Immune biomarker evaluation of sequential tyrosine kinase inhibitor and nivolumab monotherapies in renal cell carcinoma: the phase I TRIBE trial.

Background: Predictive biomarkers for immune checkpoint blockade in the second-line treatment of metastatic renal cell carcinoma (mRCC) are lacking. Materials and methods: Patients with histologically confirmed RCC who started nivolumab after at least 4 months of tyrosine kinase inhibitors (TKIs) were recruited for this study. Serial tissue and blood samples were collected for immune biomarker evaluation. The primary endpoint was to determine the association of specific T-cell subsets with clinical outcomes tested using Wilcoxon rank sum for clinical benefit rate (CBR) and log-rank test for progression-free survival (PFS). Results: Twenty patients were included in this trial with a median age of 64 years and followed-up for a median of 12 months. The median PFS for patients who received TKI was 13.8 months, while for those subsequently treated with nivolumab following TKI therapy, the median PFS was 2.6 months. CBR of nivolumab was 20% with two partial responses. Functionally active programmed cell death protein 1+ CD4+ T cells were enriched in non-responders (q = 0.003) and associated with worse PFS on nivolumab (P = 0.04). Responders showed a significant reduction in the effector CD4+T-cell (TEF) fraction compared to non-responders at 3 months on nivolumab (0.40 versus 0.80, P = 0.0005). CD127+CD4+ T cells were enriched in patients who developed immune-related adverse effects (q = 0.003). Using in-house validated multiplex immunohistochemistry for six markers, we measured tumour-associated immune cell densities in tissue samples. Responders to nivolumab showed a significantly higher mean of immune cell densities in tissue samples compared to non-responders (346 versus 87 cells/mm2, P = 0.04). Conclusions: In this small study, analysis of tissue-based and peripheral blood immune cell subsets predicted clinical outcomes of nivolumab. Further studies are warranted with larger populations to validate these observations.

PD-L1 and immune infiltrates are differentially expressed in distinct subgroups of gastric cancer.

This study investigates the association of PD-L1 expression and immune cell infiltrates and their impact on clinical outcome, in addition to their overlap with microsatellite instability (MSI), HER2 and ATM molecular subgroups of gastric cancer (GC). PD-L1 membrane expression on tumour cells (TC) and infiltrating immune cells (IC), CD3 + T-lymphocytes, CD8+ cytotoxic T-cells, ATM and HER2 were assessed by immunohistochemistry (IHC) in the ACRG (Asian Cancer Research Group) GC cohort (N = 380). EBV status was determined using in situ hybridization and MSI status was performed using PCR and MLH1 IHC. The PD-L1 segment was associated with increased T-cell infiltrates, while the MSI-high segment was enriched for PD-L1, CD3, and CD8. Multivariate analysis confirmed PD-L1 positivity, high CD3 and high CD8 as independent prognostic factors for both disease-free survival and overall survival (all p < 0.05). Patients with MSI-high tumours had better overall survival by both univariate and multivariate analysis. The ATM-low and HER2-high subgroups differed markedly in their immune profile; the ATM-low subgroups enriched for MSI, PD-L1 positivity and CD8 + T-cells, while the HER2 segment was enriched for MSS, with no enrichment for immune markers. Hence, we demonstrate a molecular profiling approach that can divide GC into four molecular subgroups, namely ATM-low, HER2-high, PD-L1 positive and MSI-high with differing levels of immune infiltrates and prognostic significance which may help to stratify patients for response to targeted therapies.

A randomized, open-label study of the efficacy and safety of AZD4547 monotherapy versus paclitaxel for the treatment of advanced gastric adenocarcinoma with FGFR2 polysomy or gene amplification.

