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.

FGF1 supports glycolytic metabolism through the estrogen receptor in endocrine-resistant and obesity-associated breast cancer.

BACKGROUND: Obesity increases breast cancer risk and breast cancer-specific mortality, particularly for people with estrogen receptor (ER)-positive tumors. Body mass index (BMI) is used to define obesity, but it may not be the best predictor of breast cancer risk or prognosis on an individual level. Adult weight gain is an independent indicator of breast cancer risk. Our previous work described a murine model of obesity, ER-positive breast cancer, and weight gain and identified fibroblast growth factor receptor (FGFR) as a potential driver of tumor progression. During adipose tissue expansion, the FGF1 ligand is produced by hypertrophic adipocytes as a stimulus to stromal preadipocytes that proliferate and differentiate to provide additional lipid storage capacity. In breast adipose tissue, FGF1 production may stimulate cancer cell proliferation and tumor progression. METHODS: We explored the effects of FGF1 on ER-positive endocrine-sensitive and resistant breast cancer and compared that to the effects of the canonical ER ligand, estradiol. We used untargeted proteomics, specific immunoblot assays, gene expression profiling, and functional metabolic assessments of breast cancer cells. The results were validated in tumors from obese mice and breast cancer datasets from women with obesity. RESULTS: FGF1 stimulated ER phosphorylation independently of estradiol in cells that grow in obese female mice after estrogen deprivation treatment. Phospho- and total proteomic, genomic, and functional analyses of endocrine-sensitive and resistant breast cancer cells show that FGF1 promoted a cellular phenotype characterized by glycolytic metabolism. In endocrine-sensitive but not endocrine-resistant breast cancer cells, mitochondrial metabolism was also regulated by FGF1. Comparison of gene expression profiles indicated that tumors from women with obesity shared hallmarks with endocrine-resistant breast cancer cells. CONCLUSIONS: Collectively, our data suggest that one mechanism by which obesity and weight gain promote breast cancer progression is through estrogen-independent ER activation and cancer cell metabolic reprogramming, partly driven by FGF/FGFR. The first-line treatment for many patients with ER-positive breast cancer is inhibition of estrogen synthesis using aromatase inhibitors. In women with obesity who are experiencing weight gain, locally produced FGF1 may activate ER to promote cancer cell metabolic reprogramming and tumor progression independently of estrogen.

Sex differences in islet stress responses support female ß cell resilience.

OBJECTIVE: Pancreatic ß cells play a key role in maintaining glucose homeostasis; dysfunction of this critical cell type causes type 2 diabetes (T2D). Emerging evidence points to sex differences in ß cells, but few studies have examined male-female differences in ß cell stress responses and resilience across multiple contexts, including diabetes. Here, we address the need for high-quality information on sex differences in ß cell and islet gene expression and function using both human and rodent samples. METHODS: In humans, we compared ß cell gene expression and insulin secretion in donors with T2D to non-diabetic donors in both males and females. In mice, we generated a well-powered islet RNAseq dataset from 20-week-old male and female siblings with similar insulin sensitivity. Our unbiased gene expression analysis pointed to a sex difference in the endoplasmic reticulum (ER) stress response. Based on this analysis, we hypothesized female islets would be more resilient to ER stress than male islets. To test this, we subjected islets isolated from age-matched male and female mice to thapsigargin treatment and monitored protein synthesis, cell death, and ß cell insulin production and secretion. Transcriptomic and proteomic analyses were used to characterize sex differences in islet responses to ER stress. RESULTS: Our single-cell analysis of human ß cells revealed sex-specific changes to gene expression and function in T2D, correlating with more robust insulin secretion in human islets isolated from female donors with T2D compared to male donors with T2D. In mice, RNA sequencing revealed differential enrichment of unfolded protein response pathway-associated genes, where female islets showed higher expression of genes linked with protein synthesis, folding, and processing. This differential expression was physiologically significant, as islets isolated from female mice were more resilient to ER stress induction with thapsigargin. Specifically, female islets showed a greater ability to maintain glucose-stimulated insulin production and secretion during ER stress compared with males. CONCLUSIONS: Our data demonstrate sex differences in ß cell gene expression in both humans and mice, and that female ß cells show a greater ability to maintain glucose-stimulated insulin secretion across multiple physiological and pathological contexts.

A three-player game model for promoting enterprise green technology innovation from the perspective of media coverage.

