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. 2021 Mar 9;24(4):102288. doi: 10.1016/j.isci.2021.102288

Fat-body brummer lipase determines survival and cardiac function during starvation in Drosophila melanogaster

Annelie Blumrich 1,2,3, Georg Vogler 3, Sandra Dresen 1,2, Soda Balla Diop 3, Carsten Jaeger 4, Sarah Leberer 1,2, Jana Grune 2,5, Eva K Wirth 2,6, Beata Hoeft 1,2, Kostja Renko 7, Anna Foryst-Ludwig 1,2, Joachim Spranger 2,6, Stephan Sigrist 8, Rolf Bodmer 3,9, Ulrich Kintscher 1,2,9,10,∗
PMCID: PMC8050372  PMID: 33889813

Summary

The cross talk between adipose tissue and the heart has an increasing importance for cardiac function under physiological and pathological conditions. This study characterizes the role of fat body lipolysis for cardiac function in Drosophila melanogaster. Perturbation of the function of the key lipolytic enzyme, brummer (bmm), an ortholog of the mammalian ATGL (adipose triglyceride lipase) exclusively in the fly's fat body, protected the heart against starvation-induced dysfunction. We further provide evidence that this protection is caused by the preservation of glycerolipid stores, resulting in a starvation-resistant maintenance of energy supply and adequate cardiac ATP synthesis. Finally, we suggest that alterations of lipolysis are tightly coupled to lipogenic processes, participating in the preservation of lipid energy substrates during starvation. Thus, we identified the inhibition of adipose tissue lipolysis and subsequent energy preservation as a protective mechanism against cardiac dysfunction during catabolic stress.

Subject areas: Molecular Physiology, Lipidomics, Metabolomics

Graphical abstract

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Highlights

  • •

    A cross talk between fat body and the heart regulates cardiac function in Drosophila

  • •

    Knockdown of fat-body brummer lipase prevents starvation-induced cardiac dysfunction

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    This involves preservation of lipid stores and maintenance of cardiac energy supply

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    Brummer-mediated preservation of fat body lipid stores involves lipolysis and lipogenesis

Molecular Physiology; Lipidomics; Metabolomics

Introduction

Lipolysis is the enzymatic hydrolysis of triacylglycerol (TAG) to glycerol and fatty acids (FAs) (Zechner, 2015). The release of FAs from TAG storage provides a major energy source in situations of energy depletion or increased energy demand, and therefore, lipolysis represents a crucial determinant of energy homeostasis (Zechner, 2015). Adipose tissue is the central lipid storage organ with the highest lipolytic activity, thereby providing a large proportion of substrates for energy production in other organs (Ahmadian et al., 2011; Dube et al., 2015). Neutral lipolysis is catalyzed by different lipases among which adipose triglyceride lipase (ATGL) has been characterized as the key lipolytic enzyme in adipose tissue (Ahmadian et al., 2011; Haemmerle et al., 2006).

The heart is an organ that likely profits from adipose-derived energy supply, since it mainly relies on noncardiac energy substrates in order to generate adequate amounts of ATP for proper excitation and contraction (Bertero and Maack, 2018). This may become specifically significant under conditions characterized by energy deficits such as starvation. So far many interactions between adipose tissue and heart have been identified that can significantly affect cardiac function, but the focus of these studies was primarily on circulating mediators secreted from adipose tissue acting on the heart, such as the adipokines adiponectin, or leptin (Shibata et al., 2005; Sweeney, 2010). Much less is known, however, whether the adipose tissue energy storage controls cardiac energy metabolism and cardiac function by providing high-energy substrates, particularly during starvation.

The Drosophila ortholog of the mammalian ATGL is the gene brummer (bmm CG5295), the key lipolytic enzyme also in the fly's adipose tissue, the fat body (Gronke et al., 2005). Loss of bmm activity in flies resulted in progressively increased lipid storage and impaired TAG mobilization (Gronke et al., 2005). In this study we used Drosophila melanogaster as a model organism to investigate the relevance of lipolysis, in particular adipose tissue lipolysis, as an energy providing process, which determines cardiac function under energy-deprived conditions. We provide evidence that reducing lipolysis by knocking down bmm gene expression, specifically in Drosophila fat body, protects against starvation-induced cardiac dysfunction and significantly prolongs lifespan. Cardiac dysfunction was apparently caused by an exhaustion of high-energy substrates during starvation, whereas perturbation of fat body lipolysis led to enlarged TAG energy stores, higher levels of whole-body energy substrates and maintenance of myocardial energy supply. This is the first study showing that inhibition of bmm-mediated adipose lipolysis protects against cardiac dysfunction induced by energy depletion, a beneficial intervention that prolongs lifespan under these conditions. This protective action likely results from the preservation of adequate energy supply for proper cardiac function.

