Pnpla3/Adiponutrin deficiency in mice does not contribute to fatty liver disease or metabolic syndrome

Abstract

PNPLA3 (adiponutrin, calcium-independent phospholipase A(2) epsilon [iPLA(2)ε]) is an adipose-enriched, nutritionally regulated protein that belongs to the patatin-like phospholipase domain containing (PNPLA) family of lipid metabolizing proteins. Genetic variations in the human PNPLA3 gene (i.e., the rs738409 I148M allele) has been strongly and repeatedly associated with fatty liver disease. Although human PNPLA3 has triacylglycerol (TAG) hydrolase and transacylase activities in vitro, its in vivo function and physiological relevance remain controversial. The objective of this study was to determine the metabolic consequences of global targeted deletion of the Pnpla3 gene in mice. We found that Pnpla3 mRNA expression is altered in adipose tissue and liver in response to acute and chronic nutritional challenges. However, global targeted deletion of the Pnpla3 gene in mice did not affect TAG hydrolysis, nor did it influence energy/glucose/lipid homoeostasis or hepatic steatosis/injury. Experimental interventions designed to increase Pnpla3 expression (refeeding, high-sucrose diet, diet-induced obesity, and liver X receptor agonism) likewise failed to reveal differences in the above-mentioned metabolic phenotypes. Expression of the Pnpla3 paralog, Pnpla5, was increased in adipose tissue but not in liver of Pnpla3-deficient mice, but compensatory regulation of genes involved in TAG metabolism was not identified. Together these data argue against a role for Pnpla3 loss-of-function in fatty liver disease or metabolic syndrome in mice.

Fig. 1.

Pnpla3 mRNA expression in animal models of fatty liver and metabolic syndrome. (A) Pnpla3 mRNA expression in brown (BAT), perigonadal (PGAT), subcutaneous (SCAT), and mesenteric (MAT) adipose tissue, and liver of overnight-fasted male FVB mice fed chow (14 kcal% fat) or high-fat diet (HFD, 42 kcal% fat) from weaning until 24 weeks of age (n = 10 per group, male, 24 week-old, overnight-fasted). (B) Pnpla3 mRNA expression in the above adipose tissue depots and liver of WT or leptin-deficient (Lep/Lep^obob) mice (n = 6 per group, male, 10 week-old, ad libitum-fed, chow diet). Pnpla3 expression is normalized to 18S rRNA and expressed relative to Pnpla3 expression in BAT of the chow-fed (A) or WT (B) control groups, respectively. * P < 0.05 by Student's t-test.

Fig. 2.

Generation of mice with global targeted deletion of Pnpla3 (Pnpla3-KO mice). (A) The native (wild-type, WT) Pnpla3 allele and targeting (knockout, KO) construct (not drawn to scale). Bacterial artificial chromosome (BAC) recombineering was used to replace 1939 bp of the native Pnpla3 gene from position -12 to +1927 including the ATG start signal and the entire exon 1 (which contains the GXSXG motif with the putative critical catalytic serine residue of the patatin domain) by a neomycin resistance cassette. Embryonic stem (ES) cell electroporation, selection, and screening were then performed using standard techniques. (B) Southern blots of ES cell clones confirming homologous recombination at upstream (BamH1) and downstream (HindIII) ends. BACs containing the WT and targeted (KO) allele are shown as controls. Lanes 1 and 2 demonstrate positive ES cell clones. Lane 3 demonstrates a negative ES cell clone. All gels were run under identical conditions. Images shown were run on separate gels. (C) Pnpla3 mRNA expression in perigonadal WAT, BAT, adrenal gland, and liver using a primer-probe set spanning exons 1–2 (Pnpla3_{Exon1_2}) (n = 7–10/group, mixed gender, 7–8 week-old, ad libitum-fed, chow diet). Similar results were obtained for primer-probe sets spanning exons 4–5 and 7–8 (data not shown). Pnpla3 expression is normalized to 18S rRNA and expressed relative to Pnpla3 expression in WAT of the control group.

Fig. 3.

TAG hydrolysis in Pnpla3 knockout (KO) mice. (A) TAG hydrolase activity was determined in cytosolic fractions of perigonadal WAT lysates from WT and KO mice (n = 6/group, male, 16–17 week-old, ad libitum-fed, chow diet) in the presence or absence of the PNPLA2/ATGL coactivator CGI-58 and/or the HSL-specific in­hibitor 76-0079. Similar results were obtained for BAT (data not shown). (B) Glycerol release over time from perigonadal WAT explants of WT and KO mice (n = 3/group, male, 8–10 week-old, ad libitum-fed, chow diet) in the absence (basal) or presence of 10 μM isoproterenol (stimulated, stim). Comparable results were obtained for NEFA release (data not shown). (C-D) mRNA expression of Pnpla2/ATGL (C) and HSL (D) in perigonadal WAT of WT and KO mice (n = 7–10/group, mixed gender, 7–8 week-old, ad libitum-fed, chow diet). Expression is normalized to 18S rRNA and expressed relative to gene expression in the WT control group. No significant differences were identified.

