Transcriptional Control of Lipid Metabolism.

Transcriptional control of lipid metabolism uses a framework that parallels the control of lipid metabolism at the protein or enzyme level, via feedback and feed-forward mechanisms. Increasing the substrates for an enzyme often increases enzyme gene expression, for example. A paucity of product can likewise potentiate transcription or stability of the mRNA encoding the enzyme or enzymes needed to produce it. In addition, changes in second messengers or cellular energy charge can act as on/off switches for transcriptional regulators to control transcript (and protein) abundance. Insects use a wide range of DNA-binding transcription factors (TFs) that sense changes in the cell and its environment to produce the appropriate change in transcription at gene promoters. These TFs work together with histones, spliceosomes, and additional RNA processing factors to ultimately regulate lipid metabolism. In this chapter, we will first focus on the important TFs that control lipid metabolism in insects. Next, we will describe non-TF regulators of insect lipid metabolism such as enzymes that modify acetylation and methylation status, transcriptional coactivators, splicing factors, and microRNAs. To conclude, we consider future goals for studying the mechanisms underlying the control of lipid metabolism in insects.

Delayed host mortality and immune response upon infection with P. aeruginosa persister cells.

Chronic infections are a heavy burden on healthcare systems worldwide. Persister cells are thought to be largely responsible for chronic infection due to their tolerance to antimicrobials and recalcitrance to innate immunity factors. Pseudomonas aeruginosa is a common and clinically relevant pathogen that contains stereotypical persister cells. Despite their importance in chronic infection, there have been limited efforts to study persister cell infections in vivo. Drosophila melanogaster has a well-described innate immune response similar to that of vertebrates and is a good candidate for the development of an in vivo model of infection for persister cells. Similar to what is observed in other bacterial strains, in this work we found that infection with P. aeruginosa persister cells resulted in a delayed mortality phenotype in Caenorhabditis elegans, Arabidopsis thaliana, and D. melanogaster compared to infection with regular cells. An in-depth characterization of infected D. melanogaster found that bacterial loads differed between persister and regular cells' infections during the early stages. Furthermore, hemocyte activation and antimicrobial peptide expression were delayed/reduced in persister infections over the same time course, indicating an initial suppression of, or inability to elicit, the fly immune response. Overall, our findings support the use of D. melanogaster as a model in which to study persister cells in vivo, where this bacterial subpopulation exhibits delayed virulence and an attenuated immune response.

Loss of Stearoyl-CoA desaturase 1 leads to cardiac dysfunction and lipotoxicity.

Diets high in carbohydrates are associated with type 2 diabetes and its co-morbidities, including hyperglycemia, hyperlipidemia, obesity, hepatic steatosis and cardiovascular disease. We used a high-sugar diet to study the pathophysiology of diet-induced metabolic disease in Drosophila melanogaster. High-sugar diets produce hyperglycemia, obesity, insulin resistance and cardiomyopathy in flies, along with ectopic accumulation of toxic lipids, or lipotoxicity. Stearoyl-CoA desaturase 1 is an enzyme that contributes to long-chain fatty acid metabolism by introducing a double bond into the acyl chain. Knockdown of stearoyl-CoA desaturase 1 in the fat body reduced lipogenesis and exacerbated pathophysiology in flies reared on high-sucrose diets. These flies exhibited dyslipidemia and growth deficiency in addition to defects in cardiac and gut function. We assessed the lipidome of these flies using tandem mass spectrometry to provide insight into the relationship between potentially lipotoxic species and type 2 diabetes-like pathophysiology. Oleic acid supplementation is able to rescue a variety of phenotypes produced by stearoyl-CoA desaturase 1 RNAi, including fly mass, triglyceride storage, gut development and cardiac failure. Taken together, these data suggest a protective role for monounsaturated fatty acids in diet-induced metabolic disease phenotypes.

Tissue-specific analysis of lipid species in Drosophila during overnutrition by UHPLC-MS/MS and MALDI-MSI.

Diets high in calories can be used to model metabolic diseases, including obesity and its associated comorbidities, in animals. Drosophila melanogaster fed high-sugar diets (HSDs) exhibit complications of human obesity including hyperglycemia, hyperlipidemia, insulin resistance, cardiomyopathy, increased susceptibility to infection, and reduced longevity. We hypothesize that lipid storage in the high-sugar-fed fly's fat body (FB) reaches a maximum capacity, resulting in the accumulation of toxic lipids in other tissues or lipotoxicity. We took two approaches to characterize tissue-specific lipotoxicity. Ultra-HPLC-MS/MS and MALDI-MS imaging enabled spatial and temporal localization of lipid species in the FB, heart, and hemolymph. Substituent chain length was diet dependent, with fewer odd chain esterified FAs on HSDs in all sample types. By contrast, dietary effects on double bond content differed among organs, consistent with a model where some substituent pools are shared and others are spatially restricted. Both di- and triglycerides increased on HSDs in all sample types, similar to observations in obese humans. Interestingly, there were dramatic effects of sugar feeding on lipid ethers, which have not been previously associated with lipotoxicity. Taken together, we have identified candidate endocrine mechanisms and molecular targets that may be involved in metabolic disease and lipotoxicity.