Results

Bmm-mediated fat body lipolysis and heart function

To investigate the effects of fat body-specific lipolysis on heart function, bmm mRNA expression was reduced using the binary UAS-GAL4 system in combination with an RNA interference (RNAi) construct. To achieve a consistent and strong knockdown of bmm in the fat body, two different bmm-RNAi constructs were combined and expressed using the fat body-specific ppl-Gal4 driver (ppl > bmmRNAi2). Fat body-specific bmm knockdown (ppl > bmmRNAi2 (fbbmmKD)) led to significantly diminished abdominal bmm mRNA levels in 2-week-old adult male flies compared with driver control flies (ppl/+) (Figure 1A). Consequently, whole-body TAG levels were significantly elevated in fbbmmKD flies (Figure 1B). This is in accordance with data from bmmmutant flies showing progressively increased lipid storage and impaired TAG mobilization (Gronke et al., 2005).

Figure 1.

Figure 1

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Starvation-induced cardiac dysfunction is rescued in fbbmmKD flies

(A) Relative bmm mRNA expression in abdomen of control (ppl/+) and fbbmmKD (ppl > bmmRNAi2) flies under fed basal conditions (N = 3, n = 2–3 each 8–11 abdomen).

(B) Whole-body triacylglycerol level normalized to fly weight (N = 3, each n = 24 flies, mean +SEM).

(C) Relative bmm mRNA expression in whole flies under fed (F) and starved (S, 72 h) conditions (N = 3, n = 3 each 10 flies).

(D) Kaplan-Meier curve of starvation survival. About 90–110 flies per group were starved and survival was examined every 3 h during daytime.

(E) Representative M-modes from SOHA recordings from fed and starved (72 h) flies. DD, diastolic diameter; SD, systolic diameter.

(F) Heart function parameters determined using SOHA of fed (F) and starved (72 h) (S) flies (N = 3, n = 10–23, mean).

(A and B) Unpaired t test, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. (D) Log rank test, ∗∗∗∗p < 0.0001. (C + F) Two-way ANOVA, Bonferroni post hoc test, arrhythmia index: Kruskal-Wallis test, Dunn's post hoc test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

We next determined cardiac function in fbbmmKD and driver control flies under basal conditions in the fed state using the semi-automated optical heartbeat analysis (SOHA) (Figures S1A and S1B). fbbmmKD flies had normal cardiac function with respect to contractility, determined as fractional shortening, heart rate, or rhythmicity compared with controls (Figure S1B). There was a moderate but significant reduction in diastolic diameter in fbbmmKD flies (Figure S1B); however, this did not affect functional parameters like fractional shortening (Figure S1B). All in all, reduced bmm-mediated fat body lipolysis seems to have no major impact on cardiac function under basal conditions.

Starvation-induced cardiac dysfunction is rescued in fbbmmKD flies

To understand the relevance of adipose tissue lipolysis as an energy-providing process for adequate cardiac function, we next asked whether impaired lipolysis and TAG mobilization due to reduced bmm-mediated fat body lipolysis would affect cardiac function during times of energy deprivation. For this, flies were starved and both starvation resistance and cardiac function were analyzed. Stimulation of lipolysis under these conditions was indirectly documented by a marked increase of bmm mRNA expression in control flies under starvation (Figure 1C). This starvation-induced up-regulation was significantly attenuated in fbbmmKD flies (Figure 1C).

Of importance, fbbmmKD flies had significantly prolonged starvation survival compared with both driver and RNAi control flies (ppl/+ and bmmRNAi/+) (Figure 1D). Next, flies were starved or received normal food for 72 h followed by analysis of cardiac function using SOHA. In accordance with the starvation-mediated decrease of survival in control flies (ppl/+ and bmmRNAi/+), starvation of these flies caused a pronounced deterioration of cardiac function characterized by a marked reduction of contractility (fractional shortening) (Figures 1E and 1F). This alteration is indicative of primarily systolic dysfunction, i.e., an insufficiency in contractile ability, and was accompanied by enlarged systolic and, to a lesser degree, diastolic cardiac diameters (Figures 1E and 1F). Noteworthy, starvation-induced cardiac dysfunction was rescued in fbbmmKD flies including a preservation of fractional shortening and maintenance of normal systolic dimensions relative to ad-lib fed flies (Figures 1E and 1F).

Lack of fat body bmm preserves cardiac ATP-linked respiration

Cardiac dysfunction under catabolic stress may result from structural damage, a process called cardiac wasting (Springer et al., 2014). To understand whether starvation-induced cardiac structural defects are responsible for cardiac dysfunction in our model, we investigated the circumferential myofibril structure in cardiac tubes. No irregularities or degradation of myofibril structures were observed using a phalloidin stain (Figure 2A), suggesting that starvation-induced impairment of cardiac contractility in controls was not caused by structural defects of the heart tube.

Figure 2.

Figure 2

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Lack of fat body bmm preserves cardiac ATP-linked respiration

(A) Representative pictures of stained F-actin (green) in cardiac tubes from control and fbbmmKD flies under fed and starved (72 h) conditions. Images were taken at the abdominal segment 2–3, where the third pair of ostial cells is located. Scale bar, 50 μm.

(B and C) (B) Analysis of the oxygen consumption rate and (C) quantification of basal, maximal, and ATP-linked respiration, and coupling efficiency (% of basal respiration used to drive ATP synthesis) from starved beating heart tubes of control (ppl/+) and fbbmmKD (ppl > bmmRNAi2) flies. (N = 3, n = 36, mean +SEM, unpaired t test, ∗∗p < 0.01, ∗∗∗∗p < 0.0001).

