The Role of Spermidine Synthase (Spds) and Spermine Synthase (Sms) in Regulating Triglyceride Storage in Drosophila

medical sciences

Article The Role of Spermidine Synthase (SpdS) and Spermine Synthase (Sms) in Regulating Triglyceride Storage in Drosophila

Tahj S. Morales †, Erik C. Avis †, Elise K. Paskowski †, Hamza Shabar, Shannon L. Nowotarski * and Justin R. DiAngelo *

Division of Science, Pennsylvania State University, Berks Campus, Reading, PA 19610, USA; [email protected] (T.S.M.); [email protected] (E.C.A.); [email protected] (E.K.P.); [email protected] (H.S.) * Correspondence: [email protected] (S.L.N.); [email protected] (J.R.D.); Tel.: +1-610-396-6005 (S.L.N.); +1-610-396-6441 (J.R.D.) † These authors contributed equally to this work.

Abstract: Polyamines are small organic cations that are important for several biological processes such as cell proliferation, cell cycle progression, and apoptosis. The dysregulation of intracellular polyamines is often associated with diseases such as cancer, diabetes, and developmental disorders. Although polyamine metabolism has been well studied, the effects of key enzymes in the polyamine pathway on lipid metabolism are not well understood. Here, we determined metabolic effects resulting from the absence of spermidine synthase (SpdS) and spermine synthase (Sms) in Drosophila. While SpdS mutants developed normally and accumulated triglycerides, Sms mutants had reduced Citation: Morales, T.S.; Avis, E.C.; viability and stored less triglyceride than the controls. Interestingly, when decreasing SpdS and Paskowski, E.K.; Shabar, H.; Sms, specifically in the fat body, triglyceride storage increased. While there was no difference in Nowotarski, S.L.; DiAngelo, J.R. The triglycerides stored in heads, thoraxes and abdomen fat bodies, abdomen fat body DNA content Role of Spermidine Synthase (SpdS) increased, and protein/DNA decreased in both SpdS- and Sms-RNAi flies, suggesting that fat body- and Spermine Synthase (Sms) in specific knockdown of SpdS and Sms causes the production of smaller fat body cells and triglycerides Regulating Triglyceride Storage in to accumulate in non-fat body tissues of the abdomen. Together, these data provide support for the Drosophila. Med. Sci. 2021, 9, 27. role that polyamines play in the regulation of metabolism and can help enhance our understanding https://doi.org/10.3390/ medsci9020027 of polyamine function in metabolic diseases.

Academic Editor: Noriyuki Murai Keywords: spermidine synthase; spermine synthase; Drosophila; triglyceride; fat body

Received: 23 March 2021 Accepted: 30 April 2021 Published: 2 May 2021 1. Introduction Metabolism-related diseases have been increasing over the past three decades, with Publisher’s Note: MDPI stays neutral obesity becoming one of the most prevalent diseases; approximately one-third of the with regard to jurisdictional claims in world’s population is considered obese [1]. Obesity is characterized by increased fat published maps and institutional afﬁl- accumulation and is associated with a number of comorbidities such as heart disease and iations. type II diabetes [2]. Due to the substantial presence of obesity globally, understanding all of the genes and biological pathways which are involved in regulating the storage of fat in humans is important to allow us to develop treatments and therapeutics for this disease. Polyamines are small, organic cations that have been shown to be important for reg- Copyright: © 2021 by the authors. ulating cell growth, proliferation, cell cycle progression, and apoptosis [3,4]. Polyamine Licensee MDPI, Basel, Switzerland. synthesis begins with the conversion of ornithine to putrescine by ornithine decarboxy- This article is an open access article lase (Odc1). Putrescine is then converted into spermidine by the enzyme spermidine distributed under the terms and synthase (SpdS) and ﬁnally, spermidine is converted into spermine by spermine syn- conditions of the Creative Commons thase (Sms) [4]. While the functions of polyamines in regulating disease states such as Attribution (CC BY) license (https:// cancer have been studied in detail [5], less is known about how polyamines function creativecommons.org/licenses/by/ in regulating lipid metabolic processes. Polyamines have been shown to be essential 4.0/).

