Lipodystrophy, Diabetes and Normal Serum Insulin in PPARγ-Deficient Neonatal Mice

Peroxisome proliferator activated receptor gamma (PPARγ) is a pleiotropic ligand activated transcription factor that acts in several tissues to regulate adipocyte differentiation, lipid metabolism, insulin sensitivity and glucose homeostasis. PPARγ also regulates cardiomyocyte homeostasis and by virtue of its obligate role in placental development is required for embryonic survival. To determine the postnatal functions of PPARγ in vivo we studied globally deficient neonatal mice produced by epiblast-restricted elimination of PPARγ. PPARγ-rescued placentas support development of PPARγ-deficient embryos that are viable and born in near normal numbers. However, PPARγ-deficient neonatal mice show severe lipodystrophy, lipemia, hepatic steatosis with focal hepatitis, relative insulin deficiency and diabetes beginning soon after birth and culminating in failure to thrive and neonatal lethality between 4 and 10 days of age. These abnormalities are not observed with selective PPARγ2 deficiency or with deficiency restricted to hepatocytes, skeletal muscle, adipocytes, cardiomyocytes, endothelium or pancreatic beta cells. These observations suggest important but previously unappreciated functions for PPARγ1 in the neonatal period either alone or in combination with PPARγ2 in lipid metabolism, glucose homeostasis and insulin sensitivity

Hepatic steatosis and normal bowel in PPARγ^-/F:MORE^+/Cre neonatal mice.

<p>(A and B) The abdominal contents reveal pale, enlarged liver (arrows) in P3 PPARγ^-/F:MORE^+/Cre mice (B) compared to normal controls (A). (C-F). H & E histology of the liver reveals microvessicular steatosis and focal hepatitis in mutant mice (D and F) but absence of steatosis or inflammation and presence of normal extramedullary hematopoiesis in control mice (C and E). Short and long arrows in E and F distinguish normal extramedullary hematopoiesis (E) from acute inflammation (F with neutrophils) in control (E) and PPARγ^-/F:MORE^+/Cre (F) mice, respectively. (G) Gastrointestinal tracts were dissected from duodenum to rectum, loosely coiled on a flat surface with duodenum central and rectum peripheral, and photographed. PPARγ-deficient small and large bowel is normal without evidence of ischemia, hemorrhage, inflammation or necrosis.</p

Lipemia with elevated serum lipids and lipoproteins in severely PPARγ deficient PPARγ^-/F:MORE^+/Cre neonatal mice.

<p>(A) Grossly lipemic serum. Abdominal skin was reflected and viewed from the deep aspect. Blood in control mice is dark red (left, arrows). Blood in mutant mice is light pink (right, arrows) reflecting lipemia. Gross lipemia was also evident in serum viewed in capillary tubes as prepared from mutant (right) compared to control (left) mice. (B) Serum non-esterified fatty acid levels reveal lipemia in P0, P1 and P3 mutant neonates. (C-E) Serum was analyzed by FPLC followed by quantitative assay for cholesterol, triglyceride, glycerol and lipoprotein particle size. (C.)Total serum cholesterol was elevated approximately 50% in mutant neonatal mice including elevation of cholesterol content in chylomicrons, VLDL and LDL but not in HDL. (D) Total serum triglyceride was elevated almost ten-fold in mutant neonatal mice with significant elevation in triglyceride content of chylomicrons, VLDL, LDL and HDL and elevation of total serum glycerol. (E) VLDL particles carrying cholesterol and triglyceride and HDL carrying triglyceride were 8%, 4% and 6% larger in diameter, respectively, in mutant neonatal mice while other values varied by less than 2%. * p < 0.05; NS, p > 0.05. “Control” indicates combined values for PPARγ^+/F:MORE^+/+, PPARγ^+/+:MORE^+/Cre and PPARγ^+/F:MORE^+/Cre neonates. The numbers of mice analyzed of each genotype is indicated in parentheses. Black asterisks, statistically significant difference between PPARγ-deficient neonates and pooled control groups. Grey asterisks, statistically significant difference between the control groups indicated. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160636#pone.0160636.s001" target="_blank">S1 Table</a>.</p

Failure to thrive and neonatal lethality in PPARγ^-/F:MORE^+/Cre mice.