(D) Fractional shortening from SOHA analysis of (ppl/+)-control flies in the presence of oligomycin (2.5 μM, blue symbols) or vehicle control (DMSO) for 10 min prior to heart function analysis. (N = 3, n = 31–39, mean, two-way ANOVA, Bonferroni post hoc test, ∗∗∗∗p < 0.0001).

(E) Fractional shortening from SOHA analysis of fbbmmKD (ppl > bmmRNAi2) flies under fed and starved conditions in the presence of oligomycin (2.5 μM, blue symbols) or vehicle control (DMSO) for 10 min prior to heart function analysis. (N = 4, n = 23–31, mean, two-way ANOVA, Bonferroni post hoc test, ∗∗∗∗p < 0.0001). OCR, oxygen consumption rate; oligo, oligomycin; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; rot/anti, rotenone/antimycin (A).

To further understand preserved cardiac function upon starvation in fbbmmKD flies, we next investigated cardiac energy metabolism in fed and starved flies (Figures 2B–2D). For this, we established a novel, reproducible technique to assess mitochondrial function in adult Drosophila hearts. In particular, oxygen consumption rate was quantified directly in fly hearts using the Seahorse XFe Analyzer. The heart tubes of starved fbbmmKD flies showed markedly higher rates of basal respiration and ATP-linked respiration accompanied by an improved coupling efficiency when compared with control flies (Figures 2B and 2C). Accordingly, when we blocked ATP-synthase by oligomycin in control flies and analyzed cardiac contractility, inhibition of ATP synthesis clearly mimicked the impairment of contractility observed under starvation (Figure 2D). Blockade of the ATP synthase by oligomycin also reduced contractility in fbbmmKD flies under fed and starved conditions (Figure 2E).

Together these data suggest that the preservation of cardiac function under starvation in fbbmmKD flies may have been caused by a starvation-resistant maintenance of adequate cardiac ATP synthesis. We next asked the question whether this improved cardiac ATP-linked respiration may have resulted from an enhanced systemic energy substrate supply under starvation. This would be consistent with observations in bmm mutant flies under starvation, which, due to their increased TAG stores, are able to maintain systemic metabolism for a prolonged period of time through sustained supply of energy substrates (Gronke et al., 2005).

Starved fbbmmKD flies have elevated whole-body energy substrates

To investigate the metabolic status and systemic energy metabolism in fed and starved flies, we first carried out metabolite profiling using mass spectrometry (Figures 3A and 3B). Principal component analysis was performed to simplify the dataset and visualize patterns (Figure 3A). The largest variance of the metabolite profile was caused by dietary intervention, i.e., food versus starvation (Figure 3A). In addition, starved fbbmmKD flies had a distinctly different metabolite profile compared with that of starved controls (Figure 3A). Hierarchical clustering of significantly regulated metabolites revealed a clear distinction between fed and starved flies independent of the genotype (Figure 3B). Of more importance, fbbmmKD flies carried significantly higher levels of metabolites involved in energy metabolism compared with starved control groups (Figure 3B). Of note, branched-chain amino acids (BCAAs) including isoleucine, leucine, and valine and distinct lipid intermediates such as glycerol myristate and oleoyl-glycerol were markedly higher in starved fbbmmKD flies, even than in fed fbbmmKD flies (Figure 3B). These data provide evidence that during starvation fbbmmKD flies are capable of maintaining higher systemic energy substrate/metabolite levels, a process likely involved in their preserved cardiac function and prolonged starvation survival.

Figure 3.

Figure 3

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Starved fbbmmKD flies have elevated whole-body energy substrates

(A and B) Analysis of control (ppl/+ and bmmRNAi/+) and fbbmmKD (ppl > bmmRNAi2) flies under fed and starved (72 h) conditions. Polar metabolite profiling using GC-MS of whole flies. (A) Principal component analysis, dashed ellipses indicate 95% confidence interval. (B) Heatmap and hierarchical clustering of 42 metabolites that were significantly regulated (two-way ANOVA, diet-genotype interaction, adjusted p < 0.05).

(C) GC-MS-based analysis of 2-week-old male bcatmut10115 and w1118 control flies. Isoleucine, leucine and valine were significantly elevated in bcatmut10115 compared with w1118. Unpaired t test, ∗∗p < 0.01.

(D) Fractional shortening from SOHA analysis of bcatmut10115. Flies were starved for 37–41 h (S) or received normal food (F). (N = 2, n = 20/18, mean). Unpaired t test, ∗p < 0.05.

(E) Fractional shortening from SOHA analysis of fed (F) and starved (72 h) (S) ppl/+ flies. Flies received branched-chain amino acids (BCAA) (BCAAs +: 10 mM Ile, 10 mM Leu, 10 mM Val) or H2O (BCAAs) as control (F: N = 3, n = 23, S: N = 3, n = 38/41, mean). Furthermore, fractional shortening from SOHA analysis of starved ppl/+ flies receiving lipid-rich BSA in saline (FA-mix) was determined. (N = 3, n = 46/44, mean). One-way ANOVA, Bonferroni post hoc test, ∗∗p < 0.01, ∗∗∗∗p < 0.0001 (BCAA). Unpaired t test, ∗∗∗p < 0.001 (FA-mix).

