is unclear. While calcineurin signaling plays important roles in pancreatic ß-cell survival, proliferation, and function, its effects on human ß-cells remain understudied. In particular, we do not understand why some calcineurin inhibitors have more profound effects on the incidence of NODAT. We compared the effects of TAC and VCS on the dynamics of insulin secretory function, programmed cell death rate, and the transcriptomic profile of human islets. We studied 2 clinically relevant doses of TAC (10 ng/mL, 30 ng/mL) and VCS (20 ng/mL, 60 ng/mL), meant to approximate the clinical trough and peak concentrations. TAC, but not VCS, caused a significant impairment of 15 mM glucose-stimulated and 30 mM KCl-stimulated insulin secretion. This points to molecular defects in the distal stages of exocytosis after voltage-gated Ca2+ entry. No significant effects on islet cell survival or total insulin content were identified. RNA sequencing showed that TAC significantly decreased the expression of 17 genes, including direct and indirect regulators of exocytosis (SYT16, TBC1D30, PCK1, SMOC1, SYT5, PDK4, and CREM), whereas VCS has less broad, and milder, effects on gene expression. Clinically relevant doses of TAC, but not VCS, directly inhibit insulin secretion from human islets, likely via transcriptional control of exocytosis machinery.