Deletion of Lkb1 in adult mice results in body weight reduction and lethality.
ABSTRACT: Liver kinase B1 (Lkb1) plays crucial roles in development, metabolism and survival. As constitutive knockout of Lkb1 in mice leads to embryonic lethality, whether Lkb1 is required for the growth and survival of adult mice is unclear. Here we address this question using a tamoxifen-inducible Lkb1 knockout (KO) mouse model: Rosa26-CreER: Lkb1flox/flox (abbreviated as Rosa-Lkb1). The Rosa-Lkb1 mice exhibited body weight reduction and died within 6 weeks after tamoxifen induction. The body weight reduction was due to reduced weight of various tissues but the brown and white adipose tissues underwent much more pronounced weight reduction relative to the overall body weight reduction. Accordingly, the Rosa-Lkb1 mice had increased blood glucose levels and were intolerant to glucose challenge. Expression levels of adipogenic and lipogenic genes in adipose tissues were also dramatically reduced by Lkb1 deletion. Additionally, Lkb1 deletion reduced lipid deposition and increased expression of mitochondrial (Pgc1a, Cox5b and Cox7a) and hepatic gluconeogenesis related genes (Pepck) in liver. Finally, the Rosa-Lkb1 mice had much reduced oxygen consumption, carbon dioxide production, and energy expenditure. These results demonstrate that Lkb1 plays an important role in maintaining body weight, liver and adipose tissue function, blood glucose homeostasis and survival in adult mice.
Project description:Vascular calcification is a common pathological feature of atherosclerosis, chronic kidney disease, vascular injury, and aging. Liver kinase B1 (LKB1) plays pivotal roles in cellular processes such as apoptosis, metabolism, and cell cycle regulation. In addition, growing evidence has indicated that LKB1 functions as a tumor suppressor gene. However, its role in vascular calcification has not been reported. LKB1<sup>flox/flox</sup> mice were hybridized with SM22-CreER<sup>T2</sup> transgenic mice and adult mice received tamoxifen to obtain smooth muscle-specific LKB1-knockout (LKB1<sup>SMKO</sup>) mice. LKB1 expression was decreased under calcifying conditions, and LKB1 overexpression had a protective effect on vascular calcification. However, high mobility group box 1 (HMGB1) overexpression partially counteracted the promotion of vascular calcification induced by LKB1 overexpression. Mechanically, LKB1 could bind to HMGB1 to promote HMGB1 degradation. Furthermore, LKB1<sup>SMKO</sup> mice showed intensified vascular calcification, which was alleviated by treatment with the HMGB1 inhibitor glycyrrhizic acid. Based on our results, LKB1 may inhibit vascular calcification via inhibiting HMGB1 expression.
Project description:<h4>Aims/hypothesis</h4>To directly assess the role of beta cell lipolysis in insulin secretion and whole-body energy homeostasis, inducible beta cell-specific adipose triglyceride lipase (ATGL)-deficient (B-Atgl-KO) mice were studied under normal diet (ND) and high-fat diet (HFD) conditions.<h4>Methods</h4>Atgl <sup>flox/flox</sup> mice were cross-bred with Mip-Cre-ERT mice to generate Mip-Cre-ERT<sup>/+</sup>;Atgl <sup>flox/flox</sup> mice. At 8 weeks of age, these mice were injected with tamoxifen to induce deletion of beta cell-specific Atgl (also known as Pnpla2), and the mice were fed an ND or HFD.<h4>Results</h4>ND-fed male B-Atgl-KO mice showed decreased insulinaemia and glucose-induced insulin secretion (GSIS) in vivo. Changes in GSIS correlated with the islet content of long-chain saturated monoacylglycerol (MAG) species that have been proposed to be metabolic coupling factors for insulin secretion. Exogenous MAGs restored GSIS in B-Atgl-KO islets. B-Atgl-KO male mice fed an HFD showed reduced insulinaemia, glycaemia in the fasted and fed states and after glucose challenge, as well as enhanced insulin sensitivity. Moreover, decreased insulinaemia in B-Atgl-KO mice was associated with increased energy expenditure, and lipid metabolism in brown (BAT) and white (WAT) adipose tissues, leading to reduced fat mass and body weight.<h4>Conclusions/interpretation</h4>ATGL in beta cells regulates insulin secretion via the production of signalling MAGs. Decreased insulinaemia due to lowered GSIS protects B-Atgl-KO mice from diet-induced obesity, improves insulin sensitivity, increases lipid mobilisation from WAT and causes BAT activation. The results support the concept that fuel excess can drive obesity and diabetes via hyperinsulinaemia, and that an islet beta cell ATGL-lipolysis/adipose tissue axis controls energy homeostasis and body weight via insulin secretion.
