Alterations in regulation of energy homeostasis in cyclic nucleotide phosphodiesterase 3B-null mice

Abstract

Cyclic nucleotide phosphodiesterase 3B (PDE3B) has been suggested to be critical for mediating insulin/IGF-1 inhibition of cAMP signaling in adipocytes, liver, and pancreatic beta cells. In Pde3b-KO adipocytes we found decreased adipocyte size, unchanged insulin-stimulated phosphorylation of protein kinase B and activation of glucose uptake, enhanced catecholamine-stimulated lipolysis and insulin-stimulated lipogenesis, and blocked insulin inhibition of catecholamine-stimulated lipolysis. Glucose, alone or in combination with glucagon-like peptide-1, increased insulin secretion more in isolated pancreatic KO islets, although islet size and morphology and immunoreactive insulin and glucagon levels were unchanged. The beta(3)-adrenergic agonist CL 316,243 (CL) increased lipolysis and serum insulin more in KO mice, but blood glucose reduction was less in CL-treated KO mice. Insulin resistance was observed in KO mice, with liver an important site of alterations in insulin-sensitive glucose production. In KO mice, liver triglyceride and cAMP contents were increased, and the liver content and phosphorylation states of several insulin signaling, gluconeogenic, and inflammation- and stress-related components were altered. Thus, PDE3B may be important in regulating certain cAMP signaling pathways, including lipolysis, insulin-induced antilipolysis, and cAMP-mediated insulin secretion. Altered expression and/or regulation of PDE3B may contribute to metabolic dysregulation, including systemic insulin resistance.

Figure 1. Targeted disruption of the murine Pde3b gene.

(A) Structure of the approximately 13-kb SalIPDE3B 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, SalI; X, XbaI; B, BstXI; N, NotI; H, HindIII. (D) Southern blot of WT, HE, and KO genomic DNA, digested with BstXI 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 Neo^r (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.

Figure 2. 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.

Figure 3. 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.

Figure 4. 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.

Figure 5. 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.

Figure 6. 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.

Figure 7. 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.

Figure 8. 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.

Figure 9. 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.

Figure 10. 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.

Figure 11. 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.