Data_Sheet_1.docx

<p>Neuropeptide Y (NPY) plays an important role in the regulation of energy homeostasis in the level of central and sympathetic nervous systems (SNSs). Genetic silencing of peripheral Y₂-receptors have anti-obesity effects, but it is not known whether pharmacological blocking of peripheral Y₂-receptors would similarly benefit energy homeostasis. The effects of a peripherally administered Y₂-receptor antagonist were studied in healthy and energy-rich conditions with or without excess NPY. Genetically obese mice overexpressing NPY in brain noradrenergic nerves and SNS (OE-NPY^DβH) represented the situation of elevated NPY levels, while wildtype (WT) mice represented the normal NPY levels. Specific Y₂-receptor antagonist, BIIE0246, was administered (1.3 mg/kg/day, i.p.) for 2 or 4.5 weeks to OE-NPY^DβH and WT mice feeding on chow or Western diet. Treatment with Y₂-receptor antagonist increased body weight gain in both genotypes on chow diet and caused metabolic disturbances (e.g., hyperinsulinemia and hypercholesterolemia), especially in WT mice. During energy surplus (i.e., on Western diet), blocking of Y₂-receptors induced obesity in WT mice, whereas OE-NPY^DβH mice showed reduced fat mass gain, hepatic glycogen and serum cholesterol levels relative to body adiposity. Thus, it can be concluded that with normal NPY levels, peripheral Y₂-receptor antagonist has no potential for treating obesity, but oppositely may even induce metabolic disorders. However, when energy-rich diet is combined with elevated NPY levels, e.g., stress combined with an unhealthy diet, Y₂-receptor antagonism has beneficial effects on metabolic status.</p

Prenatal Metformin Exposure in a Maternal High Fat Diet Mouse Model Alters the Transcriptome and Modifies the Metabolic Responses of the Offspring

<div><p>Aims</p><p>Despite the wide use of metformin in metabolically challenged pregnancies, the long-term effects on the metabolism of the offspring are not known. We studied the long-term effects of prenatal metformin exposure during metabolically challenged pregnancy in mice.</p><p>Materials and Methods</p><p>Female mice were on a high fat diet (HFD) prior to and during the gestation. Metformin was administered during gestation from E0.5 to E17.5. Male and female offspring were weaned to a regular diet (RD) and subjected to HFD at adulthood (10-11 weeks). Body weight and several metabolic parameters (e.g. body composition and glucose tolerance) were measured during the study. Microarray and subsequent pathway analyses on the liver and subcutaneous adipose tissue of the male offspring were performed at postnatal day 4 in a separate experiment.</p><p>Results</p><p>Prenatal metformin exposure changed the offspring's response to HFD. Metformin exposed offspring gained less body weight and adipose tissue during the HFD phase. Additionally, prenatal metformin exposure prevented HFD-induced impairment in glucose tolerance. Microarray and annotation analyses revealed metformin-induced changes in several metabolic pathways from which electron transport chain (ETC) was prominently affected both in the neonatal liver and adipose tissue.</p><p>Conclusion</p><p>This study shows the beneficial effects of prenatal metformin exposure on the offspring's glucose tolerance and fat mass accumulation during HFD. The transcriptome data obtained at neonatal age indicates major effects on the genes involved in mitochondrial ATP production and adipocyte differentiation suggesting the mechanistic routes to improved metabolic phenotype at adulthood.</p></div

Weight of the fetuses (E18.5).

<p>Body weight of litters at E18.5; ctr1-4 denotes prenatal control litters and met1-4 prenatal metformin litters, Litter size 8–9. ** = P<0.01 by Mann-Whitney test when individual fetal weights are tested (pooled results from two separate studies).</p

Absolute (g) and relative (% of BW) tissue weights of the offspring.

<p>The weights (g) of iWAT, g/eWAT, rWAT and mWAT and liver after seven weeks of HFD consumption (upper rows of the table). Tissue weights relative to the body weight (%; lower rows of the table). P-values by Student's t-test or Mann-Whitney test. <i>NS</i> =  not significant.</p><p>Absolute (g) and relative (% of BW) tissue weights of the offspring.</p

Reduced collagen gel contraction by <i>Mmp13^−/−</i> mouse skin fibroblasts.