To clarify whether increased systemic metabolite levels mediate cardioprotection in starved fbbmmKD flies, we first started to investigate the role of BCAAs. For this we utilized a Drosophila model with increased systemic BCAA levels. The branched-chain amino acid transferase (BCAT) mediates the initial step in BCAA catabolism and regulates BCAA levels (Neinast et al., 2019). Accordingly, bcatmut10115 flies exhibit increased systemic BCAA levels (Figure 3C) similarly to starved fbbmmKD flies. When we challenged these flies with prolonged starvation, cardiac systolic function (fractional shortening) was still significantly impaired (Figures 3D and S2A), despite the high levels of BCAAs (Figure 3C). Likewise, addition of BCAAs did not improve fractional shortening of starved control flies (ppl/+) (Figures 3E and S2B, left). These data make it unlikely that BCAAs are involved in the protection against starvation-induced cardiac dysfunction, even in fbbmmKD flies.

Of interest, adding a fatty acid (FA) mixture containing distinct FAs (C16:0; C18:0, C18:1, C18:2, C18:3), instead of BCAAs, led to a significantly improved fractional shortening and partial rescue in starved ppl/+ flies (Figures 3E and S2B, right), compared with saline alone. Together with the increased levels of lipid intermediates (Figure 3B), such as glycerol-myristate or oleoyl-glycerol, these results point toward a possible role of lipids in fbbmmKD flies as the rescuing high-energy substrate for the starved myocardium.

fbbmmKD flies maintain high levels of glycerolipids and phosphatidylcholines under starvation

In order to gain more precise insights into the regulation and relevance of lipids in starved-fbbmmKD flies, we conducted an LC-MS-based lipidomics analysis in fed and starved flies. In the principal component analysis, a marked variance of the lipid profile was caused by diet in control flies, which was absent in fbbmmKD flies (Figure 4A). Overall lipid abundance was significantly decreased in starved control flies (Figure 4B). In contrast, starved fbbmmKD flies maintained their whole-body lipid abundance on the pre-starved, fed level (Figure 4B). A closer look at distinct lipid groups revealed that the strongest regulation was present in the group of glycerolipids (Figure 4C). Here, fed and starved fbbmmKD flies had dramatically higher levels than both groups of control flies, whereas in other lipid groups significant regulation was only detectable in comparison with either driver or RNAi control flies (Figure 4C). Next, a detailed lipid class analysis showed that the observed glycerolipid increase in fbbmmKD flies was mainly caused by the maintenance of high diacylglycerol (DAG) and TAG levels under starvation (Figure 4D). In addition, phosphatidylcholine levels were preserved on the fed level in fbbmmKD flies (Figure 4D).

Figure 4.

Figure 4

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fbbmmKD flies maintain high levels of glycerolipids and phosphatidylcholines under starvation

Analysis of control (ppl/+ and bmmRNAi/+) and fbbmmKD (ppl > bmmRNAi2) flies under fed (F) and starved (72 h) (S) conditions. Lipid samples from whole flies were analyzed by LC-MS.

(A) Principal component analysis, dashed ellipses indicate 95% confidence interval.

(B) Overall lipid abundance as percent of lipid concentration in fed control (ppl/+) flies.

(C) Abundance of distinct lipid groups: fatty acyls, glycerolipids, glycerophospholipids, and sphingolipids as percent of lipid concentration in fed control (ppl/+) flies.

(D) Abundance of distinct lipid classes: CAR, acylcarnitines; FA, fatty acids; DG, diacylglycerol; TG, triacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; SM, sphingomyelin, as percent of lipid concentration in fed control (ppl/+) flies. (B-D) Tukey post hoc test was used to determine significant differences between groups. Different letters were used to indicate significant (p < 0.05) differences between groups.

These data show that perturbation of fat body lipolysis in Drosophila results in the preservation of lipid stores, a process that is likely to lead to improved compensation for the energy deficit caused by starvation.

fbbmmKD flies preserve high levels of saturated short fatty acyl chains under starvation

In contrast to the TAG increase in starved fbbmmKD flies, higher DAG levels remained unexplained. Perturbation of TAG hydrolysis should actually result in higher TAG levels and reduced DAGs, as previously described by Ahmadian and colleagues in adipose tissue-specific ATGL-deficient mice (Ahmadian et al., 2011). Thus, the question arose whether in addition to defective lipolysis the lack of bmm regulates other lipid metabolic pathways. High DAG levels in starved fbbmmKD flies could also be the result of a stimulated new synthesis or de novo lipogenesis. It has recently been shown that a lipogenic phenotype is characterized by the increased occurrence of short fatty acyl chains with increased lipid saturation (Lee et al., 2020; Rysman et al., 2010). Detailed analysis of fatty acyl chain length and their degree of saturation revealed that, in contrast to control flies, fbbmmKD flies maintain high levels of saturated short-chain fatty acids under starvation (Figure 5A). Further analysis of key lipogenic genes in Drosophila such as dLipin, a phosphatidate phosphatase converting phosphatidate to DAG (Donkor et al., 2007; Ugrankar et al., 2011), dTorsin, a repressor of dLipin (Heier and Kuhnlein, 2018), Acetyl-CoA-Carboxylase (ACC), and Midway (mdy), a DAG acyltransferase converting DAG to TAG (Buszczak et al., 2002; Heier and Kuhnlein, 2018), showed that some of these genes are differentially regulated in control and fbbmmKD flies, among which mdy was most strikingly up-regulated in starved fbbmmKD flies (Figure 5B). Finally, fat body-specific knockdown of mdy in fbbmmKD flies reversed the protective cardiac phenotype of fbbmmKD flies under starvation (Figure 5C).

