Figure 1.
Targeting construct to generate RIαB mice.
A, The upper diagram shows a simplified restriction map of a 6.6 kb fragment of the RIα genomic sequence encoding exons 9, 10 and 11 that was subcloned into Bluescript KS+. The middle diagram is the targeted allele (RIαB), which contains the B site mutation (G324D) in exon 11 (as indicated by an asterisk) and a loxP-neomycin resistance cassette inserted into a BglII site in intron 10. The bottom diagram is the Cre modified mutant RIα allele containing a single loxP site (RIαB/Cre). Restrictions sites: EcoRI, E; BglII, B; XhoI, X. B, A Southern blot of EcoRV-digested genomic ES cell DNA using a 3′ SphI/EcoRI genomic probe (as indicated by the black box). This strategy recognizes 6.6 kb fragment corresponding to the WT allele and a 7.5 kb fragment corresponding to the mutant RIα allele, RIαB. An asterisk indicates the correctly targeted clone. C, Identification of the B site mutation (as indicated by the asterisk) was confirmed by an Hph1-mediated restriction digest of a 153 bp PCR fragment generated with a primer set that amplifies within the site B cAMP domain of a WT (lane 1) and site B mutant plasmid vector (lane 2). In WT cells, Hph1 digestion of the 153 bp PCR product yields two products (126 bp and 27 bp) (lanes 3, 4, 5), whereas Hph1 digestion of the 153 bp amplicon from RIαB ES cells, which carry a single mutant allele and a single WT allele, results in three fragments: 153 bp (mutant), 126 bp (WT) and 27 bp (WT).
Figure 2.
Cre recombinase-mediated activation of the RIαB allele in ES cells decreases PKA activity and forskolin-stimulated CRE-luciferase reporter expression.
A, Transfection of ES cells with a Cre expression vector (pOG231) results in the expression of mutant RNA transcripts. Levels of mutant transcripts were determined by RT-PCR, restriction digestion, and Southern analysis as described in Materials and Methods. The 53 bp fragment indicates WT and the 140 bp fragment indicates mutant RIα transcripts. The small upper band present in lane 1 (WT) is due to incomplete digestion with BstN1, and was subtracted from lane 2 (RIαB) to determine the level of mutant RNA in these cells (<2%). The level of mutant transcript in RIαB/Cre ES cells is ∼49%. B, Detection of Cre-mediated recombination events in RIαB/Cre ES cells was determined by PCR using primers 1 and 2 (as shown in the figure) to amplify across the specified regions. Lane 1 is a WT control, Lane 2 is positive control for recombination. Lane 3 represents RIαB ES cells, which contain a single band due to presence of single WT RIα allele (the PCR conditions did not allow amplification through the floxed-neor cassette). Lane 4 represents RIαB/Cre ES cells, which contain a recombined allele (356 bp) and WT allele (309 bp). C, Kinase assay of basal and total activity in WT, RIαB, and RIαB/Cre ES cells. All samples were done in triplicate in the absence (basal PKA activity) or presence of 5 µΜ cAMP (total PKA activity) using Kemptide as substrate. Kinase activity that was not PKA-specific was measured in the presence of PKI and subtracted from basal and total values. Data values are represented as mean ± SEM. D, Representative CRE-luciferase assay. WT, RIαB, and RIαB/Cre ES cell lines were transfected with the CRE-dependent α168-luciferase reporter and then stimulated with forskolin (10 uM) for 14 h before an assay for luciferase activity as described in Materials and Methods. Transfections were done in triplicate.
Figure 3.
Cre-mediated activation of RIαB allele specifically in the liver decreases PKA activity in vivo.