<p>(<b>A</b>) Skin fibroblasts (MSF) established from wild type (WT) and MMP-13 knockout (<i>Mmp13^−/−</i>) mice were cultured in mechanically unloaded (floating) 3D collagen gel at density 2×10⁵/ml for 24 h in the presence of 0.5% FCS, 10% FCS or 0.5% FCS+TGF-β (5 ng/ml), as indicated. The cells were fixed, stained with fluorescently labeled phalloidin and Hoechst, and photographed with 20× magnification to observe morphological appearance. In contrast to <i>Mmp13^−/−</i> MSF, WT fibroblasts displayed stellate morphology with numerous thick cell extensions in response to TGF-β or 10% FCS (Scale bar = 10 µm). (<b>B</b>) WT and <i>Mmp13^−/−</i> MSF were cultured in mechanically unloaded 3D collagen gel at density 5×10⁵/ml for 24 and 48 h in the presence of 10% FCS. Contraction of collagen gels was measured from digital images of the gels and is shown as relative to the original gel size. (*<i>P</i><0.005 compared to control, Independent samples T-test, n = 4) (<b>C</b>) WT and <i>Mmp13^−/−</i> MSF were cultured in attached 3D collagen gel at density 5×10⁵/ml for 72 h in the presence of 10% FCS. Subsequently the gels were detached from the well walls and contraction was quantified after 24 h. (*<i>P</i><0.005 compared to control. Independent samples T-test, n = 3). (<b>D</b>) MSF were cultured for 72 h in 3D collagen gel in the presence 10% FCS. Equal aliquots of conditioned media were analyzed in gelatinase zymography.</p

Heatmaps of the liver and SAT gene expression.

<p>Hepatic gene expression profile of vehicle and metformin exposed male offspring at the age of 4 days, n = 6 in both groups. Genes with adjusted P-value <0.05 and LogFC cut-off ≥0.5 are shown (A). SAT gene expression profile of vehicle and metformin exposed male offspring at the age of 4 days, n = 6 in the control offspring, n = 5 in the metformin offspring. Genes with adjusted P-value <0.01 and LogFC cut-off ≥1 are shown (B).</p

The effect of HFD and metformin treatment on the metabolism of the dams.

<p>Body weight during pre-gestational HFD period (A). Fasting blood glucose after three weeks of HFD consumption (B). Body weight development during E0.5–17.5 (C). Blood glucose on the second week (E11.5–12.5) of the gestation (D). * = P<0.05 by Student's t-test.</p

qPCR validation on the microarray-based gene expression profile in the neonatal liver and SAT.

<p>The expression of <i>Fh1</i> (fumarate hydratase 1), <i>Ndufs4</i> (NADH dehydrogenase (ubiquinone) Fe-S protein 4), <i>Etfdh</i> (electron transferring flavoprotein, dehydrogenase), <i>Uqcrh</i> (ubiquinol-cytochrome c reductase hinge protein), <i>Atp5c1</i> (ATP synthase, H⁺transporting, mitochondrial F1 complex, gamma polypeptide 1), <i>Ivd</i> (isovaleryl coenzyme A dehydrogenase) and <i>Acaa2</i> (acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase)) in the neonatal liver (A) by qPCR. n = 6 in both groups. * = P<0.05, ** = P<0.01 and *** = P<0.001. The expression of <i>Ucp1</i> (uncoupling protein 1), <i>Cidea</i> (cell death-inducing DNA fragmentation factor, alpha subunit-like effector A), <i>Cpt1b</i> (carnitine palmitoyltransferase 1b) (B), <i>Ndufs4</i> (NADH dehydrogenase (ubiquinone) Fe-S protein 4), <i>Etfdh</i> (electron transferring flavoprotein, dehydrogenase), <i>Cox7b</i> (cytochrome c oxidase subunit VIIb), <i>Cycs</i> (cytochrome c), <i>Ivd</i> (isovaleryl coenzyme A dehydrogenase), <i>AdipoQ</i> (adiponectin), <i>Acaa2</i> (acetyl-Coenzyme A acyltransferase 2 (mitochondrial 3-oxoacyl-Coenzyme A thiolase)) and <i>Ces3</i> (carboxylesterase 1D) in the neonatal SAT (C) by qPCR. n = 5 in Met and 6 in Ctr group. * = P<0.05 and *** = P<0.001 by Student's t-test or Mann-Whitney test. <i>Ucp1, Cidea</i> and <i>Cpt1b</i> presented separately due to a different scale in the expression levels.</p

The effect of prenatal metformin exposure on the body weight of the offspring during RD and HFD phases.

<p>Female (A) and male offspring (B) during RD and HFD. Black circles (•)  =  Control offspring, open circles (○)  =  Metformin offspring. * = P<0.05 by 2-way RM-ANOVA and Sidak's multiple comparisons test. The body weight change during the HFD in the female (C) and male (D) offspring. n = 13–16, * = P<0.05 by Mann-Whitney test.</p

Glucose tolerance of the offspring during RD and HFD.

<p>GTT of the female (A) and male offspring (B) at 10 weeks of age during RD and after 5–6 weeks of HFD. Black circles (•)  =  Control offspring on RD, open circles (○)  =  Metformin offspring on RD, Black triangles (▴)  =  Control offspring on HFD, open triangles (Δ)  =  Metformin offspring on HFD. n = 12–16. AUC values of the GTT of the female (C) and male (D) offspring. ** = P<0.01 and *** = P<0.001 by 2-way RM-ANOVA and Sidak's multiple comparisons test. P-value of interaction 0.063 in C and <0.05 in D.</p