Gender-specific effects on food intake but no inhibition of age-related fat accretion in transgenic mice overexpressing human IGFBP-2 lacking the Cardin-Weintraub sequence motif

IGFBP-2 affects growth and metabolism and is thought to impact on energy homeostasis and the accretion of body fat via its heparin binding domains (HBD). In order to assess the function of the HBD present in the linker domain (HBD1) we have generated transgenic mice overexpressing mutant human IGFBP-2 lacking the PKKLRP sequence and carrying a PNNLAP sequence instead. Transgenic mice expressed high amounts of human IGFBP-2, while endogenous IGFBP-2 or IGF-I serum concentrations were not affected. In both genders we performed a longitudinal analysis of growth and metabolism including at least 4 separate time points between the age of 10 and 52 weeks. Body composition was assessed by nuclear magnetic resonance (NMR) analysis. Food intake was recorded by an automated online-monitoring. We describe negative effects of mutant human IGFBP-2 on body weight, longitudinal growth and lean body mass (p < 0.05). Very clearly, negative effects of mutant IGFBP-2 were not observed for fat mass accretion throughout life. Instead, relative fat mass was increased in transgenic mice of both genders (p < 0.05). In male mice transgene expression significantly increased absolute mass of total body fat over all age groups (p < 0.05). Food intake was increased in female but decreased in male transgenic mice at an age of 11 weeks. Thus our study clearly provides gender- and time-specific effects of HBD1-deficient hIGFBP-2 (H1d-BP-2) on fat mass accretion and food intake. While our data are in principal agreement with current knowledge on the role of HB-domains for fat accretion we now may also speculate on a role of HBD1 for the control of eating behavior.     

Overexpression of ubiquitous 6-phosphofructo-2-kinase in the liver of transgenic mice results in weight gain

Fructose 2,6-bisphosphate (Fru-2,6-P₂) is an important metabolite that controls glycolytic and gluconeogenic pathways in several cell types. Its synthesis and degradation are catalyzed by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2). Four genes, designated Pfkfb1-4, codify the different PFK-2 isozymes. The Pfkfb3 gene product, ubiquitous PFK-2 (uPFK-2), has the highest kinase/bisphosphatase activity ratio and is associated with proliferation and tumor metabolism. A transgenic mouse model that overexpresses uPFK-2 under the control of the phosphoenolpyruvate carboxykinase promoter was designed to promote sustained and elevated Fru-2,6-P₂ levels in the liver. Our results demonstrate that in diet-induced obesity, high Fru-2,6-P₂ levels in transgenic livers caused changes in hepatic gene expression profiles for key gluconeogenic and lipogenic enzymes, as well as an accumulation of lipids in periportal cells, and weight gain.     

The role of 5‐HT2c receptor on corticosterone‐mediated food intake

Corticosterone plays an important role in feeding behavior. However, its mechanism remains unclear. Therefore, the present study aimed to investigate the effect of corticosterone on feeding behavior. In this study, cumulative food intake was increased by acute corticosterone administration in a dose‐dependent manner. Administration of the 5‐HT_2c receptor agonist m‐chlorophenylpiperazin (mCPP) reversed the effect of corticosterone on food intake. The anorectic effects of mCPP were also blocked by the 5‐HT_2c receptor antagonist RS102221 in corticosterone‐treated mice. Both corticosterone and mCPP increased c‐Fos expression in hypothalamic nuclei, but not the nucleus of the solitary tract. RS102221 inhibited c‐Fos expression induced by mCPP, but not corticosterone. In addition, mCPP had little effect on TH and POMC levels in the hypothalamus. Furthermore, mCPP antagonized decreasing effect of the leptin produced by corticosterone. Taken together, our findings suggest that 5‐HT_2c receptors and leptin may be involved in the effects of corticosterone‐induced hyperphagia.     

