HIGHLIGHTED ARTICLE GENETICS | INVESTIGATION

Glucose Induces Sensitivity to Oxygen Deprivation and Modulates Insulin/IGF-1 Signaling and Lipid Biosynthesis in Caenorhabditis elegans Anastacia M. Garcia,* Mary L. Ladage,*,1 Dennis R. Dumesnil,*,1 Khadiza Zaman,* Vladimir Shulaev,* Rajeev K. Azad,*,†,2 and Pamela A. Padilla*,2

*Department of Biological Sciences, and †Department of Mathematics, University of North Texas, Denton, Texas 76203 ORCID ID: 0000-0001-8532-0254 (P.A.P.)

ABSTRACT Diet is a central environmental factor that contributes to the phenotype and physiology of individuals. At the root of many human health issues is the excess of calorie intake relative to calorie expenditure. For example, the increasing amount of dietary sugars in the human diet is contributing to the rise of obesity and type 2 diabetes. Individuals with obesity and type 2 diabetes have compromised oxygen delivery, and thus it is of interest to investigate the impact a high-sugar diet has on oxygen deprivation responses. By utilizing the Caenorhabditis elegans genetic model system, which is anoxia tolerant, we determined that a glucose-supplemented diet negatively impacts responses to anoxia and that the insulin-like signaling pathway, through fatty acid and ceramide synthesis, modulates anoxia survival. Additionally, a glucose-supplemented diet alters lipid localization and initiates a positive chemotaxis response. Use of RNA-sequencing analysis to compare gene expression responses in animals fed either a standard or glucose-supplemented diet revealed that glucose impacts the expression of genes involved with multiple cellular processes including lipid and carbohydrate metabolism, stress responses, cell division, and extracellular functions. Several of the genes we identified show homology to human genes that are differentially regulated in response to obesity or type 2 diabetes, suggesting that there may be conserved gene expression responses between C. elegans fed a glucose-supplemented diet and a diabetic and/or obesity state observed in humans. These findings support the utility of the C. elegans model for understanding the molecular mechanisms regulating dietary-induced metabolic diseases. KEYWORDS sugar diet; oxygen deprivation; insulin signaling; gene expression; C. elegans

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HE chronic and excessive intake of calories relative to daily energy expenditure often results in major heath issues such as obesity, metabolic syndrome, and type 2 diabetes (Ogden et al. 2012; Sonestedt et al. 2012). A recent change in the Western human diet, in comparison to traditional diets of the past, has been an increase in dietary saturated fats and sugars (e.g., sugar-sweetened beverage intake rose 135% between 1977 and 2001) (de Koning et al. 2011). Glucose, which is an essential component

Copyright © 2015 by the Genetics Society of America doi: 10.1534/genetics.115.174631 Manuscript received January 15, 2015; accepted for publication March 2, 2015; published Early Online March 10, 2015. Supporting information is available online at http://www.genetics.org/lookup/suppl/ doi:10.1534/genetics.115.174631/-/DC1. 1 These authors contributed equally to this work. 2 Corresponding authors: University of North Texas, Department of Biological Sciences, Denton, TX 76203. E-mail: [email protected]; and University of North Texas, Department of Biological Sciences, Denton, TX 76203. E-mail: [email protected]

of metabolism and energy production, induces pancreatic b-cells to secrete insulin, which in turn facilitates the import of glucose into tissues such as muscle and adipose. However, a chronic overabundance of glucose and/or fructose will have deleterious effects on cellular and tissue functions. For example, an excess of dietary sugar leads to the inability of cells to respond correctly to insulin (insulin resistance), resulting in a decrease in glucose uptake by cells and an increase in glucose remaining within the circulatory system (hyperglycemia) (Brownlee 2001; Szablewski 2011). An excess of dietary sugars increases adipose tissue, triglycerides, and low-density lipoproteins (Szablewski 2011). An additional consequence of hyperglycemia is an increase in the glycosylation of proteins, which can negatively affect protein, tissue, and organ function. In humans, the vascular system is impacted by hyperglycemia at the micro- and macrovascular levels, affecting blood supply and oxygen delivery to organs. Additionally, lower limb function can be compromised, often resulting in amputation (Frisbee

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2007a,b). Although many hyperglycemia-induced physiological and cellular changes are well studied, the impact these changes have on stress responses is not well understood. Given that oxygen delivery can be compromised in the tissues of individuals with obesity and type 2 diabetes, it is of interest to investigate the impact of oxygen deprivation in the context of a high-sugar diet. Genetic model systems are instrumental for elucidating the molecular basis for pathological states that are relevant to humans. The nematode Caenorhabditis elegans has been effective in identifying molecular mechanisms, genotypes, and environmental conditions that regulate metabolism, lifespan, and stress responses (Albert and Riddle 1988; Powell-Coffman 2010; Kenyon 2011; Hashmi et al. 2013; Murphy and Hu 2013; Runkel et al. 2014). Through these efforts, the insulin signaling pathway and its role in stress responses and longevity have been well studied. In C. elegans the insulin/IGF-1-mediated signaling (IIS) pathway involves the daf-2 and daf-16 genes, which encode the insulin/IGF-1 receptor protein and a FOXO/fork-head transcription factor, respectively (Riddle et al. 1981; Albert and Riddle 1988; Larsen et al. 1995; Kimura et al. 1997; Lin et al. 1997; Larsen 2001). The activated insulin receptor (DAF-2) interacts with insulin-like ligands (e.g., INS-7) and signals AGE-1/PI3/AKT to repress nuclear translocation of the transcription factor DAF-16 (Kenyon 2010). Reduced IIS signaling, due to food deprivation or a mutation in daf-2, induces localization of DAF-16 to the nucleus where it links with other nuclear factors to induce expression of a variety of genes (Dorman et al. 1995). DAF-16 activation promotes dauer formation, increased lifespan, fat metabolism, stress responses, innate immunity, and oxygen deprivation tolerance (Kenyon et al. 1993; McElwee et al. 2003; Murphy et al. 2003; Mendenhall et al. 2006a; Oliveira et al. 2009). C. elegans has evolved the ability to survive low-oxygen environments and has therefore been utilized as a genetic model system to understand molecular responses to oxygen deprivation (Gray et al. 2004; Powell-Coffman 2010; Padilla et al. 2011; Pocock 2011; Ma et al. 2013). C. elegans survive severe oxygen deprivation (1 day of anoxia, ,0.001 kPa O2, standard OP50 Escherichia coli diet, 20°) by entering into a hypometabolic state referred to as suspended animation. Adult hermaphrodites exposed to 1 day of anoxia and then allowed to recover in a normoxic environment will display normal anatomy and behaviors (Paul et al. 2000; Van Voorhies and Ward 2000; Jiang et al. 2001; Padilla et al. 2002). Wild-type adult hermaphrodites exposed to long-term anoxia (.3 days) have a very low survival rate with the few survivors displaying abnormal motility and anatomy (Padilla et al. 2002; LaRue and Padilla 2011). However, daf-2(e1370) mutants are resistant to long-term anoxia exposure as well as the combination of oxygen deprivation and increased temperature (Scott et al. 2002; Mendenhall et al. 2006b; LaRue and Padilla 2011). A mutation in the hyl-2 gene, which encodes a ceramide synthase, leads to changes in cellular ceramide species and an anoxia sensitivity phenotype (Menuz et al. 2009). Although ceramides are also implicated in ischemic responses, the functional role ceramides

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have in anoxia survival is not well understood (Argraves et al. 2011; Novgorodov and Gudz 2011). Of interest are the recent findings that ceramides are associated with impaired insulin action and the incidence of type 2 diabetes (Galadari et al. 2013; Lopez et al. 2013; Larsen and Tennagels 2014; Turpin et al. 2014). Several studies have shown that a high glucose diet impacts the metabolism, development, and stress responses of C. elegans (Lee et al. 2009; Schlotterer et al. 2009; Choi 2011; Mondoux et al. 2011) and that pathways and cellular processes involving glucose metabolism are conserved in metazoans (Berninsone 2006; Forsythe et al. 2006; Hashmi et al. 2013; Kitaoka et al. 2013). Here, we use C. elegans to examine the impact a glucose diet has on oxygen deprivation responses and gene expression. We determined that a glucose-supplemented diet reduces the ability of adult C. elegans to survive anoxia exposure. Modulation of the insulin-like signaling pathway through the daf2(e1370) mutation leads to anoxia resistance in animals fed a high-glucose diet. However, this anoxia survival is suppressed by mutations in daf-16, fat (fatty acid metabolism), hyl-2 (ceramide synthesis), or sod (super oxide dismutase). To examine the impact a glucose diet has on gene expression, we used RNA sequencing (RNA-seq) analysis and determined that a glucose-supplemented diet alters the expression of several genes, including those that are important for metabolism and stress responses. These findings underscore the impact environment and genotype have on stress responses.

