Featured ArticlesVolume 6 | No. 3 | March 2017
|Glucagon-CreER Mouse Line Generated by CRISPR-Cas9Pancreatic α-cells secrete glucagon to increase plasma glucose levels in times of fasting and in opposition to insulin action. α-cell dysfunction contributes to dysglycemia in type 1 and type 2 diabetes mellitus. Here, Ackermann et al. describe a novel Glucagon-CreERT2 gene-addition mouse line that was generated via CRISPR-Cas9 assisted gene targeting, does not disrupt glucagon expression from the targeted allele, and recapitulates endogenous glucagon and glucagon-like peptide 1 expression. This will be a useful tool to perform specific genetic manipulations in murine α-cells. This system allows for time-controlled and cell type-specific gene ablation, gene mutation, or gene activation, depending on the design of the loxP-modified gene of interest.|
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Objective: α-cells are the second most prominent cell type in pancreatic islets and are responsible for producing glucagon to increase plasma glucose levels in times of fasting. α-cell dysfunction and inappropriate glucagon secretion occur in both type 1 and type 2 diabetes. Thus, there is growing interest in studying both normal function and pathophysiology of α-cells. However, tools to target gene ablation or activation specifically of α-cells have been limited, compared to those available for ß-cells. Previous Glucagon-Cre and Glucagon-CreER transgenic mouse lines have suffered from transgene silencing, and the only available Glucagon-CreER “knock-in” mouse line results in glucagon haploinsufficiency, which can confound the interpretation of gene deletion analyses. Therefore, we sought to develop a Glucagon-CreERT2 mouse line that would maintain normal glucagon expression and would be less susceptible to transgene silencing.
Methods: We utilized CRISPR-Cas9 technology to insert an IRES-CreERT2 sequence into the 3' UTR of the Glucagon (Gcg) locus in mouse embryonic stem cells (ESCs). Targeted ESC clones were then injected into mouse blastocysts to obtain Gcg-CreERT2 mice. Recombination efficiency in GCG+ pancreatic α-cells and glucagon-like peptide 1 positive (GLP1+) enteroendocrine L-cells was measured in Gcg-CreERT2;Rosa26-LSL-YFP mice injected with tamoxifen during fetal development and adulthood.
Results: Tamoxifen injection of Gcg-CreERT2;Rosa26-LSL-YFP mice induced high recombination efficiency of the Rosa26-LSL-YFP locus in perinatal and adult α-cells (88% and 95%, respectively), as well as in first-wave fetal α-cells (36%) and adult enteroendocrine L-cells (33%). Mice homozygous for the Gcg-CreERT2 allele were phenotypically normal.
Conclusions: We successfully derived a Gcg-CreERT2 mouse line that expresses CreERT2 in pancreatic α-cells and enteroendocrine L-cells without disrupting preproglucagon gene expression. These mice will be a useful tool for performing temporally controlled genetic manipulation specifically in these cell types.[Hide abstract]
|Glucagon-like peptide-2 controls intestinal amino acid transport Enteroendocrine cells (EECs) have evolved in part to control the efficiency of nutrient absorption through secretion of specialized peptide hormones. Lee and colleagues examined whether and how acute glucagon-like peptide 2 (GLP-2) signaling augments intestinal amino acid (AA) absorption in mice. They identified candidate AA transporters transducing GLP-2 action and demonstrate that loss of GLP-2 receptor signaling in Glp2r-/- mice leads to reduced AA absorption. They show that GLP-2 robustly activates the mTORC1 pathway in the murine small intestine. Collectively, these findings demonstrate that GLP-2 controls energy and AA absorption through pathways engaging AA uptake, linking L cell GLP-2 to physiological mechanisms controlling intestinal AA sensing and absorption in vivo.|
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Objective: Glucagon-like peptide-2 (GLP-2) is co-secreted with GLP-1 from gut endocrine cells, and both peptides act as growth factors to expand the surface area of the mucosal epithelium. Notably, GLP-2 also enhances glucose and lipid transport in enterocytes; however, its actions on control of amino acid (AA) transport remain unclear. Here we examined the mechanisms linking gain and loss of GLP-2 receptor (GLP-2R) signaling to control of intestinal amino acid absorption in mice.
