Alterations in regulation of energy homeostasis in cyclic nucleotide phosphodiesterase 3B–null mice (original) (raw)
Targeted disruption of Pde3b. Pde3b–/– mice were generated as described in Methods (Figure 1, 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 1D). PCR carried out with A and R primers or A and E primers (Figure 1, 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 1E).
Targeted disruption of the murine Pde3b gene. (A) Structure of the approximately 13-kb Sal_I_PDE3B WT genomic fragment containing 5′-untranslated region and exon 1. (B) WT and disrupted KO gene fragments near exon 1. (C) Pde3b targeting vector. S, _Sal_I; X, _Xba_I; B, _Bst_XI; N, _Not_I; H, _Hind_III. (D) Southern blot of WT, HE, and KO genomic DNA, digested with _Bst_XI and hybridized to 32P-labeled probe 0.4 (see A and C). (E) PCR amplification of WT, HE, and KO genomic DNA using the specific primers A, E, and R indicated in A and B (A and E for lane 1; A and R for lane 2). (F) RT-PCR amplification of mRNA from WT, HE, and KO livers with primers described in Methods targeted for PDE3A (lane 1), PDE3B (lane 2), and Neor (lane 3). M, molecular weight marker. (G) Quantitation of cyclophilin A (Cyc), PDE3A, and PDE3B mRNAs in WT, HE, and KO mouse livers by real-time RT-PCR. Data were normalized to the quantity of WT cyclophilin A mRNA, taken as 100 AU. Values represent mean ± SEM (n = 4 per genotype in triplicate assays). Data were similar in 2 other groups of WT and KO mice and 1 group of HE mice. **P < 0.01. (H and I) Northern blot of WT and KO mRNAs, using probe B for mPDE3B (fat pads and liver; H) or probe A for mPDE3A (heart; I) as described in Methods. M, male; F, female; W, WT mRNA; K, KO mRNA.
As shown in Figure 1F, 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 1G, 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 1H). 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 1I). Northern blots with Clontech MTN filters revealed PDE3 mRNAs of sizes similar to those shown in Figure 1, H and I (data not shown). As also shown in Figure 1G, 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 1, 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 2A, 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 2, 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 2C). Immunoreactive approximately 135-kd PDE3B was not detected in homogenates (T) or membrane fractions (M) of adipose tissue and livers from KO mice (Figure 2D). 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.
PDE activity and Western blots. (A and B) PDE activities in epididymal adipocytes prepared from 4-month-old WT and KO mice are presented as total, PDE3, and PDE4 activities. Values represent mean ± SEM of duplicate assays (n = 3 mice per group), which were repeated with similar results. **P < 0.01. (C and D) PDE3 activities (C) and protein expression (D) in adipose tissue and liver. Total cell lysate (T) as well as cytosol (C) and membrane fractions (M) were prepared from gonadal adipose tissue and livers from 4-month-old WT and KO mice (n = 10–11 mice per group). (C) PDE3 activities (pmol cAMP hydrolyzed/min/mg protein) are expressed as mean ± SEM (duplicate assays). (D) Western blots, each lane 30 μg protein, except membrane fractions (75 μg) for β-actin detection. Results are representative of 3 experiments. R, PDE3B recombinant protein. Immunoblotting was performed using a monoclonal anti–β-actin antibody and affinity-purified rabbit antibodies raised against N-terminal (3B N-T; RKDERERDTPAMRSPPP, aa 2–18) or C-terminal (3B C-T; NASLPQADEIQVIEEADEEE, aa 1076–1095) sequences of PDE3B.
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 3A) or a 60%-fat diet (data not shown), the weight of gonadal adipose tissue was lower in KO than WT mice (Figure 3A) 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 3, 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 3D).