Materials and Methods Strains and culture conditions

The wild-type Bristol strain (N2) and mutant strains were cultured using NGM plates seeded with E. coli (OP50, HT115, or DPTS-OP50, as noted in each experiment) and raised at 20° as described (Brenner 1974). Animals were maintained on OP50 unless otherwise noted. Genetic mutants used for these studies include: daf-2(e1370), daf-16(mu86), daf-2(e1370); daf-16(mu86), fat-5(tm420); fat-6(tm331), daf-2(e1370); fat-5(tm420); fat-6(tm331), fat-6(tm331); fat-7(wa36), daf-2(e1370); fat-6(tm331); fat-7(wa36), hyl-2(tm2031), daf-2(e1370); hyl-2(tm2031), daf-2(e1370); sod-2(sj173); sod-3(sj134), glp-1(e2141), oga-1(ok1207), ced-3(n717), hif-1(ia4), egl-9(sa307), che-3(n2150), and odr-3(e1124). Adult hermaphrodites were allowed to lay eggs on a plate and the subsequent offspring developed on the specified diet. Anatomical markers used to identify developmental stage include gonad morphology or hours post L4-adult molt. Adult animals used in the assays were collected 22–26 hr post the L4-adult molt. To feed animals a sugar-supplemented diet, NGM plates were made (60 mm 3 15 mm Petri dishes filled with 10 ml NGM); the plates were allowed to solidify for at least 1 day before being seeded with 300 ml of appropriate glucose (D-(+)-glucose, Sigma) stock solution, prepared in diH2O, to reach the final desired glucose concentration. The concentration of glucose used depended on the specific experiment. For example, to make the 0.5% glucose plates, 300 ml

of a 925-mM stock was used, (50 mg/plate). Glucose solution was spread evenly, fully covering the entire plate, and allowed to dry and reach equilibrium in the media for at least 24 hr Plates were seeded with 300 ml of E. coli (OD600nm, 0.6 # 0.9) that was spread evenly, covering the plate. The bacteria solution was allowed to dry completely before animals were placed on the media. Freshly made plates were used within a 7-day period. Anoxia and paraquat exposure

Animals were placed into anoxia in BD Biobag type A anaerobic environmental chambers (BD Biosciences, Rockville MD) at 20° (Padilla et al. 2002, 2011). Anoxic conditions were verified using resazurin indicators (,0.001 kPa of O2 detection limit). Any trials that failed to reach anoxic conditions within 1.5 hr were nulled. After anoxia treatment, animals were allowed to recover for 1 day and the surviving animals were examined for an unimpaired or impaired (abnormal movement or morphology) phenotype. For oxidative stress assays using paraquat, 200 ml of a 150 mM of freshly made aqueous paraquat solution (Sigma-Aldrich) was distributed onto small NGM breeder plates seeded with OP50 E. coli. The plates contained either 0.5% glucose or no glucose. Media were allowed to dry before 10 synchronized 1-day-old adult hermaphrodites were transferred to each plate and kept at 20°. Animals were assayed on a daily basis to examine survival rate. Five independent plates were assayed. Oil Red O staining

To detect lipid levels in C. elegans of the specified genotypes, we used the Oil Red O method as described (O’Rourke et al. 2009). Briefly, 200–500 synchronized 1-day-old adult animals were collected and washed three times with 13 PBS. To permeabilize the cuticle, worms were resuspended in 120 ml of 13 PBS to which an equal volume of 23 MRWB buffer containing 2% paraformaldehyde was added. Samples were rocked for 1 hr at room temperature. Animals were then allowed to settle by gravity, supernatant was removed, and animals were washed with 13 PBS to remove paraformaldehyde. Worms were resuspended in 60% isopropanol and rocked for 15 min at room temperature to dehydrate. Animals were then allowed to settle, supernatant was removed, and 1 ml of 60% Oil Red O dye was added. Animals were rocked overnight at room temperature in the dye. After allowing worms to settle, dye was removed, samples were washed one time with 13 PBS and then resuspended in 200 ml of 13 PBS to which 0.01% Triton X-100 was added. Animals were mounted on 2% agarose slides and imaged with a Zeiss microscope and color camera. The total number of animals stained was N $ 200 for each of three independent experiments. At least 10 animals per experiment and genotype were randomly imaged and assayed for the presence of Oil Red O in the pharynx region. Fatty acid supplementation

Fatty acid supplementation was conducted as described (Deline et al. 2013). Briefly, NGM containing 0.1% Tergitol (NP-40) was autoclaved and cooled to 55°, after which oleic acid sodium

salt (sodium oleate, Nu-Chek Prep) was added to a final concentration of 300 mM. The supplemented plates were allowed to dry in the dark for 2–4 days and then seeded with 300 ml of OP50 E. coli (OD600nm, 0.6 # 0.9) that was allowed to dry in the dark for an additional 1–2 days. Embryos were then added to supplemented plates and incubated at 20° until they reached day 1 of adulthood. On day 1 of adulthood, animals were transferred to glucose-supplemented plates for 1 hr before being exposed to 24 hr of anoxia while on the glucose-supplemented plates. Carbohydrate chemotaxis experiments

Animals of the specified genotypes were raised on a diet of OP50 E. coli or DPTS OP50 E. coli (depending on the bacteria used in the assay). NGM plates were divided in two hemispheres, one containing the attractant to be tested plus OP50 E. coli and the other containing only OP50 E. coli. For each assay, 200 ml of the attractant solution (1.85 M D-glucose, 1.85 M sucrose, 1.85 M fructose, or 5 ml of isoamyl alcohol) was added to one hemisphere of the plate, allowed to soak into the NGM, and then seeded with an equally sized lawn of bacteria; the other hemisphere contained only a spot of bacteria. Synchronized 1-day-old adult hermaphrodites were collected, washed three times in M9 buffer, and 50–100 worms were pipetted onto the NGM agar at an equal distance from each food source. After 3 hr, the animals were assayed for presence in one of three locations: bacteria, attractant-supplemented bacteria, or agar. For each independent experiment, three attractant assay plates, three positive control plates (isoamyl alcohol, a known volatile attractant) (Bargmann et al. 1993), and three negative control plates (no attractant, two bacteria-only spots) were assayed simultaneously. This experiment was repeated three independent times. RNA isolation

Young nongravid adults were collected for mRNA isolation. We opted to collect animals by an egg laying procedure rather than a hypochlorite protocol to minimize potential exposure to stress (L1 starvation) or reactive oxygen species (ROS) induction by hypochlorite solution. Thus, to grow a large population of animals, gravid adults were allowed to lay eggs on several plates for 2–4 hr All adults were washed off with M9, and the embryos that remained on the plate were allowed to hatch. L1 animals were collected and placed on respective media plates (OP50 only or OP50 supplemented with 0.5% glucose). Approximately 10,000 animals (per trial) were grown to young adulthood, past the L4 molt but prior to egg production, before collection by gravity separation with M9 in 15-ml conical tubes. The animals were washed three times with M9 and allowed to settle by gravity separation to remove bacteria. Excess M9 was removed, and four volumes of Trizol per volume of animals were added. The tubes were immediately frozen in liquid nitrogen, thawed, and briefly vortexed; this step was repeated and then followed by a 5-min incubation at room temperature. To each tube, 0.2 ml of chloroform per 1 ml of Trizol was added, mixed gently for 15 sec, incubated at room temperature for 2–3 min, and then centrifuged at 12,000 3 g for 15 min

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at 4°. The colorless upper phase was transferred to an RNase-free tube. RNA was then purified using an Ambion PureLink RNA Mini kit (cat. no. 12183018A). Following RNA purification, the RNA was treated with DNase I (Life Technologies cat. no. 12185010), quantified using a NanoDrop spectrophotometer, and stored at 280°. RNA was isolated from three independent experiments. RNA-seq analysis

