Growth hormone and dexamethasone stimulate lipolysis and activate adenylyl cyclase in rat adipocytes by selectively shifting Gi alpha2 to lower density membrane fractions.

GH, in the presence of glucocorticoid, produces a delayed increase in lipolysis in rat adipose tissue, but the biochemical mechanisms that account for this action have not been established. Other lipolytic agents rapidly activate adenylyl cyclase (AC) and the resulting production of cAMP initiates a chain of reactions that culminates in the activation of hormone-sensitive lipase. We compared responses of segments of rat epididymal fat or isolated adipocytes to 30 ng/ml GH and 0.1 microg/ml dexamethasone (Dex) with 0.1 ng/ml isoproterenol (ISO), which evoked a similar increase in lipolysis. All measurements were made during the fourth hour after the addition of GH+Dex or immediately after the addition of ISO to cells or tissues that had been preincubated for 3 h without hormone. Although no significant increases in cAMP were discernible in homogenates of GH+Dex-treated tissues, Rp-cAMPS (Rp-adenosine 3'5'-phosphothioate), a competitive inhibitor of cAMP, was equally effective in decreasing lipolysis induced by GH+Dex or ISO. The proportion of PKA that was present in the active form was determined by measuring the incorporation of 32P from [gamma-32P]ATP into kemptide in the absence and presence of saturating amounts of cAMP. GH+Dex and ISO produced similar increases in protein kinase A activity in tissue extracts. Treatment with GH+Dex did not change the total forskolin-stimulated AC present in either a crude membrane pellet sedimented at 16K x g or a less dense membrane pellet sedimented at 100K x g, but doubled the AC activity in the 16K pellet when assayed in the absence of forskolin. To evaluate possible effects on G proteins, pellets obtained from centrifugation of adipocyte homogenates at 16K x g and 100K x g were solubilized and subjected to PAGE and Western analysis. GH+Dex decreased Gi alpha2 by 44% (P < 0.02) in the 16K pellets and increased it by 52% (P < 0.01) in the 100K pellets. Gs alpha in the 16K pellet was unaffected by GH+Dex and was decreased (P < 0.05) in the 100K pellet. Sucrose density fractionation of the 16K pellets revealed a similar GH+Dex-dependent shift of Gi alpha2 to less dense fractions as determined by both Western analysis and [32P]NAD ribosylation catalyzed by pertussis toxin. No such changes were seen in the distribution of Gs alpha or 5'-nucleotidase. Colchicine (100 microM) blocked the GH+Dex-dependent shift of Gi alpha2 from the 16K to the 100K pellet and blocked the lipolytic effects of GH+Dex, but not those of ISO. We conclude that by modifying the relationship between AC and Gi alpha2, GH+Dex relieves some inhibition of cAMP production and consequently increases lipolysis.

Functional GIP receptors are present on adipocytes.

In addition to its important role in maintaining glucose homeostasis, it has recently become apparent that glucose-dependent insulinotropic polypeptide (GIP) is also involved in different steps of lipid metabolism. GIP has been shown to stimulate the release of lipoprotein lipase from fat, as well as increase the rate of fat incorporation into adipose tissue. Moreover, GIP has been shown to increase the clearance rate of chylomicrons in the circulation and to inhibit the action of glucagon. Despite evidence for GIP effects on fat tissue, GIP receptors have not been identified in fat cells or tissues. The present study was undertaken to identify GIP receptors in isolated adipocytes, as well as to identify GIP receptors in the established fat cell line, differentiated 3T3-L1. RNAse protection analysis demonstrated the presence of GIP receptor transcripts in rat adipocytes. A polyclonal GIP receptor antiserum directed at the N-terminus of the receptor detected the presence of GIP receptors in both rat fat and differentiated 3T3-L1 cells by Western blot analysis. Moreover, [125I] GIP binding assays revealed both specific and displaceable GIP binding sites in differentiated 3T3-L1 cells (IC50 = 10(-9) M). When undifferentiated 3T3-L1 cells, which appear to express relatively few GIP receptors, were incubated in the presence of GIP, no effect on intracellular cAMP accumulation was detected. In contrast, the inclusion of 10 nM GIP in the incubation medium increased cAMP accumulation in rat fat cells and differentiated 3T3-L1 cells. This increase in cAMP accumulation was abolished with the specific GIP receptor antagonist GIP(7-30)NH2. The results of these studies indicate that GIP receptors are present in fat cells and are up-regulated when 3T3-L1 cells undergo differentiation to become adipocytes. Furthermore, the increase in intracellular cAMP accumulation detected upon ligand binding indicates that these receptors are functional.

GIP biology and fat metabolism.

The gastrointestinal hormone, gastric inhibitory polypeptide (GIP), is synthesized and released from the duodenum and proximal jejunum postprandially. Its release depends upon several factors including meal content and pre-existing health status (ie. obesity, diabetes, age, etc.). It was initially discovered and named for its gastric acid inhibitory properties. However, its more physiologically relevant role appears to be as an insulinotropic agent with a stimulatory effect on insulin release and synthesis. Accordingly, it was later renamed glucose-dependent insulinotropic polypeptide because its action on insulin release depends upon an increase in circulating levels of glucose. GIP is considered to be one of the principle incretin factors of the enteroinsular axis. The GIP receptor is a G-protein-coupled receptor belonging to the family of secretin/VIP receptors. GIP receptor mRNA is widely distributed in peripheral organs, including the pancreas, gut, adipose tissue, heart, adrenal cortex, and brain, suggesting it may have other functions in addition to the ones mentioned above. An overactive enteroinsular axis has been suggested to play a role in the pathogenesis of diabetes and obesity. In addition to stimulating insulin release, GIP has been shown to amplify the effect of insulin on target tissues. In adipose tissue, GIP has been reported to (1) stimulate fatty acid synthesis, (2) enhance insulin-stimulated incorporation of fatty acids into triglycerides, (3) increase insulin receptor affinity, and (4) increase sensitivity of insulin-stimulated glucose transport. In addition, although controversial, lipolytic properties of GIP have been proposed. The mechanism of action of GIP-induced effects on adipocytes is unknown, and it is unclear whether these effects of GIP on adipocytes are direct or indirect. However, there is now evidence that GIP receptors are expressed on adipocytes and that these receptors respond to GIP stimulation. Given the location of its release and the timing of its release, GIP is an ideal anabolic agent and expanding our understanding of its physiology will be needed to determine its exact role in the etiology of diabetes mellitus and obesity.