Figure 5.

Figure 5

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fbbmmKD flies preserve high levels of saturated short fatty acyl chains under starvation

(A) Relative abundance of fatty acyl chain length and average degree of saturation as median composition (%) by sample group (see methods for further details).

(B) Relative mRNA expression in whole flies under fed (F) and starved (72 h) (S) conditions. dACC, acetyl-CoA carboxylase; mdy, midway. Two-way ANOVA, Bonferroni post hoc test, ∗∗∗∗p < 0.0001.

(C) Fractional shortening from SOHA analysis of fbbmmKD (ppl > bmmRNAi2) and ppl > bmmRNAi2;mdy RNAi flies under starved conditions. (N = 3, n = 36–42, mean, unpaired t test, ∗∗∗∗p < 0.0001).

In summary, these results suggest that the observed higher lipid abundance in starved fbbmmKD flies may not only result from defective lipolysis but also may involve the stimulation of lipogenesis under starvation.

Discussion

The role of adipose tissue lipolysis as an energy-providing process for other organs of high-energy demand, such as the heart, is only incompletely understood. This may become particularly important during situations of energy deprivation. Here, we show in Drosophila melanogaster that blockade of fat body lipolysis during starvation maintains local lipid stores, likely resulting from the regulation of lipolytic and potentially lipogenic pathways. Together, these processes mediate higher systemic energy substrate levels, which protected the fly heart against starvation-induced ATP deprivation accompanied by an improvement of contractile function and life-prolonging effects during starvation.

First, we investigated the cardiac phenotype of fbbmmKD flies under basal conditions. No major differences were detected between control and fbbmmKD flies. This is in contrast to the previously observed contractile deficits in bmm heterozygotes (Diop et al., 2015). The systemic and/or cardiac loss of bmm/ATGL-mediated lipolysis as observed in bmm heterozygotes seems to be deleterious for heart function and must be distinguished from the specific loss in adipose tissue/fat body. This is in line with data from mice showing that systemic loss of ATGL results in severe cardiac dysfunction and premature death (Haemmerle et al., 2006). On the contrary, adipose tissue-specific deletion of ATGL protects the heart against pressure-induced heart failure (Kintscher et al., 2020; Salatzki et al., 2018).

Under food deprivation control flies exhibited a reduced lifespan and clear contractile deficits in the heart, a phenomenon previously described in rodents after prolonged starvation (Lee et al., 2015). Perturbation of fat body lipolysis during starvation in fbbmmKD flies resulted in a significant lifespan extension and maintenance of regular cardiac function. Starvation resistance with lifespan extension has been previously described in bmm mutant flies by Grönke and colleagues (2005). Bmm mutants exhibited increased storage fat and markedly outlived control flies under food-deprived conditions (Gronke et al., 2005). Our data now demonstrate that this previously observed improved starvation resistance with lifespan extension is mediated by the reduction of bmm-mediated lipolysis specifically in the fat body. Since adult starvation resistance in Drosophila mainly depends on total stored calories (Djawdan et al., 1998; Kezos et al., 2017), this study may suggest that reduced fat body lipolysis and subsequent decelerated TAG mobilization results in a gain of caloric storage, sustained energy fueling, maintenance of cardiac function and prolonged survival under starvation (Gronke et al., 2005).

Maintenance of normal cardiac function under starvation in fbbmmKD flies was associated with a distinct metabolomic and lipidomic profile compared with control flies. Profiles in control flies appear to correspond to a state of advanced starvation (McCue, 2010). The fbbmmKD flies, on the other hand, seem to be protected from fast consumption of energy substrates and exhibit a metabolite/lipid pattern recently described for earlier stages of starvation (Holecek et al., 2001). A closer look at the metabolite profile showed significantly increased levels of BCAAs and lipid intermediates in fbbmmKD flies. However, genetically increased BCAAs in bcat mutant flies or exogenous re-supply of BCAAs were not able to rescue contractile function in control flies, in contrast to a partial rescue by re-supply of FAs. Further MS-based lipidomics revealed the maintenance of high glycerolipid levels in fbbmmKD flies making it likely that lipid energy substrates compensate for the starvation-induced energy deficit and maintain cardiac contractile function.

