DPP-4 inhibition with linagliptin ameliorates the progression of premature aging in klotho−/− mice

Background

The potential of anti-aging effect of DPP-4 inhibitors is unknown. This study was performed to determine whether linagliptin, a DPP-4 inhibitor, could protect against premature aging in klotho?/? mice.

Methods

Klotho?/? mice exhibit multiple phenotypes resembling human premature aging, including extremely shortened life span, cognitive impairment, hippocampal neurodegeneration, hair loss, muscle atrophy, hypoglycemia, etc. To investigate the effect of linagliptin on these aging-related phenotypes, male klotho?/? mice were divided into two groups: (1) control group fed the standard diet, and (2) linagliptin group fed the standard diet containing linagliptin. Treatment with linagliptin was performed for 4 weeks. The effect of linagliptin on the above mentioned aging-related phenotypes was examined.

Results

Body weight of klotho?/? mice was greater in linagliptin group than in control group (11.1 ± 0.3 vs 9.9 ± 0.3 g; P < 0.01), which was associated with greater gastrocnemius muscle weight (P < 0.01) and greater kidney weight (P < 0.05) in linagliptin group. Thus, linagliptin significantly prevented body weight loss in klotho?/? mice. Survival rate of klotho?/? mice was greater in linagliptin group (93%) compared to control group (67%), although the difference did not reach statistical significance (P = 0.08). None of linagliptin-treated klotho?/? mice had alopecia during the treatment (P < 0.05 vs control klotho?/? mice). Latency of klotho?/? mice in passive avoidance test was larger in linagliptin group than in control group (P < 0.05), indicating the amelioration of cognitive impairment by linagliptin. Cerebral blood flow of klotho?/? mice was larger in linagliptin group than in control group (P < 0.01), being associated with greater cerebral phospho-eNOS levels (P < 0.05) in linagliptin group. Neuronal cell number in hippocampal CA1 region was greater in linagliptin group than in control group (P < 0.05). Linagliptin group had greater cerebral phospho-Akt (P < 0.05) and phospho-CREB (P < 0.05) than control group. Thus, linagliptin ameliorated brain aging in klotho?/? mice. The degree of hypoglycemia in klotho?/? mice was less in linagliptin group than in control group, as estimated by the findings of OGTT.

Conclusions

Out work provided the evidence that DPP-4 inhibition with linagliptin slowed the progression of premature aging in klotho?/? mice, and provided a novel insight into the potential role of DPP-4 in the mechanism of premature aging.

     

Dietary phosphatidylcholine supplementation reduces atherosclerosis in Ldlr−/− male mice2

Choline is an essential nutrient required for various biological processes. Eggs, dairy, and meat are rich in phosphatidylcholine (PC), whereas cereal and legumes are rich in free choline. Excess dietary choline leads to increase plasma trimethylamine N-oxide (TMAO). Epidemiological studies suggest that plasma TMAO is a biomarker for atherosclerosis and it has been suggested that a lower intake of eggs and meat would reduce choline consumption and thus reduce atherosclerosis development. To investigate whether the form of dietary choline influences atherosclerosis development in Ldlr^−/−, we randomly fed Ldlr^−/−male mice (aged 8 – 10 wk) one of the three 40% (calories) high fat diets (with 0.5% w/w of cholesterol): Control (0.1% w/w free-choline, CON), choline-supplemented (0.4% free-choline, CS), or PC-supplemented (0.1% free-choline and 0.3% choline from PC, PCS). After 12-wk of dietary intervention, the animals were euthanized and tissues and blood collected. Aortic atherosclerotic plaque area, plasma choline, lipid metabolites, and spleen and peripheral blood cell phenotypes were quantified. Surprisingly, the PCS group had significantly lower atherosclerotic lesions while having 2-fold higher plasma TMAO levels compared with both CON and CS groups (P<0.05). In the fasting state, we found that PCS decreased plasma very low-density lipoprotein-cholesterol (VLDL-C) and apolipoprotein B48 (APOB48), and increased plasma high-density lipoprotein-cholesterol (HDL-C). However, very low-density lipoprotein (VLDL) secretion was not affected by dietary treatment. We observed lower levels of circulating pro-atherogenic chemokines in the PCS group. Our study suggests that increased dietary PC intake does not induce a pro-atherogenic phenotype.     

