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. 2017 Jan 17;13(1):e1006555.
doi: 10.1371/journal.pgen.1006555. eCollection 2017 Jan.

Cardiomyocyte Regulation of Systemic Lipid Metabolism by the Apolipoprotein B-Containing Lipoproteins in Drosophila

Sunji Lee  1 , Hong Bao  2 , Zachary Ishikawa  1 , Weidong Wang  3 , Hui-Ying Lim  1   2
Affiliations

Cardiomyocyte Regulation of Systemic Lipid Metabolism by the Apolipoprotein B-Containing Lipoproteins in Drosophila

Sunji Lee et al. PLoS Genet. .

Abstract

The heart has emerged as an important organ in the regulation of systemic lipid homeostasis; however, the underlying mechanism remains poorly understood. Here, we show that Drosophila cardiomyocytes regulate systemic lipid metabolism by producing apolipoprotein B-containing lipoproteins (apoB-lipoproteins), essential lipid carriers that are so far known to be generated only in the fat body. In a Drosophila genetic screen, we discovered that when haplo-insufficient, microsomal triglyceride transfer protein (mtp), required for the biosynthesis of apoB-lipoproteins, suppressed the development of diet-induced obesity. Tissue-specific inhibition of Mtp revealed that whereas knockdown of mtp only in the fat body decreases systemic triglyceride (TG) content on normal food diet (NFD) as expected, knockdown of mtp only in the cardiomyocytes also equally decreases systemic TG content on NFD, suggesting that the cardiomyocyte- and fat body-derived apoB-lipoproteins serve similarly important roles in regulating whole-body lipid metabolism. Unexpectedly, on high fat diet (HFD), knockdown of mtp in the cardiomyocytes, but not in fat body, protects against the gain in systemic TG levels. We further showed that inhibition of the Drosophila apoB homologue, apolipophorin or apoLpp, another gene essential for apoB-lipoprotein biosynthesis, affects systemic TG levels similarly to that of Mtp inhibition in the cardiomyocytes on NFD or HFD. Finally, we determined that HFD differentially alters Mtp and apoLpp expression in the cardiomyocytes versus the fat body, culminating in higher Mtp and apoLpp levels in the cardiomyocytes than in fat body and possibly underlying the predominant role of cardiomyocyte-derived apoB-lipoproteins in lipid metabolic regulation. Our findings reveal a novel and significant function of heart-mediated apoB-lipoproteins in controlling lipid homeostasis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Genetic screening identifies Mtp as a gene for determining systemic lipid metabolism.
(A) Schematic of the results of the genetic screen showing the location of the mtp gene (blue symbol) on chromosome 2L. Red bars indicate deletions in the three deficiency lines investigated here: Df(2L)ED1378, Df(2L)BSC333, and Df(2L)Exel7080. (B) Whole-body TG levels of newly eclosed control (w1118) flies and deficiency lines on NFD or HFD. For each genotype, a 1:1 ratio of males:females was analyzed. P-values are from Student’s t-test and are between control w1118 and deficiency lines within NFD or HFD, or between NFD and HFD for the same genotype. (C) Whole-body TG levels of newly eclosed control flies (Arm-Gal4), and flies with whole-body KD of mtp (Arm-Gal4>mtpRNAi) on NFD and HFD. For each genotype, a 1:1 ratio of males:females was analyzed. P-values are from Student’s t-test and are between Gal4 control and Gal4-mediated RNAi lines within NFD or HFD, or between NFD and HFD for the same genotype. (D) Whole-body TG levels of newly eclosed control flies (Da-Gal4), and flies with whole-body KD of mtp (Da-Gal4>mtpRNAi) on NFD and HFD. For each genotype, a 1:1 ratio of males:females was analyzed. P-values are from Student’s t-test and are between Gal4 control and Gal4-mediated RNAi lines within NFD or HFD, or between NFD and HFD for the same genotype. In (B), (C) and (D), TG levels (μg/μl) were normalized to total protein (μg/μl). Results are expressed as the fold change in whole fly normalized TG compared with that of the wild-type w1118 or Gal4 control flies on NFD (set to 1.0). Results are the mean ± SEM of the indicated number of flies (N) analyzed over at least 5 independent experiments. (E) Colorimetric assay of food intake in control w1118 third instar larvae and third instar larvae with Gal4 driver only (Arm-Gal4) or with whole body knockdown of mtp using Arm-Gal4 (Arm-Gal4>mtpRNAi). Results are the mean ± SEM of the indicated number of larvae (N) analyzed over 3 independent experiments. P-values are from Student’s t-test.