<p>(A) PPARγ^-/F:MORE^+/Cre mice (right) show dermal hypopigmentation and an interscapular concavity (thick arrow, right panel) corresponding to the position of the interscapular brown fat pad in normal mice (thick arrow, left panel). Thin arrows designate retinal pigment indicating the paleness of this PPARγ^-/F:MORE^+/Cre neonate is not due to albinism. (B-D) Interscapular adipose tissue was virtually absent in PPARγ^-/F:MORE^+/Cre P3 neonates. The interscapular region contains BAT in control (dashed outline, B), but not in mutant (dashed outline, C) P3 neonates. Occasional mutants retained small amounts of interscapular tissue consisting primarily of white adipose tissue (WAT)-like tissue (dashed outline, D). (E) Body weight of neonatal mice from PPARγ^F/F x PPARγ^<b>+/-</b>:MORE^+/Cre matings. PPARγ^-/F:MORE^+/Cre mice are slightly smaller than littermates at birth (* p < 0.002), subsequently fail to gain weight in spite of apparently normal suckling and the appearance of a milk spot and are markedly smaller than littermates at P3 (*** p = 0.00001). In contrast, littermates gain weight daily (** p < 0.0003 vs. previous day). (F) Organ weights showing selective hepatomegaly in P3 PPARγ^-/F:MORE^+/Cre mice (* p < 0.05; NS, p > 0.05). (G) Neonatal lethality of PPARγ^-/F:MORE^+/Cre mice. The majority of PPARγ^-/F:MORE^+/Cre mice were euthanized at humane endpoints or found dead between P5 and P13 (* p < 0.0001). In E and F “Control” indicates combined results for PPARγ^+/F:MORE^+/+, PPARγ^-/F:MORE^+/+ and PPARγ^+/F:MORE^+/Cre neonates.</p

Lipodystrophy in PPARγ^-/F:MORE^+/Cre neonatal mice.

<p>Axillary, inguinal and subcutaneous WAT is significantly diminished and lacks mature adipocytes in PPARγ^-/F:MORE^+/Cre mice. (A-E) Skin was removed intact from euthanized P3 mice, restrained in a flattened position, fixed in 10% NBF and photographed from the deep aspect. PPARγ^-/F:MORE^+/Cre mice show greatly diminished axillary, thoracic and inguinal WAT (arrows in A) that contained wispy fibrovascular connective tissue, decreased mature adipocytes, and normal-appearing blood vessels (A, C, E, G, I) in place of well-formed WAT in controls (A, B, D PPARγ^-/F:MORE^+/+; F, H PPARγ^+/F:MORE^+/Cre;). Control (D) but not mutant (E) skin also showed refractile collections of subcutaneous adipose tissue that were diffusely distributed both superficial and deep (arrows in F and H) to the panniculus carnosus and were largely lacking in mutant skin (G and I). Dashed lines in A indicate the planes of microtome sectioning used for the histologic images presented in F-I. Long and short arrows in C indicate grossly lipemic and non-lipemic blood, respectively. Dashed rectangles in F and G indicate the areas shown at higher power in the insets. Scale bars: B, C 3 mm; D, E 1 mm.</p

Diabetes mellitus and relative insulin deficiency in severely PPARγ deficient PPARγ^-/F:MORE^+/Cre neonatal mice.

<p>(A) Serum glucose levels reveal severe diabetes mellitus in P1 and P3, but not P0, mutant neonates. (B) Serum BHA levels reveal ketonemia in P1 and P3, but not P0, mutant neonates. (C) Normal serum insulin in diabetic P3 mutant mice. (D) IHC for insulin in pancreatic islet beta cells of wild type and severely PPARγ-deficient PPARγ^-/F:MORE^+/Cre neonatal mice. Comparable staining is observed in pancreas from P0 (upper panels) mutant (right) and control (left) mice. At P3 (lower panels), pancreas from mutant mice (right) shows less intense and more diffuse staining for insulin than pancreas from control mice (left). Mutant pancreas reproducibly showed greener counter staining than wild type likely reflecting systemic effects on pancreatic cells in mutant neonates even at P0. In A-C: * p < 0.05; NS, p > 0.05.</p