Med. Sci. 2021, 9, 27. https://doi.org/10.3390/medsci9020027 https://www.mdpi.com/journal/medsci Med. Sci. 2021, 9, 27 2 of 9

for adipocyte differentiation, because treating 3T3-L1 preadipocytes in culture with the Odc1 inhibitor α-difluoromethylornithine (DFMO) or the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) inhibits differentiation of these cells into mature adipocytes [6–8]. Moreover, treating mature adipocytes in culture with APCHA decreases triglyceride levels [8]. Interestingly, in mice fed a high-fat diet, injection of either spermidine or spermine reduced weight gain [9,10], suggesting that polyamines can function to limit fat accumulation in an intact organism. The fruit fly, Drosophila melanogaster, has recently emerged as a model organism to study human obesity and other metabolic diseases due to its similar genetics and physiology to humans [11,12]. Flies also make polyamines and the enzymes involved in their synthesis are conserved with humans [13]. Our lab has previously shown that flies heterozygous for the Odc1 gene accumulate triglycerides, which is due to a combination of increasing numbers of fat cells as well as the amount of triglyceride stored in fat body cells [14]. In addition, decreasing Odc1 results in increased expression of the genes coding for the major lipid synthesis enzymes, fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC)[14]. While the metabolic functions of fly Odc1 have been described, whether SpdS or Sms function in Drosophila to regulate lipid storage is unknown. In this study, we characterized the roles of the polyamine synthesis enzymes SpdS and Sms in regulating lipid storage in Drosophila. Decreasing SpdS throughout the entire organism, or specifically in the fat body, resulted in the accumulation of triglycerides. However, while Sms mutants had blunted fat storage, fat body-specific decreases in Sms led to lipid accumulation. Interestingly, the lipid accumulation phenotypes that resulted from fat body-specific decreases in either SpdS or Sms resulted from an increase in triglycerides in non-fat body cells or tissues of the abdomen, because triglyceride storage per abdomen fat body and combined head–thorax was unchanged in SpdS-RNAi and Sms-RNAi flies. However, decreasing SpdS and Sms in the fat body led to an increase in the number of fat cells present in the abdomen fat body that were smaller in size than control fat body cells. Together, these data suggest a role for both SpdS and Sms in regulating the storage of triglycerides and strengthen the link between polyamines and lipid homeostasis.

2. Materials and Methods 2.1. Fly Genetics The fly lines utilized in these experiments and their Bloomington Stock Center ID (listed in parentheses) are as follows: SpdS -/-: y[1] w[*]; Mi{y[+Dint2]=MIC}SpdS[MI00898]/TM3, Sb[1] Ser[1] (BL#34119), SpdS -/- control: y[1]w[1] (BL#1495), Sms -/-: w[*]; P{GSV1}Sms[C909] (BL#43396), Sms -/- control: w1118 (BL#3605), UAS-Sms-RNAi: y[1] sc[*] v[1] sev[21]; P{TRiP.HMC03665}attP40 (BL#52924), UAS-SpdS-RNAi: y[1]sc[*]v[1] sev[21]; P{TRiP.HMC04307}attP40 (BL#56011), UAS-EGFP-RNAi: y[1]sc[*]v[1]sev[21]; P{y[+t7.7]v[+t1.8]=VALIUM20-EGFP.shRNA.1}attP40 (BL#41555), yolk-Gal4 [15]. The SpdS and Sms mutant alleles used here are considered hypomorphic as SpdS and Sms expression, respectively, is decreased, but not completely ablated (data not shown). Flies were raised in a 12 h:12 h light:dark cycle at 25 ◦C on a standard cornmeal–sucrose medium (100 mL Karo Lite Corn Syrup (ACH Food Companies, Inc., Oakbrook Terrace, IL, USA), 65 g cornmeal, 40 g sucrose, 9 g Drosophila agar (Genesee Scientiﬁc, San Diego, CA, USA), and 25 g whole yeast in 1.25 L water).