Gonadal fat weight, adipocyte diameters, and liver TG and FAS content in WT and _Pde3b_-KO mice. (A) WT and KO mice, housed 2 per cage and with free access to food and water, were fed normal chow. Caloric contents were 10%, 20%, and 70% fat, protein, and carbohydrate, respectively. For weight measurements, gonadal fat pads were collected from 6-month-old WT and KO mice (n = 6–8 mice per group). Data (mean ± SEM) were similar in 2 other experiments. (B and C) Diameter of epididymal adipocytes from age-matched 5-month-old WT and KO mice. (B) Cell diameter. Values are mean ± SEM (n = 357 WT and 447 KO cells). (C) Diameter distribution. The numbers of adipocytes with 5-μm intervals in their diameters were counted and plotted. Results are representative of 4 experiments. (D) Liver TG and FAS content. Liver TG content of 3- to 3.5-month-old male WT and KO mice was measured as described in Methods. Values are mean ± SEM (n = 18 per group). FAS content was determined by Western blot of liver homogenates (45 μg protein/lane) from WT and KO mice. Immunodetection was performed with anti-FAS antibody, and FAS bands were quantified. Values are mean ± SEM (n = 3 per group). *P < 0.05; **P < 0.01.
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 4, CL and ISO increased lipolysis, i.e., serum glycerol (Figure 4A) and FFA (Figure 4B) levels, to a greater extent in KO than in WT mice. In isolated adipocytes (Figure 4C), 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 4D). After fasting for 20 hours, serum glycerol (Figure 5A) and FFA (Figure 5B) levels were increased in KO mice. As shown in Figure 5C, 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 5, A and B). Insulin inhibited CL-stimulated lipolysis in adipocytes from fed and fasted WT, but not KO, mice (Figure 5C).
Effects of ISO, CL, and insulin on lipolysis in intact mice and adipocytes. (A and B) ISO or CL (1.0 mg/kg each) in PBS, or PBS alone, was injected i.p. (10 ml/kg) into 6-month-old WT and KO mice. After 20 minutes, serum glycerol (A) and FFA (B) levels were quantified. Values are mean ± SEM (n = 4 per group). Differences between serum glycerol or FFA concentrations after PBS alone (basal) and after drug administration are shown. Basal values in WT and KO mice were 20.5 ± 2.7 and 23.7 ± 1.2 mg/dl glycerol, respectively, and 1.49 ± 0.21 and 1.13 ± 0.25 mM FFA, respectively. Data were similar in 3 other experiments. (C and D) Adipocytes (0.4 ml, 5% [vol/vol]) prepared from epididymal fad pads of 6-month-old WT and KO mice were incubated for 60 minutes at 37°C in Krebs-Ringer–HEPES buffer alone or with the indicated concentrations of ISO, CL, or insulin (Ins). Lipolysis was measured as glycerol accumulation in the medium. Data are mean ± SEM of 3 incubations (duplicate assays). (C) Basal glycerol values were 0.13 ± 0.02 and 0.12 ± 0.01 nEq/h/103 cells for WT and KO, respectively. Data were similar in 3 other experiments. (D) Basal glycerol values were 0.24 ± 0.13 and 0.13 ± 0.08 nEq/h/103 cells for WT and KO, respectively. Data were similar in 2 other experiments. *P < 0.05; **P < 0.01.
Effects of fasting on lipolysis in intact mice and adipocytes. (A and B) WT and KO mice (4 months old) were fed ad libitum (NF) or fasted for 20 hours (F). Serum glycerol (A) and FFA (B) levels, determined as described in Methods, are presented as mean ± SEM (n = 6–7 mice per group). Results were similar to those from another group of fed and fasted WT and KO mice. (C) WT and KO mice (5 months old) were fasted for 20 hours before preparation of adipocytes, which were incubated for 60 minutes at 37°C with the indicated concentrations of CL and insulin. Increases from basal glycerol values are shown. Basal values were 0.16 ± 0.01 and 0.12 ± 0.00 nEq/h/103 cells for WT fed and fasted adipocytes, respectively, and 0.09 ± 0.01 and 0.09 ± 0.01 nEq/h/103 cells for KO fed and fasted adipocytes, respectively. Data are mean ± SEM of 3 incubations (duplicate assays). *P < 0.05; **P < 0.01.