Drosophila as a model to study obesity and metabolic disease.

Excess adipose fat accumulation, or obesity, is a growing problem worldwide in terms of both the rate of incidence and the severity of obesity-associated metabolic disease. Adipose tissue evolved in animals as a specialized dynamic lipid storage depot: adipose cells synthesize fat (a process called lipogenesis) when energy is plentiful and mobilize stored fat (a process called lipolysis) when energy is needed. When a disruption of lipid homeostasis favors increased fat synthesis and storage with little turnover owing to genetic predisposition, overnutrition or sedentary living, complications such as diabetes and cardiovascular disease are more likely to arise. The vinegar fly Drosophila melanogaster (Diptera: Drosophilidae) is used as a model to better understand the mechanisms governing fat metabolism and distribution. Flies offer a wealth of paradigms with which to study the regulation and physiological effects of fat accumulation. Obese flies accumulate triacylglycerols in the fat body, an organ similar to mammalian adipose tissue, which specializes in lipid storage and catabolism. Discoveries in Drosophila have ranged from endocrine hormones that control obesity to subcellular mechanisms that regulate lipogenesis and lipolysis, many of which are evolutionarily conserved. Furthermore, obese flies exhibit pathophysiological complications, including hyperglycemia, reduced longevity and cardiovascular function - similar to those observed in obese humans. Here, we review some of the salient features of the fly that enable researchers to study the contributions of feeding, absorption, distribution and the metabolism of lipids to systemic physiology.

A Drosophila model of high sugar diet-induced cardiomyopathy.

Diets high in carbohydrates have long been linked to progressive heart dysfunction, yet the mechanisms by which chronic high sugar leads to heart failure remain poorly understood. Here we combine diet, genetics, and physiology to establish an adult Drosophila melanogaster model of chronic high sugar-induced heart disease. We demonstrate deterioration of heart function accompanied by fibrosis-like collagen accumulation, insulin signaling defects, and fat accumulation. The result was a shorter life span that was more severe in the presence of reduced insulin and P38 signaling. We provide evidence of a role for hexosamine flux, a metabolic pathway accessed by glucose. Increased hexosamine flux led to heart function defects and structural damage; conversely, cardiac-specific reduction of pathway activity prevented sugar-induced heart dysfunction. Our data establish Drosophila as a useful system for exploring specific aspects of diet-induced heart dysfunction and emphasize enzymes within the hexosamine biosynthetic pathway as candidate therapeutic targets.

Role of fat body lipogenesis in protection against the effects of caloric overload in Drosophila.

The Drosophila fat body is a liver- and adipose-like tissue that stores fat and serves as a detoxifying and immune responsive organ. We have previously shown that a high sugar diet leads to elevated hemolymph glucose and systemic insulin resistance in developing larvae and adults. Here, we used stable isotope tracer feeding to demonstrate that rearing larvae on high sugar diets impaired the synthesis of esterified fatty acids from dietary glucose. Fat body lipid profiling revealed changes in both carbon chain length and degree of unsaturation of fatty acid substituents, particularly in stored triglycerides. We tested the role of the fat body in larval tolerance of caloric excess. Our experiments demonstrated that lipogenesis was necessary for animals to tolerate high sugar feeding as tissue-specific loss of orthologs of carbohydrate response element-binding protein or stearoyl-CoA desaturase 1 resulted in lethality on high sugar diets. By contrast, increasing the fat content of the fat body by knockdown of king-tubby was associated with reduced hyperglycemia and improved growth and tolerance of high sugar diets. Our work supports a critical role for the fat body and the Drosophila carbohydrate response element-binding protein ortholog in metabolic homeostasis in Drosophila.

Polygenic adaptation to overnutrition reveals a role for cholinergic signaling in longevity.

Overnutrition by high-sugar (HS) feeding reduces both the lifespan and healthspan across taxa. Pressuring organisms to adapt to overnutrition can highlight genes and pathways important for the healthspan in stressful environments. We used an experimental evolution approach to adapt four replicate, outbred population pairs of Drosophila melanogaster to a HS or control diet. Sexes were separated and aged on either diet until mid-life, then mated to produce the next generation, allowing enrichment for protective alleles over time. All HS-selected populations increased their lifespan and were therefore used as a platform to compare allele frequencies and gene expression. Pathways functioning in the nervous system were overrepresented in the genomic data and showed evidence for parallel evolution, although very few genes were the same across replicates. Acetylcholine-related genes, including the muscarinic receptor mAChR-A, showed significant changes in allele frequency in multiple selected populations and differential expression on a HS diet. Using genetic and pharmacological approaches, we show that cholinergic signaling affects Drosophila feeding in a sugar-specific fashion. Together, these results suggest that adaptation produces changes in allele frequencies that benefit animals under conditions of overnutrition and that it is repeatable at the pathway level.