Fig 2
Fig 2. Fat body and cardiomyocyte Mtp regulate systemic lipid metabolism differently on normal food diet and high fat diet.
(A–C) Whole-body TG levels of newly-eclosed flies with Gal4 drivers only (controls) or with fat body-specific knockdown of mtp using R4-Gal4 (A), ppl-Gal4 (B), or Lsp2-Gal4 (C) on NFD (blue) or HFD (red). (D-E) Whole-body TG levels of newly-eclosed flies with Gal4 drivers only (controls) or with cardiomyocyte-specific knockdown of mtp using Hand-Gal4 (D), or TinC-Gal4 (E) on NFD (blue) or HFD (red). (F) Whole-body TG levels of newly-eclosed flies with Gal4 drivers only (controls) or with muscle-specific knockdown of mtp using Mhc-Gal4 on NFD (blue) or HFD (red). In all cases, TG levels (μg/μl) were normalized to total protein (μg/μl). Results are expressed as the fold change in whole fly normalized TG compared with that of the Gal4 control flies on NFD (set to 1.0). Results are the mean ± SEM of the indicated number of flies (N) analyzed over at least 5 independent experiments. P-values are from Student’s t-test and are between Gal4 control and Gal4-mediated RNAi lines within NFD or HFD, or between NFD and HFD for the same genotype.
Fig 3
Fig 3. Fat body and cardiomyocyte apoLpp regulate systemic lipid metabolism differently on normal food diet and high fat diet.
(A) Whole-body TG levels of newly-eclosed flies with Gal4 driver only (control) or with fat body-specific knockdown of apoLpp using ppl-Gal4 (A) on NFD (blue) or HFD (red). (B, C) Whole-body TG levels of newly-eclosed flies with Gal4 drivers only (controls) or with cardiomyocyte-specific knockdown of apoLpp using Hand-Gal4 (B), or TinC-Gal4 (C) on NFD (blue) or HFD (red). (D) RT-PCR analysis of mtp mRNA levels in whole flies with Arm-Gal4 driver only (control) or with Arm-Gal4-induced overexpression of full-length mtp (mtp+). Actin serves as an internal control. (E) Whole-body TG levels of newly-eclosed flies with Gal4 driver only (control), with cardiomyocyte-specific overexpression of mtp+ using TinC-Gal4, or with cardiomyocyte-specific overexpression of mtp+ and apoLppRNAi using TinC-Gal4 on NFD (blue) or HFD (red). In A-C and E, TG levels (μg/μl) were normalized to total protein (μg/μl). Results are expressed as the fold change in whole fly normalized TG compared with that of the Gal4 control flies on NFD (set to 1.0). Results are the mean ± SEM of the indicated number of flies (N) analyzed over at least 5 independent experiments. P-values are from Student’s t-test and are between Gal4 control and Gal4-mediated RNAi lines within NFD or HFD, or between NFD and HFD for the same genotype.
Fig 4
Fig 4. Inhibition of Mtp or apoLpp in fat body or cardiomyocytes promotes intestinal lipid accumulation on normal food diet and high fat diet.