Methods: Absorption, transport, and clearance of essential AAs, specifically lysine, were measured in vivo by Liquid Chromatography triple quadrupole Mass Spectrometry (LC-MS/MS) and ex vivo with Ussing chambers using intestinal preparations from Glp2r+/+ and Glp2r-/- mice. Immunoblotting determined jejunal levels of protein components of signaling pathways (PI3K-AKT, and mTORC1-pS6-p4E-BP1) following administration of GLP-2, protein gavage, and rapamycin to fasted Glp2r+/+ and Glp2r-/- mice. Expression of AA transporters from full thickness jejunum and 4F2hc from brush border membrane vesicles (BBMVs) was measured by real-time PCR and immunoblotting, respectively.
Results: Acute administration of GLP-2 increased basal AA absorption in vivo and augmented basal lysine transport ex vivo. GLP-2-stimulated lysine transport was attenuated by co-incubation with wortmannin, rapamycin, or tetrodotoxin ex vivo. Phosphorylation of mTORC1 effector proteins S6 and 4E-BP1 was significantly increased in wild-type mice in response to GLP-2 alone, or when co-administered with protein gavage, and abolished following oral gavage of rapamycin. In contrast, activation of GLP-1R signaling did not enhance S6 phosphorylation. Disruption of GLP-2 action in Glp2r-/- mice reduced lysine transport ex vivo and attenuated the phosphorylation of S6 and 4E-BP1 in response to oral protein. Moreover, the expression of cationic AA transporter slc7a9 in response to refeeding, and the abundance of 4F2hc in BBMVs following protein gavage, was significantly attenuated in Glp2r-/- mice.
Conclusions: These findings reveal an important role for GLP-2R signaling in the physiological and pharmacological control of enteral amino acid sensing and assimilation, defining an enteroendocrine cell-enterocyte axis for optimal energy absorption.[Hide abstract]
|Bezafibrate ameliorates diabetes in TallyHo mice Bezafibrate (BEZ) is a member of the fibrate group that possesses the unique feature of activating all known peroxisome proliferator-activated receptors (PPARs). PPARs are transcription factors regulating crucial genes involved in fatty acid metabolism and insulin sensitivity. To study whether BEZ could also ameliorate conditions associated with fatty liver and type 2 diabetes, Franko et al. used the TallyHo mouse model, which is characterized by elevated plasma lipid levels and body weight, high fat mass, steatosis, and intermediate to severe diabetes. Their data demonstrated that BEZ ameliorates impaired glucose metabolism in TallyHo mice via decreased hepatic fat content and suppressed hepatic gluconeogenesis in association with increased mitochondrial mass and elevated metabolic flexibility.|
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Objective: Recently, we have shown that Bezafibrate (BEZ), the pan-PPAR (peroxisome proliferator-activated receptor) activator, ameliorated diabetes in insulin deficient streptozotocin treated diabetic mice. In order to study whether BEZ can also improve glucose metabolism in a mouse model for fatty liver and type 2 diabetes, the drug was applied to TallyHo mice.
Methods: TallyHo mice were divided into an early (ED) and late (LD) diabetes progression group and both groups were treated with 0.5% BEZ (BEZ group) or standard diet (SD group) for 8 weeks. We analyzed plasma parameters, pancreatic beta-cell morphology, and mass as well as glucose metabolism of the BEZ-treated and control mice. Furthermore, liver fat content and composition as well as hepatic gluconeogenesis and mitochondrial mass were determined.
Results: Plasma lipid and glucose levels were markedly reduced upon BEZ treatment, which was accompanied by elevated insulin sensitivity index as well as glucose tolerance, respectively. BEZ increased islet area in the pancreas. Furthermore, BEZ treatment improved energy expenditure and metabolic flexibility. In the liver, BEZ ameliorated steatosis, modified lipid composition and increased mitochondrial mass, which was accompanied by reduced hepatic gluconeogenesis.