BACKGROUND: Approximately 5%-10% of gastric cancers have a fibroblast growth factor receptor-2 (FGFR2) gene amplification. AZD4547 is a selective FGFR-1, 2, 3 tyrosine kinase inhibitor with potent preclinical activity in FGFR2 amplified gastric adenocarcinoma SNU16 and SGC083 xenograft models. The randomized phase II SHINE study (NCT01457846) investigated whether AZD4547 improves clinical outcome versus paclitaxel as second-line treatment in patients with advanced gastric adenocarcinoma displaying FGFR2 polysomy or gene amplification detected by fluorescence in situ hybridization. PATIENTS AND METHODS: Patients were randomized 3:2 (FGFR2 gene amplification) or 1:1 (FGFR2 polysomy) to AZD4547 or paclitaxel. Patients received AZD4547 80 mg twice daily, orally, on a 2 weeks on/1 week off schedule of a 21-day cycle or intravenous paclitaxel 80 mg/m2 administered weekly on days 1, 8, and 15 of a 28-day cycle. The primary end point was progression-free survival (PFS). Safety outcomes were assessed and an exploratory biomarker analysis was undertaken. RESULTS: Of 71 patients randomized (AZD4547 n = 41, paclitaxel n = 30), 67 received study treatment (AZD4547 n = 40, paclitaxel n = 27). Among all randomized patients, median PFS was 1.8 months with AZD4547 and 3.5 months with paclitaxel (one-sided P = 0.9581); median follow-up duration for PFS was 1.77 and 2.12 months, respectively. The incidence of adverse events was similar in both treatment arms. Exploratory biomarker analyses revealed marked intratumor heterogeneity of FGFR2 amplification and poor concordance between amplification/polysomy and FGFR2 mRNA expression. CONCLUSIONS: AZD4547 did not significantly improve PFS versus paclitaxel in gastric cancer FGFR2 amplification/polysomy patients. Considerable intratumor heterogeneity for FGFR2 gene amplification and poor concordance between FGFR2 amplification/polysomy and FGFR2 expression indicates the need for alternative predictive biomarker testing. AZD4547 was generally well tolerated.

SELECT-2: a phase II, double-blind, randomized, placebo-controlled study to assess the efficacy of selumetinib plus docetaxel as a second-line treatment of patients with advanced or metastatic non-small-cell lung cancer.

BACKGROUND: Combination of selumetinib plus docetaxel provided clinical benefit in a previous phase II trial for patients with KRAS-mutant advanced non-small-cell lung cancer (NSCLC). The phase II SELECT-2 trial investigated safety and efficacy of selumetinib plus docetaxel for patients with advanced or metastatic NSCLC. PATIENTS AND METHODS: Patients who had disease progression after first-line anti-cancer therapy were randomized (2 : 2 : 1) to selumetinib 75 mg b.i.d. plus docetaxel 60 or 75 mg/m2 (SEL + DOC 60; SEL + DOC 75), or placebo plus docetaxel 75 mg/m2 (PBO + DOC 75). Patients were initially enrolled independently of KRAS mutation status, but the protocol was amended to include only patients with centrally confirmed KRAS wild-type NSCLC. Primary end point was progression-free survival (PFS; RECIST 1.1); statistical analyses compared each selumetinib group with PBO + DOC 75 for KRAS wild-type and overall (KRAS mutant or wild-type) populations. RESULTS: A total of 212 patients were randomized; 69% were KRAS wild-type. There were no statistically significant improvements in PFS or overall survival for overall or KRAS wild-type populations in either selumetinib group compared with PBO + DOC 75. Overall population median PFS for SEL + DOC 60, SEL + DOC 75 compared with PBO + DOC 75 was 3.0, 4.2, and 4.3 months, HRs: 1.12 (90% CI: 0.8, 1.61) and 0.92 (90% CI: 0.65, 1.31), respectively. In the overall population, a higher objective response rate (ORR; investigator assessed) was observed for SEL + DOC 75 (33%) compared with PBO + DOC 75 (14%); odds ratio: 3.26 (90% CI: 1.47, 7.95). Overall the tolerability profile of SEL + DOC was consistent with historical data, without new or unexpected safety concerns identified. CONCLUSION: The primary end point (PFS) was not met. The higher ORR with SEL + DOC 75 did not translate into prolonged PFS for the overall or KRAS wild-type patient populations. No clinical benefit was observed with SEL + DOC in KRAS wild-type patients compared with docetaxel alone. No unexpected safety concerns were reported. TRIAL IDENTIFIER: Clinicaltrials.gov NCT01750281.