Project description:Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the production of PGD<sub>2</sub> in adipocytes and is selectively induced by a high-fat diet (HFD) in adipose tissue. In this study, we investigated the effects of HFD on obesity and insulin resistance in two distinct types of adipose-specific L-PGDS gene knockout (KO) mice: fatty acid binding protein 4 (fabp4, aP2)-Cre/L-PGDS <sup>flox/flox</sup> and adiponectin (AdipoQ)-Cre/L-PGDS <sup>flox/flox</sup> mice. The L-PGDS gene was deleted in adipocytes in the premature stage of the former strain and after maturation of the latter strain. The L-PGDS expression and PGD<sub>2</sub> production levels decreased in white adipose tissue (WAT) under HFD conditions only in the aP2-Cre/L-PGDS <sup>flox/flox</sup> mice, but were unchanged in the AdipoQ-Cre/L-PGDS <sup>flox/flox</sup> mice. When fed an HFD, aP2-Cre/L-PGDS <sup>flox/flox</sup> mice significantly reduced body weight gain, adipocyte size, and serum cholesterol and triglyceride levels. In WAT of the HFD-fed aP2-Cre/L-PGDS <sup>flox/flox</sup> mice, the expression levels of the adipogenic, lipogenic, and M1 macrophage marker genes were decreased, whereas those of the lipolytic and M2 macrophage marker genes were enhanced or unchanged. Insulin sensitivity was improved in the HFD-fed aP2-Cre/L-PGDS <sup>flox/flox</sup> mice. These results indicate that PGD<sub>2</sub> produced by L-PGDS in premature adipocytes is involved in the regulation of body weight gain and insulin resistance under nutrient-dense conditions.
Project description:Serine/threonine kinase 11, commonly known as liver kinase b1 (Lkb1), is a tumor suppressor that regulates cellular energy metabolism and stem cell function. Satellite cells are skeletal muscle resident stem cells that maintain postnatal muscle growth and repair. Here, we used MyoD(Cre)/Lkb1(flox/flox) mice (called MyoD-Lkb1) to delete Lkb1 in embryonic myogenic progenitors and their descendant satellite cells and myofibers. The MyoD-Lkb1 mice exhibit a severe myopathy characterized by central nucleated myofibers, reduced mobility, growth retardation, and premature death. Although tamoxifen-induced postnatal deletion of Lkb1 in satellite cells using Pax7(CreER) mice bypasses the developmental defects and early death, Lkb1 null satellite cells lose their regenerative capacity cell-autonomously. Strikingly, Lkb1 null satellite cells fail to maintain quiescence in noninjured resting muscles and exhibit accelerated proliferation but reduced differentiation kinetics. At the molecular level, Lkb1 limits satellite cell proliferation through the canonical AMP-activated protein kinase/mammalian target of rapamycin pathway, but facilitates differentiation through phosphorylation of GSK-3?, a key component of the WNT signaling pathway. Together, these results establish a central role of Lkb1 in muscle stem cell homeostasis, muscle development, and regeneration.
Project description:Single nucleotide polymorphisms in intron 1 of the fat mass and obesity-associated (FTO) gene were found to be associated with an increased risk of adult obesity. Enhanced FTO expression in mice leads to hyperphagia, increased fat mass, and higher body weight. Neuronal-specific FTOâ??deleted mice have an identical lean body weight phenotype to global FTO-deleted mice. The physiological role of adipose FTO in the homeostasis of energy regulation remains to be elucidated. We used microarrays to elucidate the metabolic pathways that are regulated by FTO in the white fat. FTO flox/flox and Adiponectin-cre FTO flox/flox (AFO) mice were fed with chow diet. White fat tissues from epididymal adipose pad were harvested under ad lib condition for RNA isolation. Three independent pools of FTO flox/flox and AFO mouse white fat RNA were included in the study.