Conclusions: Our data showed that BEZ ameliorates diabetes probably via reduced steatosis, enhanced hepatic mitochondrial mass, improved metabolic flexibility and elevated hepatic insulin sensitivity in TallyHo mice, suggesting that BEZ treatment could be beneficial for patients with NAFLD and impaired glucose metabolism.[Hide abstract]
|The Roles of Adiponectin-Induced Ceramidase Signaling in Lipid and Glucose HomeostasisAdiponectin is capable of inducing ceramidase activity through its receptors AdipoR1 and AdipoR2. To gain further insights into the local physiological consequences of adiponectin and AdipoR-induced ceramidase activation, Holland, Xia, and colleagues have generated models that inducibly express AdipoR1 or AdipoR2. They used these models to determine which adiponectin receptor may have the most beneficial effects on glucose tolerance. Their data suggest that the lowering of ceramides is a critical player in adiponectin-induced improvements in non-alcoholic fatty liver disease and hepatic insulin resistance in mice with diet-induced obesity.|
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Objective: Adiponectin and the signaling induced by its cognate receptors, AdipoR1 and AdipoR2, have garnered attention for their ability to promote insulin sensitivity and oppose steatosis. Activation of these receptors promotes the deacylation of ceramide, a lipid metabolite that appears to play a causal role in impairing insulin signaling.
Methods: Here, we have developed transgenic mice that overexpress AdipoR1 or AdipoR2 under the inducible control of a tetracycline response element. These represent the first inducible genetic models that acutely manipulate adiponectin receptor signaling in adult mouse tissues, which allows us to directly assess AdipoR signaling on glucose and lipid metabolism.
Results: Overexpression of either adiponectin receptor isoform in the adipocyte or hepatocyte is sufficient to enhance ceramidase activity, whole body glucose metabolism, and hepatic insulin sensitivity, while opposing hepatic steatosis. Importantly, metabolic improvements fail to occur in an adiponectin knockout background. When challenged with a leptin-deficient genetic model of type 2 diabetes, AdipoR2 expression in adipose or liver is sufficient to reverse hyperglycemia and glucose intolerance.
Conclusions: These observations reveal that adiponectin is critical for AdipoR-induced ceramidase activation which enhances hepatic glucose and lipid metabolism via rapidly acting “cross-talk” between liver and adipose tissue sphingolipids.[Hide abstract]
|Effects of a natriuretic peptide in the regulation of adipose tissue and metabolismNatriuretic peptides (NPs) have metabolic effects like promotion of lipolysis in human adipocytes. Importantly, obesity, diabetes, and the metabolic syndrome are all characterized by reduced levels of circulating NP. In the present study, Glöde and colleagues used an optimized designer NP (CD-NP, or Cenderitide) and analyzed the effect of long-term CD-NP in the context of diet-induced obesity. Their findings indicate that the positive effects of NPs on metabolism are only transient and that long-term treatment with NPs might even exacerbate obesity in overweight or obese subjects when accompanied with excessive calorie intake.|
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Objective: Obesity is defined as an abnormal increase in white adipose tissue (WAT) and is a major risk factor for type 2 diabetes and cardiovascular disease. Brown adipose tissue (BAT) dissipates energy and correlates with leanness. Natriuretic peptides have been shown to be beneficial for brown adipocyte differentiation and browning of WAT.
Methods: Here, we investigated the effects of an optimized designer natriuretic peptide (CD-NP) on murine adipose tissues in vitro and in vivo.
Results: In murine brown and white adipocytes, CD-NP activated cGMP production, promoted adipogenesis, and increased thermogenic markers. Consequently, mice treated for 10 days with CD-NP exhibited increased “browning” of WAT. To study CD-NP effects on diet-induced obesity (DIO), we delivered CD-NP for 12 weeks. Although CD-NP reduced inflammation in WAT, CD-NP treated DIO mice exhibited a significant increase in body mass, worsened glucose tolerance, and hepatic steatosis. Long-term CD-NP treatment resulted in an increased expression of the NP scavenging receptor (NPR-C) and decreased lipolytic activity.