FGFR2 amplification has prognostic significance in gastric cancer: results from a large international multicentre study.

BACKGROUND: In preclinical gastric cancer (GC) models, FGFR2 amplification was associated with increased tumour cell proliferation and survival, and drugs targeting this pathway are now in clinical trials. METHODS: FGFR2 FISH was performed on 961 GCs from the United Kingdom, China and Korea, and the relationship with clinicopathological data and overlap with HER2 amplification were analysed. RESULTS: The prevalence of FGFR2 amplification was similar between the three cohorts (UK 7.4%, China 4.6% and Korea 4.2%), and intratumoral heterogeneity was observed in 24% of FGFR2 amplified cases. FGFR2 amplification was associated with lymph node metastases (P<0.0001). FGFR2 amplification and polysomy were associated with poor overall survival (OS) in the Korean (OS: 1.83 vs 6.17 years, P=0.0073) and UK (OS: 0.45 vs 1.9 years, P<0.0001) cohorts, and FGFR2 amplification was an independent marker of poor survival in the UK cohort (P=0.0002). Co-amplification of FGFR2 and HER2 was rare, and when high-level amplifications did co-occur these were detected in distinct areas of the tumour. CONCLUSION: A similar incidence of FGFR2 amplification was found in Asian and UK GCs and was associated with lymphatic invasion and poor prognosis. This study also shows that HER2 and FGFR2 amplifications are mostly exclusive.

PDH kinase inhibitors: a novel therapy for Type II diabetes?

The pyruvate dehydrogenase multienzyme complex catalyses the oxidative decarboxylation of pyruvate, which is an important regulatory step in oxidative metabolism. Phosphorylation of the E1 (pyruvate decarboxylase) subunit on one of three specific serine residues results in loss of enzyme activity. Four dedicated PDHK (pyruvate dehydrogenase kinase) isoenzymes have been identified, each of which display a distinct tissue-specific expression profile, and have differential regulatory properties. Thus PDHK play a key role in controlling the balance between glucose and lipid oxidation according to substrate supply. Increasing glucose oxidation by inhibiting PDHK may be an effective mechanism to increase glucose utilization; additionally, increasing pyruvate oxidation may further contribute to lowering of glucose level by decreasing the supply of gluconeogenic substrates. A number of PDHK inhibitors are now available to enable this mechanism to be evaluated as a therapy for diabetes. The isoenzyme selectivity profile of AZD7545 and related compounds will be described and evidence for their non-ATP-competitive mode of action presented. These compounds increase PDH activity in vivo, and when dosed chronically, improve glycaemic control in Zucker rats. Furthermore, glucose lowering has been demonstrated in the hyperglycaemic Zucker diabetic fatty rat. This result supports the hypothesis that inhibition of PDHK may be an effective therapy for Type II diabetes.

AZD7545, a novel inhibitor of pyruvate dehydrogenase kinase 2 (PDHK2), activates pyruvate dehydrogenase in vivo and improves blood glucose control in obese (fa/fa) Zucker rats.

PDH (pyruvate dehydrogenase) is a key enzyme controlling the rate of glucose oxidation, and the availability of gluconeogenic precursors. Activation of PDH in skeletal muscle and liver may increase glucose uptake and reduce glucose production. This study describes the properties of AZD7545, a novel, small-molecule inhibitor of PDHK (PDH kinase). In the presence of PDHK2, AZD7545 increased PDH activity with an EC(50) value of 5.2 nM. In rat hepatocytes, the rate of pyruvate oxidation was stimulated 2-fold (EC(50) 105 nM). A single dose of AZD7545 to Wistar rats increased the proportion of liver PDH in its active, dephosphorylated form in a dose-related manner from 24.7 to 70.3% at 30 mg/kg; and in skeletal muscle from 21.1 to 53.3%. A single dose of 10 mg/kg also significantly elevated muscle PDH activity in obese Zucker (fa/fa) rats. Obese, insulin-resistant, Zucker rats show elevated postprandial glucose levels compared with their lean counterparts (8.7 versus 6.1 mM at 12 weeks old). AZD7545 (10 mg/kg) twice daily for 7 days markedly improved the 24-h glucose profile, by eliminating the postprandial elevation in blood glucose. These results suggest that PDHK inhibitors may be beneficial agents for improving glucose control in the treatment of type 2 diabetes.