We performed next-generation RNA-seq experiments and analysis to decipher the expression pattern of genes differentially regulated by glucose in wild-type C. elegans. For a reliable statistical analysis, RNA sampling was done in three biological replicates both for wild-type and glucose-fed C. elegans. The RNA sequencing was done at the University of Texas Southwestern Genomics Core facility, which resulted in generation of 33.5 M, 30.9 M, and 33.9 M single-end reads 50 bp in length for the three wild-type replicates (mean 32.8 million reads), and 34 M, 32.5 M, and 32.1 M single-end reads 50 bp in length for the three glucose-fed replicates (mean 32.9 million reads). These deep sequencing raw transcriptome data were then used for a comprehensive quantitative analysis of differential gene regulation in glucose-fed C. elegans. We employed the frequently used, publicly available Tuxedo suite of programs for RNA-seq analysis, which includes the software Bowtie (Langmead et al. 2009), Tophat (Trapnell et al. 2009), and Cufflinks (Trapnell et al. 2010). Bowtie is an ultrahighthroughput sequence read aligner that takes the FASTQ files of sequence reads as input and performs alignment of reads onto the reference genome. Tophat utilizes the services of Bowtie to first obtain the read alignment and then parse the alignment to infer the exon–exon splice junctions. A database of putative junctions is built by examining the “islands” of reads aligning contiguously onto exonic locations. The splice junctions are confirmed by aligning the reads against this database; the two fragments of “junction” reads aligning to different genome locations provide the hints of 59 and 39 splice sites. Cufflinks utilizes the read alignment provided by Tophat to perform the differential expression analysis of transcripts. We used the C. elegans genome build WS195 at National Center for Biotechnology Information as the reference genome for obtaining the read alignment using Bowtie and the splice junctions using Tophat. Cufflinks was used to identify the genes in glucosefed C. elegans with significant alteration in their expressions compared to wild-type C. elegans. This is accomplished by examining the difference in their abundance under the two conditions. The abundance of a transcript is measured in terms of fragments per kilobase of transcript per million fragments mapped (FPKM), normalized for the transcript length and total number of cDNA fragments for a sample replicate (Trapnell et al. 2010). The difference in expression is obtained as the logarithm of fold change in abundance between the two conditions. Cufflinks performs a statistical significance test for differential expression of each transcript based on a negative binomial model estimated from the data (Trapnell et al. 2010). The transcripts passing the significance test [false discovery rate (FDR) adjusted P-value to be ,0.05] (Trapnell et al.

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2010) were examined further for their abundance fold change between the conditions. Custom programs were developed for further parsing of these data and classifying the differentially expressed genes based on functional annotation at WormBase database (http://www.wormbase.org/). The differentially expressing genes were further grouped into different functional classes using gene ontology-based hierarchical functional classification and the Database for Annotation, Visualization, and Integrated Discovery (DAVID) functional classification tool (http://david.abcc.ncifcrf.gov/home.jsp). We used the Gene List Venn Diagram program (http://genevenn.sourceforge. net) to compare our RNA-seq dataset with other gene expression datasets. Quantitative RT-PCR

Animals were collected at day 1 of adulthood for mRNA isolation. RNA extraction was performed as described above. Reverse transcription to generate cDNA was performed using the SuperScript III first-strand synthesis system for RT-PCR (Invitrogen, cat. no. 18080-051). Quantitative RT-PCR was carried out using a Viia7 Real-Time PCR system (Applied Biosystems) and following primer validation, results were analyzed using the comparative Ct method (Applied Biosystems Viia7 Real-Time PCR system, booklet 3). As per Hoogewijs et al. (2008), mRNA levels of Y45F10D.4 was the most consistent control for normalization. The average of at least two technical replicates was used for each independent experiment. Primer sequences are available upon request.

Results A glucose-supplemented diet impacts stress response

Although it is known that both genotype and environment will influence the responses to oxygen deprivation, less is understood regarding the impact diet has on oxygen deprivation responses (Scott et al. 2002; Mendenhall et al. 2009; Powell-Coffman 2010; LaRue and Padilla 2011). We determined that a glucose-supplemented diet reduces the ability of N2 wild-type C. elegans to survive severe oxygen deprivation (anoxia) (Figure 1A). As the concentration of glucose increased, the ability to survive anoxia decreased (Figure 1A). While conducting the anoxia sensitivity assays, we also examined whether the animal showed an impaired phenotype (abnormal movement or morphology) after anoxia exposure. A lower concentration of glucose (0.0625%) did not reduce anoxia survival rate but did increase impairment in the animal following anoxia exposure. The animals fed a glucose-supplemented diet had a decreased ability to survive shorter bouts of anoxia (Figure 1B). The animals were also sensitive to anoxia when fed a fructosesupplemented diet prior to anoxia treatment, indicating that both sugars negatively impact oxygen deprivation response (Supporting Information, Figure S1A). It is possible that glucose is altering the osmotic environment and thus leading to increased sensitivity to anoxia. To determine if other carbohydrate molecules that likely alter the osmotic environment

will also alter anoxia survival, we fed animals a mannitol- or sucrose-supplemented diet prior to anoxia exposure. The animals fed these carbohydrates survived anoxia, suggesting that osmotic stress per se does not alter anoxia survival (Figure S1A). The metabolism of sucrose requires an enzyme, which may be rate limiting, to cleave sucrose into fructose and glucose; this may be why sucrose did not suppress anoxia survival. C. elegans are maintained in the laboratory by feeding the standard OP50 E. coli strain (Brenner 1974), which is the E. coli strain used in these experiments. To assess if the bacteria’s capacity to import glucose affected the response C. elegans had to a glucose-supplemented diet, we used the E. coli glucose-transporter mutant strain DPTS-OP50. The anoxia sensitivity was not as pronounced in the animals fed 0.5% glucose with the glucose-transporter mutant strain of E. coli (DPTS-OP50), thus suggesting that the uptake and/or metabolism of glucose by the bacteria will also impact anoxia survival (Figure 1C). There was, however, a significant decrease in anoxia survival when the animals were fed a DPTS-OP50 diet supplemented with higher concentrations of glucose (2%) (Figure 1C). These data indicate that a glucose-supplemented diet alters anoxia survival and that the specific bacteria strain used as a food source does affect this response. There is evidence that a high-glucose diet increases the level of ROS (Schlotterer et al. 2009), and so we wanted to determine if a glucose-supplemented diet increased sensitivity to paraquat (an ROS generator). We found that adults raised on a 0.5% glucose diet were more sensitive to paraquat (Figure 1D), indicating that anoxia may not be the only stress response negatively affected by a glucose-supplemented diet. Insulin signaling, lipid biosynthesis, and super oxide dismutase modulates the anoxia response of glucose-fed animals

A mutation in the insulin-like receptor gene, daf-2, results in several well-studied phenotypes such as increased lifespan and stress responses. The daf-2(e1370) animals fed a glucosesupplemented diet have a higher anoxia survival rate in comparison to wild-type animals (Figure 2A). The daf-2(e1370) animal’s sensitivity to anoxia did increase when fed a higher concentration of glucose (.1%). The ability for daf-2(e1370) animals to survive anoxia, after being fed a glucose diet (0.5%), is daf-16 dependent (Figure 2B). Another known genetic mutant that survives long-term anoxia, glp-1(e2141), is not resistant to anoxia when fed a glucose-supplemented diet (Figure S1B) (Mendenhall et al. 2009; LaRue and Padilla 2011). Furthermore, the daf-2(e1370); glp-1(e131) double mutant fed a glucose-supplemented diet did not have a significantly higher anoxia survival phenotype in comparison to daf-2(e1370) animals. (Figure S1B). These data indicate that anoxia resistance per se does not minimize the negative impact of a glucosesupplemented diet. Other genetic mutations we examined did not enhance anoxia survival in animals fed a glucosesupplemented diet (HIF-1 signaling, programmed cell death, or O-linked N-acetylglucosamine modifications) (Figure S1C). DAF-16 activation promotes increased lifespan, fat metabolism, stress responses, innate immunity, and anoxia toler-