The question now arises how lipid energy substrates for the rescue of the starved myocardium can be provided at all in the absence of TAG hydrolysis. One explanation would be that bmm knockdown in fat body leads to deceleration of lipolysis but TAG hydrolysis still occurs at low level providing energy substrates for cardiac energy production. This maintenance of lipolysis in fbbmmKD flies could be mediated by alternative fat body lipases including hormone-sensitive lipase (dHSL) or doppelganger von brummer (dob) (Gronke et al., 2005; Heier and Kuhnlein, 2018). Another explanation could be the regulation of other lipid metabolic pathways in fbbmmKD flies. Previous reports documented that adipose tissue lipolysis is tightly coupled to lipogenesis (Mottillo et al., 2014; Schreiber et al., 2015). In contrast to our data, in these studies pharmacological induction of adipose tissue lipolysis in mice fed a normal chow diet induced lipogenesis and absence of adipose tissue lipolysis decreased lipogenesis under high-fat diet feeding (Mottillo et al., 2014; Schreiber et al., 2015). In our study, a lipogenic lipid and gene expression profile was induced in fbbmmKD flies under starvation. These data are consistent with a previously observed up-regulation of the fatty acid synthase gene expression in bmm mutant flies (Diop et al., 2015). It appears that, under conditions of energy depletion, perturbation of lipolysis may be able to induce lipogenesis to rescue systemic energy supply, whereas under conditions of energy excess the shutdown of lipolysis also stops lipogenesis. At first glance, the simultaneous occurrence of lipogenesis and energy production via fatty acid oxidation seems paradoxical. However, previous studies described the simultaneous induction of both processes in skeletal muscle, memory T cells, and in brown adipose tissue (O'Sullivan et al., 2014; Solinas et al., 2004; Yu et al., 2002). One could therefore speculate that the improved cardiac energy supply during starvation in fbbmmKD flies is mediated by decelerated lipolysis and simultaneously elevated lipogenesis, which together result in the continued supply of lipid-based energy substrates.

Finally, our data point toward mdy as a potential target gene under lipolytic regulation. Mdy encodes an acyl coenzyme A, structurally related to mammalian DAG-acyltransferase-1 (DGAT-1), with a crucial function in TAG formation (Buszczak et al., 2002; Heier and Kuhnlein, 2018). We show that mdy is involved in the protective cardiac phenotype of fbbmmKD flies. Whether other lipogenic genes are involved in these processes, and how the lack of bmm mediates mdy up-regulation, requires further investigations.

Energy depletion not only occurs in situations of exogenous shortage of food resources (McCue, 2010) but also plays a crucial role in the clinical setting of catabolic stress during cachexia (Petruzzelli and Wagner, 2016). Of more importance, it is now well established that cancer-mediated cachexia is closely accompanied by cardiac dysfunction and heart failure (Springer et al., 2014). Here, we show in Drosophila melanogaster that blockade of fat body lipolysis during starvation maintains local energy stores and higher systemic energy substrate levels. This protects the fly heart against starvation-induced ATP deprivation associated with an improvement of contractile function and life-prolonging effects during starvation. These results may be also used to expand future studies toward protective new approaches in cachexia-associated cardiac dysfunction.

Limitations of the study

Our study has distinct limitations. Inhibition of lipolysis fbbmmKD flies likely results in multiple metabolic changes during starvation. Our data only provides an indirect link between the metabolic profile of fbbmmKD flies and the cardiac phenotype. Further mechanistic proof, e.g., that lipogenesis is involved, would require additional experiments. Second, metabolomics and lipidomic analyses were performed in whole flies and not on the tissue level. Third, by focusing on bmm we did not clarify whether other interventions that increase lipid stores in flies would result in similar phenotypes.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Anna Foryst-Ludwig (anna.foryst@charite.de).

Materials availability

Available through lead contact.

Data and code availability

Metabolomics and lipidomics data are available at Mendeley data: https://dx.doi.org/10.17632/jpnds6gr9d.1.

Methods

All methods can be found in the accompanying transparent methods supplemental file.

Acknowledgments

This study was supported by the BMBF (German Ministry of Education and Research): BfR1328-564 and German Centre for Cardiovascular Research (DZHK) BER 5.4 PR. A.B. is supported by the DZHK;BER 5.4 PR. U.K. is supported by the DZHK; BER 5.4 PR, the Deutsche Forschungsgemeinschaft (DFG – KI 712/10-1), the BMBF/BfR1328-564 and the Einstein Foundation/ Foundation Charité (EVF-BIH-2018-440). R.B. is supported by grants R01 HL54732 and P01 AG033456 from NIH. We thank scivisto for the support with the graphical abstract.

Author contributions

A.B. substantially contributed to conception and design of the study, acquisition of data, and data analysis and interpretation; drafted the article; and revised the article critically for important intellectual content. G.V. contributed substantially to conception and design of the study and data analysis and interpretation. S.D. substantially contributed to conception and design of the study, acquisition of data, and data analysis and interpretation and revised the article critically for important intellectual content. S.B.D. substantially contributed to conception and design of the study and data analysis and interpretation and revised the article critically. C.J. substantially contributed to conception of the study, acquisition of data, and data analysis and interpretation (MS analysis). J.G., S.L., E.K.W., B.H., K.R. contributed to conception of the study, acquisition of data, and data analysis and interpretation (J.G., S.L., B.H.: Drosophila, SOHA; E.K.W., K.R.: Seahorse). A.F.L., J.S., S.S. substantially contributed to conception and design of the study and data analysis and interpretation and revised the article critically for important intellectual content. R.B. and U.K. substantially contributed to conception and design of the study and data analysis and interpretation, drafted the article, and revised the article critically for important intellectual content, and substantially contributed to acquisition of funding. All authors finally approved the version to be published.