Prevention of TGFβ induction attenuates angII-stimulated vascular biglycan and atherosclerosis in Ldlr−/− mice

Angiotensin II (angII) accelerates atherosclerosis, but the mechanisms are not fully understood. The aim of this study was to determine whether TGFβ is required for angII-induced atherosclerosis. Ldlr-null mice fed a normal chow diet were infused with angII or saline for 28 days. A single injection of TGFβ neutralizing antibody 1D11 (2 mg/kg) prevented angII-induction of TGFβ1 levels, and strikingly attenuated angII-induced accumulation of aortic biglycan content. To study atherosclerosis, mice were infused with angII or saline for 4 weeks, and then fed Western diet for a further 6 weeks. 1D11 had no effect on systolic blood pressure or plasma cholesterol; however, angII-infused mice that received 1D11 had reduced atherosclerotic lesion area by 30% (P < 0.05). Immunohistochemical analyses demonstrated that angII induced both lipid retention and accumulation of biglycan and perlecan which colocalized with apoB. 1D11 strikingly reduced the effect of angII on biglycan but not perlecan. 1D11 decreased total collagen content (P < 0.05) in the lesion area without changing plaque inflammation markers (CD68 and CD45). Thus, this study demonstrates that neutralization of TGFβ attenuated angII stimulation of biglycan accumulation and atherogenesis in mice, suggesting that TGFβ-mediated biglycan induction is one of the mechanisms underlying angII-promoted atherosclerosis.     

Gene expression changes in the secondary palate and mandible of Prdm16 −/− mice

Loss of Prdm16 expression in the mouse leads to a complete cleft of the secondary palate. We have now determined changes in gene expression in the secondary palates of Prdm16 ^?/? fetuses in an attempt to reveal the mechanism(s) leading to the failure of palate closure in these mice. Defined pathway-based polymerase chain reaction arrays were used to analyze the expression of genes associated with the extracellular matrix and the transforming growth factor-β and bone morphogenetic protein signaling networks, perturbations of which can lead to palatal clefting. Loss of Prdm16 expression in the secondary palate leads to alterations in numerous genes within these groups, many of which have been linked to chondrogenesis and osteogenesis. The expression of several genes linked to bone development was significantly changed in the developing secondary palate. Analysis of gene expression in the mandibles of Prdm16 ^?/? fetuses revealed similar alterations in the same gene set. These data suggest that one function of Prdm16 is the regulation of genes that play a role in the differentiation of mesenchymal cells into chondro-/osteocytes.     

Counteract of bone marrow of blotchy mice against the increases of plasma copper levels induced by high-fat diets in LDLR−/− mice

BackgroundBone marrow of blotchy mouse (blotchy marrow) reflects the function of transmembrane domain and relevant intramembrane sites of ATP7A in myeloid cells. By chronic infusion of angiotensin II, we previously found that blotchy marrow plays a minor role in regulating plasma copper. Moreover, the recipients of blotchy marrow presented a moderate reduction of plasma lipids and inflammatory mediator production. Little is known about whether these changes are a specific response to angiotensin II or reveal a more general role of ATP7A.Objective and designWe investigated if blotchy marrow reduces plasma lipids and inflammatory mediators induced by high-fat diets. To test this hypothesis, blotchy and control marrows were reconstituted to the recipient mice (irradiated male LDLR−/− mice), followed by high-fat-diet feeding for 4 months. At the end points, plasma metals (copper, zinc and iron), lipid profiling (cholesterol, triglyceride, phospholipids and lipoprotein) and six inflammatory mediators (lymphotacin, MCP3, MCP5, TIMP1, VEGF-A and IP-10) were measured. Parallel experiments were performed using male LDLR−/− mice fed either high-fat diets or chow diets for 4 months.ResultsIn addition to hyperlipidemia and low-grade inflammation, high-fat diets selectively increased plasma copper concentration compared to chow diets in LDLR−/− mice. After high-fat-diet feeding, the recipients with blotchy marrow showed a decrease in plasma copper (p < 0.01) and an increase in plasma iron (p < 0.05). The recipients with blotchy marrow also presented decreases in cholesterol (p < 0.01) and phospholipids (p < 0.05) in plasma. Surprisingly, plasma levels of MCP3 (p < 0.05), MCP5 (p < 0.05), TIMP1 (p < 0.01), VEGF-A (p < 0.01) and IP-10 (p < 0.01) were significantly increased in the recipients with blotchy marrow compared to controls; the increased levels of MCP3, MCP5 and TIMP1 were more than 50%.ConclusionOur studies showed that blotchy marrow counteracts the increased copper levels induced by high-fat diets, indicating that circulating myeloid cells can regulate blood copper levels via ATP7A. Moreover, transplantation of blotchy marrow followed by high-fat diets leads to a decrease in lipid profile and an increase in inflammatory mediator production. Overall, blotchy marrow mediates divergent responses to angiotensin II and high-fat diets in vivo.     