Estrogen receptor alpha is required for mammary development and the induction of mammary hyperplasia and epigenetic alterations in the aromatase transgenic mice

Aromatase transgenic mice exhibit hyperplastic and dysplastic changes, attesting to the importance of local estrogen in breast carcinogenesis. These mice also show increased levels of the estrogen receptor and β (ER, ERβ) suggesting that this receptor may play an important role in the initiation of estrogen-mediated mammary hyperplasia observed in these mice. To address the specific role of ER in the mammary development and in the induction of estrogen-mediated hyperplasia in aromatase transgenic mice, we have generated MMTV-aromatase × ER knockout cross (referred as aromatase/ERKO). Even though ERβ is expressed in aromatase/ERKO mice, lack of ER leads to impaired mammary growth in these mice. The data suggest that ER plays an important role in the mammary gland development as well as in the induction of mammary hyperplasia in aromatase transgenic mice. Lack of ER expression in the aromatase/ERKO mice resulted in a decrease in the expression of Cyclin D1, PCNA and TGFβ relative to the aromatase parental strain. The studies involving aromatase/ERKO mice show that lack of ER results in impaired mammary development even in the presence of continuous tissue estrogen, suggesting estrogen/ER-mediated actions are critical for mammary development and carcinogenesis.     

Adiponectin downregulates its own production and the expression of its AdipoR2 receptor in transgenic mice

Adiponectin (ApN) is an adipokine whose expression and plasma levels are inversely related to obesity and insulin-resistant states. The in vivo effects of a chronic expression of exogenous ApN restricted to adipose tissue are unclear. Moreover, the regulatory effects of ApN on its own expression and on that of its receptors are still unknown. In this study, we generated transgenic (Tg) mice with moderate expression of exogenous ApN targeted to adipose tissue (native full-length ApN being placed under control of the adipocyte promoter aP2). After a transient overexpression of ApN in young pups, we intriguingly observed a reduction of ApN mRNA levels and protein content in fat depots, together with a decrease of circulating ApN in adult mice. As a result, the phenotype of these adult mice included glucose intolerance, insulin resistance, and increased adiposity. Reduced expression of ApN in fat tissue was associated with diminished expression of uncoupling protein 2 involved in energy dissipation, and higher expression of fatty acid synthase, a key enzyme of lipogenesis, and of TNFalpha implicated in insulin resistance. Concomitantly, the expression of the ApN receptor AdipoR2 that mediates action of full-length ApN was downregulated, while that of AdipoR1 was unaffected. In agreement with the in vivo studies, recombinant ApN added to the culture medium of 3T3-F442A adipocytes caused a decrease in AdipoR2 and ApN mRNA levels. This treatment did not affect the expression of AdipoR1. Eventually, we demonstrated a contrario that AdipoR2 (but not R1) was specifically upregulated in fat of ApN(-/-) mice. Our in vivo and in vitro data provide evidence for a novel regulatory feedback loop by which ApN downregulates its own production and the expression of its AdipoR2 receptor.     

Loss of Bak enhances lymphocytosis but does not ameliorate thrombocytopaenia in BCL-2 transgenic mice