Declaration of interests

The authors declare no competing interests.

Published: April 23, 2021

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2021.102288.

Supplemental information

Document S1. Transparent methods, Figures S1 and S2, and Table S1
mmc1.pdf (1.1MB, pdf)

References

  1. Ahmadian M., Abbott M.J., Tang T., Hudak C.S., Kim Y., Bruss M., Hellerstein M.K., Lee H.Y., Samuel V.T., Shulman G.I. Desnutrin/ATGL is regulated by AMPK and is required for a brown adipose phenotype. Cell Metab. 2011;13:739–748. doi: 10.1016/j.cmet.2011.05.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bertero E., Maack C. Metabolic remodelling in heart failure. Nat. Rev. Cardiol. 2018;15:457–470. doi: 10.1038/s41569-018-0044-6. [DOI] [PubMed] [Google Scholar]
  3. Buszczak M., Lu X., Segraves W.A., Chang T.Y., Cooley L. Mutations in the midway gene disrupt a Drosophila acyl coenzyme A: diacylglycerol acyltransferase. Genetics. 2002;160:1511–1518. doi: 10.1093/genetics/160.4.1511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Diop S.B., Bisharat-Kernizan J., Birse R.T., Oldham S., Ocorr K., Bodmer R. PGC-1/Spargel counteracts high-fat-diet-induced obesity and cardiac lipotoxicity downstream of TOR and brummer ATGL lipase. Cell Rep. 2015;10:1572–1584. doi: 10.1016/j.celrep.2015.02.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Djawdan M., Chippindale A.K., Rose M.R., Bradley T.J. Metabolic reserves and evolved stress resistance in Drosophila melanogaster. Physiol. Zool. 1998;71:584–594. doi: 10.1086/515963. [DOI] [PubMed] [Google Scholar]
  6. Donkor J., Sariahmetoglu M., Dewald J., Brindley D.N., Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns. J. Biol. Chem. 2007;282:3450–3457. doi: 10.1074/jbc.M610745200. [DOI] [PubMed] [Google Scholar]
  7. Dube J.J., Sitnick M.T., Schoiswohl G., Wills R.C., Basantani M.K., Cai L., Pulinilkunnil T., Kershaw E.E. Adipose triglyceride lipase deletion from adipocytes, but not skeletal myocytes, impairs acute exercise performance in mice. Am. J. Physiol. Endocrinol. Metab. 2015;308:E879–E890. doi: 10.1152/ajpendo.00530.2014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gronke S., Mildner A., Fellert S., Tennagels N., Petry S., Muller G., Jackle H., Kuhnlein R.P. Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila. Cell Metab. 2005;1:323–330. doi: 10.1016/j.cmet.2005.04.003. [DOI] [PubMed] [Google Scholar]
  9. Haemmerle G., Lass A., Zimmermann R., Gorkiewicz G., Meyer C., Rozman J., Heldmaier G., Maier R., Theussl C., Eder S. Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase. Science. 2006;312:734–737. doi: 10.1126/science.1123965. [DOI] [PubMed] [Google Scholar]
  10. Heier C., Kuhnlein R.P. Triacylglycerol metabolism in Drosophila melanogaster. Genetics. 2018;210:1163–1184. doi: 10.1534/genetics.118.301583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Holecek M., Sprongl L., Tilser I. Metabolism of branched-chain amino acids in starved rats: the role of hepatic tissue. Physiol. Res. 2001;50:25–33. [PubMed] [Google Scholar]
  12. Kezos J.N., Cabral L.G., Wong B.D., Khou B.K., Oh A., Harb J.F., Chiem D., Bradley T.J., Mueller L.D., Rose M.R. Starvation but not locomotion enhances heart robustness in Drosophila. J. Insect Physiol. 2017;99:8–14. doi: 10.1016/j.jinsphys.2017.03.004. [DOI] [PubMed] [Google Scholar]
  13. Kintscher U., Foryst-Ludwig A., Haemmerle G., Zechner R. The role of adipose triglyceride lipase and cytosolic lipolysis in cardiac function and heart failure. Cell Rep. Med. 2020;1:100001. doi: 10.1016/j.xcrm.2020.100001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lee S.R., Ko T.H., Kim H.K., Marquez J., Ko K.S., Rhee B.D., Han J. Influence of starvation on heart contractility and corticosterone level in rats. Pflugers Arch. 2015;467:2351–2360. doi: 10.1007/s00424-015-1701-9. [DOI] [PubMed] [Google Scholar]
  15. Lee Y., Lai H.T.M., de Oliveira Otto M.C., Lemaitre R.N., McKnight B., King I.B., Song X., Huggins G.S., Vest A.R., Siscovick D.S. Serial biomarkers of de novo lipogenesis fatty acids and incident heart failure in older adults: the cardiovascular health study. J. Am. Heart Assoc. 2020;9:e014119. doi: 10.1161/JAHA.119.014119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. McCue M.D. Starvation physiology: reviewing the different strategies animals use to survive a common challenge. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2010;156:1–18. doi: 10.1016/j.cbpa.2010.01.002. [DOI] [PubMed] [Google Scholar]