A, Kinase assay on tissue homogenates from WT and RIαB mice measured in the absence (basal) or presence (total) of 5 µΜ cAMP using kemptide as the substrate. Non-PKA specific activity was measured in the presence of PKI and subtracted from basal and total values. Data values are represented as mean ± SEM; n = 3 animals per group. B, Representative PCR analysis of genomic DNA of brain (B), heart (H), kidney (K), liver (L) pancreas (P) and skeletal muscle (SM) were examined for Cre-mediated recombination of the RIαB allele in WT, RIαB and RIαB/Alb-cre mice. A 309 bp fragment indicates WT RIα, whereas a 365 bp fragment indicates the recombined mutant RIα allele, which was restricted to the liver. C, Kinase assay on liver homogenates from WT, RIαB, and RIαB/Alb-cre measured in the absence (basal) or presence (total) of 5 µΜ cAMP using Kemptide as the substrate. Non-PKA specific activity was measured in the presence of PKI and subtracted from basal and total values. Data values are represented as mean ± SEM; n = 3 animals per group. D, Representative western analysis and quantitation of pixel density on film exposures from WT, RIαB/Alb-Cre and RIαB mouse liver extracts. Data values in bar graph are represented as mean ± SEM. n = 4 replicates.
Figure 4.
Liver-specific PKA inhibition does not alter fasting-regulated gene expression.
mRNA levels of enzymes required for gluconeogenesis in the 24 hr fasted state (Fast) or after being fasted for 24 hr then allowed access to food for 6 hr (Fed) in WT, RIαB, and RIαB/Alb-cre mice. Levels of each mRNA were measured by quantitative real time PCR as described in Materials and Methods. Data are expressed relative to the results of a fasted WT control animal. Each value represents the mean ± SEM; n = 3 animals per group. No significant differences were found in gene induction. PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1α), PEPCK (phosphoenolpyruvate carboxykinase), G6Pase (glucose-6-phosphatase), GCK (glucokinase), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), and GLUT2 (glucose transporter type 2).
Table 1.
Plasma glucose, insulin, and metabolite concentrations in 24 hr fasted or 24 hr fasted and 6 hr refed animals.
Figure 5.
RIαB/Alb-cre mice exhibit increases in glucose disposal.
A, Blood glucose levels were determined in 24 hr fasted WT, RIαB, and RIαB/Alb-cre mice. n = 10. B, Glucose tolerance test was performed in 24 hr fasted WT, RIαB, and RIαB/Alb-cre mice injected i.p. with 2 mg of glucose per gram of body weight. Blood glucose levels were determined at the indicated time points. Over the first 60 min, the glucose disposal of RIαB/Alb-cre was significantly enhanced compared to RIαB alone (p<.001). n = 7. C, WT, RIαB, and RIαB/Alb-cre mice were injected with 0.75 mU of insulin per kilogram of body weight at time 0 and blood glucose levels were determined at the indicated time points. Values are presented as means ± SEM. n = 10.
Figure 6.
RIαB/Alb-cre mice and RIαB mice on the RIIα KO background exhibit increases in glucose disposal.
A, Representative kinase assay of basal and total kinase activity in WT, RIαB, RIαB/Alb-Cre,RIIαKO, RIαB:RIIαKO, and RIαB/Alb-Cre:RIIαKO liver. All samples were done in triplicate in the absence (basal) or presence (total) of 5 µΜ cAMP using Kemptide as substrate. Kinase activity that was not PKA-specific was measured in the presence of PKI and subtracted from basal and total values. Data values are represented as mean ± SEM. At least three mice from each genotype were used for the kinase assay. B, Glucose tolerance tests on 24 hr fasted WT, RIαB and RIαB/Alb-Cre mice all crossed onto the RIIα KO background are shown. Two mg of glucose per gram of body weight was injected i.p. and blood glucose levels were determined at the indicated time points. Values are presented as means ± SEM. n = 5. Glucose levels in RIαB/Alb-cre animals were significantly reduced compared to WT and RIαB during the first 30 min. P<0.01.
Table 2.
Phenotypes Observed by Activating the RIαB Allele with Various Cre Driver Mouse Lines.