Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression.

Inducible expression systems are indispensable for precise regulation and in-depth analysis of biological process. Binary Tet-On system has been widely employed to regulate transgenic expression by doxycycline. Previous pig models with tetracycline regulatory elements were generated through random integration. This process often resulted in uncertain expression and unpredictable phenotypes, thus hindering their applications. Here, by precise knock-in of binary Tet-On 3G elements into Rosa26 and Hipp11 locus, respectively, a double knock-in reporter pig model was generated. We characterized excellent properties of this system for controllable transgenic expression both in vitro and in vivo. Two attP sites were arranged to flank the tdTomato to switch reporter gene. Single or multiple gene replacement was efficiently and faithfully achieved in fetal fibroblasts and nuclear transfer embryos. To display the flexible application of this system, we generated a pig strain with Dox-inducing hKRASG12D expression through phiC31 integrase-mediated cassette exchange. After eight months of Dox administration, squamous cell carcinoma developed in the nose, mouth, and scrotum, which indicated this pig strain could serve as an ideal large animal model to study tumorigenesis. Overall, the established pig models with controllable and switchable transgene expression system will provide a facilitating platform for transgenic and biomedical research.

Early memory deficits and extensive brain network disorganization in the AppNL-F/MAPT double knock-in mouse model of familial Alzheimer's disease.

A critical challenge in current research on Alzheimer's disease (AD) is to clarify the relationship between network dysfunction and the emergence of subtle memory deficits in itspreclinical stage. The AppNL-F/MAPT double knock-in (dKI) model with humanized ß-amyloid peptide (Aß) and tau was used to investigate both memory and network dysfunctions at an early stage. Young male dKI mice (2 to 6 months) were tested in three tasks taxing different aspects of recognition memory affected in preclinical AD. An early deficit first appeared in the object-place association task at the age of 4 months, when increased levels of ß-CTF and Aß were detected in both the hippocampus and the medial temporal cortex, and tau pathology was found only in the medial temporal cortex. Object-place task-dependent c-Fos activation was then analyzed in 22 subregions across the medial prefrontal cortex, claustrum, retrosplenial cortex, and medial temporal lobe. Increased c-Fos activation was detected in the entorhinal cortex and the claustrum of dKI mice. During recall, network efficiency was reduced across cingulate regions with a major disruption of information flow through the retrosplenial cortex. Our findings suggest that early perirhinal-entorhinal pathology is associated with abnormal activity which may spread to downstream regions such as the claustrum, the medial prefrontal cortex and ultimately the key retrosplenial hub which relays information from frontal to temporal lobes. The similarity between our findings and those reported in preclinical stages of AD suggests that the AppNL-F/MAPT dKI model has a high potential for providing key insights into preclinical AD.

FGF21 does not require adipocyte AMP-activated protein kinase (AMPK) or the phosphorylation of acetyl-CoA carboxylase (ACC) to mediate improvements in whole-body glucose homeostasis.

OBJECTIVE: Fibroblast growth factor 21 (FGF21) shows great potential for the treatment of obesity and type 2 diabetes, as its long-acting analogue reduces body weight and improves lipid profiles of participants in clinical studies; however, the intracellular mechanisms mediating these effects are poorly understood. AMP-activated protein kinase (AMPK) is an important energy sensor of the cell and a molecular target for anti-diabetic medications. This work examined the role of AMPK in mediating the glucose and lipid-lowering effects of FGF21. METHODS: Inducible adipocyte AMPK ß1ß2 knockout mice (iß1ß2AKO) and littermate controls were fed a high fat diet (HFD) and treated with native FGF21 or saline for two weeks. Additionally, HFD-fed mice with knock-in mutations on the AMPK phosphorylation sites of acetyl-CoA carboxylase (ACC)1 and ACC2 (DKI mice) along with wild-type (WT) controls received long-acting FGF21 for two weeks. RESULTS: Consistent with previous studies, FGF21 treatment significantly reduced body weight, adiposity, and liver lipids in HFD fed mice. To add, FGF21 improved circulating lipids, glycemic control, and insulin sensitivity. These effects were independent of adipocyte AMPK and were not associated with changes in browning of white (WAT) and brown adipose tissue (BAT). Lastly, we assessed whether FGF21 exerted its effects through the AMPK/ACC axis, which is critical in the therapeutic benefits of the anti-diabetic medication metformin. ACC DKI mice had improved glucose and insulin tolerance and a reduction in body weight, body fat and hepatic steatosis similar to WT mice in response to FGF21 administration. CONCLUSIONS: These data illustrate that the metabolic improvements upon FGF21 administration are independent of adipocyte AMPK, and do not require the inhibitory action of AMPK on ACC. This is in contrast to the anti-diabetic medication metformin and suggests that the treatment of obesity and diabetes with the combination of FGF21 and AMPK activators merits consideration.

The diabetes medication Canagliflozin reduces cancer cell proliferation by inhibiting mitochondrial complex-I supported respiration.

OBJECTIVE: The sodium-glucose transporter 2 (SGLT2) inhibitors Canagliflozin and Dapagliflozin are recently approved medications for type 2 diabetes. Recent studies indicate that SGLT2 inhibitors may inhibit the growth of some cancer cells but the mechanism(s) remain unclear. METHODS: Cellular proliferation and clonogenic survival were used to assess the sensitivity of prostate and lung cancer cell growth to the SGLT2 inhibitors. Oxygen consumption, extracellular acidification rate, cellular ATP, glucose uptake, lipogenesis, and phosphorylation of AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase, and the p70S6 kinase were assessed. Overexpression of a protein that maintains complex-I supported mitochondrial respiration (NDI1) was used to establish the importance of this pathway for mediating the anti-proliferative effects of Canagliflozin. RESULTS: Clinically achievable concentrations of Canagliflozin, but not Dapagliflozin, inhibit cellular proliferation and clonogenic survival of prostate and lung cancer cells alone and in combination with ionizing radiation and the chemotherapy Docetaxel. Canagliflozin reduced glucose uptake, mitochondrial complex-I supported respiration, ATP, and lipogenesis while increasing the activating phosphorylation of AMPK. The overexpression of NDI1 blocked the anti-proliferative effects of Canagliflozin indicating reductions in mitochondrial respiration are critical for anti-proliferative actions. CONCLUSION: These data indicate that like the biguanide metformin, Canagliflozin not only lowers blood glucose but also inhibits complex-I supported respiration and cellular proliferation in prostate and lung cancer cells. These observations support the initiation of studies evaluating the clinical efficacy of Canagliflozin on limiting tumorigenesis in pre-clinical animal models as well epidemiological studies on cancer incidence relative to other glucose lowering therapies in clinical populations.