2.2. Protein, Triglyceride and DNA Assays One-week-old whole female flies or dissected cuticles from abdomens with fat bodies attached from one-week-old female flies, or combined heads and thoraxes with legs and wings attached from one-week-old female flies, were homogenized in lysis buffer (140 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.1% Triton-X with 1X protease inhibitor cocktail (Millipore- Sigma, Burlington, MA, USA)). Following homogenization, samples were centrifuged at 4 ◦C for 15 min at 16,000× g. Proteins, triglycerides, and DNA were measured as previously described [16]. Proteins were measured using the Pierce BCA Protein Assay kit Med. Sci. 2021, 9, x FOR PEER REVIEW 5 of 5

(Millipore-Sigma, Burlington, MA, USA)). Following homogenization, samples were centrifuged at 4 °C for 15 min at 16,000× g. Proteins, triglycerides, and DNA were measured as previously described [16]. Proteins were measured using the Pierce BCA Protein Assay Med. Sci. 2021, 9, 27 3 of 9 kit (ThermoFisher, Waltham, MA), and triglycerides were measured using the Infinity Triglyceride kit (ThermoFisher) in all samples. Triglyceride measurements were normalized to total protein content. In fat body samples, DNA content was measured using the (ThermoFisher,Quant-iT double-stranded Waltham, MA, DNA USA), high-sensitivity and triglycerides kit, were according measured to using the manufacturer’s the Infinity in- Triglyceridestructions (ThermoFisher). kit (ThermoFisher) in all samples. Triglyceride measurements were normalized to total protein content. In fat body samples, DNA content was measured using the Quant- iT double-stranded DNA high-sensitivity kit, according to the manufacturer’s instructions 2.3. Statistics (ThermoFisher). The results are expressed as the mean ± standard error (SE). Comparisons between 2.3.experimental Statistics and control conditions were made using unpaired Student’s t-tests or one- wayThe analysis results of are variance expressed (ANOVA), as the mean as ±describedstandard in error the (SE). figure Comparisons legends. p between < 0.05 was con- experimentalsidered statistically and control significant. conditions were made using unpaired Student’s t-tests or one-way analysis of variance (ANOVA), as described in the figure legends. p < 0.05 was considered statistically3. Results significant. 3.3.1. Results SpdS and Sms Mutants have Altered Triglyceride Storage 3.1. SpdSIn order and Sms to Mutantsstudy the Have effects Altered of Triglyceride SpdS and StorageSms on fly lipid metabolism, we character- izedIn mutants order to of study SpdS the (SpdS effectsMI00898 of SpdS, referredand Sms toon here fly lipidas SpdS metabolism, -/-) and weSms characterized (Smsc909, referred to MI00898 c909 mutantshere as ofSmsSpdS -/-()SpdS and compared, referred them to here to yw as SpdSand -/-w)1118 and flies,Sms respectively.(Sms , referred SpdS to here-/- flies had as Sms -/-) and compared them to yw and w1118 flies, respectively. SpdS -/- flies had similar similar viability and developmental progression compared to controls (data not shown). viability and developmental progression compared to controls (data not shown). However, SpdSHowever, -/- adults SpdS appeared -/- adults larger appeared by eye, especially larger by in eye, the abdomenespecially (Figure in the1A). abdomen It is possible (Figure 1A). thatIt is anypossible increase that in any size increase in SpdS -/- inflies size was in SpdS due to-/- increased flies was lipid due storage.to increased To test lipid this storage. hypothesis,To test this triglyceride hypothesis, content triglyceride was measured content in wasSpdS measured -/- flies and in comparedSpdS -/- flies to controls. and compared Triglycerideto controls.content Triglyceride was noticeably content increasedwas noticeably in SpdS -/-increasedflies (Figure in SpdS1B), indicating -/- flies (Figure a role 1B), in- fordicatingSpdS in a limitingrole for fatSpdS accumulation in limiting in fat flies. accumulation in flies.

FigureFigure 1. 1.Spermidine Spermidine synthase synthase(SpdS (SpdS) mutant) mutant flies have flies augmentedhave augmented triglyceride triglyceride storage. (A storage.) One-week- (A) One- oldweek-old female femaleSpdS -/- SpdSflies had -/- flies a larger had abdomen a larger comparedabdomen tocomparedyw controls. to yw A representative controls. A representative image is shown.image is (B shown.) Triglycerides (B) Triglycerides were measured were in SpdS measured -/- mutants in SpdS by homogenizing -/- mutants by pairs homogenizing of one-week-old pairs of adultone-week-old female flies adult and ywfemalecontrols. flies Triglyceride and yw controls. measurements Triglyceride were normalized measurements by dividing were normalized by total by proteindividing content. by total Bars protein represent content. means Bars± S.E. represent (n = 23–25). means * p < ± 0.05 S.E. using (n = Student’s23–25). * tp-test < 0.05 comparing using Student’s SpdSt-test -/- comparingto yw controls. SpdS -/- to yw controls.