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 6A, and in agreement with previous results by us and others (5, 12, 13, 19, 20), 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. (A) Insulin accumulation during incubation of isolated pancreatic islets for 1 hour with 3 or 16.7 mM glucose and without or with 100 nM GLP-1 as indicated. Data are mean ± SEM (n = 4). Inset: Immunoreactive PDE3B was detected by Western blots of WT, not KO, homogenates. (B) CL (1.0 mg/kg) in PBS or PBS alone was injected i.p. (10 ml/kg) into 6-month-old WT and KO mice. At the indicated times, differences from time 0 (basal) in serum insulin levels were quantified. Basal insulin levels in WT and KO mice were 1.4 ± 0.2 and 2.6 ± 0.1 ng/ml, respectively. Data (mean ± SEM; n = 4 mice per group) are representative of 3 experiments. (C and D) ISO or CL (1.0 mg/kg) in PBS or PBS alone were injected i.p. or i.v. (10 ml/kg) into 3- (i.p) or 4-month-old (i.v.) WT and KO mice. After 20 minutes, tail blood was collected, and changes from basal serum insulin (C) and blood glucose (D) levels were measured. Basal values were similar in PBS-treated WT and KO mice. Data (mean ± SEM; n = 4–5 mice per group) are representative of 3 experiments. *P < 0.05; **P < 0.01; †_P < 0.001.
In intact mice, administration of β3 receptor agonists are known to induce insulin secretion (21, 22). Administration of i.p. CL increased serum insulin levels to a greater extent in KO than in WT mice in a time- (Figure 6B) 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 6C), 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, 22), 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 6A, 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 5), 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 6, C and D). As also shown in Figure 6C, 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 6D). 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 7, 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 4 and 5). 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 7C), serum insulin levels were higher in older (9-month-old) KO males (Figure 7D) 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 6A).
ITTs and GTTs. (A and B) Male 5-month-old WT and KO mice were fasted overnight for 20 hours prior to i.p. injection (10 ml/kg) of insulin (0.5 U/kg) in PBS or PBS alone, and blood glucose (A) and serum FFA (B) levels were measured at the indicated times. Glucose concentrations (A) are reported relative to those at time 0. Basal glucose values in WT and KO mice were 70 ± 3 and 66 ± 3 mg/dl, respectively. Data (mean ± SEM; n = 6 mice per group) were similar in 8 (A) and 2 (B) other experiments. (C and D) Male 9-month-old WT and KO mice were fasted overnight, with free access to water. At the indicated times after i.p. injections (10 ml/kg) of PBS alone or of glucose (2 g/kg) in PBS, blood glucose (C) and serum insulin (D) levels were quantified. Data (mean ± SEM; n = 6 mice per group) were similar in 2 other experiments. *P < 0.05; **P < 0.01.
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 1, 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.
Hyperinsulinemic-euglycemic clamps
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 3D, TG content and FAS expression were significantly increased in livers from _Pde3b_-KO mice. As shown in Figure 8A, cAMP content was increased in liver extracts from KO mice, consistent with increased phosphorylation of PKA substrates (Figure 8B) and cAMP regulatory element–binding protein (CREB) (Figure 8C). 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 8C). Expression of PGC-1α, PEPCK, and glucose-6-phosphatase mRNAs were also significantly increased in fasted KO compared with fasted WT livers (Table 2), consistent with enhanced glucose production in KO livers. In addition, expression of tribbles 3 (TRB3) protein (Figure 8C) and mRNA (Table 2) 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 9A, 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 9B), 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 2). 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. (A) Hepatic cAMP content. cAMP was measured in liver extracts from 5-month-old WT and _Pde3b_-KO mice either fed or fasted for 6 hours as described in Methods. Values on the y axis represent nanomoles of cAMP per gram of liver. Data (mean ± SEM; n = 4 per group), which represent duplicate assays, were similar in a second group of fasted and nonfasted WT and KO mice. (B and C) Western blotting of liver lysates (50 μg protein/lane) was performed as described in Methods. n = 4 fed, 3 fasted mice per group. Data from a second, identical group of fed (n = 4) and 6-hour fasted (n = 3) WT and KO mice were similar. (B) Immunodetection with anti-PKA substrates antibody, which detects substrate proteins phosphorylated by cAMP-dependent protein kinase. (C) Immunodetection with anti–phospho-CREB (pS133); anti-CREB; anti–PGC-1α; anti-PEPCK; anti-TRB3; anti–SOCS-3; and anti-actin antibodies.