Bax and Bak are critical effectors of apoptosis. Although both are widely expressed and usually functionally redundant, recent studies suggest that Bak has particular importance in certain cell types. Genetic and biochemical studies indicate that Bak activation is prevented primarily by Mcl-1 and Bcl-x_L, whereas Bax is held in check by all pro-survival Bcl-2 homologues, including Bcl-2 itself. In this study, we have investigated whether loss of Bak or elevated Mcl-1 modulates haemopoietic abnormalities provoked by overexpression of Bcl-2. The Mcl-1 transgene had little impact, probably because the expression level was insufficient to effectively reduce Bak activation. However, loss of Bak enhanced lymphocytosis in vavP-BCL-2 transgenic mice and increased resistance of their thymocytes to some cytotoxic agents, implying that Bak-specific signals can be triggered in certain lymphoid populations. Nevertheless, lack of Bak had no significant impact on thymic abnormalities in vavP-BCL-2tg mice, which kinetic analysis suggested was due to accumulation of self-reactive thymocytes that resist deletion. Intriguingly, although Bak^−/− mice have elevated platelet counts, Bak^−/−vavP-BCL-2 mice, like vavP-BCL-2 littermates, were thrombocytopaenic. To clarify why, the vavP-BCL-2 platelet phenotype was scrutinised more closely. Platelet life span was found to be elevated in vavP-BCL-2 mice, which should have provoked thrombocytosis, as in Bak^−/− mice. Analysis of bone marrow chimaeric mice suggested the low platelet phenotype was due principally to extrinsic factors. Following splenectomy, blood platelets remained lower in vavP-BCL-2 than wild-type mice. However, in Rag1^−/− BCL-2tg mice, platelet levels were normal, implying that elevated lymphocytes are primarily responsible for BCL-2tg-induced thrombocytopaenia.     

The role of 5‐HT2A receptor and 5‐HT2A/5‐HT1A receptor interaction in the suppression of catalepsy

In the present study, the 5‐HT_2A and 5‐HT_1A receptors functional activity and 5‐HT_2A receptor gene expression were examined in the brain of ASC/Icg and congenic AKR.CBAD13Mit76C mouse strains (genetically predisposed to catalepsy) in comparison with the parental catalepsy‐resistant AKR/J and catalepsy‐prone CBA/Lac mouse strains. The significantly reduced 5‐HT_2A receptor functional activity along with decreased 5‐HT_2A receptor gene expression in the frontal cortex was found in all mice predisposed to catalepsy compared with catalepsy‐resistant AKR/J. 5‐HT_2A agonist DOI (0.5 and 1 mg/kg, i.p.) significantly reduced catalepsy in ASC/Icg and CBA/Lac, but not in AKR.CBAD13Mit76C mice. Essential increase in 5‐HT_1A receptor functional activity was shown in catalepsy‐prone mouse strains in comparison with catalepsy‐resistant AKR/J mice. However, in AKR.CBAD13Mit76C mice it was lower than in ASC/Icg and CBA/Lac mice. The inter‐relation between 5‐HT_2A and 5‐HT_1A receptors in the regulation of catalepsy was suggested. This suggestion was confirmed by prevention of DOI anticataleptic effect in ASC/Icg and CBA/Lac mice by pretreatment with 5‐HT_1A receptor antagonist p‐MPPI (3 mg/kg, i.p.). At the same time, the activation of 5‐HT_2A receptor led to the essential suppression of 5‐HT_1A receptor functional activity, indicating the opposite effect of 5‐HT_2A receptor on pre‐ and postsynaptic 5‐HT_1A receptors. Thus, 5‐HT_2A/5‐HT_1A receptor interaction in the mechanism of catalepsy suppression in mice was shown.     

Global deletion of lipocalin 2 does not reverse high-fat diet-induced obesity resistance in stearoyl-CoA desaturase-1 skin-specific knockout mice

Over the past century, obesity has developed into a paramount health issue that affects millions of people worldwide. Obese individuals have an increased risk to develop other metabolic disorders, such as insulin resistance and atherosclerosis, among others. Previously we determined that mice lacking stearoyl-CoA desaturase-1 (SCD1) enzyme specifically in the skin (SKO) were lean and protected from high-fat diet induced adiposity. Additionally, lipocalin 2 (Lcn2) mRNA was found to be 27-fold higher in the skin of SKO mice compared to control mice. Given reports suggesting that Lcn2 plays a role in protection against diet-induced weight gain, adiposity and insulin resistance, we hypothesized that deletion of Lcn2 alongside the skin-specific SCD1 deficiency would diminish the obesity resistance observed in SKO mice. To test this, we developed mice lacking SCD1 expression in the skin and also lacking Lcn2 expression globally and surprisingly, these mice did not gain significantly more weight than the SKO mice under high-fat diet conditions. Therefore, we conclude that Lcn2 does not mediate the protection against high-fat diet-induced adiposity observed in SKO mice.     