ance (Kenyon et al. 1993; McElwee et al. 2003; Murphy et al. 2003; Mendenhall et al. 2006a; Oliveira et al. 2009). We used RNA interference (RNAi) to screen through a subset of DAF-16 regulated genes and determined that knockdown of fat-7, which encodes a D-9 desaturase involved with lipid biosynthesis, suppresses the daf-2(e1370) glucose-fed anoxia resistance phenotype (Figure 2C). The biosynthetic pathway involved with the catalysis of short-chain fatty acids to polyunsaturated fatty acids is known in C. elegans (Watts 2009). By using RNAi, we determined that knockdown of genes that code for enzymes involved in lipid biosynthesis also suppresses the daf-2(e1370) glucose-fed anoxia resistance phenotype (Figure 2C). The fat-5; fat-6 or fat-6; fat-7 double mutations also suppress the ability for daf-2(e1370) animals to survive anoxia after being fed a glucose-supplemented diet (Figure 2D). Note that the fat5(tm420) and fat-6(tm331) are deletion alleles and most likely null mutations. The fat-7(wa36) is a base pair mutation leading to a premature stop codon that eliminates two transmembrane domains and is most likely a strong reduction-offunction allele (Brock et al. 2007). Other studies have shown that a mutation in the hyl-2 gene, which encodes a ceramide synthase, affects anoxia survival (Menuz et al. 2009). The hyl2(tm2031) mutation, which is a deletion allele, also suppresses the ability for glucose-fed daf-2(e1370) animals to survive anoxia (Figure 2D). These results indicate that modulation of lipid biosynthetic pathways influence anoxia survival in animals fed a glucose-supplemented diet. Another set of genetic mutations that suppresses the daf-2(e1370) glucose-fed anoxia resistance phenotype are mutations in the superoxide dismutase genes sod-2 and sod-3 (Figure 2D). Together, this indicates that daf-2(e1370) resistance to anoxia exposure when fed a glucose-supplemented diet may require multiple pathways. In C. elegans a high-sugar diet increases lipid stores, as detected by Nile Red staining (Nomura et al. 2010; Zheng et al. 2014). Using Oil Red O staining to detect lipids, we also observed that N2 wild-type animals fed a glucose-supplemented diet, relative to an OP50-only diet, have a significant increase in lipid droplets in the anterior (head and pharynx) region of the animal (Figure 3). The hyl-2(tm2031) animals fed a glucose-supplemented diet, compared to those fed a standard diet, did not have a significant accumulation of lipid droplets in the head/pharynx region of the animal (Figure 3C). Also, compared to N2 animals, hyl-2(tm2031) animals appeared to have an overall reduction in Oil Red O staining. As others have reported, daf-2(e1370) animals have an increase in lipids (Figure 3) (Ashrafi et al. 2003). In some, but not all, of the animals carrying the daf-2(e1370) allele and fed a standard OP50 diet, lipid droplets in the head/pharynx region were observed (Figure 3C). Together, these results further support the idea that a glucose-supplemented diet acts as an obesity mimetic in C. elegans and that both diet and genotype affect lipid distribution within the animal. We examined whether the lipid biosynthesis mutants have an overall sensitivity to anoxia when fed a standard OP50 diet and determined that with the exception of the

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Figure 1 A glucose-supplemented diet negatively impacts C. elegans ability to survive anoxia and paraquat exposure. (A) A diet supplemented with glucose (.0.125%) reduces the ability of C. elegans to survive exposure to 1 day of anoxia. Bar indicates a significant decrease in the number of animals alive in comparison to animals not fed glucose prior to anoxia exposure. The * indicates that animals fed an OP50-only diet had a significant increase in unimpaired animals after anoxia treatment in comparison to all animals fed a glucose diet (one-way ANOVA, Bonferonni multiple comparisons, P , 0.05; at least three independent experiments were conducted, with n $ 100). (B) Animals fed a glucose-supplemented diet are sensitive to decreased anoxia exposures. The * indicates that there was a significant decrease in survivorship in animals fed a glucose diet prior to anoxia exposure in comparison to animals fed a glucose-supplemented diet and not exposed to anoxia. The bar indicates a significant decrease in unimpaired phenotype in comparison to control animals not fed a glucose-supplemented diet (one-way ANOVA, Bonferonni multiple comparisons, P , 0.05; three independent experiments were conducted, with n $ 100). (C) The E. coli strain and glucose impact anoxia survival. OP50 is the standard C. elegans food; HT115 is the parent strain used for RNAi, and DPTS-OP50 has a mutation in a glucose transporter. The * indicates that there is a decrease in survival relative to control animals not fed a glucose-supplemented diet. The ** indicates a significant increase in impaired animals after anoxia exposure in comparison to animals not fed a glucose-supplemented diet (one-way ANOVA, Bonferonni multiple comparisons, P , 0.05; three independent experiments were conducted, with n $ 100). (D) Animals fed a glucose-supplemented diet are sensitive to the ROS generator paraquat. The 1-day-old adult animals were placed on OP50 E. coli food containing paraquat or 0.5% glucose and paraquat. Asterisk denotes a significant difference in percentage of survival (twoway ANOVA, P , 0.001). For all experiments, error bar equals standard deviation.

fat-6; fat-7 double mutant, all animals survive 1 day of anoxia treatment when fed a normal OP50 diet (Figure 4A). The hyl-2(tm2031) mutant, fed a standard OP50 diet, is sensitive to 2 days of anoxia exposure (Menuz et al. 2009) but this sensitivity to anoxia is suppressed by daf-2(e1370) (Figure 4B). The daf-2(e1370) mutant can survive 3 days of anoxia, yet this phenotype is suppressed by either hyl2(tm2031) or a glucose-supplemented diet (Figure 4B). These data suggest that both diet and ceramide biosynthesis can alter the ability of daf-2 animals to be resistant to long bouts of anoxia treatment but are not essential for daf-2

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animals to survive shorter bouts of anoxia. The long-term anoxia resistance phenotype observed in the daf-2(e1370) mutant (fed a standard diet) is not suppressed by mutations in sod-2; sod-3, fat-6; fat-7, or fat-5; fat-6 (Figure 4B). To determine if supplemental feeding of a specific fatty acid could rescue the anoxia sensitivity induced by glucosesupplementation, animals were raised on control media or on media supplemented with 0.3 mM sodium oleate from the L1 larval stage to 1-day-old adults (Deline et al. 2013). Oleate supplementation did rescue the anoxia sensitivity of daf-2; fat6; fat-7 mutant animals but not for the other genotypes tested

Figure 2 The insulin signaling pathway modulates glucose-fed anoxia survival rate. (A) The daf-2(e1370) animals, in comparison to wild-type animals, survive anoxia better after being fed a glucose-supplemented diet. Bar indicates that there was a significant increase in survivorship for daf-2(e1370) animals in comparison to wild-type animals given the same diet and anoxia exposure (two-way ANOVA, Bonferonni multiple comparisons, P , 0.05). (B) A mutation in daf-16 or knockdown via RNAi suppresses the ability of daf-2(e1370) animals to survive anoxia after being fed a glucose-supplemented diet. The * indicates a significant increase in survivorship for daf-2(e1370) animals fed a glucose-supplemented diet prior to anoxia exposure in comparison to other animals shown with the specified genotypes (one-way ANOVA, Bonferonni multiple comparisons, P , 0.05). (C) RNAi of genes involved with lipid biosynthesis suppresses the ability of glucose-fed daf-2(e1370) animals to survive 1 day of anoxia (0.5% glucose). Bar indicates that the number of animals alive is significantly different in comparison to daf-2(e1370) animals fed empty vector as a control (one-way ANOVA, Bonferonni multiple comparisons, P , 0.05). (D) Genetic mutations affecting lipid or ceramide biosynthesis suppress the ability of daf-2(e1370) animals to survive anoxia after being fed a glucose-supplemented diet. Bar indicates that the number of animals alive is significantly different in comparison to daf-2(e1370) animals fed empty vector as a control (one-way ANOVA, Bonferonni multiple comparisons, P , 0.05). For all experiments, hermaphrodites were examined; error bar equals standard deviation; at least three independent experiments were conducted, with n $ 100.

(Figure 4C). This may be due to the fact that fatty acid composition differs between the different mutants (Brock et al. 2007; Shi et al. 2013). To improve uptake of the oleate, we opted to not chronically feed the animals both glucose and oleate simultaneously; rather, animals were raised on the fatty acid supplemented media to maximize uptake and then transferred as adults to 0.5% glucose media and allowed to eat for 1 hr before being exposed to 24 hr of anoxia (Deline et al. 2013). This difference in methodology may explain why the

daf-2; fat mutants fed glucose for 1 hr, as opposed to being raised on glucose, had a higher anoxia survival rate (Figure 4C compared to Figure 2D). The 1-hr feeding of glucose was sufficient to induce the anoxia sensitivity in N2 and daf-2; hyl-2(tm2031) animals, and the addition of oleate did not suppress this sensitivity (Figure 4C). The glucose-fed daf-2 mutant survives anoxia as expected and oleate supplementation did not significantly alter survival rate (Figure 4C). This suggests that the hyl-2 mutation leads to an increased

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Figure 3 Glucose-supplemented diet induces lipid accumulation. Oil Red O staining was used to localize lipids within the animal. (A) Animals of specified genotypes fed a standard diet or a glucose-supplemented diet were stained with Oil Red O. Representative images of whole animals are shown. (B) Enlarged image of the anterior region of the animals. Lipids can be detected in the intestine, oocyte/germline, and pharynx regions. Arrow points to the posterior region of the terminal bulb of the pharynx, near the pharyngeal–intestinal valve. Note that glucose fed animals (N2, daf-2, and daf-2; hyl-2) contain lipid droplets in the pharynx region. For each genotype, there is more Oil Red O staining in glucose-fed animals relative to control-fed animals. (C) The presence or absence of lipids within the anterior region (head and pharynx) of the animal was assayed. At least 10 animals from three independent experiments were randomly imaged and assayed for the presence of lipid droplets, detected by Oil Red O, in the anterior and pharynx regions. Error bar equals standard deviation; bar indicates there was a significant difference in glucose-fed animals in comparison to OP50-fed control animals (one-way ANOVA, Bonferonni multiple comparisons, P , 0.05).