Alterations in insulin-signaling components in livers from _Pde3b_-KO mice. Western blots of liver lysates (50 μg protein/lane) were performed as described in Methods. n = 4 fed, 3 fasted mice per group. Data from an identical group of nonfasted (n = 4) and 6-hour-fasted (n = 3) WT and KO mice were similar. (A) Immunodetection with anti-phosphotyrosine (pY), anti–IRS-1, anti–insulin receptor (IR), anti–phospho-PKB, anti-PKB, anti–phospho-FKHRL1 (pS253), anti-FKHRL1, anti–phospho–GSK-3 (pY216), and anti–GSK-3 antibodies. (B) Immunodetection with anti–phospho–IRS-1 (pS612), anti–phospho–IRS-1 (pS307), anti–phospho-JNK, anti-JNK, anti–phospho-ERK, and anti-ERK antibodies.
Real-time RT-PCR quantification of selected mRNAs in livers of fasted and fed WT and 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 10A). Although basal lipogenesis was reduced in adipocytes from KO mice, insulin-stimulated lipogenesis was significantly enhanced in KO adipocytes (Figure 10B), which was explained in part by an increase in the expression of FAS (Figure 10C). As shown in Figure 10D, 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. -month-old WT and _Pde3b_-KO mice. (A and B) Adipocytes (0.2 ml 15% cells [vol/vol] in A; 1 ml 2.0% cells [vol/vol] in B) were incubated with the indicated concentrations of insulin for 10 minutes (A; n = 3) and 3 hours (B; n = 5). Uptake of 2-[1-3H]-deoxyglucose (A) and incorporation of D-[3H]-glucose into lipids (B) were measured as described in Methods and expressed as fold increase relative to nonstimulated cells. In KO adipocytes, basal lipogenesis was 56% ± 0.08% (mean ± SEM; P = 0.0001) that of WT adipocytes. Data (mean ± SEM) are from 5 independent experiments, each of which used adipocytes pooled from 2–3 mice. *P < 0.05; **P < 0.01. (C) Western blot analysis of FAS from WT and KO mice (20 μg protein/lane; n = 3) using anti-FAS antibody. (D) Adipocytes (each batch consisted of adipocytes from 2 of a total 6 WT and 6 KO mice) were incubated for 10 minutes with or without 1 nM insulin. Adipocyte fractions, prepared as described in Methods, were subjected to Western blotting with antibody recognizing PKB phosphorylated on serine 473 and, after stripping, with anti-PKB antibody. One Western blot representative of 3 is shown. PKB/phosphorylated PKB bands from 6 WT and 6 KO mice were quantified.
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 (26–28). One such adipokine, adiponectin, enhances insulin sensitivity in peripheral tissues, especially liver (27, 29, 30). In mice on normal chow (Figure 11A) or before or after (Figure 11B) 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 11A). Adiponectin mRNA expression was increased in adipose tissue from _Pde3b_-KO mice fed normal chow (Figure 11C). 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 11D) in both WT and KO mice, indicating appropriate responses to acute increases in cAMP.
Adiponectin expression. (A and B) Serum adiponectin concentrations were quantified in WT and KO mice, either fed or after fasting for 20 hours, fed normal chow (A; 6 months of age) or at the start (at 2 months of age) and end (after 14 weeks) of a 60%-fat diet (B). Data (mean ± SEM; n = 6–9 mice per group) were similar in 2 other experiments for A. (C) Adiponectin mRNA from epidydimal fat pads was amplified via real-time quantitative RT-PCR of total RNA as described in Supplemental Methods. Data (mean ± SEM; n = 4 mice per group) were similar in 2 other experiments. Inset: Agarose gel electrophoresis of adiponectin real-time RT-PCR products. A, adiponectin; C, cyclophilin A. Data from 1 other experiment were similar. (D) At the indicated times after i.p. injection (10 ml/kg) of CL (1.0 mg/kg) in PBS or PBS alone administered to 4- to 5-month old WT and KO mice, serum adiponectin levels were measured. Data (mean ± SEM) represent the percent change relative to basal values at time 0 (n = 4–9 mice per group). Basal adiponectin values were 22.2 ± 0.7 and 41.0 ± 2.0 μg/ml in WT and KO, respectively. Note: Values at every time point following CL injection were significantly lower (P < 0.01) except at 10 minutes in KO mice in the case of adiponectin. Data from 1 other experiment were similar. *P < 0.05; **P < 0.01.