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In contrast to SpdS -/- flies, but consistent with previous reports, Sms -/- flies had a In contrast to SpdS -/- flies, but consistent with previous reports, Sms -/- flies had a 47%47% reductionreduction in in viability viability (data (data not not shown shown [17 ]).[17]). However, However, the the adult adultSms Sms -/- flies -/- flies that that did didemerge emerge looked looked smaller smaller by eyeeye and and had had thinner thinner abdomens abdomens (Figure (Figure2A). To 2A). determine To determine whetherwhether the the thinner thinner abdomen abdomen was was due due to decreased to decrea fatsed storage, fat storage, triglyceride triglyceride content wascontent was measuredmeasured in inSms Sms -/- -/-flies. flies.Sms Sms -/- flies -/- flies had lowerhad lower triglyceride triglyceride levels comparedlevels compared to controls to controls (Figure(Figure2B), 2B), a phenotypea phenotype opposite opposite of the of SpdSthe SpdS -/- flies -/- (Figureflies (Figure1B). Together, 1B). Together, these data these data combinedcombined with with the theSpdS SpdS -/- data-/- data described described above above suggest suggest a role fora role both forSpdS bothand SpdSSms andin Sms in regulatingregulating lipid lipid metabolism metabolism in Drosophila in Drosophila. .

FigureFigure 2. 2.Spermine Spermine synthase synthase(Sms (Sms) mutant) mutant ﬂies storeflies lessstore triglyceride less triglyceride than control than ﬂies. control (A) One-week-flies. (A) One-

oldweek-old female Smsfemale -/- ﬂies Sms displayed -/- flies displayed a smallerabdomen a smaller compared abdomen to comparedw1118 controls. to w1118 A representative controls. A repre- imagesentative is shown. image (isB )shown. Triglycerides (B) Triglycerides were measured were in measuredSms -/- mutants in Sms by -/- homogenizing mutants by homogenizing pairs of one-week-oldpairs of one-week-old adult female adult ﬂies andfemalew1118 fliescontrols. and w1118 Triglyceride controls. measurements Triglyceride were measurements normalized by were nor- dividingmalized by by total dividing protein by content. total protein Bars represent content. means Bars± representS.E. (n = 22–25).means * ±p S.E.< 0.05 (nusing = 22–25). Student’s * p < 0.05 tusing-test comparing Student’sSms t-test -/- tocomparingw1118 controls. Sms -/- to w1118 controls.

3.2.3.2. SpdSSpdS and and Sms Sms Act Act in thein the Fat Fat Body Body to Regulate to Regulate Organismal Organismal Lipid Storage Lipid Storage The Sms -/- flies had reduced viability and other developmental defects [17]; therefore, it is possibleThe Sms that -/- theflies lower had lipidreduced levels viability in these and flies other may be developmental secondary to one defects of these [17]; there- developmentalfore, it is possible defects. that To the rule lower out thislipid possibility, levels in these as well flies as to may assess be whethersecondarySpdS to andone of these Smsdevelopmentalact in the fat body defects. (the majorTo rule lipid out storage this possibility, organ in the as fly) well to regulate as to assess lipid metabolism, whether SpdS and weSms used act RNAin the interference fat body (the (RNAi) major to lipid decrease storageSpdS organor Sms inspecifically the fly) to inregulate the adult lipid fat metabo- body.lism, RNAiwe used knockdown RNA interference of SpdS in the (RNAi) fly fat bodyto decrease resulted SpdS in increased or Sms triglyceride specifically levels in the adult (Figurefat body.3A), RNAi consistent knockdown with the lipidof SpdS storage in the phenotype fly fat body of the resultedSpdS -/- inflies increased (Figure1 triglycerideB). Inlevels contrast, (Figure the fat3A), body-specific consistent RNAiwith the knockdown lipid storage of Sms phenotyperevealed increased of the SpdS triglyceride -/- flies (Figure Sms -/- content1B). In (Figurecontrast,3B), the which fat body-specific is the opposite RNAi of the knockdownphenotype of Sms and revealed suggests increased that the triglyc- blunted lipid storage phenotype in the Sms -/- flies may be indirect or secondary to a eride content (Figure 3B), which is the opposite of the Sms -/- phenotype and suggests that developmental defect. Overall, these data suggest that both SpdS and Sms normally act in thethe fly blunted fat body lipid to regulate storage the phenotype storage of triglycerides.in the Sms -/- flies may be indirect or secondary to a developmental defect. Overall, these data suggest that both SpdS and Sms normally act in the fly fat body to regulate the storage of triglycerides.