Conclusions: NP effects differed depending on the duration of treatment raising questions about the rational of natriuretic peptide treatment in obese patients.[Hide abstract]
|Attenuated secretion of GIP does not alleviate hyperphagic obesity and insulin resistance Glucose-dependent insulinotropic polypeptide (GIP) promotes adipogenesis and lipid accumulation in adipocytes. Therefore, there has been some debate over whether GIP agonists used to improve glucose homeostasis may increase adiposity while GIP antagonists could promote weight loss. In the present study, Shimazu-Kuwahara et al. examined whether complete ablation of GIP production could reduce weight gain in the absence of the adipocyte hormone leptin, a condition that results in extreme hyperphagia, obesity, hyperinsulinemia, and insulin resistance, in both mice and humans. They found that Lepob/ob mice became equally obese and insulin resistant whether or not GIP was present, suggesting that GIP antagonism is unlikely to be effective at improving metabolism in extreme obesity associated with defective leptin action.|
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Objective: Glucose-dependent insulinotropic polypeptide (GIP) is released during meals and promotes nutrient uptake and storage. GIP receptor knockout mice are protected from diet induced weight gain and thus GIP antagonists have been proposed as a treatment for obesity. In this study, we assessed the role of GIP in hyperphagia induced obesity and metabolic abnormalities in leptin deficient (Lepob/ob) mice.
Methods: We crossbred GIP-GFP knock-in homozygous mice (GIPgfp/gfp) that have complete GIP knockout, and mice heterozygous for the ob mutation (Lepob/+) mice to generate Lepob/+/GIP+/+, Lepob/ob/GIP+/+, and Lepob/ob/GIPgfp/gfp mice. Male animals were weighed weekly and both oral glucose and insulin tolerance testing were performed to assess glucose homeostasis and circulating profiles of GIP and insulin. Body composition was evaluated by computerized tomography (CT) scan and analyses of indirect calorimetry and locomotor activity were performed.
Results: Postprandial GIP levels were markedly elevated in Lepob/ob/GIP+/+ mice compared to Lepob/+/GIP+/+ controls and were undetectable in Lepob/ob/GIPgfp/gfp mice. Insulin levels were equivalently elevated in both Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice compared to controls at 8 weeks of age but the hyperinsulinemia was marginally reduced in Lepob/ob/GIPgfp/gfp by 21 weeks, in association with amelioration of glucose intolerance. Both Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice remained equivalently insulin resistant. Body weight gain and subcutaneous and visceral fat volume of both Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice were significantly higher than that of Lepob/+/GIP+/+ mice, while no significant differences were seen between Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice. Locomotor activity and energy expenditure were decreased in both Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice compared to control Lepob/+/GIP+/+ mice, while no significant differences were seen between Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice. There was no significant difference in fat oxidation among the three groups. Fat content in liver was significantly lower in Lepob/ob/GIPgfp/gfp compared to Lepob/ob/GIP+/+ mice, while that of control Lepob/+/GIP+/+ mice was the lowest.
Conclusions: Our results indicate that GIP knockout does not prevent excess weight gain and metabolic derangement in hyperphagic leptin deficient mice.[Hide abstract]
|Relevance of PCSK1 variants for childhood obesity and glucose metabolismVariants in the proprotein convertase subtilisin kexin type 1 (PCSK1) gene encoding proprotein convertase 1/3 (PC1/3) contribute to polygenic obesity risk. Löffler, Behrendt, and colleagues identify two new variants and investigate their clinical relevance together with eight known variants with regard to obesity and glucose metabolism. Their findings of a novel heterozygous loss of function PCSK1 variant in a subject with severe early onset obesity, polycystic ovary syndrome, hypertension, and increased proinsulin levels, and associations of PCSK1 single nucleotide polymorphisms with obesity and insulin resistance in a large pediatric cohort, support the role of PCSK1 variants contributing to obesity in children.|
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Objective: Variants in Proprotein Convertase Subtilisin/Kexin Type 1 (PCSK1) may be causative for obesity as suggested by monogenic cases and association studies. Here we assessed the functional relevance in experimental studies and the clinical relevance through detailed metabolic phenotyping of newly identified and known PCSK1 variants in children.
