Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

University of Cologne

Intrauterine growth restriction is associated with impaired lung function in adulthood. It is unknown whether such impairment of lung function is linked to the transforming growth factor (TGF)-β system in the lung. Therefore, we investigated the effects of IUGR on lung function, expression of extracellular matrix (ECM) components and TGF-β signaling in rats. IUGR was induced in rats by isocaloric protein restriction during gestation. Lung function was assessed with direct plethysmography at postnatal day (P) 70. Pulmonary activity of the TGF-β system was determined at P1 and P70. TGF-β signaling was blocked in vitro using adenovirus-delivered Smad7. At P70, respiratory airway compliance was significantly impaired after IUGR. These changes were accompanied by decreased expression of TGF-β1 at P1 and P70 and a consistently dampened phosphorylation of Smad2 and Smad3. Furthermore, the mRNA expression levels of inhibitors of TGF-β signaling (Smad7 and Smurf2) were reduced, and the expression of TGF-β-regulated ECM components (e.g. collagen I) was decreased in the lungs of IUGR animals at P1; whereas elastin and tenascin N expression was significantly upregulated. In vitro inhibition of TGF-β signaling in NIH/3T3, MLE 12 and endothelial cells by adenovirus-delivered Smad7 demonstrated a direct effect on the expression of ECM components. Taken together, these data demonstrate a significant impact of IUGR on lung development and function and suggest that attenuated TGF-β signaling may contribute to the pathological processes of IUGR-associated lung disease.

Inhibition of TGF-β signaling and decreased apoptosis in IUGR-associated lung disease in rats.

Early postnatal hyperalimentation has long-term implications for obesity and developing renal disease. Suppressor of cytokine signaling (SOCS) 3 inhibits phosphorylation of signal transducer and activator of transcription (STAT) 3 and ERK1/2 and thereby plays a pivotal role in mediating leptin resistance. In addition, SOCS-3 is induced by both leptin and inflammatory cytokines. However, little is known about the intrinsic-renal leptin synthesis and function. Therefore, this study aimed to elucidate the implications of early postnatal hyperalimentation on renal function and on the intrinsic-renal leptin signaling. Early postnatal hyperalimentation in Wistar rats during lactation was induced by litter size reduction at birth (LSR) either to LSR10 or LSR6, compared with home cage control male rats. Assessment of renal function at postnatal day 70 revealed decreased glomerular filtration rate and proteinuria after LSR6. In line with this impairment of renal function, renal inflammation and expression as well as deposition of extracellular matrix molecules, such as collagen I, were increased. Furthermore, renal expression of leptin and IL-6 was up-regulated subsequent to LSR6. Interestingly, the phosphorylation of Stat3 and ERK1/2 in the kidney, however, was decreased after LSR6, indicating postreceptor leptin resistance. In accordance, neuropeptide Y (NPY) gene expression was down-regulated; moreover, SOCS-3 protein expression, a mediator of postreceptor leptin resistance, was strongly elevated and colocalized with NPY. Thus, our findings not only demonstrate impaired renal function and profibrotic processes but also provide compelling evidence of a SOCS-3-mediated intrinsic renal leptin resistance and concomitant up-regulated NPY expression as an underlying mechanism.

Early postnatal hyperalimentation impairs renal function via SOCS-3 mediated renal postreceptor leptin resistance.

Intrauterine growth restriction (IUGR) is a risk factor for impairment of lung function in adolescence and adulthood. Inflammatory and proliferative processes linking IUGR and perturbed extracellular matrix (ECM) as an underlying mechanism have not been addressed so far. Therefore, in this study, we aimed to investigate the developmental regulation of inflammatory and profibrotic processes in the lung subsequent to IUGR. IUGR was induced in rats by isocaloric protein restriction during gestation. Lung function was assessed with direct plethysmography at postnatal day (P) 28 and P70. Lungs were obtained at P1, P42, and P70 for assessment of mRNA, protein expression, immunohistochemistry, and gelatinolytic activity. Both respiratory system resistance and compliance were impaired subsequent to IUGR at P28 and this impairment was even more pronounced at P70. In line with these results, the expression of ECM components and metabolizing enzymes was deregulated. The deposition of collagen was increased at P70. In addition, the expression of inflammatory cytokines and both the activity and the expression of target genes of Stat3 signaling were dynamically regulated, with unaltered or decreased expression at P1 and significantly increased expression art P70. Taken together, these data give evidence for an age-dependent impairment of lung function as a result of a developmentally regulated increase in inflammatory and profibrotic processes subsequent to IUGR.

