Phospholipases D1 and D2 Suppress Appetite and Protect against Overweight

<div><p>Obesity is a major risk factor predisposing to the development of peripheral insulin resistance and type 2 diabetes (T2D). Elevated food intake and/or decreased energy expenditure promotes body weight gain and acquisition of adipose tissue. Number of studies implicated phospholipase D (PLD) enzymes and their product, phosphatidic acid (PA), in regulation of signaling cascades controlling energy intake, energy dissipation and metabolic homeostasis. However, the impact of PLD enzymes on regulation of metabolism has not been directly determined so far. In this study we utilized mice deficient for two major PLD isoforms, PLD1 and PLD2, to assess the impact of these enzymes on regulation of metabolic homeostasis. We showed that mice lacking PLD1 or PLD2 consume more food than corresponding control animals. Moreover, mice deficient for PLD2, but not PLD1, present reduced energy expenditure. In addition, deletion of either of the PLD enzymes resulted in development of elevated body weight and increased adipose tissue content in aged animals. Consistent with the fact that elevated content of adipose tissue predisposes to the development of hyperlipidemia and insulin resistance, characteristic for the pre-diabetic state, we observed that <i>Pld1</i>^<i>-/-</i> and <i>Pld2</i>^<i>-/-</i> mice present elevated free fatty acids (FFA) levels and are insulin as well as glucose intolerant. In conclusion, our data suggest that deficiency of PLD1 or PLD2 activity promotes development of overweight and diabetes.</p></div

Free fatty acids in circulation are elevated in mice deficient for PLD1 or PLD2.

<p>A) Free fatty acids (FFAs), B) Glycerol, C) Triglycerides in circulation of 18-weeks old mice with indicated genotypes. Data represented as mean +/- S.E.M., n = 9 males for control (black), n = 6 males for <i>Pld1</i>^<i>-/-</i> (orange), n = 8 males for <i>Pld2</i>^<i>-/-</i> (green). *p<0.05, **p<0.01, ***p<0.001</p

Deletion of Pld1 or Pld2 promotes insulin resistance and glucose intolerance.

<p>A) Insulin tolerance test in <i>Pld1</i>^<i>-/-</i>, <i>Pld2</i>^<i>-/-</i> and control mice at 16 weeks old. n = 7 for control (black), n = 6 for <i>Pld1</i>^<i>-/-</i> (orange), n = 8 for <i>Pld2</i>^<i>-/-</i> (green). B) Area under the curve for the insulin tolerance test. C) Glucose tolerance test in <i>Pld1</i>^<i>-/-</i>, <i>Pld2</i>^<i>-/-</i> and control mice at 18 weeks old. n = 12 for control (black), n = 10 for <i>Pld1</i>^<i>-/-</i> (orange), n = 12 for <i>Pld2</i>^<i>-/-</i> (green). D) Area under the curve for the glucose tolerance test. E) Insulin levels in circulation of mice with indicated genotypes at 20 weeks old. n = 8 males for control (black), n = 6 males for <i>Pld1</i>^<i>-/-</i> (orange), n = 8 males for <i>Pld2</i>^<i>-/-</i> (green). Data represented as mean +/- S.E.M., *p<0.05, **p<0.01, ***p<0.001</p

Deletion of <i>Pld1</i> or <i>Pld2</i> does not affect satiety response.

<p>A) 24 hours cumulative food intake of 20-weeks old mice with indicated genotypes. B) Food intake at different time-points after overnight fasting of 20-weeks old mice with indicated genotypes. Data represented as mean +/- S.E.M., n = 6 females for control (black), n = 4 females for <i>Pld1</i>^<i>-/-</i> (orange), n = 6 females for <i>Pld2</i>^<i>-/-</i> (green). *p<0.05, **p<0.01, ***p<0.001</p

mRNA expression in hypothalamus of neuropeptides controlling food intake.

<p>Relative expression of mRNA in hypothalamus of <i>Pld1</i>^<i>-/-</i>, <i>Pld2</i>^<i>-/-</i> and control mice. The analyzed targets are presented as those with known orexigenic effect: neuropeptide Y (Npy), neuropeptide Y receptor 1 (Npyr1), Agouti Related Neuropeptide (AgRp), Hypocretin (Hcrt), Galanin (Gal); those with anorexigenic effect: Pro-opiomelanocortin (Pomc), Cocaine-amphetamine-regulated transcript (Cart), Corticotropin-releasing factor (Crf), Neuromedin U (Nmu); and those involved in the metabolism of GABA and glutamate: Glutamate-ammonia ligase (Glul), Glutamate decarboxylase (Gad1), Glutaminase (Gls) and 4-aminobutyrate aminotransferase (Abat). Data represented as mean +/- S.E.M., n = 6 males for control (black), n = 5 males for <i>Pld1</i>^<i>-/-</i> (orange), n = 6 males for <i>Pld2</i>^<i>-/-</i> (green). *p<0.05, **p<0.01, ***p<0.001</p

Deletion of <i>Pld1</i> or <i>Pld2</i> promotes food intake.

