Targeted disruption of Pde3b.
Pde3b–/– mice were generated as described in Methods (Figure , A–C). Mice used for experiments reported here were progeny of at least 6–7 backcrosses of heterozygous (HE) F1 mice with JAX 129/SvJ (pTyrc-ch/pTyrc substrain) mice. Genotyping of the F2 generation mice by Southern blot analyses demonstrated the predicted 6.5-kb restriction fragment in KO mice, and both 11.5-kb (WT) and 6.5-kb (KO) fragments in HE mice (Figure D). PCR carried out with A and R primers or A and E primers (Figure , A and B) amplified the expected approximately 3.0-kb band with genomic DNA from the KO and HE mice or the approximately 3.3-kb band from WT and HE mice, respectively (Figure E).
Targeted disruption of the murine Pde3b gene.
As shown in Figure F, using appropriately designed primers, PDE3A transcripts were amplified in mRNA from WT, HE, and KO mice; PDE3B transcripts were amplified in mRNA from WT and HE, but not KO mice; and neomycin resistance gene–targeted (Neor-targeted) mRNA was amplified in mRNA from HE and KO mice. As shown in Figure G, real-time quantitative 1-step RT-PCR using SYBR Green as the reporter fluorophore and primers from exons 2 and 3 indicated that PDE3B mRNA amplification in KO and HE livers was approximately 5% and 65%, respectively, that of WT liver. In Northern blots, PDE3B probe B (from the portion of the Pde3b gene replaced by Neor) detected approximately 5.5-kb transcripts in mRNA from fat pads and liver of WT, but not KO, mice (Figure H). In contrast, 2 heart mRNA transcripts (approximately 7.5 and 4.5 kb) were detected using PDE3A probe A with mRNA from both the WT and KO mice (Figure I). Northern blots with Clontech MTN filters revealed PDE3 mRNAs of sizes similar to those shown in Figure , H and I (data not shown). As also shown in Figure G, the amount of PDE3A mRNA was much lower than that of PDE3B mRNA in WT livers and was not increased in HE or KO livers. Thus, results in Figure , G and I, indicate no evidence of compensatory changes in expression of PDE3A in KO mice (at least in liver and heart).
PDE3 activity in homogenates of isolated adipocytes was assessed by measurement of cAMP hydrolysis in the absence or presence of cilostamide, a specific PDE3 inhibitor (ref. 2
; selective PDE3A and PDE3B inhibitors are not available). As shown in Figure A, PDE3 activity was virtually absent in KO adipocytes, indicating that PDE3B is the PDE3 isoform expressed in adipocytes. In isolated WT adipocytes, PDE3B accounted for almost one-half of the total PDE activity; and PDE4, somewhat less (Figure , A and B). PDE4 activity is that activity inhibited by 10 μM rolipram, a selective PDE4 inhibitor (2
). PDE4 activities were similar in WT and KO adipocytes, indicating that there was no compensatory increase in PDE4 in KO adipocytes. PDE3B accounted for most of the PDE3 activity in adipose tissue and liver (Figure C). Immunoreactive approximately 135-kd PDE3B was not detected in homogenates (T) or membrane fractions (M) of adipose tissue and livers from KO mice (Figure D). As shown in Supplemental Figure 1 (supplemental material available online with this article; doi:10.1172/JCI24867DS1), residual PDE3 activity in livers from PDE3B KO mice could be accounted for by PDE3A; lower molecular weight material not associated with PDE3 activity was detected with anti-PDE3B (C-T) in membrane fractions from WT and KO mice.
Characteristics of Pde3b-KO mice.