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FigureFigure 3. 3.Decreasing Decreasing either eitherSpdS SpdSor Sms orin Sms the fat in bodythe fat increases body increases triglyceride triglyceride storage. Triglycerides storage. Triglycerides A yolk-Gal4>SpdS-RNAi yolk-Gal4>EGFP-RNAi werewere measured measured in ( in) ( one-week-oldA) one-week-old female female yolk-Gal4>SpdS-RNAiflies and flies and yolk-Gal4>EGFP-RNAi controls (n = 49–60) and (B) one-week-old female yolk-Gal4>Sms-RNAi flies and yolk-Gal4>EGFP- controls (n = 49–60) and (B) one-week-old female yolk-Gal4>Sms-RNAi flies and yolk-Gal4>EGFP- RNAi controls (n = 19–20). Triglyceride measurements were normalized by dividing by total protein RNAi controls (n = 19–20). Triglyceride measurements were normalized by dividing by total pro- content. Bars represent means ± S.E. * p < 0.05 using Student’s t-test comparing yolk-Gal4>SpdS-RNAi tein content. Bars represent means ± S.E. * p < 0.05 using Student’s t-test comparing yolk- or yolk-Gal4>Sms-RNAi flies to the appropriate yolk-Gal4>EGFP-RNAi controls. Gal4>SpdS-RNAi or yolk-Gal4>Sms-RNAi flies to the appropriate yolk-Gal4>EGFP-RNAi controls. 3.3. SpdS and Sms Control Fat Body Cell Number and Size 3.3.To SpdS better and understand Sms Control the Fat mechanism Body Cell of Number how SpdS andand SizeSms act in the fly fat body to regulateTo lipid better metabolism, understand we dissected the mechanism combined headsof how and SpdS thoraxes and asSms well act as abdomenin the fly fat body to fat bodies from SpdS-RNAi and Sms-RNAi flies and measured the amount of triglyceride storedregulate in each lipid combined metabolism, head–thorax we dissected and abdomen combined fat body heads preparation. and thoraxes Interestingly, as well as abdomen therefat bodies were no from differences SpdS-RNAi in triglyceride and Sms-RNAi levels in SpdSflies -andRNAi measuredor Sms-RNAi thefat amount bodies orof triglyceride head–thoraxstored in each preparations combined and head–thorax their respective and controls abdomen (Figure fat4A–C). body This preparation. suggests that Interestingly, thethere increased were triglycerideno differences storage in phenotype triglyceride of whole levelsSpdS-RNAi in SpdSand-RNASms-RNAii or Smsflies-RNAi arises fat bodies or fromhead–thorax excess lipids preparations being stored in and non-fat their body respective cells/tissues controls in the abdomen.(Figure 4A–C). Moreover, This we suggests that havethe previouslyincreased shown triglyceride that fat body storage DNA phenotype content can be of used whole as a surrogateSpdS-RNAi measurement and Sms-RNAi flies for fat body cell number, and protein/DNA can be used as an indication of cell size [16,18]; therefore,arises from we measured excess lipids fat body being DNA stored content in and non- proteinfat body in fat bodiescells/tissues with decreased in the SpdSabdomen. More- orover,Sms levels.we have Interestingly, previously in bothshownSpdS-RNAi that fatand bodySms-RNAi DNA contentfat bodies, can fat be body used DNA as a surrogate contentmeasurement increased for compared fat body to cell control number, flies (Figure and prot4D,E),ein/DNA but protein/DNA can be used decreased as an indication of comparedcell size to[16,18]; controls therefore, (Figure4F,G). we Together,measured these fat databody suggest DNA acontent potential and role protein of SpdS in fat bodies andwithSms decreasedin regulating SpdS the numberor Sms andlevels. size Interestingly, of abdomen fat bodyin both cells. SpdS-RNAi and Sms-RNAi fat bodies, fat body DNA content increased compared to control flies (Figure 4D,E), but protein/DNA decreased compared to controls (Figure 4F,G). Together, these data suggest a potential role of SpdS and Sms in regulating the number and size of abdomen fat body cells.