Developmental regulation of inflammatory cytokine-mediated Stat3 signaling: the missing link between intrauterine growth restriction and pulmonary dysfunction?

Intrauterine growth restriction (IUGR) is intimately linked with postnatal catch-up growth, leading to impaired lung structure and function. However, the impact of catch-up growth induced by early postnatal hyperalimentation (HA) on the lung has not been addressed to date.

Prevention of early postnatal hyperalimentation protects against activation of transforming growth factor-β/bone morphogenetic protein and interleukin-6 signaling in rat lungs after intrauterine growth restriction.

Regulation of placental nutrient transport significantly affects fetal development and may modify intrauterine growth restriction (IUGR) and fetal programming. We hypothesized that placental nutrient transporters are differentially affected both by utero-placental insufficiency and prenatal surgical stress. Pregnant rats underwent bilateral uterine artery and vein ligation (LIG), sham operation (SOP) or no operation (controls, C) on gestational day E19. Placentas were obtained by caesarean section 4 h (LIG, n=20 placentas; SOP, n=24; C, n=12), 24 h (LIG, n=28; SOP, n=20; C, n=12) and 72 h (LIG, n=20; SOP, n=20; C, n=24) after surgery. Gene and protein expression of placental nutrient transporters for fatty acids (h-FABP, CD36), amino acids (SNAT1, SNAT2) and glucose (GLUT-1, Connexin 26) were examined by qRT-PCR, western blot and immunohistochemistry. Interestingly, the mean protein expression of h-FABP was doubled in placentas of LIG and SOP animals 4, 24 (SOP significant) and 72 h (SOP significant) after surgery. CD36 protein was significantly increased in LIG after 72 h. SNAT1 and SNAT2 protein and gene expressions were significantly reduced in LIG and SOP after 24 h. Further significantly reduced proteins were GLUT-1 in LIG (4 h, 72 h) and SOP (24 h), and Connexin 26 in LIG (72 h). In conclusion, placental nutrient transporters are differentially affected both by reduced blood flow and stress, probably modifying the already disturbed intrauterine milieu and contributing to IUGR and fetal programming. Increased fatty acid transport capacity may affect energy metabolism and could be a compensatory reaction with positive effects on brain development. J. Cell. Biochem. 117: 1594-1603, 2016. © 2015 Wiley Periodicals, Inc.

Increased Rat Placental Fatty Acid, but Decreased Amino Acid and Glucose Transporters Potentially Modify Intrauterine Programming.

Childhood obesity is a risk factor for asthma, but the molecular mechanisms linking both remain elusive. Since obesity leads to chronic low-grade inflammation and affects metabolic signaling we hypothesized that postnatal hyperalimentation (pHA) induced by maternal high-fat-diet during lactation leads to early-onset obesity and dysregulates pulmonary adipocytokine/insulin signaling, resulting in metabolic programming of asthma-like disease in adult mice. Offspring with pHA showed at postnatal day 21 (P21): (1) early-onset obesity, greater fat-mass, increased expression of IL-1β, IL-23, and Tnf-α, greater serum leptin and reduced glucose tolerance than Control (Ctrl); (2) less STAT3/AMPKα-activation, greater SOCS3 expression and reduced AKT/GSK3β-activation in the lung, indicative of leptin resistance and insulin signaling, respectively; (3) increased lung mRNA of IL-6, IL-13, IL-17A and Tnf-α. At P70 body weight, fat-mass, and cytokine mRNA expression were similar in the pHA and Ctrl, but serum leptin and IL-6 were greater, and insulin signaling and glucose tolerance impaired. Peribronchial elastic fiber content, bronchial smooth muscle layer, and deposition of connective tissue were not different after pHA. Despite unaltered bronchial structure mice after pHA exhibited significantly increased airway reactivity. Our study does not only demonstrate that early-onset obesity transiently activates pulmonary adipocytokine/insulin signaling and induces airway hyperreactivity in mice, but also provides new insights into metabolic programming of childhood obesity-related asthma.