Newborn KO pups exhibited no obvious physical defects. Their growth and development, general behavior, and activity levels were similar to those of WT mice, and KO mice of both sexes were fertile. Most experiments were carried out with groups of age-matched WT and KO mice, whose dates of birth varied by 0–5 days; the mice were usually older than 3–4 months. Pde3b-KO mice were slightly heavier than their WT counterparts (Supplemental Table 1) and exhibited variations in coat color from white to yellowish brown. When mice were fed normal chow (Figure A) or a 60%-fat diet (data not shown), the weight of gonadal adipose tissue was lower in KO than WT mice (Figure A) and also represented a lesser percentage of body weight (data not shown). The mean cell diameter of KO adipocytes was significantly smaller than that of WT adipocytes, and the cell diameter distribution was shifted to a lower range (Figure , B and C). Weights of livers (Supplemental Table 1) as well as hearts and pancreata (data not shown) did not differ between WT and KO mice. TG content, however, was significantly increased in livers in KO mice and was associated with increased expression of fatty acid synthase (FAS) (Figure D).
Gonadal fat weight, adipocyte diameters, and liver TG and FAS content in WT and Pde3b-KO mice.
Insulin- and catecholamine-mediated regulation of lipolysis is altered in Pde3b-KO mice.
To assess the impact of the absence of adipocyte PDE3B on catecholamine-induced lipolysis in vivo, CL 316,243 (CL; a specific β3
receptor agonist — β3
receptors are the predominant subtype in rodent adipocytes; ref. 18
) and isoproterenol (ISO; a general β receptor agonist) were administered by i.p. injection, and their effects on serum glycerol and FFA levels were measured. Basal levels of glycerol and FFA were similar in fed WT and KO mice. As shown in Figure , CL and ISO increased lipolysis, i.e., serum glycerol (Figure A) and FFA (Figure B) levels, to a greater extent in KO than in WT mice. In isolated adipocytes (Figure C), CL, which at low concentrations was more effective than ISO, also stimulated lipolysis to a greater extent in adipocytes isolated from KO mice than from WT mice. Consistent with current ideas that activation of PDE3B and reduction of cAMP are critical in the antilipolytic action of insulin (7
), insulin inhibited catecholamine-activated lipolysis in WT, but not KO, adipocytes (Figure D). After fasting for 20 hours, serum glycerol (Figure A) and FFA (Figure B) levels were increased in KO mice. As shown in Figure C, CL stimulated lipolysis to a greater extent in adipocytes from fed KO mice than fed WT mice. CL-stimulated lipolysis was further enhanced in adipocytes from fasted WT, but not KO, mice, perhaps due to the fact that lipolytic pathways were activated to a greater extent in fasted KO than WT mice (Figure , A and B). Insulin inhibited CL-stimulated lipolysis in adipocytes from fed and fasted WT, but not KO, mice (Figure C).
Effects of ISO, CL, and insulin on lipolysis in intact mice and adipocytes.
Effects of fasting on lipolysis in intact mice and adipocytes.
Insulin secretion is increased in Pde3b-KO mice.
To more directly assess the effects of the absence of PDE3B in pancreatic β cells, we examined glucose-stimulated insulin secretion by pancreatic islets isolated from WT and KO mice. As shown in Figure A, and in agreement with previous results by us and others (5
), in the presence of 16.7 mM glucose, without or with GLP-1, glucose-stimulated insulin secretion was increased to a greater extent in KO than in WT islets, indicative of increased content of, and/or increased responsiveness to, cAMP in Pde3b
-deficient islets. Immunohistochemical analyses of pancreata from WT and KO mice showed no significant differences in islet size and expression/localization of insulin, glucagon, the β cell–specific transcription factor PDX-1, glucose transporter–2 (GLUT-2), and β cell glucokinase (data not shown).
Differences in insulin secretion from pancreatic islets, serum insulin concentrations, and blood glucose disposal in WT and Pde3b-KO mice.
In intact mice, administration of β3
receptor agonists are known to induce insulin secretion (21
). Administration of i.p. CL increased serum insulin levels to a greater extent in KO than in WT mice in a time- (Figure B) and concentration-dependent (data not shown) manner. Maximal increases were observed within 20 minutes after injection. Administration of ISO also increased serum insulin levels to a greater extent in KO mice (Figure C), but ISO was less effective than CL. Because stimulatory effects of β3
receptor agonists on insulin secretion are thought to be indirect and related to the presence of white adipose tissue depots (21
), the effects of CL are presumably related to production and/or release of an adipocytokine(s) or incretin(s). Taken together with the results shown in Figure A, these observations suggest that in Pde3b
-KO mice, there is increased secretion of, and/or enhanced islet-responsiveness to, these agents.