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FigureFigure 4. 4.Decreasing Decreasing either eitherSpdS SpdSor orSms Smsin in the the fat fat body body increases increases the the number number but but decreases decreases the the size size ofof fat fat body body cells. cells. (A (A)) Total Total triglyceride triglyceride was was measured measured in in samples samples of of combined combined heads heads andand thoraxesthoraxes dissected from one-week-old female yolk-Gal4>SpdS-RNAi and yolk-Gal4>Sms-RNAi flies and yolk- dissected from one-week-old female yolk-Gal4>SpdS-RNAi and yolk-Gal4>Sms-RNAi flies and yolk- Gal4>EGFP-RNAi controls (n = 24–48). A one-way ANOVA was used to compare head–thorax tri- Gal4>EGFP-RNAi controls (n = 24–48). A one-way ANOVA was used to compare head–thorax glycerides in yolk-Gal4>SpdS-RNAi and yolk-Gal4>Sms-RNAi flies, and yolk-Gal4>EGFP-RNAi con- triglyceridestrols. (B,C) Total in yolk-Gal4>SpdS-RNAi triglyceride, (D,E) totaland DNA,yolk-Gal4>Sms-RNAi and (F,G) total proteinflies, and contentsyolk-Gal4>EGFP-RNAi were measured in controls.fat bodies (B, Cdissected) Total triglyceride, from one-week-old (D,E) total female DNA, yolk-Gal4>SpdS-RNAi and (F,G) total protein flies contents and yolk-Gal4>EGFP- were measured in fatRNAi bodies controls dissected (n = from 21–26) one-week-old and fat bodies female dissectedyolk-Gal4>SpdS-RNAi from one-week-oldflies female and yolk-Gal4>EGFP-RNAi yolk-Gal4>Sms-RNAi controlsflies and (n yolk-Gal4>EGFP-RNAi= 21–26) and fat bodies controls dissected (n from = 54–55). one-week-old Protein measurements female yolk-Gal4>Sms-RNAi were normalizedflies by and yolk-Gal4>EGFP-RNAidividing by total DNAcontrols content (n to= determine 54–55). Protein the size measurements of each fat body were cell. normalized Bars represent by dividing means by ± totalS.E. DNA * p < 0.05 content using to determineStudent’s t the-test size comparing of each fatyolk-Gal4>SpdS-RNAi body cell. Bars represent or yolk-Gal4>Sms-RNAi means ± S.E. * p < flies 0.05 to usingthe appropriate Student’s t-test yolk-Gal4>EGFP-RNAi comparing yolk-Gal4>SpdS-RNAi controls. or yolk-Gal4>Sms-RNAi flies to the appropriate yolk-Gal4>EGFP-RNAi controls.