Naringenin alleviates nonalcoholic steatohepatitis in middle-aged Apoe−/−mice: role of SIRT1

BackgroundNaringenin is naturally isolated from citrus fruits possessing many pharmacological activities. However, little is known about the effect of naringenin on nonalcoholic steatohepatitis (NASH) in the model of metabolic syndrome.PurposeThe present study is aimed to investigate the effect of naringenin on NASH in 12-mo-old male ApoE^−/− mice and its possible underlying mechanism.MethodsIn vivo, 12-mo-old male ApoE^−/− mice were administrated with naringenin by intragastric gavage for 12 weeks. At the end of experiment, the blood samples and liver tissues were collected. Metabolic parameters in serum, levels of triglyceride, cholesterol and hydroxyproline, activities of antioxidant enzymes, and content of inflammatory cytokines (TNF-α and IL-6) in liver were examined by corresponding assay kits. Pathological changes in liver were observed by hematoxylin-eosin, oil red O, masson's trichrome, picro-sirius red and senescence β-galactosidase staining. Dihydroethidium was used for detection of reactive oxygen species (ROS). In vitro, AML-12 cells were treated with oleic acid in the presence or absence of naringenin for 24 h. Transfection of SIRT1 siRNA was also conducted in vitro. Lipid accumulation, cellular ROS generation, malondialdehyde content, antioxidant enzyme activities and secretion levels of TNF-α and IL-6 were examined. Both in vivo and in vitro, gene expressions were detected by real-time PCR or western blot.ResultsNaringenin administration improved metabolic parameters, suppressed hepatic steatosis, regulated expression of genes involved in lipid metabolism (FASN, SCD1, PPARα and CPT1α), reduced hepatic fibrosis and cell senescence, inhibited hepatic inflammation as evidenced by the decreased macrophage recruitment and content of TNF-α and IL-6, and reduced hepatic oxidative stress by suppressing ROS generation and normalizing activities of antioxidant enzymes. Notably, naringenin administration increased hepatic SIRT1 protein expression and activity along with the increased deacetylation of liver kinase B1 (LKB1), PGC1α and NF-κB. In vitro study, the benefits of naringenin on lipid accumulation, oxidative stress and inflammation were diminished by SIRT1 siRNA transfection.ConclusionsThese results indicate that naringenin administration may be a potential curative therapy for NASH treatment and the activation of hepatic SIRT1-mediated signaling cascades is involved in its beneficial effects.     

Properties of the permeability transition in VDAC1−/− mitochondria

Opening of the permeability transition pore (PTP), a high-conductance mitochondrial channel, causes mitochondrial dysfunction with Ca²⁺ deregulation, ATP depletion, release of pyridine nucleotides and of mitochondrial apoptogenic proteins. Despite major efforts, the molecular nature of the PTP remains elusive. A compound library screening led to the identification of a novel high affinity PTP inhibitor (Ro 68-3400), which labeled a ∼32 kDa protein that was identified as isoform 1 of the voltage-dependent anion channel (VDAC1) [A.M. Cesura, E. Pinard, R. Schubenel, V. Goetschy, A. Friedlein, H. Langen, P. Polcic, M.A. Forte, P. Bernardi, J.A. Kemp, The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore. J. Biol. Chem. 278 (2003) 49812–49818]. In order to assess the role of VDAC1 in PTP formation and activity, we have studied the properties of mitochondria from VDAC1^−/− mice. The basic properties of the PTP in VDAC1^−/− mitochondria were indistinguishable from those of strain-matched mitochondria from wild-type CD1 mice, including inhibition by Ro 68-3400, which labeled identical proteins of 32 kDa in both wild-type and VDAC1^−/− mitochondria. The labeled protein could be separated from all VDAC isoforms. While these results do not allow to exclude that VDAC is part of the PTP, they suggest that VDAC is not the target for PTP inhibition by Ro 68-3400.     

