No abstract

Somatostatin has been demonstrated to negatively regulate pancreatic growth in vivo. In this study we used the AR4-2J rat pancreatic acinar tumor cell line to investigate the effect of a stable somatostatin analog, SMS 201-995 (SMS) on cell proliferation. SMS induced an antiproliferative effect on both serum or epidermal growth factor (EGF)-induced cell proliferation; exposure of the cells for 48 h to SMS caused a slight inhibition of serum-induced proliferation (maximal inhibition, 26%) and abolished the growth-promoting effect of EGF. Maximal effect was observed with 10 nM SMS, and half-maximal (IC50) effect with 0.06-0.1 nM SMS. Binding studies with an iodinated derivative of SMS, [125I-Tyr3]SMS, revealed the presence of a single class of high affinity binding sites on AR4-2J plasma membranes with an equilibrium dissociation constant of 0.2 +/- 0.03 nM and a binding site number of 1.1 +/- 0.07 pmol/mg protein. Addition of the nonhydrolyzable GTP analog, guanosine 5-[gamma-thio] triphosphate (GTP gamma S), increased the rate of dissociation of the specifically bound peptide in agreement with the coupling of somatostatin receptors with a GTP-binding regulatory protein. The good agreement between the IC50 for SMS inhibition of cell proliferation and the apparent Kd for binding indicates that the characterized binding sites are the somatostatin receptors that mediate the antiproliferative effect of SMS. When cells were grown in serum-free medium EGF stimulated AR4-2J cell proliferation with half-maximal (ED50) and maximal effects at 0.6 and 10 nM EGF, respectively. This stimulatory effect of EGF was mediated by specific receptors, since binding studies with [125I]EGF indicated that AR4-2J cells contained a single class of EGF receptors (13,000 sites/cell), with an affinity constant for [125I]EGF (Kd = 0.9 +/- 0.09 nM) close to the ED50 for EGF stimulation of cell growth. To examine if SMS-induced growth inhibition involved a cAMP-dependent mechanism we first studied the effect of SMS on cAMP production. SMS had no effect on basal cAMP, but completely inhibited VIP-stimulated cAMP production with an IC50 of 0.2 nM. Pertussis toxin, which is known to abolish the inhibitory effect of somatostatin on adenylate cyclase activity in AR4-2J cells, did not reverse the ability of SMS to inhibit cell proliferation as well as EGF-induced cell proliferation. These data indicate that the antiproliferative effect of SMS does not involve the GTP-binding protein-mediated negative coupling of somatostatin receptors to adenylate cyclase.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of insulin on [6-l4 C] glucose metabolism was assessed in vitro and in vivo in epididymal adipose tissue and diaphragm of rats fed either a low-fat (9 per cent fat cal.) or a high-fat diet (72 per cent fat cal.). In vitro, diaphragm of fat-fed rats showed a lower glucose uptake than that of rats fed the low-fat diet, but had identical glycogen labeling and lactic acid production and a strongly reduced I4 CO2 production. Responsiveness of these pathways to insulin was unaltered by the fat content of the diet. The adipose tissue of fat-fed rats versus that of rats fed the low-fat diet showed: a higher lactic acid production and more efficient glycerogenesis and glycogenesis, all of these pathways being responsive to insulin; a lower glucose uptake and a strongly depressed fatty acid labeling, these two pathways being unresponsive to insulin. In-vivo labeling of glycogen in diaphragm in both basal and insulin-stimulated conditions was identical in the two groups of rats. In adipose tissue the amount of t4 C sequestered in the gh/ceride-glycerol moiety was the same in the two groups in basal and insulin-stimulated conditions, whereas the labeling of the fatty acid moiety and its increment with insulin were reduced by more than 99 per cent by the high-fat diet.These results show that alterations in fat content of the diet lead to differences in response to insulin that are pathway-and organspecific. DIABETES 27:114-20, February, 1978.A high-fat diet in rats causes dramatic changes in peripheral tissue glucose metabolism both in vitro and in vivo. 1 " 5 In addition, an impaired tolerance to intravenously 6 or orally 7 administered glucose and a reduced effect of exogenous 6 or endogenous 7 insulin

We have previously shown that somatostatin promotes the stimulation of a membrane tyrosine phosphatase activity in pancreatic cells. To gain insight into the mechanism of somatostatin action, we purified somatostatin-receptor complexes from somatostatin 28-prelabelled rat pancreatic plasma membranes by immunoaffinity chromatography using immobilized antibodies raised against the N-terminal part of somatostatin 28, somatostatin 28 (1-14), which is not involved in receptor-binding-site recognition. After SDS gel electrophoresis a band with a molecular mass of 87 kDa was identified in the affinity-purified material as the somatostatin receptor. The 87 kDa protein was not observed when the membrane receptors were solubilized in a free unoccupied or somatostatin 14-occupied form, or when nonimmune serum replaced the anti-[somatostatin 28 (1-14)] anti-serum. Somatostatin 14 inhibited the appearance of the 87 kDa protein in the same range of concentrations that inhibit radioligand binding on pancreatic membranes. After somatostatin 28 treatment of membranes, purified somatostatin receptor preparations exhibited an elevated tyrosine phosphatase activity that dephosphorylated phosphorylated epidermal growth factor receptor and poly(Glu,Tyr). This activity was related to the presence of somatostatin receptors in purified material. It was increased by dithiothreitol and inhibited by orthovanadate. In purified material containing somatostatin receptors, anti-[Src homology 2 domains (SH2)]-containing tyrosine phosphatase SHPTP1 polyclonal antibodies identified a protein of 66 kDa which was not detected in the absence of somatostatin receptor. Furthermore, the anti-SHPTP1 antibodies immunoprecipitated specific somatostatin receptors from somatostatin-prelabelled pancreatic membranes and from untreated membranes. These results indicate that a 66 kDa tyrosine phosphatase related to SHPTP1 co-purifies with the pancreatic somatostatin receptors, and suggest that this protein is associated with somatostatin receptors at the membrane level.

Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of 125I-[Tyr11]somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites (Kd = 0.55 +/- 0.06 nM) with a maximal binding capacity of 258 +/- 20 fmol/10(6) cells. Somatostatin receptor structure was analyzed by covalently cross-linking 125I-[Tyr11]somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide (Mr 80,000) containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation (IC50 = 0.4 nM) was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. We conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein Ni to inhibit adenylate cyclase.

The binding of 125I-[Tyr11]somatostatin to guinea pig pancreatic acini was saturable and temperature, protein, and radioligand concentration dependent. Dissociation rate was very slow (t1/2 = 193 +/- 24 min). Scatchard analysis revealed a single class of binding sites with a Kd of 0.28 +/- 0.02 nM and a maximal binding capacity of 72 +/- 10.6 fmol/mg prot. There was a strong correlation between binding capacity and extracellular calcium concentration. Incubating acini in EGTA-containing medium with no added Ca2+ caused a 50% decrease in maximal binding capacity with a decrease in receptor affinity. Furthermore, in the absence of calcium, bound somatostatin was rapidly released (t1/2 = 14 +/- 1 min). Subcellular fractionation studies and acid treatment of acini incubated with the tracer showed that most of the somatostatin binding sites were located on the cell surface. Agents that altered cellular calcium in pancreatic acini, such as analogues of cholecystokinin and cholinergic agents, also inhibited the binding of 125I-[Tyr11]somatostatin by a calcium-dependent process. We conclude that somatostatin binds to specific plasma membrane receptors to form a slowly reversible complex that is highly reactive with calcium. Cell calcium-mobilizing agents decrease the affinity of acinar somatostatin receptors for somatostatin.

We have previously demonstrated the presence of specific binding sites for somatostatin on plasma membranes from pancreatic acinar cells. In the present study we attempted to characterize the fate of receptor-bound 125I-[Tyr11]somatostatin. Internalization of somatostatin was rapid (reaching a plateau at 20% of the cell-associated specific radioactivity) and temperature dependent. To follow the processing of bound somatostatin, acini were incubated with 125I-[Tyr11]somatostatin at 5 degrees C during 16 h then, after washing, incubated at 37 degrees C for 90 min in fresh medium. Surface-bound somatostatin decreased rapidly, whereas radioactivity increased in the cell interior and the incubation medium. Intracellular and membrane-bound radioactivity was mainly intact 125I-[Tyr11]somatostatin. Degradation occurred at the plasma membrane level and led to iodotyrosine production. After 15 min of incubation, 15% of the initially surface-bound 125I-[Tyr11]somatostatin was compartmentalized within the cell, mainly in the microsomal fraction. After 30 min, a significant increase in radioactivity appeared in the nuclear fraction. These results indicate that the major part of somatostatin cellular degradation takes place at the plasma membrane level. Within the cell, somatostatin is routed to the nucleus via particular fractions sedimenting with microsomal vesicles.

A phosphotyrosyl protein phosphatase (PTPase) activity has been characterized in the plasma membranes of confluent AR42J pancreatic tumor cells using 32P-labeled poly(Glu, Tyr) as substrate. Membrane PTPase activity exhibited an apparent Michaelis constant of 3 microM and an apparent maximal velocity of 0.9 nmol.min-1.mg-1. It was inhibited by orthovanadate, zinc, poly(Glu,Tyr) and was stimulated by EDTA and dithiothreitol. Gel filtration of solubilized plasma membranes gave a peak of enzyme activity at a relative molecular weight of 70,000. Plasma membrane PTPase activity was changed during AR42J cell growth. At the beginning of culture, the control PTPase activity was minimal. Over the 5 days of culture, PTPase activity increased to reach a maximum (3.5-fold over control activity) preceding confluency by 2 days. Then the high level of PTPase activity was sustained until confluency. Incubation of the cells with the stable somatostatin analogue SMS 201-995 (SMS) resulted in a rapid and transient activation of crude membrane PTPase activity. Activation reached a maximum level within 5 min of addition and return to control levels within 20 min. The effect of SMS was dose dependent with half-maximal and maximal activation occurring at 6 pM and 0.1 nM SMS respectively.