Objective: The objective of this study was to explore the game relationship among enterprise, the government, and the public under the new media environment, so as to provide decision-making reference for improving enterprise green technology innovation and promoting economy high-quality development with new media participation. Methods: This study constructs a three-subject evolutionary game model of enterprise, government, and public based on multi-agent relationship analysis and evolutionary game theory. In addition, the derivation of an evolutionary equilibrium strategy and numerical simulation analysis is carried out to comprehensively explore the evolution trajectory of green technology innovation system under the new media environment. Findings: (1) The system may have four stable evolutionary strategies: (1,0,0), (0,0,1), (1,0,1), and (1,1,1). (2) The initial strategy probability of various actors would affect the system evolution speed but not the evolution result, and the authenticity of new media reports is an important factor determining the system evolution of green technology innovation. (3) Numerical simulation results show that a fair and just new media environment can effectively constrain the traditional production behavior of enterprise, actively guide the public to participate in supervision, and play an alternative role to government regulation to a certain extent. Value: This study explores the evolutionary balance strategy of green technology innovation system under the new media environment, which not only enriches relevant theories of media environment governance but also has important reference value for promoting enterprises' green technology innovation and establishing an environmental governance system jointly governed by government, enterprise, public, and media.

Dynamic Ins2 Gene Activity Defines ß-Cell Maturity States.

Transcriptional and functional cellular specialization has been described for insulin-secreting ß-cells of the endocrine pancreas. However, it is not clear whether ß-cell heterogeneity is stable or reflects dynamic cellular states. We investigated the temporal kinetics of endogenous insulin gene activity using live cell imaging, with complementary experiments using FACS and single-cell RNA sequencing, in ß-cells from Ins2GFP knockin mice. In vivo staining and FACS analysis of islets from Ins2GFP mice confirmed that at a given moment, ∼25% of ß-cells exhibited significantly higher activity at the evolutionarily conserved insulin gene, Ins2. Live cell imaging over days captured Ins2 gene activity dynamics in single ß-cells. Autocorrelation analysis revealed a subset of oscillating cells, with mean oscillation periods of 17 h. Increased glucose concentrations stimulated more cells to oscillate and resulted in higher average Ins2 gene activity per cell. Single-cell RNA sequencing showed that Ins2(GFP)HIGH ß-cells were enriched for markers of ß-cell maturity. Ins2(GFP)HIGH ß-cells were also significantly less viable at all glucose concentrations and in the context of endoplasmic reticulum stress. Collectively, our results demonstrate that the heterogeneity of insulin production, observed in mouse and human ß-cells, can be accounted for by dynamic states of insulin gene activity.

Release of moth pheromone compounds from Nicotiana benthamiana upon transient expression of heterologous biosynthetic genes.

BACKGROUND: Using genetically modified plants as natural dispensers of insect pheromones may eventually become part of a novel strategy for integrated pest management. RESULTS: In the present study, we first characterized essential functional genes for sex pheromone biosynthesis in the rice stem borer Chilo suppressalis (Walker) by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana, including two desaturase genes CsupYPAQ and CsupKPSE and a reductase gene CsupFAR2. Subsequently, we co-expressed CsupYPAQ and CsupFAR2 together with the previously characterized moth desaturase Atr∆11 in N. benthamiana. This resulted in the production of (Z)-11-hexadecenol together with (Z)-11-hexadecenal, the major pheromone component of C. suppressalis. Both compounds were collected from the transformed N. benthamiana headspace volatiles using solid-phase microextraction. We finally added the expression of a yeast acetyltransferase gene ATF1 and could then confirm also (Z)-11-hexadecenyl acetate release from the plant. CONCLUSIONS: Our results pave the way for stable transformation of plants to be used as biological pheromone sources in different pest control strategies.

A low-sugar diet enhances Drosophila body size in males and females via sex-specific mechanisms.

In Drosophila, changes to dietary protein elicit different body size responses between the sexes. Whether these differential body size effects extend to other macronutrients remains unclear. Here, we show that lowering dietary sugar (0S diet) enhanced body size in male and female larvae. Despite an equivalent phenotypic effect between the sexes, we detected sex-specific changes to signalling pathways, transcription and whole-body glycogen and protein. In males, the low-sugar diet augmented insulin/insulin-like growth factor signalling pathway (IIS) activity by increasing insulin sensitivity, where increased IIS was required for male metabolic and body size responses in 0S. In females reared on low sugar, IIS activity and insulin sensitivity were unaffected, and IIS function did not fully account for metabolic and body size responses. Instead, we identified a female-biased requirement for the Target of rapamycin pathway in regulating metabolic and body size responses. Together, our data suggest the mechanisms underlying the low-sugar-induced increase in body size are not fully shared between the sexes, highlighting the importance of including males and females in larval studies even when similar phenotypic outcomes are observed.

Green Chemistry Production of Codlemone, the Sex Pheromone of the Codling Moth (Cydia pomonella), by Metabolic Engineering of the Oilseed Crop Camelina (Camelina sativa).