Brain phenotype of transgenic mice overexpressing cystathionine β-synthase

Background

The cystathionine β-synthase (CBS) gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS) cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA) metabolism, a pathway important for several brain physiological processes.

Methodology/Principal Findings

Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1) expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line.

Conclusion/Significance

We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.     

Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR−/− and apoE−/− mice

The role of farnesoid X receptor (FXR) in the development of atherosclerosis has been unclear. Here, LDL receptor (LDLR^−/−) or apolipoprotein E (apoE^−/−) female or male mice were fed a Western diet and treated with a potent synthetic FXR agonist, WAY-362450. Activation of FXR blocked diet-induced hypertriglyceridemia and elevations of non-HDL cholesterol and produced a near complete inhibition of aortic lesion formation. WAY-362450 also induced small heterodimer partner (SHP) expression and repressed cholesterol 7α-hydroxylase (CYP7A1) and sterol 12 α-hydroxylase (CYP8B1) expression. To determine if SHP was essential for these protective activities, LDLR^−/−SHP^−/− and apoE^−/−SHP^−/− mice were similarly treated with WAY-362450. Surprisingly, a notable sex difference was observed in these mice. In male LDLR^−/−SHP^−/− or apoE^−/−SHP^−/− mice, WAY-362450 still repressed CYP7A1 and CYP8B1 expression by 10-fold and still strongly reduced non-HDL cholesterol levels and aortic lesion area. In contrast, in the female LDLR^−/−SHP^−/− or apoE^−/−SHP^−/− mice, WAY-362450 only slightly repressed CYP7A1 and CYP8B1 expression and did not reduce non-HDL cholesterol or aortic lesion size. WAY-362450 inhibition of hypertriglyceridemia remained intact in LDLR^−/− or apoE^−/− mice lacking SHP of both sexes. These results suggest that activation of FXR protects against atherosclerosis in the mouse, and this protective effect correlates with repression of bile acid synthetic genes, with mechanistic differences between male and female mice.     

Sclerostin antibody treatment rescues the osteopenic bone phenotype of TGFβ inducible early gene-1 knockout female mice

Deletion of TGFβ inducible early gene-1 (TIEG) in mice results in an osteopenic phenotype that exists only in female animals. Molecular analyses on female TIEG knockout (KO) mouse bones identified increased expression of sclerostin, an effect that was confirmed at the protein level in serum. Sclerostin antibody (Scl-Ab) therapy has been shown to elicit bone beneficial effects in multiple animal model systems and human clinical trials. For these reasons, we hypothesized that Scl-Ab therapy would reverse the low bone mass phenotype of female TIEG KO mice. In this study, wildtype (WT) and TIEG KO female mice were randomized to either vehicle control (Veh, n = 12/group) or Scl-Ab therapy (10 mg/kg, 1×/wk, s.c.; n = 12/group) and treated for 6 weeks. Following treatment, bone imaging analyses revealed that Scl-Ab therapy significantly increased cancellous and cortical bone in the femur of both WT and TIEG KO mice. Similar effects also occurred in the vertebra of both WT and TIEG KO animals. Additionally, histomorphometric analyses revealed that Scl-Ab therapy resulted in increased osteoblast perimeter/bone perimeter in both WT and TIEG KO animals, with a concomitant increase in P1NP, a serum marker of bone formation. In contrast, osteoclast perimeter/bone perimeter and CTX-1 serum levels were unaffected by Scl-Ab therapy, irrespective of mouse genotype. Overall, our findings demonstrate that Scl-Ab therapy elicits potent bone-forming effects in both WT and TIEG KO mice and effectively increases bone mass in female TIEG KO mice.     

Rag-dependent and Rag-independent mechanisms of Notch1 rearrangement in thymic lymphomas of Atm−/− and scid mice

