Immunohistochemical evidence for the existence of a dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32) in brown adipose tissue of pigs.

Meister, Björn; Fried, Gabriel; Hökfelt, Tomas; Hemmings, Hugh C.; Greengard, Paul

doi:10.1073/pnas.85.22.8713

Cited by 20 publications

(16 citation statements)

References 27 publications

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“…One explanation of these results is that PP1 in fully differentiated adipocytes is regulated by DARPP-32 binding, and stimulation of PP1 by insulin occurs through disinhibition of the phosphatase. Although DARPP-32 is considered to be the neuronal homologue of Inh-1, both pig brown fat and bovine adipose tissue have previously been reported to contain DARPP-32 (28,29). DARPP-32 immunoreactivity was also detected in primary rat adipocytes (Fig.…”

Section: Discussionmentioning

confidence: 63%

The Regulation of Glycogen Synthase by Protein Phosphatase 1 in 3T3-L1 Adipocytes

Brady¹,

Nairn

Saltiel³

1997

Journal of Biological Chemistry

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The stimulation of glycogen-targeted protein phosphatase 1 (PP1), glycogen synthase, and glycogen synthesis by insulin was examined during the differentiation of 3T3-L1 fibroblasts into adipocytes. Insulin treatment barely changed the low levels of glycogen synthesis measured in fibroblasts. Following differentiation into adipocytes, insulin increased glycogen synthesis up to 40-fold. After further culturing of the adipocytes for a week, insulin stimulated glycogen accumulation 700-fold. Differentiation of 3T3-L1 cells also resulted in the increased expression of glycogen synthase and in increases in both total glycogen synthase activity and -fold stimulation by insulin. While the levels of PP1 protein were unchanged by differentiation, PP1 specific activity decreased over 60%, although sensitivity to insulin treatment was augmented. Concurrently, levels of the PP1 inhibitor protein DARPP-32 were dramatically induced upon 3T3-L1 adipogenesis. DARPP-32 in both 3T3-L1 and primary rat adipocytes was exclusively localized to the particulate fractions, including the glycogen-enriched pellet. PP1 activity from 3T3-L1 adipocytes exhibited a kinetic lag in vitro, which was not present in fibroblast extracts. Insulin pretreatment of the adipocyte cells overcame the in vitro lag in PP1 activity, resulting in up to 5-fold stimulation of PP1 activity being measured at early assay time points. These results suggest that in 3T3-L1 adipocytes, DARPP-32 may maintain glycogen-targeted PP1 activity in a low basal state, priming the phosphatase for stimulation by insulin.Protein phosphorylation plays a critical role in the regulation of lipid and glucose metabolism (1). As the major anabolic hormone regulating glucose utilization and storage, insulin exerts many of its effects by promoting the net dephosphorylation of enzymes such as glycogen synthase, glycogen phosphorylase, and phosphorylase kinase, resulting in the stimulation of glycogen synthesis. Several lines of evidence indicate that these insulin-stimulated dephosphorylations are catalyzed by protein phosphatase 1 (PP1).1 This phosphatase is found in nearly all cellular compartments and is thought to be targeted intracellularly by specific proteins (2, 3). Such targeting proteins have been identified that localize PP1 to the nucleus (4), the sarcoplasmic reticulum (5), glycogen, and myofibrils (reviewed in Ref. 3). The best characterized of these are the mammalian glycogen-targeting proteins. Three related members have been described: G M , isolated from skeletal muscle (6, 7), the hepatic G L protein (8, 9), and the recently cloned PTG protein (10, 11). Unlike the extremely restricted expression of G M and G L , PTG is highly expressed in the major insulinresponsive tissues, including fat, skeletal muscle, heart, and liver. 2 PP1 activity is controlled by targeting proteins in several ways. First, by localizing PP1 to subcellular compartments, such as glycogen, targeting proteins serve to increase PP1 specific activity against co-localized substrates. The two glycogen-ta...

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Section: Discussionmentioning

confidence: 63%

The Regulation of Glycogen Synthase by Protein Phosphatase 1 in 3T3-L1 Adipocytes

Brady¹,

Nairn

Saltiel³

1997

Journal of Biological Chemistry

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“…DARPP-32 expression is dramatically induced upon differentiation of 3T3-L1 fibroblasts into adipocytes and correlates with a decrease in PP1 basal activity and increase in stimulation by insulin. 3 Furthermore, DARPP-32 is expressed in pig brown fat (24), bovine adipose tissue (25), and in primary rat adipocytes, where it has been reported to be dephosphorylated in response to insulin (23). PP1 bound to PTG was resistant to inhibition by DARPP-32 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Role of Protein Targeting to Glycogen (PTG) in the Regulation of Protein Phosphatase-1 Activity