(A-C’) Representative confocal images of Nile Red-stained lipid droplets in the unfixed intestines of third instar larvae on NFD. (A, A’), control larvae (ppl-Gal4); (B, B’), larvae with fat body-specific KD of mtp (ppl-Gal4>mtpRNAi); and (C, C’), larvae with fat body-specific KD of apoLpp (ppl-Gal4>apoLppRNAi). (A, B, C) Lower magnification (10X) images with scale bars representing 100 μm. (A’, B’, C’) Insets represent the magnified (20X) portion of the intestines (yellow boxes) with scale bar representing 70 μm. Arrows in insets indicate lipid droplets within the enterocytes. (D-F’) Representative confocal images of Nile Red-stained lipid droplets in the unfixed intestines of third instar larvae on HFD. (D, D’), control larvae (ppl-Gal4); (E, E’), larvae with fat body-specific KD of mtp (ppl-Gal4>mtpRNAi); and (F, F’), larvae with fat body-specific KD of apoLpp (ppl-Gal4>apoLppRNAi). (D, E, F) Lower magnification (10X) images with scale bars representing 100 μm. (D’, E’, F’) Insets represent the magnified (20X) portion of the intestines (yellow boxes) with scale bar representing 70 μm. Arrows in insets indicate lipid droplets within the enterocytes. (G-I’) Representative confocal images of Nile Red-stained lipid droplets in the unfixed intestines of third instar larvae on NFD. (G, G’), control larvae (Hand-Gal4); (H, H’), larvae with cardiomyocyte-specific KD of mtp (Hand-Gal4>mtpRNAi); and (I, I’), larvae with cardiomyocyte-specific KD of apoLpp (Hand-Gal4>apoLppRNAi). (G, H, I) Lower magnification (10X) images with scale bars representing 100 μm. (G’, H’, I’) Insets represent the magnified (20X) portion of the intestines (yellow boxes) with scale bar representing 70 μm. Arrows in insets indicate lipid droplets within the enterocytes. (J-L’) Representative confocal images of Nile Red-stained lipid droplets in the unfixed intestines of third instar larvae on HFD. (J, J’), control larvae (Hand-Gal4); (K, K’), larvae with cardiomyocyte-specific KD of mtp (Hand-Gal4>mtpRNAi); and (L, L’), larvae with cardiomyocyte-specific KD of apoLpp (Hand-Gal4>apoLppRNAi). (J, K, L) Lower magnification (10X) images with scale bars representing 100 μm. (J’, K’, L’) Insets represent the magnified (20X) portion of the intestines (yellow boxes) with scale bar representing 70 μm. Arrows in insets indicate lipid droplets within the enterocytes.
Fig 5
Fig 5. High fat diet alters relative Mtp expression levels in the cardiomyocytes and fat body.
(A, B) Relative mRNA levels of Mtp in larval fat body (A) and larval hearts (B) on NFD and HFD. Results are expressed as the fold difference compared with the NFD condition. Values were normalized with gapdh. ***p < 0.001, by two-tailed paired t-test analyzed over 3 independent experiments. (C) Relative mRNA levels of Mtp in in larval heart relative to larval fat body on HFD. Results are expressed as the fold difference. Values were normalized with gapdh. *p < 0.05, ***p < 0.001, by two-tailed paired t-test analyzed over 3 independent experiments. (D) Relative mRNA levels of Mtp in larval heart relative to larval fat body on NFD. Results are expressed as the fold difference. Values were normalized with gapdh. *p < 0.05, ***p < 0.001, by two-tailed paired t-test analyzed over 3 independent experiments. (E-E”) Representative confocal images of Mtp (E), Boca (E’), and Mtp and Boca (E”) stainings of the hearts in the Hand-Gal4 control third instar larvae on NFD. Scale bars represent 40 μm. Insets in E-E” represent the same portion of the heart tube (yellow boxes). Arrows in inset E mark Mtp and DAPI nuclear staining in three cardiomyocytes whereas arrows in inset E’ and inset E” mark Boca and DAPI staining and Mtp and Boca staining in the same three cardiomyocytes, respectively. (F-F”) Representative confocal images of Mtp (F), Boca (F’), and Mtp and Boca (F”) stainings of the hearts in the Hand-Gal4 control third instar larvae on HFD. Scale bars represent 40 μm. Insets in F-F” represent the same portion of the heart tube (yellow boxes). Arrows in inset F mark Mtp and DAPI nuclear staining in four cardiomyocytes whereas arrows in inset F’ and inset F” mark Boca and DAPI staining and Mtp and Boca staining in the same four cardiomyocytes, respectively. (G-G”) Representative confocal images of Mtp (G), Boca (G’), and Mtp and Boca (G”) stainings of the hearts in the Hand-Gal4−mediated mtp KD third instar larvae on HFD. Scale bars represent 40 μm. Insets in G-G” represent the same portion of the heart tube (yellow boxes). Arrows in inset C mark Mtp and DAPI nuclear staining in two cardiomyocytes whereas arrows in inset G’ and inset G” mark Boca and DAPI staining and Mtp and Boca staining in the same two cardiomyocytes, respectively.