The pathways of thymic lymphomagenesis are classified as Rag-dependent or -independent according to their dependence on recombination-activating gene (Rag1/2) proteins. The role of the two-lymphoma pathways in oncogene rearrangements and the connection between lymphoma pathways and rearrangement mechanisms, however, remain obscure. We compared the incidence and latency of thymic lymphomas, and associated rearrangements of the representative oncogene Notch1 among Rag2^?/?, ataxia telangiectasia mutated (Atm)^?/?, and severe combined immune deficiency (scid) mice combined with Rag2 deficiency. Contrary to expectations, Rag2^?/? mice were prone to thymic lymphoma development, suggesting the existence of a Rag2-independent lymphoma pathway in Rag2^?/? mice. The lymphoma incidence in Rag2^?/?Atm^?/? mice was lower than that in Atm^?/? mice, but higher than that in Rag2^?/? mice, indicating that Atm^?/? mice develop lymphomas through both pathways. Scid mice developed lymphomas with an incidence and latency similar to Rag2^?/?scid mice, suggesting that Rag2-mediated V(D)J recombination-driven events are not necessarily required for lymphomagenesis in scid mice. Notch1 rearrangement mechanisms were classified as Rag2-dependent or Rag2-independent based on the presence of recombination signal-like sequences at rearranged sites. In Rag2^?/? lymphomas, Notch1 must be rearranged independently of Rag2 function, implying that Rag2^?/? mice are susceptible to lymphomagenesis due to the presence of other rearrangement mechanisms. The results in Atm^?/? mice suggest that Notch1 was rearranged through both lymphoma pathways. In scid mice, the frequency of Rag2-mediated rearrangements was relatively low compared with that in wild-type mice, suggesting that the Rag2-independent lymphoma pathway prevails in the development of thymic lymphomas in scid mice. Thus, two rearrangement mechanisms underlie the lymphoma pathways and constitute the mechanistic bases for lymphomagenesis, thereby providing the molecular criteria for distinguishing between Rag2-dependent and Rag2-independent lymphoma pathways.     

Characterization of intestinal inflammation and identification of related gene expression changes in mdr1a−/− mice

Multidrug resistance targeted mutation (mdr1a (-/-) ) mice spontaneously develop intestinal inflammation. The aim of this study was to further characterize the intestinal inflammation in mdr1a (-/-) mice. Intestinal samples were collected to measure inflammation and gene expression changes over time. The first signs of inflammation occurred around 16 weeks of age and most mdr1a (-/-) mice developed inflammation between 16 and 27 weeks of age. The total histological injury score was the highest in the colon. The inflammatory lesions were transmural and discontinuous, revealing similarities to human inflammatory bowel diseases (IBD). Genes involved in inflammatory response pathways were up-regulated whereas genes involved in biotransformation and transport were down-regulated in colonic epithelial cell scrapings of inflamed mdra1 (-/-) mice at 25 weeks of age compared to non-inflamed FVB mice. These results show overlap to human IBD and strengthen the use of this in vivo model to study human IBD. The anti-inflammatory regenerating islet-derived genes were expressed at a lower level during inflammation initiation in non-inflamed colonic epithelial cell scrapings of mdr1a (-/-) mice at 12 weeks of age. This result suggests that an insufficiently suppressed immune response could be crucial to the initiation and development of intestinal inflammation in mdr1a (-/-) mice.     

Slow accumulation of mutations in Xpc−/− mice upon induction of oxidative stress

XPC is one of the key DNA damage recognition proteins in the global genome repair route of the nucleotide excision repair (NER) pathway. Previously, we demonstrated that NER-deficient mouse models Xpa^?/? and Xpc^?/? exhibit a divergent spontaneous tumor spectrum and proposed that XPC might be functionally involved in the defense against oxidative DNA damage. Others have mechanistically dissected several functionalities of XPC to oxidative DNA damage sensitivity using in vitro studies. XPC has been linked to regulation of base excision repair (BER) activity, redox homeostasis and recruitment of ATM and ATR to damage sites, thereby possibly regulating cell cycle checkpoints and apoptosis. XPC has additionally been implicated in recognition of bulky (e.g. cyclopurines) and non-bulky DNA damage (8-oxodG). However, the ultimate contribution of the XPC functionality in vivo in the oxidative DNA damage response and subsequent mutagenesis process remains unclear. Our study indicates that Xpc^?/? mice, in contrary to Xpa^?/? and wild type mice, have an increased mutational load upon induction of oxidative stress and that mutations arise in a slowly accumulative fashion. The effect of non-functional XPC in vivo upon oxidative stress exposure appears to have implications in mutagenesis, which can contribute to the carcinogenesis process. The levels and rate of mutagenesis upon oxidative stress correlate with previous findings that lung tumors in Xpc^?/? mice overall arise late in the lifespan and that the incidence of internal tumors in XP-C patients is relatively low in comparison to skin cancer incidence.