Reducing ether lipids improves Drosophila overnutrition-associated pathophysiology phenotypes via a switch from lipid storage to beta-oxidation.

High-calorie diets increase the risk of developing obesity, cardiovascular disease, type-two diabetes (T2D), and other comorbidities. These "overnutrition" diets also promote the accumulation of a variety of harmful lipids in the heart and other peripheral organs, known as lipotoxicity. However, the mechanisms underlying lipotoxicity and its influence on pathophysiology remain unknown. Our study uses genetics to identify the role of ether lipids, a class of potential lipotoxins, in a Drosophila model of overnutrition. A high-sugar diet (HSD) increases ether lipids and produces T2D-like pathophysiology phenotypes, including obesity, insulin resistance, and cardiac failure. Therefore, we targeted ether lipid biosynthesis through the enzyme dihydroxyacetonephosphate acyltransferase (encoded by the gene DHAPAT). We found that reducing DHAPAT in the fat body improved TAG and glucose homeostasis, cardiac function, respiration, and insulin signaling in flies fed a HSD. The reduction of DHAPAT may cause a switch in molecular signaling from lipogenesis to fatty acid oxidation via activation of a PPARα-like receptor, as bezafibrate produced similar improvements in HS-fed flies. Taken together, our findings suggest that ether lipids may be lipotoxins that reduce fitness during overnutrition.

dHNF4 regulates lipid homeostasis and oogenesis in Drosophila melanogaster.

The fly genome contains a single ortholog of the evolutionarily conserved transcription factor hepatocyte nuclear factor 4 (HNF4), a broadly and constitutively expressed member of the nuclear receptor superfamily. Like its mammalian orthologs, Drosophila HNF4 (dHNF4) acts as a critical regulator of fatty acid and glucose homeostasis. Because of its role in energy storage and catabolism, the insect fat body controls non-autonomous organs including the ovaries, where lipid metabolism is essential for oogenesis. The present paper used dHNF4 overexpression (OE) in the fat bodies and ovaries to investigate its potential roles in lipid homeostasis and oogenesis. When the developing fat body overexpressed dHNF4, animals exhibited reduced size and failed to pupariate, but no changes in body composition were observed. Conditional OE of dHNF4 in the adult fat body produced a reduction in triacylglycerol content and reduced oogenesis. Ovary-specific dHNF4 OE increased oogenesis and egg-laying, but reduced the number of adult offspring. The phenotypic effects on oogenesis that arise upon dHNF4 OE in the fat body or ovary may be due to its function in controlling lipid utilization.

Meep, a Novel Regulator of Insulin Signaling, Supports Development and Insulin Sensitivity via Maintenance of Protein Homeostasis in Drosophila melanogaster.

Insulin signaling is critical for developmental growth and adult homeostasis, yet the downstream regulators of this signaling pathway are not completely understood. Using the model organism Drosophila melanogaster, we took a genomic approach to identify novel mediators of insulin signaling. These studies led to the identification of Meep, encoded by the gene CG32335 Expression of this gene is both insulin receptor- and diet-dependent. We found that Meep was specifically required in the developing fat body to tolerate a high-sugar diet (HSD). Meep is not essential on a control diet, but when reared on an HSD, knockdown of meep causes hyperglycemia, reduced growth, developmental delay, pupal lethality, and reduced longevity. These phenotypes stem in part from Meep's role in promoting insulin sensitivity and protein stability. This work suggests a critical role for protein homeostasis in development during overnutrition. Because Meep is conserved and obesity-associated in mammals, future studies on Meep may help to understand the role of proteostasis in insulin-resistant type 2 diabetes.

Similar effects of high-fructose and high-glucose feeding in a Drosophila model of obesity and diabetes.

As in mammals, high-sucrose diets lead to obesity and insulin resistance in the model organism Drosophila melanogaster (called Drosophila hereafter). To explore the relative contributions of glucose and fructose, sucrose's component monosaccharides, we compared their effects on larval physiology. Both sugars exhibited similar effects to sucrose, leading to obesity and hyperglycemia. There were no striking differences resulting from larvae fed high glucose versus high fructose. Some small but statistically significant differences in weight and gene expression were observed that suggest Drosophila is a promising model system for understanding monosaccharide-specific effects on metabolic homeostasis.