sensitivity to anoxia in glucose-fed animals in comparison to the fat mutants (Figure 4C). C. elegans display positive chemotaxis behavior to a glucose-supplemented diet

Many organisms use chemotaxis to seek out food sources, and it is known that C. elegans can sense their environment and specific food sources. To further examine the response C. elegans

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has to a glucose diet, we set up an assay to determine if the animals distinguish between a spot of food that is the standard OP50 E. coli diet or a spot of OP50 E. coli mixed with a specific sugar. We found that a significantly higher number of animals selected the E. coli diet supplemented with a sugar compared to the control diet (Figure 5A). This suggests that C. elegans can sense specific carbohydrates within their diet. In these experiments, isoamyl alcohol was used as a positive control for

Figure 4 Lipid biosynthesis impacts anoxia survival. (A) Animals of the specified genotype were fed a standard OP50 diet and exposed to 1 day of anoxia. The fat6; fat-7 double mutant is sensitive to 1 day of anoxia exposure. The * indicates a significant decrease in survival compared to other genotypes (one-way ANOVA, Bonferonni multiple comparisons, P , 0.001). (B) Animals of the specified genotype were exposed to 2 or 3 days of anoxia. All animals were fed a standard OP50 diet with the exception of daf-2(e1370) as indicated. The * indicates that hyl-2(tm2031) animals exposed to 2 days of anoxia have a significant decrease in survival. The bar indicates that hyl-2(tm2031) or a glucose-supplemented diet suppresses the ability of daf-2(e1370) animals to survive 3 days of anoxia. (one-way ANOVA, Bonferonni multiple comparisons, P , 0.001). (C) Animals of the specified genotype were raised on a standard OP50 diet or a diet supplemented with oleate. Animals were then transferred to a glucose-supplemented diet, allowed to eat the glucose-supplemented diet for 2 hr, and then exposed to 24 hr of anoxia. The * indicates a significant increase in anoxia survival when fed oleate (one-way ANOVA, Bonferonni multiple comparisons, P , 0.001). For all experiments, hermaphrodites were examined; error bar equals standard deviation; at least three independent experiments were conducted, with n $ 100.

chemotaxis. Disruption of lipid biosynthesis did not alter the animal’s ability to chemotax toward a glucose-supplemented OP50 diet (Figure 5B). However, the statistically significant difference between food choices was diminished in the hyl2(tm2031) animals (P , 0.05) in comparison to other genotypes (P , 0.001). The ability to sense a glucose diet was examined in two genetic mutants that are known to have altered chemotaxis and environmental sensing. A mutation in odr-3, a gene required for chemotaxis and odorant avoidance, impacts the ability to sense isoamyl alcohol (the positive control), yet had no impact on the ability of worms to select bacteria supplemented with glucose as a food source (Figure 5C). A significant number of odr-3(e1124) animals selected to feed on the bacteria supplemented with glucose. The che-3 gene encodes a dynein heavy chain that affects the integrity of sensory cilia, thus affecting neuronal function. A mutation in che-3(n2150) did impact the ability of worms to select bacteria supplemented with glucose, suggesting that C. elegans use neuronal functions to sense glucose (Figure 5D). As expected, the che-3(n2150) animals did not sense and chemotax toward the isoamyl alcohol (positive control). Note that, unlike N2 or lipid mutants, the odr-3(e1124) and che-3(n2150) animals were found on other regions of the plate in which no food was present (agar region); these data were included in the analysis. Together, these results indicate that C. elegans can sense a glucose diet and that disruption of nervous system function via sensory cilia can alter this feeding behavior. Transcriptional changes induced by a glucose-supplemented diet

To identify gene expression changes associated with a glucose-supplemented diet, we used RNA-seq to compare the transcript profile of wild-type adults (young, nongravid)

raised on a glucose-supplemented diet relative to those raised on an OP50-only diet. The glucose-supplemented diet impacted the gene expression of 2370 of the 20,375 genes analyzed; 1850 genes were up-regulated and 520 genes were downregulated (Table S1, Figure 6A). The fold increase in expression values (normalized read count, FPKM; see Materials and Methods) for significantly up-regulated genes in animals fed a glucosesupplemented diet ranged from 1.34 to 136.99 (Table S2). On the other hand, the fold decrease in expression of down-regulated genes in animals fed a glucose diet ranged from 1.34 to 9.63 (Table S2). Genes differentially regulated by a glucose diet were classified and evaluated using PANTHER (http://www.pantherdb.org), WormBase annotation (http://www.wormbase.org), and DAVID (http://david.abcc.ncifcrf.gov/home.jsp) (Chen et al. 2005; Huang da et al. 2009; Mi et al. 2013). The PANTHER classification system combines gene function, ontology, and pathways to analyze large datasets (Mi et al. 2013). Based on the PANTHER biological processes classification, genes involved with metabolic processes were the gene class that displayed the highest number of alterations in gene expression in response to a glucosesupplemented diet (Figure 6, B and C). PANTHER was also used to classify the differentially expressed genes identified from the RNA-seq experiment based on cellular processes (Figure S2), molecular function (Figure S3), and protein class (Figure S4). Using WormBase, PANTHER, and KEGG, we further classified some of the genes that were up-regulated (Table S3) or down-regulated (Table S4). Several genes that are known or predicted to be involved with lipid biology were up-regulated in response to glucose supplementation (Table S3); these genes included those that are involved with lipid processes, lipid binding, or lipid transport. This list includes genes predicted to encode stearoyl-CoA desaturase (delta-9 desaturase) (fat-5),

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Figure 5 C.elegans chemotax toward a sugar-supplemented OP50 E. coli diet. Adult animals, of the specified genotypes, were allowed to roam on the plate for 2–4 hr (20°) before the number of animals grazing on a specific diet was quantified. Data are represented here as the percentage of animals grazing on the food choice indicated. Isoamyl alcohol is a positive control and the no-attractant plate contained identical bacterial spots (negative control). (A) N2 animals were assayed to determine if they do or do not select bacteria supplemented with a sugar. Bar indicates the diet in which a significant number of animals grazed on OP50 bacteria + indicated attractant in comparison to the OP50-only diet (two-way ANOVA, Bonferroni multiple comparisons, P , 0.001). (B) Animals of the specified genotypes were assayed to determine if they do or do not select bacteria supplemented with glucose. There is a significant increase in animals, of all genotypes, to select a glucose-supplemented diet (***P , 0.0001, **P , 0.01, *P , 0.05, two-way ANOVA, Bonferroni multiple comparisons). (C) The odr-3(e1124) and (D) che-3(n2150) animals were assayed to determine if the mutation affected the ability to select a glucose-supplemented diet. The * indicates a significant increase in animals’ selection of a glucose-supplemented diet (two-way ANOVA, Bonferroni multiple comparisons, P , 0.001). Error bar equals standard deviation; at least three independent experiments were conducted, with n $ 100 animals.

long-chain acyl-CoA synthetase (acs-2, acs-15, acs-18), and ceramide glycosyl transferase (cgt-1), indicating that a glucosesupplemented diet induces genes involved with lipid biosynthesis and modification. Using the PANTHER program, we were able to classify genes classes (Table S3). There were 112 genes (6% of the up-regulated genes) that were classified by PANTHER to be involved with some aspect of lipid metabolism. Zhang et al. (2013) documented lipid genes in the C. elegans genome. We used the Gene List Venn Diagram program (http://genevenn.sourceforge.net) to compare the genes that were-up-regulated by glucose to the dataset from Zhang et al. (2013) and identified 56 of the 471 genes to be up-regulated by glucose (11.9%) (Table S3). Genes that are known or predicted to be involved with carbohydrate metabolism (e.g., glycolytic genes gpd-2, gpd-3, or gluconeogenesis

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genes PEPCK pck-1, pck-3) or carbohydrate transport (sugar transporter swt-1) were also up-regulated in response to glucose. There were also a number of up-regulated genes that are predicted to be involved with the transfer of glycosyl groups, in addition to other genes that have catalytic activities (e.g., acyl transfer, oxidoreductase, hydrolase) (Table S3). The idea that a glucose-supplemented diet is stressful for the animal is supported by the fact that stress response genes were induced (e.g., chaperones, antioxidants, apoptotic processes, cellular defense, toxic substance response) (Table S3). Genes that are known or predicted to be part of the extracellular matrix (Gene Ontology, GO: 0031012) or extracellular region (GO: 0005576) were the largest class of differentially expressed genes when analysis was based on cellular component (Figure S2A, Table S3).