Pde3b-KO mice are insulin resistant.
As shown in Supplemental Table 1, blood glucose and serum concentrations of glycerol, TGs, insulin, and FFAs were not significantly different in fed WT and KO mice. Although serum glycerol and FFA levels were increased in fasted KO mice (Figure ), blood glucose and serum insulin levels were not changed (data not shown). Results of a number of experiments, however, indicated that KO mice were insulin resistant. Thus, despite the very high serum insulin concentrations in KO mice within 20 minutes after i.p. administration of CL, reduction in, or disposal of, blood glucose was not greater in CL-treated KO mice (Figure , C and D). As also shown in Figure C, within 10–20 minutes after i.v. injection of CL, serum insulin levels were also increased to a significantly greater extent in KO than in WT mice, although the differences between WT and KO mice were smaller following i.v. injection than i.p. injection. Despite the approximately 40-fold increase in serum insulin levels in the KO mice following i.v. administration of CL, there was much less removal or disposal of glucose in CL-treated KO mice than in CL-treated WT mice (Figure D). In addition, during insulin tolerance tests (ITTs) following i.p. injection of insulin to male mice, insulin was much less effective in reducing blood glucose and serum FFA levels in male KO than WT mice (Figure , A and B). Although effects of insulin on glucose disposal were similar in female WT and KO mice, insulin was less effective in reducing FFA levels in KO females than in WT females (data not shown). These results are consistent with the lack of the antilipolytic action of insulin in isolated adipocytes from KO mice (Figures and ). Furthermore, although clearance of blood glucose was similar in WT and KO mice during glucose tolerance tests (GTTs) following administration of i.p. glucose loads (Figure C), serum insulin levels were higher in older (9-month-old) KO males (Figure D) and females (data not shown); the increase in insulin secretion was not observed during GTTs in younger mice. The increase in serum insulin levels during GTTs observed in older KO mice is indicative of an altered exocytotic or secretory response to a glucose load of pancreatic β cells that lack PDE3B, and is consistent with the increased responsiveness of isolated KO islets to glucose and GLP-1 (Figure A).
To gain insight into the possible tissue loci of the insulin resistance observed in the different settings, i.e., i.p. ITTs, i.p. GTTs, and i.p. administration of CL, hyperinsulinemic-euglycemic clamps were performed. As shown in Table , after 16 hours of fasting, basal glucose, insulin, and endogenous glucose production (EGP) were not significantly different in WT and KO mice, and clamp blood glucose levels also were not significantly different between WT and KO mice (122 ± 3 and 119 ± 1 mg/dl). Under the hyperinsulinemic clamp conditions, KO mice exhibited slightly lower whole-body glucose uptake and reduced uptake in brown adipose tissue than in WT mice (P < 0.09), but these differences were not statistically significant. The steady-state glucose infusion rate required to maintain euglycemia was significantly higher in WT (159 ± 13 μmol/min/kg) than in KO mice (86 ± 7 μmol/min/kg; P < 0.01), suggesting impaired insulin responsiveness in KO mice. Insulin-induced suppression of EGP was markedly lower in KO mice (30.9% ± 8.6% in KO versus 88.7% ± 6.7% in WT mice; P < 0.01), suggesting that insulin does not effectively inhibit hepatic glucose output in KO mice. Thus, in regard to dysregulation of glucose homeostasis in KO mice, the liver appears to play a critical role.
Insulin and cAMP signaling, PPARγ coactivator–1α/phosphoenolpyruvate carboxykinase expression, inflammation markers, and lipid metabolism are altered in livers and adipocytes from KO mice.