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4. Discussion In this study, we uncovered a role for SpdS and Sms enzymes in regulating lipid metabolism in the fruit fly, Drosophila melanogaster. While SpdS -/- flies accumulated triglycerides, Sms -/- flies were lean compared to controls. Moreover, previous studies using an independent Sms mutant in Drosophila have shown that the loss of Sms reduces viability, and our data are consistent with this model because we observed a 47% reduction in viability in our Sms mutants (data not shown [17]). Loss of Sms also results in neuronal deterioration due to impaired autophagy flux and mitochondrial function [17]. Therefore, it is possible that the lower triglyceride storage phenotype that we observed in Sms -/- flies here was secondary to a defect that emerges during development (a defect that may be dependent on the levels of polyamines in these animals). Consistent with this hypothesis, when Sms expression is decreased, specifically in the adult fat body, we showed that triglycerides accumulate, which is similar to the phenotypes observed in both SpdS -/- and SpdS-RNAi flies. This suggests that SpdS and Sms both act in the Drosophila fat body to regulate the storage of triglycerides. Previous studies from our lab have shown that while Odc1 mutants in Drosophila are not viable, Odc1 heterozygous flies have excess triglyceride storage, and part of this phenotype arises from increased fat cell number [14]. This increase in fat cell number is consistent with the SpdS-RNAi and Sms-RNAi fat body phenotypes described here, suggesting that these three polyamine synthesis enzymes may work in concert to regulate lipid metabolism in the fly fat body (but whether the production of the polyamines themselves contributes to regulating lipid metabolism in the fat body is unclear). However, Odc1 heterozygous flies have a stronger lipid accumulation phenotype than SpdS-RNAi and Sms-RNAi flies, much of which comes from increased triglyceride storage in each fat cell [14], indicating that Odc1 may have additional roles in regulating lipid metabolism and may act independently of SpdS and Sms to carry out these additional metabolic functions. Additional experiments aimed at determining the genetic and biochemical interactions that may occur among Odc1, SpdS and Sms in the fly fat body, including determining the levels of each of the polyamines in fat body cells with decreased Odc1, SpdS or Sms, may provide insight into the roles of these enzymes in regulating lipid homeostasis. Here, we have shown that SpdS-RNAi and Sms-RNAi flies have normal head–thorax and abdomen fat body triglyceride levels, despite having increased triglycerides when examining whole flies. This suggests that when SpdS and Sms levels were decreased in fat body cells, alterations in triglyceride storage occurred in non-fat body cells of the abdomen, such as the ovaries, which store lipids to support reproduction [19]. While abdomen fat body triglyceride storage was normal in SpdS-RNAi and Sms-RNAi flies, fat body cell number was increased compared to control fat bodies. Interestingly, the fat body cells from these flies were also smaller, as indicated by a decreased protein/DNA ratio, and the combination of more fat body cells that are smaller could explain the normal triglyceride storage phenotype observed in the dissected abdomen fat bodies. It is possible that decreasing SpdS or Sms may alter fat cell differentiation, proliferation, and/or apoptosis to control fat body cell size and number, because polyamines have been well established to regulate these cellular processes [3,4]. Inhibiting ornithine decarboxylase and spermine synthase in cultured 3T3-L1 preadipocytes blocks differentiation into mature adipocytes [6–8]. Moreover, the depletion of spermidine in 3T3-L1 preadipocytes was also shown to inhibit adipogenesis [20]. These findings are inconsistent with the increased fat cell number observed in SpdS-RNAi and Sms-RNAi flies, suggesting that altering cell proliferation and/or differentiation may not be the mechanism whereby decreased SpdS or Sms expression may alter fat body cell size or number. It is possible that the loss of SpdS or Sms in the fat body may decrease apoptosis to result in an increase in the number of fat body cells. Polyamines have been shown to either promote or inhibit apoptosis, depending on the cell type and environmental condition [21,22]. Experiments aimed at measuring cell proliferation and apoptosis in fat bodies with decreased SpdS and Sms expression and Med. Sci. 2021, 9, 27 8 of 9

further characterizing the triglyceride storage phenotypes in these ﬂies may help clarify these outstanding questions. The dysregulation of polyamines has been shown to affect glucose and lipid metabolism and diet-induced obesity in numerous model systems [23]. In fact, increased polyamines in white adipose tissue, liver, or muscle have been linked to resistance to diet-induced obesity [24,25]. Moreover, in mice with diet-induced obesity, increases to either spermidine or spermine were shown to cause weight loss [10]. In the study described here, we have characterized the roles of SpdS and Sms in regulating the storage of triglycerides in the Drosophila fat body, thereby expanding the correlation between the polyamines and lipid metabolism. Due to the high conservation of the polyamine synthesis enzymes in most organisms, from E. coli to humans [3], the results described here will hopefully be broadly applicable (especially for those people with Snyder–Robinson Syndrome, a disease which arises from mutations in spermine synthase). The results of this study support the role of the polyamine pathway in regulating triglyceride storage and adds to our growing understanding of the genes and pathways important for controlling overall lipid homeostasis. Additionally, these studies highlight polyamines as potential therapeutic targets for metabolic disorders such as obesity and type II diabetes.

Author Contributions: Conceptualization, S.L.N. and J.R.D.; methodology, J.R.D.; validation, E.C.A., E.K.P., H.S. and J.R.D.; formal analysis, E.C.A., E.K.P., H.S. and J.R.D.; investigation, E.C.A., E.K.P., H.S. and J.R.D.; resources, J.R.D.; data curation, J.R.D.; writing—original draft preparation, T.S.M.; writing—review and editing, T.S.M., E.C.A., E.K.P., H.S., S.L.N., and J.R.D.; visualization, T.S.M., S.L.N. and J.R.D.; supervision, S.L.N. and J.R.D.; project administration, S.L.N. and J.R.D.; funding acquisition, S.L.N. and J.R.D. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available with the article. Acknowledgments: Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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