Synthetic pheromones have been used for pest control over several decades. The conventional synthesis of di-unsaturated pheromone compounds is usually complex and costly. Camelina (Camelina sativa) has emerged as an ideal, non-food biotech oilseed platform for production of oils with modified fatty acid compositions. We used Camelina as a plant factory to produce mono- and di-unsaturated C12 chain length moth sex pheromone precursors, (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid, by introducing a fatty acyl-ACP thioesterase FatB gene UcTE from California bay laurel (Umbellularia californica) and a bifunctional ∆9 desaturase gene Cpo_CPRQ from the codling moth, Cydia pomonella. Different transgene combinations were investigated for increasing pheromone precursor yield. The most productive Camelina line was engineered with a vector that contained one copy of UcTE and the viral suppressor protein encoding P19 transgenes and three copies of Cpo_CPRQ transgene. The T2 generation of this line produced 9.4% of (E)-9-dodecenoic acid and 5.5% of (E,E)-8,10-dodecadienoic acid of the total fatty acids, and seeds were selected to advance top-performing lines to homozygosity. In the T4 generation, production levels of (E)-9-dodecenoic acid and (E,E)-8,10-dodecadienoic acid remained stable. The diene acid together with other seed fatty acids were converted into corresponding alcohols, and the bioactivity of the plant-derived codlemone was confirmed by GC-EAD and a flight tunnel assay. Trapping in orchards and home gardens confirmed significant and specific attraction of C. pomonella males to the plant-derived codlemone.

Biosynthesis of the Sex Pheromone Component (E,Z)-7,9-Dodecadienyl Acetate in the European Grapevine Moth, Lobesia botrana, Involving ∆11 Desaturation and an Elusive ∆7 Desaturase.

The European grapevine moth, Lobesia botrana, uses (E,Z)-7,9-dodecadienyl acetate as its major sex pheromone component. Through in vivo labeling experiments we demonstrated that the doubly unsaturated pheromone component is produced by ∆11 desaturation of tetradecanoic acid, followed by chain shortening of (Z)-11-tetradecenoic acid to (Z)-9-dodecenoic acid, and subsequently introduction of the second double bond by an unknown ∆7 desaturase, before final reduction and acetylation. By sequencing and analyzing the transcriptome of female pheromone glands of L. botrana, we obtained 41 candidate genes that may be involved in sex pheromone production, including the genes encoding 17 fatty acyl desaturases, 13 fatty acyl reductases, 1 fatty acid synthase, 3 acyl-CoA oxidases, 1 acetyl-CoA carboxylase, 4 fatty acid transport proteins and 2 acyl-CoA binding proteins. A functional assay of desaturase and acyl-CoA oxidase gene candidates in yeast and insect cell (Sf9) heterologous expression systems revealed that Lbo_PPTQ encodes a ∆11 desaturase producing (Z)-11-tetradecenoic acid from tetradecanoic acid. Further, Lbo_31670 and Lbo_49602 encode two acyl-CoA oxidases that may produce (Z)-9-dodecenoic acid by chain shortening (Z)-11-tetradecenoic acid. The gene encoding the enzyme introducing the E7 double bond into (Z)-9-dodecenoic acid remains elusive even though we assayed 17 candidate desaturases in the two heterologous systems.

Multi-Functional Desaturases in Two Spodoptera Moths with ∆11 and ∆12 Desaturation Activities.

The beet armyworm, Spodoptera exigua, uses (Z,E)-9,12-tetradecadienyl acetate as the major component of its sex pheromone. Previous isotope-labeling experiments demonstrated an unusual ∆12 desaturase activity involved in the biosynthesis of this compound; however, the putative ∆12 desaturase gene has not been reported to date. In the present study, we confirmed this ∆12 desaturation pathway by in vivo labeling experiments, and characterized candidate desaturase genes in a yeast heterologous expression system. We demonstrated that a pheromone gland-specific desaturase, SexiDes5, uses palmitic acid and the subsequently chain-shortened product (Z)-9-tetradecenoic acid as substrates to produce (Z)-11-hexadecenoic and (Z,E)-9,12-tetradecadienoic acids, respectively. In addition, the homologous desaturase SlitDes5 from the congeneric Spodoptera litura had analogous functions.

Large number of putative chemoreception and pheromone biosynthesis genes revealed by analyzing transcriptome from ovipositor-pheromone glands of Chilo suppressalis.