Early-onset obesity dysregulates pulmonary adipocytokine/insulin signaling and induces asthma-like disease in mice.

One major risk factor for childhood overweight is maternal obesity. The underlying molecular mechanisms are ill-defined, and effective prevention strategies are missing.

Dietary intervention in obese dams protects male offspring from WAT induction of TRPV4, adiposity, and hyperinsulinemia.

Individuals with intrauterine growth restriction (IUGR) are at risk for chronic lung disease. Using a rat model, we showed in our previous studies that altered lung structure is related to IL-6/STAT3 signaling. As neuropeptide Y (NPY), a coneurotransmitter of the sympathetic nervous system, regulates proliferation and immune response, we hypothesized that dysregulated NPY after IUGR is linked to IL-6, impaired myofibroblast function, and alveolar growth. IUGR was induced in rats by isocaloric low-protein diet; lungs were analyzed on embryonic day (E) 21, postnatal day (P) 3, P12, and P23. Finally, primary neonatal lung myofibroblasts (pnF) and murine embryonic fibroblasts (MEF) were used to assess proliferation, apoptosis, migration, and IL-6 expression. At E21, NPY and IL-6 expression was decreased, and AKT/PKC and STAT3/AMPKα signaling was reduced. Early reduction of NPY/IL-6 was associated with increased chord length in lungs after IUGR at P3, indicating reduced alveolar formation. At P23, however, IUGR rats exhibited a catch-up of body weight and alveolar growth coupled with more proliferating myofibroblasts. These structural findings after IUGR were linked to activated NPY/PKC, IL-6/AMPKα signaling. Complementary, IUGR-pnF showed increased survival, impaired migration, and reduced IL-6 compared with control-pnF (Co-pnF). In contrast, NPY induced proliferation, migration, and increased IL-6 synthesis in fibroblasts. Additionally, NPYmice showed reduced IL-6 signaling and less proliferation of lung fibroblasts. Our study presents a novel role of NPY during alveolarization: NPY regulates) IL-6 and lung STAT3/AMPKα signaling, and) proliferation and migration of myofibroblasts. These new insights in pulmonary neuroimmune interaction offer potential strategies to enable lung growth.

Novel role of NPY in neuroimmune interaction and lung growth after intrauterine growth restriction.

Childhood obesity is associated with renal diseases. Maternal obesity is a risk factor linked to increased adipocytokines and metabolic disorders in the offspring. Therefore, we studied the impact of maternal obesity on renal-intrinsic insulin and adipocytokine signaling and on renal function and structure. To induce maternal obesity, female mice were fed a high-fat diet (HFD) or a standard diet (SD; control group) prior to mating, during gestation, and throughout lactation. A third group of dams was fed HFD only during lactation (HFD-Lac). After weaning at postnatal day (P)21, offspring of all groups received SD. Clinically, HFD offspring were overweight and insulin resistant at P21. Although no metabolic changes were detected at P70, renal sodium excretion was reduced by 40%, and renal matrix deposition increased in the HFD group. Mechanistically, two stages were differentiated. In the early stage (P21), compared with the control group, HFD showed threefold increased white adipose tissue, impaired glucose tolerance, hyperleptinemia, and hyperinsulinemia. Renal leptin/Stat3-signaling was activated. In contrast, the Akt/ AMPKα cascade and Krüppel-like factor 15 expression were decreased. In the late stage (P70), although no metabolic differences were detected in HFD when compared with the control group, leptin/Stat3-signaling was reduced, and Akt/AMPKα was activated in the kidneys. This effect was linked to an increase of proliferative (cyclinD1/D2) and profibrotic (ctgf/collagen IIIα1) markers, similar to leptin-deficient mice. HFD-Lac mice exhibited metabolic changes at P21 similar to HFD, but no other persistent changes. This study shows a link between maternal obesity and metabolic programming of renal structure and function and intrinsic-renal Stat3/Akt/AMPKα signaling in the offspring.

Christina Vohlen

Translational Experimental Pediatrics,
Experimental Pulmonology,
Department of Pediatrics and Adolescent Medicine,
Translational Experimental Pediatrics, Experimental Pulmonology, Department of Pediatrics and Adolescent Medicine

University of Cologne

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes