Enhanced lipogenesis through Pparγ helps cavefish adapt to food scarcity

Abstract

Nutrient availability varies seasonally and spatially in the wild. While many animals, such as hibernating animals or migrating birds, evolved strategies to overcome periods of nutrient scarcity,^1,2 the cellular mechanisms of these strategies are poorly understood. Cave environments represent an example of nutrient-deprived environments, since the lack of sunlight and therefore primary energy production drastically diminishes the nutrient availability.³ Here, we used Astyanax mexicanus, which includes river-dwelling surface fish and cave-adapted cavefish populations, to study the genetic adaptation to nutrient limitations.^4-9 We show that cavefish populations store large amounts of fat in different body regions when fed ad libitum in the lab. We found higher expression of lipogenesis genes in cavefish livers when fed the same amount of food as surface fish, suggesting an improved ability of cavefish to use lipogenesis to convert available energy into triglycerides for storage into adipose tissue.^10-12 Moreover, the lipid metabolism regulator, peroxisome proliferator-activated receptor γ (Pparγ), is upregulated at both transcript and protein levels in cavefish livers. Chromatin immunoprecipitation sequencing (ChIP-seq) showed that Pparγ binds cavefish promoter regions of genes to a higher extent than surface fish and inhibiting Pparγ in vivo decreases fat accumulation in A. mexicanus. Finally, we identified nonsense mutations in per2, a known repressor of Pparγ, providing a possible regulatory mechanism of Pparγ in cavefish. Taken together, our study reveals that upregulated Pparγ promotes higher levels of lipogenesis in the liver and contributes to higher body fat accumulation in cavefish populations, an important adaptation to nutrient-limited environments.

Keywords: Astyanax mexicanus; adaptation; cavefish; fat storage; lipid metabolism; lipogenesis; per2; pparγ.

Figure 1.. Cavefish display more body fat in various areas of the body compared to surface fish.

A) Total body fat comparison (fat mass/total body weight) between adult (1yr old) surface fish, and Pachón and Tinaja cavefish using EchoMRI (n = 9,10,10 respectively). B) H&E staining of fish head sections of the three fish populations (Surface, Pachón, Tinaja). The sagittal sections were performed across the eye area of the head, the upper panel showing the entire section and the lower panel showing the region indicated with a white box in the upper panel, revealing that the orbit (eye socket) in cavefish is filled with adipocytes (n = 5 per population, scale bar = 1 mm in the upper panel, scale bar = 100 μm in the bottom panel). C) Transverse H&E staining of fish trunk sections close to the anal fin of the three fish populations (n = 5 per population, scale bar = 1 mm). D) Quantification of fat area to the whole transverse trunk section area in surface fish, Pachón, and Tinaja cavefish using “Convert to Mask” in ImageJ (n = 5 per population). E) Total lipid content (%) in surface fish, Pachón, and Tinaja cavefish (n = 10 per population) using the Folch method. Cartoon highlighting sampling areas for total lipid content quantification (H = head, D = dorsal part, V = ventral part, black lines indicate the boundaries of sampling). Significances calculated with Wilcoxon test, ** p < 0.01.

Figure 2.. Lipogenesis genes are upregulated and fatty acid profile is altered in the liver of Pachón cavefish compared to surface fish.

A) Experimental design schematic for RNA-seq analysis of subadult (4 months) Pachón and surface fish (n = 3 per population and condition). B) GO-term comparison and analysis of upregulated genes in refed Pachón and surface fish livers. Green arrows indicate the key lipid anabolic pathways, fatty acid biosynthesis and triglyceride biosynthesis process. C) Heatmap of lipogenesis genes in fasted and refed surface fish and Pachón cavefish. D) Relative expression (RT-qPCR) of fatty acid biosynthesis genes in livers of 4-day fasted and refed surface fish and Pachón cavefish (n = 14–15, wilcoxon-test). E) Expression of scd in livers of 4-day fasted and refed surface fish and Pachón cavefish (n = 3) TPM: transcript per million. F) Fatty acid profiles of two monounsaturated fatty acids (n = 6 Wilcoxon test) data from Medley et al. G) Refed Pachón cavefish livers have a higher desaturation index (C16:1n7/C16, and C18:1n9/C18) than surface fish (n = 6, Wilcoxon test) data from Medley et al, * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3.. pparγ transcripts and Pparγ protein is upregulated in cavefish livers.

A) pparγ mRNA expression level comparison between surface fish and Pachón cavefish under two feeding conditions: 4-day fasted and refed. TPM indicates transcript per million reads (n = 3 for each group, *** p < 0.001). B) Immunostaining of Pparγ (magenta), E-cadherin (yellow), and DAPI (turquoise) in liver sections of surface fish and Pachón cavefish. No primary ab indicates no primary antibody control. Scale bar = 30 μm. C) Quantification of mean fluorescent intensity of Pparγ staining (n=3 for surface fish and Pachón livers. 187–317 hepatocytes were randomly selected from each fish liver sample for intensity measurement (Wilcoxon test, * p < 0.05). D) Venn diagram of Pparγ ChIP-seq peaks within 3kb of predicted transcription start sites in surface and Pachón cavefish livers. E) Comparison of Pparγ ChIP-seq peak height (in log2 normalized read number) between surface fish and Pachón cavefish (576 peaks with Pparγ canonical binding sites. wilcoxon test, ** p < 0.01). F) Examples of Ppary ChIP-seq peaks on known lipogenesis target genes (mgat1a, cd36).

Figure 4.. Pharmacological manipulation of pparγ and mutations of per2 in Astyanax populations.

A) Representative images of Nile red staining of surface fish and Pachón cavefish at 11 dpf raised under control conditions (0.2% DMSO) and PPARγ inhibitor, GW9662 (40 μM). Arrows indicate the locations of adipose tissue. Scale bar = 500 μm. B) Adipose tissue area comparison between surface fish and Pachón cavefish at 11dpf raised under control condition and Pparγ inhibitor (n = 21, 9, 32, 45 from left to right). Significance was calculated with Wilcoxon test: *p<0.05. C) Comparison of the onset of adipogenesis between surface fish and Pachón cavefish at 11dpf raised under control conditions and Pparγ inhibitor (n = 46 surface DMSO; n = 73 surface inhibitor treatment; n = 32 Pachón DMSO; n = 46 Pachón inhibitor treatment). Significance was calculated with Chi-square test: n.s. p > 0.05; *** p < 0.001. D) Schematic depiction of the splice variant of per2 leading to a skipping of Exon 21 and a premature stop codon in Exon 22 in Pachón and Tinaja cavefish. The 7 bp nucleotide insertion in Molino per2 Exon 13 also leads to a premature stop codon. The dominant transcript of per2 in Phreatichthys andruzzii (Somalian cavefish) carries a premature stop codon in Exon 18. The filled dark grey box (Tr), representing 225 bp nucleotides, shows the location of a transposon-derived sequence, which incorporated into the transcript leading to a premature stop codon. The middle panel shows the amino acid sequences near the stop codon. (tv_1: transcript variant 1; tv_3: transcript variant 3, which is the most abundant transcript). Right: schematic graphic of Per2 in Mexican cavefish and Somalian cavefish. PPAR: homology to predicted Pparγ binding domain. CRY: homology to Cry1 interacting region. PAS: homology to Per-Arnt-Sim domain; CK1: homology to casein kinase binding domain. Numbers indicate the amino acid number from N-terminal (left) to C-terminal (right). E) Gel images of per2 cDNA amplification in various tissues of Astyanax populations (F:fin; L:liver; B:brain; A:adipose tissue; H:heart; M:muscle).