Figure 6 RNA-sequencing analysis reveals differentially regulated genes in response to a glucose-supplemented diet. (A) Scatter plot shows expression values (normalized read counts, FPKM; see Materials and Methods) for transcripts in animals fed a standard OP50 E. coli diet (x-axis) vs. those in a glucose-supplemented diet (y-axis); data are shown on log scale. Transcripts that were up-regulated or down-regulated in response to a glucose diet are shown. (B and C) Genes were categorized based on Gene Ontology (GO) annotations for biological functions; the proportion for each category is displayed in the pie chart; B shows the gene expression profile for those up-regulated in glucose condition, and C shows the gene expression profile for those down-regulated in glucose condition. For both B and C, the GO classification analysis was performed using PANTHER.

There are a fewer number of genes that were downregulated in response to a glucose-supplemented diet (Table S1), and only a small number of these genes are predicted to be involved with lipid or carbohydrate biology (Table S4). Although, two of the genes that code for the glycolytic enzyme GPD (gpd-2 and gpd-3) were up-regulated in the glucose-fed animals, the other two genes that code for the GPD glycolytic enzyme (gpd-1 and gpd-4) were down-regulated in response to glucose. The mRNA expression of daf-16 itself was not differentially regulated by a glucose diet. Additionally, genes (sod-2/3 and hyl-2) that suppress the daf-2(e1370) glucosefed anoxia resistant phenotype were not differentially regulated by a glucose diet. However, there was a down-regulation of several genes (daf-18, ins-8, daf-15, dct-16, and smg-1) involved with the insulin-like signaling pathway, indicating that a glucose diet can impact the mRNA expression of genes

involved with insulin signaling. There are many labs that have identified genes that are potentially regulated by DAF-16 (Murphy et al. 2003; Murphy 2006; Lee et al. 2009; Riedel et al. 2013). Given that daf-2(e1370) animals survive anoxia and N2 animals do not survive anoxia when fed a glucose diet, it is of interest to identify genes that are expressed in daf2(e1370) and down-regulated by a glucose diet. We identified 11 genes that were found by others to be regulated in a DAF16-dependent manner and that we found to be down-regulated in N2-fed glucose (Table S4). A number of genes that were down-regulated by glucose supplementation were classified as nucleobase-containing compound associated with metabolic process (GO: 0006139), which contain genes predicted to be involved with DNA or RNA metabolic processes (Table S4). Examples of genes within this classification include those known or predicted to be involved with DNA repair, DNA replication, DNA recombination, RNA splicing, RNA transcription, RNA processing, or nucleobase metabolism. Furthermore, if one looks at the genes that are classified by protein class (Figure S4B), the largest grouping was for the nucleic acid binding protein class (PC00171). We also found that a large group of genes known or predicted to be involved with the biological process of cell cycle were downregulated (Table S4); many of these genes are also classified as being in the DNA or chromosome binding protein class. Together, these data indicate that glucose supplementation alters many processes and is likely a stress for the animal. We used quantitative RT-PCR to verify a few genes identified by the RNA-seq approach. Three of the four genes tested had a significant difference in mRNA change between the glucose supplemented compared to the OP50 fed (Figure S5). We used BLASTp analysis to identify the human proteins that have high identity and query coverage in local alignment to the most differentially expressed C. elegans genes (.25-fold upregulated or .2.5-fold down-regulated) in glucose-fed animals. With this classification, there are 63 genes that are up-regulated .25-fold (Table S5); 31 of these genes (49%) are predicted or known to encode a collagen protein. Several of these genes (17/63; 27%) did not have a significant similarity to any human genes. The other genes were known or predicted to be involved with various processes including metabolism or stress responses. There were 20 genes that were .2.5-fold downregulated in glucose-fed animals (Table S6); 4 of these genes (20%) did not have a significant similarity to any human genes. There were two cytochrome P450 genes (cyp-25A1 and cyp35D1) that were down-regulated in response to a glucose diet. Evaluation of differentially expressed genes (Table S2) indicates that five other cytochrome P450 genes were actually up-regulated (cyp-13B1, cyp-33C5, cyp-32A1, cyp-13A12, cyp33C8); cyp-13A12 has been implicated in responses to O2 deprivation and reoxygenation (Ma et al. 2013). The gene T19H12.6 that was down-regulated has not been studied in C. elegans but is predicted to encode a protein that is part of the gamma glutamyl transferase/peptidase (GGT) family (PTHR11686) and shows identity to the human GGT1. The GGT proteins cleave g-glutamyl peptide bonds in glutathione

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and transfer the g-glutamyl moiety to acceptors; they are associated with diseases such as diabetes and metabolic syndrome (Hong et al. 2014). A positive association exists between insulin and GGT (Konya et al. 1990), and GGT1 was found to be down-regulated in whole blood gene expression analysis of obese subjects with type 2 diabetes after bariatric surgery or weight loss (Berisha et al. 2011). This suggests that there may be some conserved genes that respond to a glucose-supplemented diet in C. elegans and genes that are associated with metabolic diseases in humans. Others used microarray analysis to identify genes that are differentially regulated when fed a 2% glucose diet (Lee et al. 2009). Of the 40 genes identified in their study, 13 of the genes were also identified by our RNA-seq analysis (Table S7). The experimental procedure in our study compared with the Lee et al. (2009) study was not identical, which may contribute to the differences in the genes identified. The studies differed in the concentration of supplemented glucose, the developmental stage (we used young adults prior to egg lay and they used 5-fluorodeoxyuridine (FUDR)-fed 1-day-old adults) and we synchronized worms using egg-lay technique instead of hypochlorite. We used BLASTp analysis to identify other C. elegans genes that are differentially regulated by a glucose diet and have similarity to human genes that are known or thought to be involved with metabolic syndrome, obesity, and/or type 2 diabetes or obesity (Table 1). We identified genes involved with cellular energy metabolism, insulin or glucose regulation, and lipid biosynthesis. Additionally, we identified human homologs that are differentially regulated in a diabetic or obese physiological state, for C. elegans genes that were up-regulated in animals fed a glucose-supplemented diet (e.g., asm-2; UGCG ceramide glucosyltransferase) (Schuchman et al. 1992; Boini et al. 2010; Blachnio-Zabielska et al. 2012; Li et al. 2012). Our findings from this study indicate that there are potentially conserved gene expression responses between C. elegans fed a glucose-supplemented diet and a diabetic and/or obesity state observed in humans.

Discussion A chronic increase in sugary substances within the human diet is associated with severe health issues (e.g., obesity, metabolic syndrome, type 2 diabetes, cardiovascular dysfunction) as well as tissue and metabolic dysfunctions (e.g., insulin resistance, hyperglycemia, increase in serum triglycerides). Dietary-induced hyperglycemia can disrupt autoregulation of blood flow, resulting in oxygen deprivation of peripheral tissue and ineffective responses to ischemic events (e.g., myocardial infarction) (Trayhurn 2013). Furthermore, microvascular and macrovascular dysfunction can lead to multiorgan and tissue damage, blindness, and amputations (Creager et al. 2003; Beckman et al. 2013; Paneni et al. 2013). In fact, vascular diseases are the principal causes of death in people with type 2 diabetes. Central to the pathology of vascular disease is disruption of blood flow and oxygen deprivation. Thus, a thorough understanding of the