  17. Mottillo E.P., Balasubramanian P., Lee Y.H., Weng C., Kershaw E.E., Granneman J.G. Coupling of lipolysis and de novo lipogenesis in brown, beige, and white adipose tissues during chronic beta3-adrenergic receptor activation. J. Lipid Res. 2014;55:2276–2286. doi: 10.1194/jlr.M050005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Neinast M., Murashige D., Arany Z. Branched chain amino acids. Annu. Rev. Physiol. 2019;81:139–164. doi: 10.1146/annurev-physiol-020518-114455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. O'Sullivan D., van der Windt G.J., Huang S.C., Curtis J.D., Chang C.H., Buck M.D., Qiu J., Smith A.M., Lam W.Y., DiPlato L.M. Memory CD8(+) T cells use cell-intrinsic lipolysis to support the metabolic programming necessary for development. Immunity. 2014;41:75–88. doi: 10.1016/j.immuni.2014.06.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Petruzzelli M., Wagner E.F. Mechanisms of metabolic dysfunction in cancer-associated cachexia. Genes Dev. 2016;30:489–501. doi: 10.1101/gad.276733.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rysman E., Brusselmans K., Scheys K., Timmermans L., Derua R., Munck S., Van Veldhoven P.P., Waltregny D., Daniels V.W., Machiels J. De novo lipogenesis protects cancer cells from free radicals and chemotherapeutics by promoting membrane lipid saturation. Cancer Res. 2010;70:8117–8126. doi: 10.1158/0008-5472.CAN-09-3871. [DOI] [PubMed] [Google Scholar]
  22. Salatzki J., Foryst-Ludwig A., Bentele K., Blumrich A., Smeir E., Ban Z., Brix S., Grune J., Beyhoff N., Klopfleisch R. Adipose tissue ATGL modifies the cardiac lipidome in pressure-overload-induced left ventricular failure. PLoS Genet. 2018;14:e1007171. doi: 10.1371/journal.pgen.1007171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Schreiber R., Hofer P., Taschler U., Voshol P.J., Rechberger G.N., Kotzbeck P., Jaeger D., Preiss-Landl K., Lord C.C., Brown J.M. Hypophagia and metabolic adaptations in mice with defective ATGL-mediated lipolysis cause resistance to HFD-induced obesity. Proc. Natl. Acad. Sci. U S A. 2015;112:13850–13855. doi: 10.1073/pnas.1516004112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Shibata R., Sato K., Pimentel D.R., Takemura Y., Kihara S., Ohashi K., Funahashi T., Ouchi N., Walsh K. Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK- and COX-2-dependent mechanisms. Nat. Med. 2005;11:1096–1103. doi: 10.1038/nm1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Solinas G., Summermatter S., Mainieri D., Gubler M., Pirola L., Wymann M.P., Rusconi S., Montani J.P., Seydoux J., Dulloo A.G. The direct effect of leptin on skeletal muscle thermogenesis is mediated by substrate cycling between de novo lipogenesis and lipid oxidation. FEBS Lett. 2004;577:539–544. doi: 10.1016/j.febslet.2004.10.066. [DOI] [PubMed] [Google Scholar]
  26. Springer J., Tschirner A., Haghikia A., von Haehling S., Lal H., Grzesiak A., Kaschina E., Palus S., Potsch M., von Websky K. Prevention of liver cancer cachexia-induced cardiac wasting and heart failure. Eur. Heart J. 2014;35:932–941. doi: 10.1093/eurheartj/eht302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sweeney G. Cardiovascular effects of leptin. Nat. Rev. Cardiol. 2010;7:22–29. doi: 10.1038/nrcardio.2009.224. [DOI] [PubMed] [Google Scholar]
  28. Ugrankar R., Liu Y., Provaznik J., Schmitt S., Lehmann M. Lipin is a central regulator of adipose tissue development and function in Drosophila melanogaster. Mol. Cell. Biol. 2011;31:1646–1656. doi: 10.1128/MCB.01335-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yu X.X., Lewin D.A., Forrest W., Adams S.H. Cold elicits the simultaneous induction of fatty acid synthesis and beta-oxidation in murine brown adipose tissue: prediction from differential gene expression and confirmation in vivo. FASEB J. 2002;16:155–168. doi: 10.1096/fj.01-0568com. [DOI] [PubMed] [Google Scholar]
  30. Zechner R. Fat FLUX: enzymes, regulators, and pathophysiology of intracellular lipolysis. EMBO Mol. Med. 2015;7:359–362. doi: 10.15252/emmm.201404846. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent methods, Figures S1 and S2, and Table S1
mmc1.pdf (1.1MB, pdf)

Data Availability Statement

Metabolomics and lipidomics data are available at Mendeley data: https://dx.doi.org/10.17632/jpnds6gr9d.1.

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