Fig. 4.

Energy homeostasis in Pnpla3 knockout (KO) mice. (A–D) Body weight in grams (g) of WT and KO mice fed chow (A), low-fat low-sucrose (LFLS) (B), low-fat high-sucrose (LFHS) (C), or high-fat high-sucrose (HFHS) (D) diets from weaning until 19 weeks of age. (E-H) Fat mass as a percent of total body mass (%) of WT and KO mice fed chow (E), LFLS (F), LFHS (G), or HFHS (H) diets from weaning until 19 weeks of age (n = 6–11/group, male). No significant differences were identified.

Fig. 5.

Glucose tolerance and insulin sensitivity in Pnpla3 knockout (KO) mice. (A–D) Glucose tolerance tests for WT and KO mice fed chow (A), low-fat low-sucrose (LFLS) (B), low-fat high-sucrose (LFHS) (C), or high-fat high-sucrose (HFHS) (D) diets (n = 6–11/group, male, 14 week-old). Mice were injected intraperitoneally with 1.75 g glucose per kg body weight following a 6 h fast, and plasma glucose was determined at the times indicated. (E–H) Insulin tolerance tests for WT and KO mice fed chow (E), LFLS (F), LFHS (G), or HFHS (H) diets (n-6–11/group, male, 16 week-old). Mice were injected intraperitoneally with regular human insulin at 0.80 units per kg of body weight following a 4 h fast, and plasma glucose was determined at the times indicated. No significant differences were identified.

Fig. 6.

Lipid homeostasis in Pnpla3 knockout (KO) mice. (A) Liver TAG content normalized to tissue weight in WT and KO mice fed low-fat low-sucrose (LFLS), low-fat high-sucrose (LFHS), or high-fat high-sucrose (HFHS) diets (n = 6–11/group, male, 19 week-old, ad libitum-fed). (B) TAG molecular species normalized to protein content in liver of WT and KO mice fed LFHS diet (n = 7–10/group, male, 19 week-old, ad libitum-fed). TAG molecular species in liver lipid extracts were determined by electrospray ionization mass spectrometric analysis using C51:3 as an internal standard (tri 17:1). TAG species listed on the x axis are identified by the total number of carbons in the fatty acid moieties and the total number of double bonds in those fatty acids moieties (i.e., C51:3 represents a glycerol backbone esterified to three fatty acids with 17 carbons and 1 double bond each). (C–D) Serum aspartate aminotransferase (AST) (C) and alanine aminotransferase (ALT) (D) in WT and KO mice fed LFLS, LFHS, or HFHS diets (n = 6–11/group, male, 19 week-old, ad libitum-fed). (E–F) In vivo cholesterol (E) and fatty acid (F) biosynthesis in liver, BAT, and perigonadal WAT of WT and KO mice (n = 5/group, male, 8–9 week-old, chow diet). Fatty acid and cholesterol biosynthesis rates were calculated as micromoles of ³H-radioactivity from [³H]H₂O incorporated into fatty acids or digitonin-precipitable sterols (DPSs), respectively, per gram of tissue per h as described in methods. * P < 0.05 by Student's t-test (for effect of diet, only comparisons in the WT group are shown).

Fig. 7.

Gene expression in adipose tissue and liver of Pnpla3 knockout (KO) mice. Pnpla3 mRNA expression in (A) perigonadal WAT and (B) liver, and (C) Pnpla5 mRNA expression in WAT of ad libitum-fed 19 week-old male WT and KO mice fed chow, low-fat low-sucrose (LFLS), low-fat high sucrose (LFHS), or high-fat high sucrose (HFHS) diet since weaning (n = 6–11/group). Genes of interest are normalized to cyclophilin as a reference gene and expressed relative to gene expression in the WT chow-fed control for each gene/tissue. Significant main effects (P < 0.05) of diet (d) and genotype (g) as well as interactions between diet and genotype (d x g) are indicated in upper right corner. Simple effects for pair-wise comparisons are indicated over the appropriate bars (g for comparison of KO vs. WT within a given diet group; d for comparison of special diet vs. chow control within a given genotype group). Expression data for additional genes are found in supplemental Table I.