Brady¹,

Printen²,

Mastick³

et al. 1997

Journal of Biological Chemistry

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We have recently cloned from 3T3-L1 adipocytes a novel glycogen-targeting subunit of protein phosphatase-1, termed PTG (Printen, J. A., Brady, M. J., and Saltiel, A. R. (1997) Science 275, 1475-1478). Differentiation of 3T3-L1 fibroblasts into highly insulin-responsive adipocytes resulted in a marked increase in PTG expression. Immobilized glutathione S-transferase (GST)-PTG fusion protein specifically bound either PP1 or phosphorylase a. Addition of soluble GST-PTG to 3T3-L1 lysates increased PP1 activity against 32 P-labeled phosphorylase a by decreasing the K m of PP1 for phosphorylase 5-fold, while having no effect on the V max of the dephosphorylation reaction. Alternatively, PTG did not affect PP1 activity against hormone-sensitive lipase. PTG was not a direct target of intracellular signaling, as insulin or forskolin treatment of cells did not activate a kinase capable of phosphorylating PTG in vivo or in vitro. Finally, PTG decreased the ability of DARPP-32 to inhibit PP1 activity from 3T3-L1 adipocyte lysates. These data cumulatively suggest that PTG increases PP1 activity against specific proteins by several distinct mechanisms.While much attention has been focused on the activation of protein kinase signaling cascades, many enzymes involved in glucose and lipid metabolism are regulated by dephosphorylation (2). As the main physiological hormone controlling glucose utilization, insulin exerts many of its effects through the activation of type 1 protein phosphatase (PP1).1 However, insulin treatment of cells results in the dephosphorylation of only a limited number of proteins, while simultaneously other proteins are phosphorylated (3). This paradox suggests that mechanisms must exist whereby insulin activates discrete pools of PP1, leading to the dephosphorylation of specific target proteins.PP1 is found in many cellular compartments, including the nucleus, plasma membrane, and glycogen particle. It is thought that the cellular localization of this enzyme is mediated by its association with targeting proteins (4, 5). We have recently identified a novel PP1 glycogen-targeting subunit from 3T3-L1 adipocytes, termed PTG for protein targeting to glycogen (1). PTG is the third member of a family of PP1 glycogen-targeting subunits, which also includes R GL , isolated from muscle (6, 7), and the hepatic G L protein (8, 9). In contrast to the restricted localization of R GL and G L , PTG is highly expressed in all insulin-sensitive tissues.2 In addition to targeting PP1 to the glycogen particle, PTG can also form complexes with PP1 substrate enzymes that regulate glycogen metabolism, namely glycogen synthase, glycogen phosphorylase, and phosphorylase kinase. Overexpression of PTG in the metabolically inactive CHO-IR cell line dramatically increased the levels of basal and insulin-stimulated glycogen synthesis (1). PTG may therefore serve as a scaffolding protein, assembling the proteins involved in glycogen metabolism and priming them for the reception of intracellular signals.The mechanism by which insulin sp...

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“…A role for dopamine in regulating the phosphorylation of DARPP-32 has been demonstrated in brain [22] and is postulated in the other tissues in which DARPP-32 is found [23]. It is likely that the phosphorylation of DARPP-32 in adipose tissue is also under catecholamine control, possibly by noradrenaline released from sympathetic nerve endings innervating the tissue and/or by other CAMPlinked neurotransmitters or hormones, such as insulin [8] or dopamine (see discussion in [7]). …”

Section: Sensitivity To Chemical Cleavagementioning

confidence: 99%

“…Recent studies have demonstrated DARPP-32 immunoreactivity in brown fat adipocytes from pig [7]. Moreover, inhibitor-1 , partially purified from rat adipose tissue, exhibited an apparent molecular mass of 32 kDa by SDSjPAGE [8].…”

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confidence: 99%

Inhibitors of protein phosphatase‐1

Strålfors¹,

Hemmings²,

Greengard³

1989

European Journal of Biochemistry

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Protein phosphatase inhibitor-1 was purified from bovine adipose tissue. The protein had an apparent molecular mass of 32 kDa by SDSjPAGE and a Stokes' radius of 3.4 nm. It was phosphorylated by CAMPdependent protein kinase on a threonyl residue; this phosphorylation was necessary for inhibition of protein phosphatase-I . Bovine adipose tissue inhibitor-1 was compared directly with rabbit skeletal muscle inhibitor-1 and with a 32 000-M,, dopamine-and CAMP-regulated phosphoprotein from bovine brain (DARPP-32), also an inhibitor of protein phosphatase-1 . By the following biochemical and immunochemical criteria, bovine adipose tissue inhibitor-I was found to be very similar and possibly identical to DARPP-32 and was clearly distinct from skeletal muscle inhibitor-1 : molecular mass by SDS/PAGE; Stokes' radii; phosphorylation on threonine residues; Staphylococcus-aureus-V8-protease-generated peptide patterns analyzed by SDSjPAGE; tryptic phosphopeptide maps analysed by two-dimensional thin-layer electrophoresisjchromatography ; elution on reverse-phase HPLC ; chymotryptic peptide maps as analysed by reverse-phase HPLC; amino acid composition; antibody recognition by immunoprecipitation and immunoblotting; effect of cyanogen bromide cleavage on protein phosphatase inhibitor activity. Based on these results we conclude that bovine brain and adipose tissue contain an identical phosphoprotein inhibitor of protein phosphatase-1 (DARPP-32), which is distinct from that of skeletal muscle (inhibitor-1).

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Immunohistochemical evidence for the existence of a dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32) in brown adipose tissue of pigs.

Cited by 20 publications

References 27 publications

The Regulation of Glycogen Synthase by Protein Phosphatase 1 in 3T3-L1 Adipocytes

The Regulation of Glycogen Synthase by Protein Phosphatase 1 in 3T3-L1 Adipocytes

Role of Protein Targeting to Glycogen (PTG) in the Regulation of Protein Phosphatase-1 Activity

Inhibitors of protein phosphatase‐1

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