Selective breeding for catalepsy changes the distribution of microsatellite D13Mit76 alleles linked to the 5-HT serotonin receptor gene in mice

Catalepsy (pronounced motor inhibition) is a natural defensive reaction against predator. Recently, the quantitative trait locus for catalepsy was mapped on mouse chromosome 13 near the 5-HT(1A) serotonin receptor gene. Here, the linkage between catalepsy and the 5-HT(1A) receptor gene was verified using breeding experiment. Selective breeding for high predisposition to catalepsy was started from backcross BC[CBA x (CBA x AKR)] generation between catalepsy-prone (CBA) and catalepsy-resistant (AKR) mouse strains. CBA and AKR strains also differed in the 5-HT(1A) receptor functional activity. A rapid increase of cataleptic percentage from 21.2% in the backcrosses to 71% in the third generation of selective breeding (S3) was shown. The fragment of chromosome 13 including the 5-HT(1A) receptor gene was marked with D13Mit76 microsatellite. Breeding for catalepsy increased the concentration of CBA-derived and decreased the concentration of AKR-derived alleles of microsatellite D13Mit76 in the S1 and S2. All mice of the S9 and S12 were homozygous for CBA-derived allele of D13Mit76 marker. Mice of the S12 showed CBA-like receptor activity. These findings indicate that selective breeding for behavior can involve selection of polymorphic variants of the 5-HT(1A) receptor gene.     

Genetic polymorphism c.1438A>G of the 5-HT<Subscript>2A</Subscript> receptor is associated with abdominal obesity in Chinese Northern Han population

Objective We examined the potential impact of the 5-hydroxytryptamine 2A receptor (5-HT_2AR) c.1438A>G promoter polymorphism on obesity and estimates of insulin, glucose as well as lipid metabolism. Methods The genotypes and allelic frequencies of the 5-HT_2AR c.1438A>G were examined with polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) in 210 patients with overweight/obesity and 216 unrelated healthy subjects. Results The genotype (AA, AG, and GG) distribution of c.1438A>G polymorphism of the 5-HT_2AR gene promoter was 35%, 46%, and 19% in patients, and 32%, 56%, 12% in controls, respectively, no significant difference was found between two groups. Association of genetic diversity of 5-HT_2AR c.1438A>G with the total body fat, fat distribution and clinical characteristics revealed that overweight/obese men carrying G allele were associated with increased body mass index (P = 0.043), waist circumference (P = 0.038), waist-to-hip ratio (P = 0.045), in comparison with patients who carrying A allele, but there were no significant difference between the c.1438A>G genotype groups in overweight/obese women. Conclusion No significant associations were detected. However, the present study suggests the possibility that an abnormal production rate of the 5-HT_2AR c.1438A>G gene product might lead to the development of abdominal obesity in men but not in women. Su Ying and Yan-Ming Sun equally contributed to this study.     

Overexpression of ABIN-2, a negative regulator of NF-kappaB, delays liver regeneration in the ABIN-2 transgenic mice

Activation of NF-kappaB is one of the earliest responses at the start of liver regeneration, and is required for hepatocyte cell cycle progression. The A20-binding inhibitor of NF-kappaB activation-2, ABIN-2, is an inhibitor of NF-kappaB. However, its effects on hepatocyte cell cycle progression are not known and its involvement in liver regeneration has not been explored. In this study, the temporal expression pattern of the mouse ABIN-2 was studied during liver regeneration induced by partial hepatectomy. We demonstrate that ABIN-2 is rapidly and transiently induced, and expression peaked at around 8h post-hepatectomy. To test that the inducible expression of ABIN-2 serves to regulate NF-kappaB during liver regeneration, transgenic mice overexpressing human ABIN-2 protein in the liver were generated. Our transgenic data demonstrated that overexpression of ABIN-2 inhibited NF-kappaB nuclear translocation, which peaked at around 2-4h post-hepatectomy, and this led to an impairment of the G1/S transition as well as a delay in hepatocyte cell cycle progression of the regenerating liver. In addition, overexpression of ABIN-2 specifically inhibited endogenous ABIN-2 mRNA induction, suggesting a negative feedback mechanism for ABIN-2 expression. In conclusion, ABIN-2 may function as a negative regulator that downregulates NF-kappaB activation during liver regeneration.     