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impact that the metabolic abnormalities, associated with type 2 diabetes, have on oxygen deprivation responses is necessary for a complete understanding of the disease process. Here, our goal is to use C. elegans as a model to determine how a glucosesupplemented diet impacts oxygen deprivation responses and gene expression. Our findings along with what others have seen indicate that a glucose-supplemented diet is a stress for C. elegans. We found that glucose alters the expression of several genes classified as stress response genes. However, this response does not enhance the capacity to survive additional stresses (anoxia, paraquat). This is inline with the idea that glucose is a stress and that an additional stress (anoxia), which can require overlapping but different responses for survival, overwhelms the system and leads to compromised viability (Figure 7). Furthermore, others have found a glucose diet reduces lifespan, dauer formation, and increases ROS. Yet, a mutation in the daf-2 insulin/IGF-1 receptor led to anoxia resistance even when fed a glucose-supplemented diet, which is not all that surprising given that the daf-2 animal is known to be resistant to stress. Many of the phenotypes associated with the daf-2(e1370) mutant are through the activation of the FOXO/forkhead transcription factor DAF-16; a mutation in daf-16 did indeed suppress the daf2(e1370) glucose-fed anoxia survival phenotype. Using a genetic approach, we identified some downstream targets of DAF-16 (fat and sod genes) and a DAF-16 independent gene (hyl-2) that are required for the glucose-fed anoxia-resistant phenotype exhibited by the daf-2(e1370) mutant. Since excess glucose is thought to increase ROS levels (Schlotterer et al. 2009), it seems appropriate that antioxidant genes such as sod-2/3, which are activated in the daf-2(e1370) animal, will protect against the glucose-supplemented diet. It is not clear what the protective role fatty acid and ceramide biosynthesis has on the daf-2(e1370) animal fed a glucose-supplemented diet and exposed to anoxia. However, considering the phenotypes exhibited by the mutations that suppress the daf-2(e1370) glucose-fed anoxia-resistant phenotype may shed some light on the mechanisms required for anoxia survival. The fat and elo mutants have distinct fatty acid composition changes, with the fat-6; fat-7 double mutant displaying a defect in the production of unsaturated fatty acids and other phenotypes such as slow growth, reduced fertility, reduced lifespan, decreased fecundity, and increased expression of genes involved in fatty acid oxidation (Brock et al. 2007; Shi et al. 2013). All of the D9 desaturase mutants (fat-5, fat-6, and fat-7) display decreased cold tolerance and increased endurance to heat stress (Horikawa and Sakamoto 2010; Savory et al. 2011). Additionally fat-2, fat-6, and fat-7 mutants display strong sensitivity to oxidative stress (Horikawa and Sakamoto 2010). It is possible that in the daf2(e1370) mutant the lipid profile is altered in such a way that is conducive for maintenance of cellular structures and function during anoxia stress (Castro et al. 2013) and that knockdown of genes involved with lipid biosynthesis would disrupt lipid homeostasis. It has recently been shown that ceramides are involved with a surveillance system that detects mitochondrial defects (Liu et al. 2014); thus, it is possible that the

Table 1 Examples of C. elegans genes, with altered gene expression subject to a glucose-supplemented diet, that may be relevant to human disease Gene

Human gene (% identity)

Brief description

T19H12.6

GGT1 (28)

g-Glutamyl traspeptidase

ant-1.4

SLC25A6/ANT3 (71)

ADP/ADT translocator

aqp-7

AQP10 (36)

Aquaglyceroporin

lbp-6

FABP4 (48)

Fatty acid binding protein

C54E4.5

MIOX (51)

Inositol oxygenase

poml-3

PON2 (30)

Paraxonase

pkc-2

PRKCA (69)

Protein kinase C

asm-2

SMPD1 (37)

Acid spingomyelinase

cgt-1

UGCG (40)

Ceramide glucosyltransferase

R11F4.1

GK (50)

Glycerol kinase

ech-7

ECHS1 (56)

Enoyl-coenzye A hydratase

acs-15

ACSL1 (36)

Long-chain fatty acid CoA ligase

fat-5

SCD (47)

Stearoyl-CoA desaturase

ins-37; ins-8 ugt-62

Insulin Glucagon

Insulin-related peptide beta type Glucagon preprotein

fut-2

FUT1 (23)

Fucosyltransferase

wrt-4

DHH, IHH, SHH

Hedgehog

clec-70

CRD-4 (28)

col-60

COL6A3 (46)

C-type carbohydrate recognition domain Collagen

Associated disease state Associated with type 2 diabetes and obesity (; Konya et al. 1990; Berisha et al. 2011) ADP/ATP translocase family that plays a fundamental role in cellular energy metabolism; interacts with SIRT4 to regulate insulin secretion in response to glucose; differentially expressed in diabetic mice (Ahuja et al. 2007; Lu et al. 2008) AQP10 is expressed in human adipose tissue, functions to regulate glycerol efflux and thus a normal adiposity (protection from obesity) (Laforenza et al. 2013) FABP4 functions as an adipokine, regulates insulin secretion during obesity; associated with pathogenesis of atherosclerosis, type 2 diabetes, metabolic and vascular disease (Engeli et al. 2013; Kralisch and Fasshauer 2013; Lamounier-Zepter et al. 2013; Kim et al. 2014; Wu et al. 2014) Increase in MIOX activity is proportional to serum glucose concentrations; diabetic mice display increased MIOX in the cortex of the kidney (Nayak et al. 2005; Lu et al. 2009; Yang et al. 2010) Encode high density lipoprotein (HDL)-related glycoproteins; PON2 has a significant protective role against macrophage triglyceride accumulation, macrophage TG biosynthesis, microsomal DGAT1 activity and macrophage oxidative stress, under high glucose concentrations in mice (Meilin et al. 2010) Type 2 diabetic rats display persistent translocation and activation of PKC in soleus muscles, and this persistent PKC activation may contribute to impaired glycogen synthesis and insulin resistance (Avignon et al. 1996) Stress is thought to activate sphingomyelinase to generate ceramide, which serves as a second messenger in initiating the apoptotic response; expression higher in obese diabetic patients (Schuchman et al. 1992; Boini et al. 2010; Blachnio-Zabielska et al. 2012Li et al. 2012;) Catalyzes the first glycosylation step in glycosphingolipid biosynthesis; lipid/sugar membrane components; synthase in central nervous system regulates body weight and energy homeostasis (Nordstrom et al. 2013) Glycerol kinase deficiency has been implicated in in insulin resistance and type 2 diabetes mellitus; enhanced adipose glycerol kinase enzymatic activity in aquaporin 7 deficient obese mice (Hibuse et al. 2005; Rahib et al. 2007) Catalyzes the second step in mitochondrial fatty acid beta-oxidation; downregulated in WT mouse heart tissue in response to ischemic post-conditioning following ischemia–reperfusion. This effect is not observed in diabetic mouse heart tissue (Zhu et al. 2012) Key role in both the synthesis of cellular lipids and the degradation of fatty acids; increased expression is associated with an inflammatory macrophage phenotype and atherosclerosis in type 1 diabetes (Kanter et al. 2012) Delta-5 desaturase activity is altered in type 2 diabetes patients (Imamura et al. 2014); Role in the onset of diet-induced peatic insulin resistance (Gutierrez-Juarez et al. 2006) Insulin regulates glucose uptake; insulin resistance results in diabetes Control of carbohydrate metabolism (Bataille and Dalle 2014; Cho et al. 2014) FUT1; Fucosyl transferase; increased activity in human serum of type 2 diabetes; indicator of liver disease; FUT7 mutation in humans associated with noninsulin dependent diabetes (Bengtson et al. 2001) Hedgehog signaling is implicated in protecting beta-cells from cytokine-induced cytotoxicity (Puri et al. 2013; Teperino et al. 2014) C-type lectin; binds sugar; implicated in innate immunity response (Hitchen et al. 1998; Feinberg et al. 2000) Collagen; deposition in obesity patients (McCulloch et al. 2014)

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Figure 7 Intersection between glucose and anoxia stress. Work from our lab and others indicates that glucose is a stress and impacts various processes. Genetic analysis has revealed processes that have a role in either resistance or sensitivity to anoxia when fed a glucose diet.

daf-2(e1370) animal requires specific ceramides to defend against the toxic effects a glucose-supplemented diet has on the mitochondria. It remains to be determined if the sod-2/3 antioxidant system will function independently or in conjunction with ceramide and fatty acid molecules in promoting glucose-fed anoxia survival in the daf-2(e1370) animal. One outstanding question is how a glucose-supplemented diet promotes anoxia sensitivity in wild-type animals. Given the results obtained by feeding C. elegans either OP50 or the DPTS-OP50 E. coli strain, we hypothesize that both the glucose and the glucose-exposed bacteria will impact the stress responses of C. elegans. We focused on examining how a glucosesupplemented diet impacts wild-type C. elegans by using RNA-seq to compare the gene expression profiles between animals fed a glucose-supplemented diet relative to a standard diet. The differential expression of specific genes, in response to different environments, does not necessarily imply functional relevance or essential function of the genes relative to the environmental change. However, we did identify differentially expressed genes that are compatible with the notion that a glucose-supplemented diet alters many cellular functions, notably metabolism (glycolysis, gluconeogenesis, and lipid biosynthesis), stress responses (chaperons and cytochrome P450 genes), protein modifications (protein glycosylation), and cellular structure (collagen genes). The fact that the stress-response genes are differentially regulated implies that this glucose-supplemented diet is a stress to the animal and that the addition of another stress, like anoxia exposure or paraquat, could overload the stress response pathways. Lee et al. (2009) used microarray analysis to identify 40 genes that were differentially regulated in C. elegans fed a 2% glucose diet in comparison to control (Schlotterer et al. 2009; Choi 2011; Mondoux et al. 2011); 13 of these 40 genes were also identified by us using the RNA-seq approach. Genes that are involved with lipid metabolism were identified by both transcriptomic approaches, further supporting the idea that a glucose-supplemented diet alters lipid metabolism in C. elegans. Our transcriptomic approach identified many more genes that were differentially regulated by a glucose-supplemented diet. This may be due to the experimental approach to detect tran-