The chemoreception role of moth ovipositor has long been suggested, but its molecular mechanism is mostly unknown. By transcriptomic analysis of the female ovipositor-pheromone glands (OV-PG) of Chilo suppressalis, we obtained 31 putative chemoreception genes (9 OBPs, 10 CSPs, 2 ORs, 1 SNMP, 8 CXEs and 1 AOX), in addition to 32 genes related to sex pheromone biosynthesis (1 FAS, 6 Dess, 10 FARs, 2 ACOs, 1 ACC, 4 FATPs, 3 ACBPs and 5 ELOs). Tissue expression profiles further revealed that CsupCSP2 and CsupCSP10 were OV-PG biased, while most chemoreception genes were highly and preferably expressed in antennae. This suggests that OV-PG employs mostly the same chemoreception proteins as in antennae, although the physiological roles of these proteins might be different in OV-PG. Of the 32 pheromone biosynthesis related genes, CsupDes4, CsupDes5 and CsupFAR2 are strongly OV-PG biased, and clustered with functionally validated genes from other moths, strongly indicating their involvement in specific step of the pheromone biosynthesis. Our study for the first time identified a large number of putative chemoreception genes, and provided an important basis for exploring the chemoreception mechanisms of OV-PG in C. suppressalis, as well as other moth species.

Putative pathway of sex pheromone biosynthesis and degradation by expression patterns of genes identified from female pheromone gland and adult antenna of Sesamia inferens (Walker).

The general pathway of biosynthesis and degradation for Type-I sex pheromones in moths is well established, but some genes involved in this pathway remain to be characterized. The purple stem borer, Sesamia inferens, employs a pheromone blend containing components with three different terminal functional groups (Z11-16:OAc, Z11-16:OH, and Z11-16:Ald) of Type-I sex pheromones. Thus, it provides a good model to study the diversity of genes involved in pheromone biosynthesis and degradation pathways. By analyzing previously obtained transcriptomic data of the sex pheromone glands and antennae, we identified 73 novel genes that are possibly related to pheromone biosynthesis (46 genes) or degradation (27 genes). Gene expression patterns and phylogenetic analysis revealed that one desaturase (SinfDes4), one fatty acid reductase (SinfFAR2), and one fatty acid xtransport protein (SinfFATP1) genes were predominantly expressed in pheromone glands, and clustered with genes involved in pheromone synthesis in other moth species. Ten genes including five carboxylesterases (SinfCXE10, 13, 14, 18, and 20), three aldehyde oxidases (SinfAOX1, 2 and 3), and two alcohol dehydrogenases (SinfAD1 and 3) were expressed specifically or predominantly in antennae, and could be candidate genes involved in pheromone degradation. SinfAD1 and 3 are the first reported alcohol dehydrogenase genes with antennae-biased expression. Based on these results we propose a pathway involving these potential enzyme-encoding gene candidates in sex pheromone biosynthesis and degradation in S. inferens. This study provides robust background information for further elucidation of the genetic basis of sex pheromone biosynthesis and degradation, and ultimately provides potential targets to disrupt sexual communication in S. inferens for control purposes.

Differential expression patterns in chemosensory and non-chemosensory tissues of putative chemosensory genes identified by transcriptome analysis of insect pest the purple stem borer Sesamia inferens (Walker).

BACKGROUND: A large number of insect chemosensory genes from different gene subfamilies have been identified and annotated, but their functional diversity and complexity are largely unknown. A systemic examination of expression patterns in chemosensory organs could provide important information. METHODOLOGY/PRINCIPAL FINDINGS: We identified 92 putative chemosensory genes by analysing the transcriptome of the antennae and female sex pheromone gland of the purple stem borer Sesamia inferens, among them 87 are novel in this species, including 24 transcripts encoding for odorant binding proteins (OBPs), 24 for chemosensory proteins (CSPs), 2 for sensory neuron membrane proteins (SNMPs), 39 for odorant receptors (ORs) and 3 for ionotropic receptors (IRs). The transcriptome analyses were validated and quantified with a detailed global expression profiling by Reverse Transcription-PCR for all 92 transcripts and by Quantitative Real Time RT-PCR for selected 16 ones. Among the chemosensory gene subfamilies, CSP transcripts are most widely and evenly expressed in different tissues and stages, OBP transcripts showed a clear antenna bias and most of OR transcripts are only detected in adult antennae. Our results also revealed that some OR transcripts, such as the transcripts of SNMP2 and 2 IRs were expressed in non-chemosensory tissues, and some CSP transcripts were antenna-biased expression. Furthermore, no chemosensory transcript is specific to female sex pheromone gland and very few are found in the heads. CONCLUSION: Our study revealed that there are a large number of chemosensory genes expressed in S. inferens, and some of them displayed unusual expression profile in non-chemosensory tissues. The identification of a large set of putative chemosensory genes of each subfamily from a single insect species, together with their different expression profiles provide further information in understanding the functions of these chemosensory genes in S. inferens as well as other insects.