Fig 6
Fig 6. High fat diet alters relative apoLpp expression levels in the cardiomyocytes and fat body.
(A, B) Relative mRNA levels of apoLpp in larval fat body (A) and larval hearts (B) on NFD and HFD. Results are expressed as the fold difference compared with the NFD condition. Values were normalized with gapdh. ***p < 0.001, by two-tailed paired t-test analyzed over 3 independent experiments. (C) Relative mRNA levels of apoLpp in in larval heart relative to larval fat body on HFD. Results are expressed as the fold difference. Values were normalized with gapdh. *p < 0.05, ***p < 0.001, by two-tailed paired t-test analyzed over 3 independent experiments. (D) Relative mRNA levels of apoLpp in larval heart relative to larval fat body on NFD. Results are expressed as the fold difference. Values were normalized with gapdh. *p < 0.05, ***p < 0.001, by two-tailed paired t-test analyzed over 3 independent experiments. (E-E”) Representative confocal images of apoLpp (E), Boca (E’), and apoLpp, Boca and DAPI (E”) in cardiomyocytes of the Hand-Gal4 control third instar larvae on NFD. Scale bars represent 40 μm. Boca marks the endoplasmic reticulum and DAPI marks the nucleus in each cardiomyocyte. (F-F”) Representative confocal images of apoLpp (F), Boca (F’), and apoLpp, Boca and DAPI (F”) in cardiomyocytes of the Hand-Gal4 control third instar larvae on HFD. White arrows in F and F” indicate the apoLpp puncta which reflect the presence of Lpp. Yellow arrows in F’ and F” indicate the endoplasmic reticulum marker Boca, and DAPI marks the nuclei in the cardiomyocytes. The magenta arrow in F” indicates the co-localization of apoLpp and Boca. Scale bars represent 40 μm. (G-G”) Representative confocal images of apoLpp (G), Boca (G’), and apoLpp, Boca and DAPI (G”) in cardiomyocytes of the Hand-Gal4-mediated mtp KD third instar larvae on HFD. White arrows in G and G” indicate the strong punctate staining of apoLpp which reflects the accumulation of Lpp. Yellow arrows in G’ and G” indicate the endoplasmic reticulum marker Boca, and DAPI marks the nuclei in the cardiomyocytes. Scale bars represent 40 μm.
Fig 7
Fig 7. A model depicting the relative contributions of Lpp derived from the fat body and cardiomyocytes in controlling systemic lipid metabolism on NFD and HFD.
(A) On NFD, Lpp derived from the cardiomyocytes play an equally important role as Lpp derived from the fat body in systemic lipid homeostasis maintenance. Higher levels of Mtp and apoLpp (blue threads) are present in the fat body than in cardiomyocytes on NFD. Both the cardiomyocyte- and fat body-derived Lpp are recruited to the intestine where they promote the uptake of dietary lipids from the enterocytes and transport the dietary lipids to peripheral tissues for energy production or for storage in the fat body. (B) HFD induces an upregulation of the relative expression of Mtp and apoLpp in the cardiomyocytes (red arrows) and downregulation of their relative expression in the fat body (green arrows), culminating in higher levels of Mtp (bold) and apoLpp (blue threads) in the cardiomyocytes than in the fat body. This could underlie the predominant role of the cardiomyocyte-derived Lpp in the determining of lipid metabolic responses to HFD (thick versus broken arrows, B).
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