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script differences (RNA-seq in comparison to microarrays) or the methodological differences in glucose feeding (0.5% glucose in comparison to 2% glucose, young nongravid adults in comparison to 1-day-old adults fed FUDR). A glucose-supplemented diet induces accumulation of lipids in the pharynx region, as seen by the Oil Red O staining of C. elegans, which may be indicative of glucose being an obesity mimetic. Furthermore, a glucose-supplemented diet induced gene expression changes in many genes predicted to be involved with lipid metabolism or function. It is possible that the excess intake of sugars disrupts fatty acid and ceramide homeostasis in a way that leads to metabolic and cellular changes resulting in sensitivity to stresses like oxygen deprivation. The recent finding that specific ceramide species are associated with type 2 diabetes (Galadari et al. 2013; Lopez et al. 2013) further implicates the role these molecules may have in metabolic dysfunction. These findings underscore the impact environment can have on gene expression that will likely impact the physiology of an organism. It may be that a combination of cellular changes will ultimately contribute to the anoxia sensitivity. In wild-type animals, the mRNA expression of daf-16 or sod2/3 was not differentially regulated by a glucose-supplemented diet. However, there was a down-regulation of daf-18, ins-8, and daf-15, which are involved in the insulin/insulin-like signaling pathway but not necessarily directly regulated by DAF16 (Murphy and Hu 2013). We identified 11 genes that are down-regulated by a glucose diet and found by others to be regulated in a DAF-16-dependent manner. Further analysis of these genes would be helpful to determine if they have a role in the daf-2(e1370) glucose-fed anoxia resistance phenotype. The idea that a glucose-supplemented diet impacts insulin signaling is in line with the finding by others that dauer formation (which is regulated by insulin signaling) is suppressed in animals fed a glucose diet (Mondoux et al. 2011). Regulation of the insulinsignaling pathway is mediated through DAF-2 and DAF-18 with a downstream affect being the activation or deactivation of DAF-16 via post-translational phosphorylation/dephosphorylation. DAF-18, the human PTEN tumor suppressor homolog, negatively regulates insulin-like signaling by dephosphorylation of

AGE-1-generated PIP3, which impacts AKT-1/AKT-2 kinase activity thus regulating DAF-16 (Ogg and Ruvkun 1998). Mutations in daf-18 can suppress the dauer constitutive and longevity phenotype in daf-2 mutants. However, it is important to recognize that a daf-18 allele may not impact an organism in the same manner as a decrease in express of daf-18. Our studies show that a glucose-supplemented diet negatively impacts oxygen deprivation survival and significantly alters gene expression profiles in C. elegans. Genetic suppression analysis of the known stress-resistant mutant daf-2(e1370) revealed that antioxidant, fatty acid biosynthesis and ceramide synthesis are essential for the animal to survive anoxia when fed a glucose-supplemented diet. In wild-type animals, many genes were up-regulated in response to a glucose-supplemented diet and a large number of these genes are known or predicted to be involved with metabolism. We suggest that many processes (e.g., metabolism, cell signaling, stress responses) are altered in animals fed a high-glucose diet and that central to the glucose-induced anoxia sensitivity is mitochondria dysfunction via disruption of lipid metabolism, ceramide homeostasis, and ROS changes.

Acknowledgments We thank J. Watts and S. J. Lee for C. elegans or E. coli strains used in this work, S. Brumbley and L. Main for valuable technical assistance, and the Caenorhabditis elegans Genetics Stock Center (CGC), which is funded by National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). This work was supported by a grant from the National Science Foundation (CAREER 0747391) to P.A.P. and research support from the University of North Texas Office of Research and Economic Development, College of Arts and Sciences, and Department of Biological Sciences to P.A.P., R.K.A., and V.S.

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GENETICS Supporting Information http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.115.174631/-/DC1

Glucose Induces Sensitivity to Oxygen Deprivation and Modulates Insulin/IGF-1 Signaling and Lipid Biosynthesis in Caenorhabditis elegans Anastacia M. Garcia, Mary L. Ladage, Dennis R. Dumesnil, Khadiza Zaman, Vladimir Shulaev, Rajeev K. Azad, and Pamela A. Padilla

Copyright © 2015 by the Genetics Society of America DOI: 10.1534/genetics.115.174631

  Figure S1   (A) A diet supplemented with fructose (0.5%) reduces the ability of C. elegans to survive when exposed to 1 day of anoxia.  (B) The long-term anoxia survival mutant glp-1(e2141) is sensitive to 1 day of anoxia exposure when fed a glucose-supplemented diet;  daf-2(e1370) suppresses the anoxia sensitivity observed in glp-1(e2141) mutants. Note that in order to conduct these experiments the  animals had to be raised at 15°C to the L3 stage and then transferred to 25°C. Bar indicates a significant increase in survival in  comparison to N2 animals (1 way ANOVA, Bonferonni Multiple Comparisons, p25 Fold Change

85 (4.59%)

0

5>10 Fold Change

522 (28.22%)

5 (.96%)

2.5X in Glucose-Fed Animals Worm Gene C07G1.7 C33G8.3

Human BLAST Result (% Identity) No Significant Similarity No Significant Similarity

C36C5.12

EPB42 (26%)

col-158

cyp-35D1 E02H4.7

COL25A1 (38%) CYP3A43/CYP3A4 (31%) CYP2A6 (29%) SRP14 (31%)

F11D5.7

SLC2A1 (23%)

cyp-25A1

F15E11.15; IQGAP3 (23%) pud-3 F19H8.5; C10orf131 (37%) mltn-10 No Significant F55G11.4 Similarity HISPPD2A (31%) lips-14

   

lips-4; fil-2

SCOC (41%)

lys-7

SLC25A14 (32%)

nhr-74

ESR1 (38%)

T19H12.6

GGT1 (28%)

T24B8.5

KIAA2026 (32%)

T24D5.6

SLC33A1 (33%)

Y44A6B.3

No Significant Similarity

Human Protein Information

Erythrocyte membrane protein, ATP-binding protein which may regulate the association of protein 3 with ankyrin. Collagen, type XXV, alpha 1 Cytochrome P450 Cytochrome P450-2A6 Signal recognition particle 14kDa Solute carrier family 2 (facilitated glucose transporter) IQ motif containing GTPase activating protein 3 Uncharacterized

IP6 kinase and PP-IP5 (also called IP7) kinase Interacts with ADP-ribosylation factor-like proteins Mitochondrial uncoupling protein, separate oxidative phosphorylation from ATP synthesis with energy dissipated as heat estrogen receptor 1, ligand-activated transcription factor composed of several domains important for hormone binding, DNA binding, and activation of transcription Gamma-glutamyltraspeptidase; catalyzes the transfer of the glutamyl moiety of glutathione to amino acids and dipeptide acceptors, expressed in tissues involved in absorption and secretion, may contribute to the etiology of diabetes Uncharacterized Acetyl-coenzyme A transporter; regulates autophagy; required for the formation of Oacetylated (Ac) ganglioside

A. M. Garcia et al. 

11 SI 

Y49G5A.1

WAP8 (33%)

Contains eight cysteines forming four disulfide bonds at the core of the protein, and functions as a protease inhibitor

                                                           

12 SI 

 

A. M. Garcia et al. 

Table S7 Genes differentially regulated by glucose- identified by two independent studies* Gene/Sequence Wormbase ID Gene Class Description Up-regulated: Unknown (C53A3.2) WBGene00016892 Not known aqp-8 (K02G10.7) WBGene00000176 Aquaporin or aquaglyceroporin related comt-4 (Y40B10A.6) WBGene00021491 Catechol-O-MethylTransferase family ugt-41 (F10D2.11) WBGene00017336 UDP-glucoronosyl/glucosyl transferase col-12 (F15H10.1) WBGene00000601 Collagen nhr-43 (C29E6.5) WBGene00003633 Nuclear hormone receptor family ptr-22 (Y80D3A.7) WBGene00004236 Patched Related family Unknown (F44G3.2) WBGene00009706 Not known far-3 (F15B9.1) WBGene00001387 Fatty acid/retinol binding protein cdr-2 (C54D10.1) WBGene00008296 Cadmium responsive amt-1 (C05E11.4) WBGene00000133 Ammonium Transporter homolog Down-Regulated: gba-4 (Y4C6B.6) WBGene00021160 Beta-glucocerebrosidase spp-5 (T08A9.9) WBGene00004990 Saposin-like protein family * Identified by this study and Lee et al, 2009 

   

A. M. Garcia et al. 

13 SI