Table 1.
Gene-specific PCR primers for conventional and quantitative real time PCR.
Figure 1.
DAR expression in human adipose tissue and adipocytes.
A, comparison of DAR expression in human pituitary (P), striatum (S) and sc adipose tissue (A), as determined by conventional RT-PCR. B, expression of selected DAR in the stromo-vascular cell (SVC) fraction and mature adipocytes, as determined by real-time PCR. Data are expressed as relative changes over SVC. Each value is a mean±SEM of 3 determinations; *, p<0.05. C, Immunoblot of selected DAR proteins in tissues and cells. Lanes: 1, pituitary; 2, proliferating primary preadipocytes; 3, proliferating LS14; 4, proliferating SW872; 5, differentiated primary adipocytes; 6, differentiated LS14; 7, differentiated SW872. Each lane was loaded with 40 µg proteins, except for the pituitary (30 µg proteins). β-actin (β-Act) was used as a loading control. Expression of D1R, D2R and D4R (panel D), and PRL vs PRLR (panel E) during adipogenesis in LS14 cells was determined by qPCR. Data are expressed as relative changes over day 0, and were calculated from the cycle threshold and efficiency measurements (Mean±SEM of 3 determinations).
Figure 2.
Expression of active arylsulfatase A (ARSA) in adipose tissue and adipocytes and confirmation of DA-S bioactivity.
A, comparison of mRNA levels of ARSA (A), ARSB (B) and ARSC (C) in sc adipose tissue, as determine by qPCR. Data are expressed as fold changes in gene expression over ARSB, and were calculated from the cycle threshold and efficiency measurements. B, ARSA activity before (light bars) and after (dark bars) differentiation of LS14 and SW872 adipocytes. Specific ARSA activity was determined by subtracting enzyme activity in the presence of silver nitrate from total enzyme activity. Each value is a mean±SEM of 4 determinations; *, p<0.05. C, both DA and DA-S inhibit PRL release from sc adipose tissue explants, whereas isoproterenol (ISO), a β-AR agonist, causes stimulation. Explants were incubated with the different compounds for 24 hr and PRL concentration in CM was determined by the Nb2 bioassay. Data are expressed as % of control. Each value is a mean±SEM of 6 determinations; *, p<0.05.
Figure 3.
DA and bromocriptine (BRO) inhibit PRL release from different types of adipocytes.
A, isolated sc mature adipocytes. B, differentiated sc primary adipocytes. C, differentiated LS14 cells. D, differentiated SW872 cells. In each case, cells were incubated with different doses of DA or bromocriptine (BRO) for 24 hr, and PRL in CM was determined by the Nb2 bioassay. Each value is a mean±SEM of 6 determinations; *, p<0.05.
Figure 4.
Both DA and DA-S suppress adipocyte PRL gene expression via the superdistal promoter.
A, diagram of the pituitary and superdistal promoters which regulate pituitary and extrapituitary PRL expression, respectively; both generate an identical PRL protein. UTR, untranslated region; SP, signal peptide. B, the superdistal PRL promoter construct driving a luciferase reporter that was stably transfected in SW872 cells. Also shown are putative transcription factor binding sites capable of responding to activation of PKA and MAPK signaling. C, inhibition of PRL gene expression by DA and DA-S after 6 hr of incubation; D, inhibition of PRL gene expression by DA and stimulation by IBMX after 24 hrs of incubation. Stably transfected SW872 cells were induced to differentiate and then incubated with increasing doses of DA or DA-S (0.1 nM to 100 nM) or with 250 µM IBMX. Luciferase activity was determined in cell lysates by luminommetry. Each value is a mean±SEM of 6 determinations; *, p<0.05.
Figure 5.
Different signaling pathways mediate DAR actions in the adipocytes.
Sc adipose explants (panel A), or differentiated SW872 cells (panel B) were incubated with 10 nM DA or 100 nM raclopride (RAC), a D2R antagonist, for 24 hr, or pre-incubated with RAC 1 hr before DA addition. PRL in CM was determined by the Nb2 bioassay. Each value is a mean±SEM of 6 determinations; *, p<0.05. Differentiated LS14 cells were incubated with 1, 10 or 100 nM DA for 30 min and intracellular cAMP (panel C) or cGMP (panel D) levels were determined by respective ELISAs. Each value is a mean±SEM of 4 determinations; *, p<0.05. E, differentiated LS14 cells were incubated with 10 nM DA, and cell lysates obtained at different times were analyzed for phosphorylate ERK1/2 (pERK1/2) and phosphorylated Akt (pAkt) by Western blotting. Total ERK1/2 and Akt served as loading controls.
Figure 6.
Effects of DA on adipokine/cytokine release.
Sc explants (panel A), isolated mature adipocytes (Panel B) or differentiated primary adipocytes (panel C) were incubated with 1 or 100 nM DA or with 10 nM of SKF38393, a D1R/D5R agonist, for 24 hr. Leptin concentration in CM was determined by ELISA. Each value is a mean±SEM of 5 determinations; *, p<0.05. D, differentiated primary adipocytes were incubated as above and adiponectin release was determined by ELISA. E, proliferating primary adipocytes were incubated with 1 or 100 nM DA or with 10 nM of SKF for 6 hr and IL-6 release was determined by ELISA.
Figure 7.
Putative transcription factor binding sites in the promoters of D1R and D2R.
Sites were identified using Genomatix MatInspector. The diagram is not drawn to scale.
Figure 8.
A model depicting the involvement of DA/DAR system in adipocyte functions.
DA can reach the adipocytes from infiltrating lymphocyte/macrophages, sympathetic nerve endings or via the circulation in the form of DA-S. DA-S can become de-conjugated to bioactive DA by ARSA which is secreted from the lysosomes. DA binds to either D2R-like or D1R-like membrane receptors. Activation of D2R causes suppression of cAMP and inhibition of PRL gene expression and release. Activation of D1R-like receptors results in the inhibition of leptin release and stimulation of adiponectin and IL-6, an effect which may be mediated via the cGMP or MAPK signaling. Other catecholamines (i.e. NE and Epi) from the circulation or sympathetic neurons activate β-AR and modulate, by yet an unknown fashion, adipokine release. See text for additional explanations. ARSA, arylsulfatase A; β-AR, β-adrenergic receptors; D2R/D1R, type 1 or type 2 dopamine receptors; DA, dopamine; DA-S, dopamine sulfate; Epi, epinephrine; NE, norepinephrine; MAPK, mitogen-activated protein kinase; PRL, prolactin.