Protection of atherogenesis in thromboxane A2 receptor-deficient mice is not associated with thromboxane A2 receptor in bone marrow-derived cells

In the previous study, we generated mice lacking thromboxane A2 receptor (TP) and apolipoprotein E, apoE(-/-)TP(-/-) mice, and reported that the double knockout mice developed markedly smaller atherosclerotic lesions than those in apoE(-/-) mice. To investigate the mechanism responsible for reduced atherosclerosis in apoE(-/-)TP(-/-) mice, we examined the role of TP in bone marrow (BM)-derived cells in the development of the atherosclerotic lesions. When we compared the function of macrophages in apoE(-/-) and in apoE(-/-)TP(-/-) mouse in vitro, there was no difference in the expression levels of cytokines and chemokines after stimulation with lipopolysaccharide. We then transplanted the BM from either apoE(-/-) or apoE(-/-)TP(-/-) mice to either apoE(-/-) or apoE(-/-)TP(-/-) mice after sublethal irradiation. After 12 weeks with high fat diet, we analyzed the atherosclerotic lesion of aortic sinus. When the BM from apoE(-/-) or apoE(-/-)TP(-/-) mice was transplanted to apoE(-/-) mice, the lesion size was almost the same as that of apoE(-/-) mice without BM transplantation. In contrast, when the BM from apoE(-/-) or apoE(-/-)TP(-/-) mice was transplanted to apoE(-/-)TP(-/-) mice, the lesion size was markedly reduced. These results indicate that the protection of atherogenesis in TP(-/-) mice is not associated with TP in BM-derived cells.     

In vivo efflux of serotonin in the dorsal raphe nucleus of 5-HT1A receptor knockout mice

In the dorsal raphe nucleus (DR), extracellular serotonin (5-HT) regulates serotonergic transmission through 5-HT1A autoreceptors. In this work we used in vivo microdialysis to examine the effects of stressful and pharmacological challenges on DR 5-HT efflux in 5-HT1A receptor knockout (5-HT1A-/-) mice and their wild-type counterparts (5-HT1A+/+). Baseline 5-HT concentrations did not differ between both lines of mice, which is consistent with a lack of tonic control of 5-HT1A autoreceptors on DR 5-HT release. (R)-(+)-8-Hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT, 0.5 mg/kg) reduced 5-HT levels to 30% of basal values in 5-HT1A+/+ mice, but not in 5-HT1A-/- mice. The selective 5-HT1B receptor agonist 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b]pyridin-5-one dihydrochloride (CP 93129, 300 micro m) reduced dialysate 5-HT to the same extent (30-40% of baseline) in the two genotypes, which suggests a lack of compensatory changes in 5-HT1B receptors in the DR of such mutant mice. Both a saline injection and handling for 3 min increased DR dialysate 5-HT in mutants, but not in 5-HT1A+/+ mice. Fluoxetine (5 and 20 mg/kg) elevated 5-HT in a dose-dependent manner in both genotypes. However, this effect was markedly more pronounced in the 5-HT1A-/- mice. The increased responsiveness of the extracellular 5-HT in the DR of 5-HT1A receptor knockout mice reflects a lack of the autoinhibitory control exerted by 5-HT1A autoreceptors.     

BAC transgenic mice to study the expression of P2X2 and P2Y1 receptors

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y₁R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y₁R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09792-9.