<p>A) 24 hours cumulative food intake of 20-weeks old mice with indicated genotypes. B) Oxygen consumption of mice with indicated genotypes at different time of the day. C) Average oxygen consumption during light and dark phase. D) Carbon dioxide production of mice with indicated genotypes at different time of the day. E) Average carbon dioxide during light and dark phase. F) Voluntary movements of mice with indicated genotypes at different time of the day. G) Average voluntary movements of mice during light and dark phase. H) Respiratory exchange rate of mice with indicated genotypes at different time of the day. I) Average respiratory exchange rate of mice during light and dark phase. Data represented as mean +/- S.E.M., n = 6 males for control (black), n = 4 males for <i>Pld1</i>^<i>-/-</i> (orange), n = 6 males for <i>Pld2</i>^<i>-/-</i> (green). *p<0.05, **p<0.01, ***p<0.001</p

Echocardiographic analyses in <i>Jun^Δmu</i> mice after TAC.

<p>All values are shown as mean ± SEM. n = 5–6 per group. p<0.05 is indicated as: # WT TAC vs WT sham; † KO TAC vs KO sham; § KO TAC vs WT TAC. HR, Heart rate; LVPWd, Left ventricular posterior wall in diastole; LVPWs, Left ventricular posterior wall in systole; LVIDd, Left ventricular internal diameter in diastole; LVIDs, Left ventricular internal diameter in systole; FS, Fractional Shortening; EF, Ejection Fraction.</p

<i>Jun^Δmu</i> mice display impaired myocardial remodeling.

<p>(a) Histological analyses. EvG staining reveals mild spontaneous fibrosis in hearts of <i>Jun^Δmu</i> mice, being markedly enhanced after TAC. (b) Relative expression of indicated fibrotic markers assessed by quantitative RT-PCR in hearts isolated from sham or TAC operated <i>Jun^Δmu</i> and <i>Jun^f/f</i> mice. Data are presented as values ± SEM. (*) p<0.05, (**) p<0.01, (***) p<0.001; n = 5 per group. (c) TUNEL staining of heart cross sections. TAC does not induce apoptosis in hearts of <i>Jun^f/f</i> mice. <i>Jun^Δmu</i> mice show slightly more apoptotic cardiomyocytes already at baseline, while the apoptotic rate markedly increased in hearts of <i>Jun^Δmu</i> mice upon TAC. (d) Quantification of TUNEL positive nuclei does in sham or TAC operated animals of indicated genotypes. Data are presented as values ± SEM. (*) p<0.05, (**) p<0.01.</p

Upregulation of extracellular matrix proteins and downregulation expression of sarcomeric associated proteins in hearts of <i>Jun^Δmu</i> mice.

<p>(a) Expression of indicated genes in hearts of sham or TAC operated mice of indicated genotypes. Data are presented as values ± SEM. (*) p<0.05, (**) p<0.01, (***) p<0.001, (****) p<0.0001; n = 5 per group. (b) Western blot analyses of Periostin abundancein hearts from sham and TAC operated <i>Jun^Δmu</i> and <i>Jun^f/f</i> mice. Tubulin was used as a loading control for the extracts. (c) Immunoflorescence staining for Periostin in cross sections of hearts from sham and TAC operated animals of indicated genotypes.</p

Generation of <i>Jun^Δmu</i> mice.

<p>(a) Southern blot analysis of genomic DNA from total heart, skeletal muscle and kidney extracts. Deleted band (Δ band) occurs only in samples from MCK-cre positive heart and skeletal muscle. (b) PCR analysis of genomic DNA. PCR in samples from <i>Jun^+/+</i> (+/+), <i>Jun^f/f</i> (f/f) and <i>Jun^Δmu</i> (f/f Cre) mice yielded a 297 bp band corresponding to the wild type allele, a 344 bp band for the floxed allele and a 450 bp for the Δ allele. (c) Quantitative RT-PCR. <i>Jun</i> mRNA levels are down-regulated in total heart extracts from <i>Jun^Δmu</i> mice. (d) Western blot analysis of c-Jun protein levels in total heart extracts of indicated genotypes. (e) Immunofluorescence of isolated mouse neonatal cardiomycoytes. Nuclear localization of c-Jun can be observed in plated neonatal <i>Jun^f/f</i> cardiomyocytes, but not <i>Jun^Δmu</i> cardiomyocytes.</p