Project description:Compelling evidence from both human and animal studies suggests a physiological link between the circadian rhythm and metabolism but the underlying mechanism is still incompletely understood. We examined the role of PPAR?, a key regulator of energy metabolism, in the control of physiological and behavioral rhythms by analyzing two strains of whole-body PPAR? null mouse models. Systemic inactivation of PPAR? was generated constitutively by using Mox2-Cre mice (MoxCre/flox) or inducibly by using the tamoxifen system (EsrCre/flox/TM). Circadian variations in oxygen consumption, CO(2) production, food and water intake, locomotor activity, and cardiovascular parameters were all remarkably suppressed in MoxCre/flox mice. A similar phenotype was observed in EsrCre/flox/TM mice, accompanied by impaired rhythmicity of the canonical clock genes in adipose tissues and liver but not skeletal muscles or the kidney. PPAR? inactivation in isolated preadipocytes following exposure to tamoxifen led to a similar blockade of the rhythmicity of the clock gene expression. Together, these results support an essential role of PPAR? in the coordinated control of circadian clocks and metabolic pathways.
Project description:Liver regeneration after injury is normally mediated by proliferation of hepatocytes, although recent studies have suggested biliary epithelial cells (BECs) can differentiate into hepatocytes during severe liver injury when hepatocyte proliferation is impaired. We investigated the effect of hepatocyte-specific β-catenin deletion in recovery from severe liver injury and BEC-to-hepatocyte differentiation. To induce liver injury, we administered choline-deficient, ethionine-supplemented (CDE) diet to three different mouse models, the first being mice with deletion of β-catenin in both BECs and hepatocytes (Albumin-Cre; Ctnnb1<sup>flox/flox</sup> mice). In our second model, we performed hepatocyte lineage tracing by injecting Ctnnb1<sup>flox/flox</sup> ; Rosa-stop<sup>flox/flox</sup> -EYFP mice with the adeno-associated virus serotype 8 encoding Cre recombinase under the control of the thyroid binding globulin promoter, a virus that infects only hepatocytes. Finally, we performed BEC lineage tracing via Krt19-Cre<sup>ERT</sup> ; Rosa-stop<sup>flox/flox</sup> -tdTomato mice. To observe BEC-to-hepatocyte differentiation, mice were allowed to recover on normal diet following CDE diet-induced liver injury. Livers were collected from all mice and analyzed by quantitative real-time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. We show that mice with lack of β-catenin in hepatocytes placed on the CDE diet develop severe liver injury with impaired hepatocyte proliferation, creating a stimulus for BECs to differentiate into hepatocytes. In particular, we use both hepatocyte and BEC lineage tracing to show that BECs differentiate into hepatocytes, which go on to repopulate the liver during long-term recovery. Conclusion: β-catenin is important for liver regeneration after CDE diet-induced liver injury, and BEC-derived hepatocytes can permanently incorporate into the liver parenchyma to mediate liver regeneration.
Project description:Although innate immunity is linked to metabolic health, the effect of leptin signaling in cells from the innate immune system on glucose homeostasis has not been thoroughly investigated. We generated two mouse models using Cre-lox methodology to determine the effect of myeloid cell-specific leptin receptor (Lepr) reconstitution and Lepr knockdown on in vivo glucose metabolism. Male mice with myeloid cell-specific Lepr reconstitution (Lyz2Cre<sup>+</sup>Lepr<sup>loxTB/loxTB</sup>) had better glycemic control as they aged compared to male mice with whole-body transcriptional blockade of Lepr (Lyz2Cre<sup>-</sup>Lepr<sup>loxTB/loxTB</sup>). In contrast, Lyz2Cre<sup>+</sup>Lepr<sup>loxTB/loxTB</sup> females only had a trend for diminished hyperglycemia after a prolonged fast. During glucose tolerance tests, Lyz2Cre<sup>+</sup>Lepr<sup>loxTB/loxTB</sup> males had a mildly improved plasma glucose profile compared to Cre<sup>-</sup> controls while Lyz2Cre<sup>+</sup>Lepr<sup>loxTB/loxTB</sup> females had a similar glucose excursion to their Cre<sup>-</sup> controls. Myeloid cell-specific Lepr knockdown (Lyz2Cre<sup>+</sup>Lepr<sup>flox/flox</sup>) did not significantly alter body weight, blood glucose, insulin sensitivity, or glucose tolerance in males or females. Expression of the cytokine interleukin 10 (anti-inflammatory) tended to be higher in adipose tissue of male Lyz2Cre<sup>+</sup>Lepr<sup>loxTB/loxTB</sup> mice (p = 0.0774) while interleukin 6 (pro-inflammatory) was lower in male Lyz2Cre<sup>+</sup>Lepr<sup>flox/flox</sup> mice (p < 0.05) vs. their respective controls. In conclusion, reconstitution of Lepr in cells of myeloid lineage has beneficial effects on glucose metabolism in male mice.