Although it is not possible to precisely determine the role of the liver in the development of insulin resistance in the KO mice, a number of changes that may contribute were identified. As shown in Figure D, TG content and FAS expression were significantly increased in livers from Pde3b
-KO mice. As shown in Figure A, cAMP content was increased in liver extracts from KO mice, consistent with increased phosphorylation of PKA substrates (Figure B) and cAMP regulatory element–binding protein (CREB) (Figure C). Furthermore, the content of PPARγ coactivator–1α (PGC-1α), a transcriptional factor regulated by CREB, and phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, was increased in KO livers, the latter especially in fasted animals (Figure C). Expression of PGC-1α, PEPCK, and glucose-6-phosphatase mRNAs were also significantly increased in fasted KO compared with fasted WT livers (Table ), consistent with enhanced glucose production in KO livers. In addition, expression of tribbles 3 (TRB3) protein (Figure C) and mRNA (Table ) was increased in fasted KO livers. Gene expression of TRB3, an inhibitor of PKB activation, is regulated by PGC-1α and PPARα, and induction of TRB3 is thought to be important in development of hepatic insulin resistance by PGC-1α (23
). As shown in Figure A, in extracts from KO livers, tyrosine phosphorylation of insulin receptor substrate–1 (IRS-1) was decreased without any change in total IRS-1. Serine phosphorylation of PKB and forkhead (Drosophila) homolog (rhabdomyosarcoma) like 1 (FKHRL1) was decreased, and tyrosine phosphorylation of glycogen synthase kinase-3 (GSK-3) was increased in KO liver extracts, consistent with decreased signaling via PKB, a critical kinase involved in insulin-mediated effects on glucose and lipid metabolism (24
). With respect to stress/inflammation signals (Figure B), in livers from KO mice, phosphorylated JNK (but not total JNK), which could be important in increased serine phosphorylation of IRS-1, was increased. As analyzed by real-time RT-PCR, levels of SOCS-3 mRNA and mRNAs of proinflammatory cytokines IL-1, IL-6, TNF-α, and plasminogen activator inhibitor–1 (PAI-1) were increased in fasted KO liver (Table ). Many of these changes could contribute to development of insulin resistance in KO mice (25
Hepatic cAMP content and gluconeogenic gene expression in nonfasted and 6-hour-fasted WT and Pde3b-KO mice.
Real-time RT-PCR quantification of selected mRNAs in livers of fasted and fed WT and KO mice
Alterations in insulin-signaling components in livers from Pde3b-KO mice.
Consistent with hyperinsulinemic-euglycemic clamps in intact mice, no significant differences in insulin-induced glucose uptake were detected in isolated adipocytes from WT and KO mice (Figure A). Although basal lipogenesis was reduced in adipocytes from KO mice, insulin-stimulated lipogenesis was significantly enhanced in KO adipocytes (Figure B), which was explained in part by an increase in the expression of FAS (Figure C). As shown in Figure D, insulin-induced phosphorylation and activation of PKB were similar in WT and KO adipocytes.
Effects of insulin on glucose uptake, lipogenesis, and activation of PKB in adipocytes from 3- to 3.
Serum adiponectin, but not leptin, is increased in KO mice.
In addition to its traditional role as a storage depot for fat, adipose tissue is an important secretory organ of multiple factors, including so-called adipokines, which affect inflammation, appetite, insulin sensitivity, metabolism, and energy expenditure (26
). One such adipokine, adiponectin, enhances insulin sensitivity in peripheral tissues, especially liver (27
). In mice on normal chow (Figure A) or before or after (Figure B) 14 weeks on a 60%-fat diet, serum adiponectin concentrations were higher in KO mice (male and female) than in WT mice and did not change significantly after an overnight fast (Figure A). Adiponectin mRNA expression was increased in adipose tissue from Pde3b
-KO mice fed normal chow (Figure C). There were no significant differences in serum leptin between WT and KO mice fed normal chow (Supplemental Table 1). Administration of CL decreased serum leptin (data not shown) and adiponectin (Figure D) in both WT and KO mice, indicating appropriate responses to acute increases in cAMP.