Results: In 52 obese children selected for elevated proinsulin levels and/or impaired glucose tolerance, we found eight known variants and two novel heterozygous variants (c.1095 + 1G > A and p.S24C) by sequencing the PCSK1 gene. Patients with the new variants presented with extreme obesity, impaired glucose tolerance, and PCOS. Functionally, c.1095 + 1G > A caused skipping of exon8 translation and a complete loss of enzymatic activity. The protein was retained within the endoplasmic reticulum (ER) causing ER stress. The p.S24C variant had no functional effect on protein size, cell trafficking, or enzymatic activity. The known variants rs6230, rs35753085, and rs725522 in the 50 end did not affect PCSK1 promoter activity. In clinical association studies in 1673 lean and obese children, we confirmed associations of rs6232 and rs6234 with BMI-SDS and of rs725522 with glucose stimulated insulin secretion and Matsuda index. We did not find the new variants in any other subjects.
Conclusions: We identified and functionally characterized two rare novel PCSK1 variants of which c.1095 + 1G > A caused complete loss of protein function. In addition to confirming rs6232 and rs6234 in PCSK1 as polygenic risk variants for childhood obesity, we describe an association of rs725522 with insulin metabolism. Our results support the contribution of PCSK1 variants to obesity predisposition in children.[Hide abstract]
|Cholinergic neurons in the hypothalamus regulate food intakeDespite evidence for the physiological roles of the cholinoceptive system in the control of food intake, the origin of cholinergic neurons remains elusive. Jeong et al. examined whether stimulation of the cholingeric pathway from the dorsomedial hypothalamus (DMH) to the arcuate nucleus (ARC) is able to regulate food intake. They found that increased cholinergic neuron activity promoted food intake. Direct stimulation of cholinergic axon terminals in the ARC also increased food intake. This orexigenic effect was completely abolished by a muscarinic receptor antagonist. Hence, the DMH-ARC pathway may play a key role in regulating energy intake.|
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Objective: Central cholinergic neural circuits play a role in the regulation of feeding behavior. The dorsomedial hypothalamus (DMH) is considered the appetite-stimulating center and contains cholinergic neurons. Here, we study the role of DMH cholinergic neurons in the control of food intake.
Methods: To selectively stimulate DMH cholinergic neurons, we expressed stimulatory designer receptors exclusively activated by designer drugs (DREADDs) and channelrhodopsins in DMH cholinergic neurons by injection of adeno-associated virus (AAV) vectors into the DMH of choline acetyltransferase (ChAT)-IRES-Cre mice. We also generated transgenic mice expressing channelrhodopsins in cholinergic neurons with the Cre-LoxP technique. To delete the Chat gene exclusively in the DMH, we injected an AAV carrying a Cre recombinase transgene into the DMH of floxed ChAT mice. Food intake was measured with and without selective stimulation of DMH cholinergic neurons.
Results: Mice lacking the Chat gene in the DMH show reduced body weight as compared to control. Chemogenetic activation of DMH cholinergic neurons promotes food intake. This orexigenic effect is further supported by experiments of optogenetic stimulation of DMH cholinergic neurons. DMH cholinergic neurons innervate pro-opiomelanocortin neurons in the arcuate nucleus of the hypothalamus (ARC). Treatment with acetylcholine (ACh) enhances GABAergic inhibitory transmission to ARC POMC neurons that is blocked by the muscarinic receptor antagonist. Direct activation of cholinergic fibers in the ARC readily stimulates food intake that is also abolished by the muscarinic receptor antagonist.
Conclusions: ACh released from DMH cholinergic neurons regulates food intake and body weight. This effect is mediated in part through regulation of ARC POMC neurons. Activation of muscarinic receptors on GABAergic axon terminals enhances inhibitory tone to ARC POMC neurons. Hence, this novel DMHACh ? ARCPOMC pathway plays an important role in the control of food intake and body weight.[Hide abstract]