Project description:Excess body weight and hyperlipidaemia cause severe health problems and have social implications. Amycenone is an active substance extracted from Yamabushitake mushrooms with no reports of its activity against excess body weight and hyperlipidaemia. This research clarifies the effects and mechanisms of action of amycenone on the inhibition of body weight excess and hyperlipidaemia attenuation using KK-<i>A<sup>y</sup></i> mice. Amycenone or water was administered to 8-week-old male KK-<i>A<sup>y</sup></i> mice by gavage for 8 weeks. Their body weight and food intake were recorded during the experiment. At the end of the experimental period, the mice were dissected, and blood samples, lipid metabolism-related organs and tissues were collected and stored for further analysis. Amycenone treatment suppressed body weight gain and improved serum levels of fasting blood glucose and non-esterified fatty acids. Additionally, serum and hepatic cholesterol and triacylglycerol levels were reduced after this treatment, whereas the phosphorylation levels of AMPK, PKA and HSL increased and the expression level of FAS decreased. The protein level of C/EBPβ and gene expression level of <i>Cpt1</i> were higher in the perirenal adipose tissue of amycenone-treated KK-<i>A<sup>y</sup></i> mice. Furthermore, amycenone phosphorylated AMPK, PKA and ACC, and PPARγ expression was lower in the mesenteric adipose tissue. The phosphorylation levels of AMPK, LKB1, PKA and ACC were also induced, and FAS expression level was reduced in the liver of the amycenone-treated group. Amycenone could reduce excess body weight and attenuate hyperlipidaemia in KK-<i>A<sup>y</sup></i> mice by inhibiting lipogenesis and promoting lipolysis through lipid metabolism pathway stimulation and fatty acid β-oxidation acceleration.
Project description:The adipocyte-derived hormone leptin acts within the central nervous system to decrease food intake and body weight and to increase renal and thermogenic brown adipose tissue sympathetic nerve activity (SNA). Previous studies have focused on hypothalamic brain regions, although recent findings have identified leptin receptors (ObR) in a distributed brain network, including the circumventricular subfornical organ (SFO), a forebrain region devoid of a blood-brain barrier. We tested the hypothesis that ObR in the SFO are functionally linked to leptin-induced decreases in food intake and body weight and increases in SNA. SFO-targeted microinjections of an adenovirus encoding Cre-recombinase in ObR(flox/flox) mice resulted in selective ablation of ObR in the SFO. Interestingly, deletion of ObR in the SFO did not influence the decreases in either food intake or body weight in response to daily systemic or cerebroventricular administration of leptin. In line with these findings, reduction in SFO ObR did not attenuate leptin-mediated increases in thermogenic brown adipose tissue SNA. In contrast, increases in renal SNA induced by systemic or cerebroventricular administration of leptin were abolished in mice with SFO-targeted deletion of ObR. These results demonstrate that ObR in the SFO play an important role in leptin-induced renal sympathoexcitation, but not in the body weight, food intake, or brown adipose tissue SNA thermogenic effects of leptin. These findings highlight the concept of a distributed brain network of leptin action and illustrate that brain regions, including the SFO, can mediate distinct cardiovascular and metabolic responses to leptin.