Effect of dietary additives on intestinal permeability in both Drosophila and a human cell co-culture.

Increased intestinal barrier permeability has been correlated with aging and disease, including type 2 diabetes, Crohn's disease, celiac disease, multiple sclerosis and irritable bowel syndrome. The prevalence of these ailments has risen together with an increase in industrial food processing and food additive consumption. Additives, including sugar, metal oxide nanoparticles, surfactants and sodium chloride, have all been suggested to increase intestinal permeability. We used two complementary model systems to examine the effects of food additives on gut barrier function: a Drosophila in vivo model and an in vitro human cell co-culture model. Of the additives tested, intestinal permeability was increased most dramatically by high sugar. High sugar also increased feeding but reduced gut and overall animal size. We also examined how food additives affected the activity of a gut mucosal defense factor, intestinal alkaline phosphatase (IAP), which fluctuates with bacterial load and affects intestinal permeability. We found that high sugar reduced IAP activity in both models. Artificial manipulation of the microbiome influenced gut permeability in both models, revealing a complex relationship between the two. This study extends previous work in flies and humans showing that diet can play a role in the health of the gut barrier. Moreover, simple models can be used to study mechanisms underlying the effects of diet on gut permeability and function.This article has an associated First Person interview with the first author of the paper.

A Complex Relationship between Immunity and Metabolism in Drosophila Diet-Induced Insulin Resistance.

Both systemic insulin resistance and tissue-specific insulin resistance have been described in Drosophila and are accompanied by many indicators of metabolic disease. The downstream mediators of insulin-resistant pathophysiology remain unclear. We analyzed insulin signaling in the fat body studying loss and gain of function. When expression of the sole Drosophila insulin receptor (InR) was reduced in larval fat bodies, animals exhibited developmental delay and reduced size in a diet-dependent manner. Fat body InR knockdown also led to reduced survival on high-sugar diets. To look downstream of InR at potential mediators of insulin resistance, transcriptome sequencing (RNA-seq) studies in insulin-resistant fat bodies revealed differential expression of genes, including those involved in innate immunity. Obesity-associated insulin resistance led to increased susceptibility of flies to infection, as in humans. Reduced innate immunity was dependent on fat body InR expression. The peptidoglycan recognition proteins (PGRPs) PGRP-SB2 and PGRP-SC2 were selected for further study based on differential expression studies. Downregulating PGRP-SB2 selectively in the fat body protected animals from the deleterious effects of overnutrition, whereas downregulating PGRP-SC2 produced InR-like phenotypes. These studies extend earlier work linking the immune and insulin signaling pathways and identify new targets of insulin signaling that could serve as potential drug targets to treat type 2 diabetes.

Seven-Up Is a Novel Regulator of Insulin Signaling.

Insulin resistance is associated with obesity, cardiovascular disease, non-alcoholic fatty liver disease, and type 2 diabetes. These complications are exacerbated by a high-calorie diet, which we used to model type 2 diabetes in Drosophila melanogaster Our studies focused on the fat body, an adipose- and liver-like tissue that stores fat and maintains circulating glucose. A gene regulatory network was constructed to predict potential regulators of insulin signaling in this tissue. Genomic characterization of fat bodies suggested a central role for the transcription factor Seven-up (Svp). Here, we describe a new role for Svp as a positive regulator of insulin signaling. Tissue-specific loss-of-function showed that Svp is required in the fat body to promote glucose clearance, lipid turnover, and insulin signaling. Svp appears to promote insulin signaling, at least in part, by inhibiting ecdysone signaling. Svp also impairs the immune response possibly via inhibition of antimicrobial peptide expression in the fat body. Taken together, these studies show that gene regulatory networks can help identify positive regulators of insulin signaling and metabolic homeostasis using the Drosophila fat body.

A high-sugar diet produces obesity and insulin resistance in wild-type Drosophila.

Insulin-resistant, 'type 2' diabetes (T2D) results from a complex interplay between genes and environment. In particular, both caloric excess and obesity are strongly associated with T2D across many genetic backgrounds. To gain insights into how dietary excess affects insulin resistance, we studied the simple model organism Drosophila melanogaster. Larvae reared on a high-sugar diet were hyperglycemic, insulin resistant and accumulated fat--hallmarks of T2D--compared with those reared on control diets. Excess dietary sugars, but not fats or proteins, elicited insulin-resistant phenotypes. Expression of genes involved in lipogenesis, gluconeogenesis and ß-oxidation was upregulated in high-sugar-fed larvae, as were FOXO targets, consistent with known mechanisms of insulin resistance in humans. These data establish a novel Drosophila model of diet-induced insulin resistance that bears strong similarity to the pathophysiology of T2D in humans.