AZD7545 is a selective inhibitor of pyruvate dehydrogenase kinase 2.

The PDH (pyruvate dehydrogenase) multi-enzyme complex catalyses a key regulatory step in oxidative glycolysis. Phosphorylation of the E1 subunit of the complex on serine residues results in the inactivation of enzyme activity. A family of four dedicated PDH kinase isoenzymes exists, each of which displays a distinct tissue-specific expression profile. AZD7545 is one of a series of PDH kinase inhibitors developed for the treatment of type 2 diabetes. The isoenzyme-selectivity profile of AZD7545 and related compounds is described and the consequences for their in vivo mode of action are discussed.

Growth hormone-dependent differentiation of 3T3-F442A preadipocytes requires Janus kinase/signal transducer and activator of transcription but not mitogen-activated protein kinase or p70 S6 kinase signaling.

The signals mediating growth hormone (GH)-dependent differentiation of 3T3-F442A preadipocytes under serum-free conditions have been studied. GH priming of cells was required before the induction of terminal differentiation by a combination of epidermal growth factor, tri-iodothyronine, and insulin. Cellular depletion of Janus kinase-2 (JAK-2) using antisense oligodeoxynucleotides (ODNs) prevented GH-stimulated JAK-2 and signal transducer and activator of transcription (STAT)-5 tyrosine phosphorylation and severely attenuated the ability of GH to promote differentiation. Although p42(MAPK)/p44(MAPK) mitogen-activated protein kinases were activated during GH priming, treatment of cells with PD 098059, which prevented activation of these kinases, did not block GH priming. However, antisense ODN-mediated depletion of mitogen-activated protein kinases from the cells showed that their expression was necessary for terminal differentiation. Similarly, although p70(s6k) was activated during GH priming, pretreatment of cells with rapamycin, which prevented the activation of p70(s6k), had no effect on GH priming. However, rapamycin did partially block epidermal growth factor, tri-iodothyronine, and insulin-stimulated terminal differentiation. By contrast, cellular depletion of STAT-5 with antisense ODNs completely abolished the ability of GH to promote differentiation. These results indicate that JAK-2, acting specifically via STAT-5, is necessary for GH-dependent differentiation of 3T3-F442A preadipocytes. Activation of p42(MAPK)/p44(MAPK) and p70(s6k) is not essential for the promotion of differentiation by GH, although these signals are required for GH-independent terminal differentiation.

Regulation of the GTP-binding protein-based antilipolytic system of sheep adipocytes by growth hormone.

Chronic exposure of sheep adipose tissue to growth hormone (GH) in vitro decreases the ability of the adenosine analogue, N6-phenylisopropyladenosine (PIA), to inhibit isoprenaline-stimulated lipolysis by a mechanism which is dependent on both gene transcription and protein serine/threonine phosphorylation. The inhibition is not due to a change in ligand binding to the adenosine receptor, the amounts of the three isoforms of the inhibitory GTP-binding protein, Gi, or the maximum (forskolin-stimulated) adenylate cyclase activity. The ability of GH to modulate the PIA-activated adenosine receptor to stimulate dissociation of heterotrimeric Gi was assessed by measurement of pertussis toxin-catalysed ADP-ribosylation of Gi; GH does not appear to alter the interaction between the activated receptor and Gi. The ability of GH to alter the ability of activated Gi to inhibit adenylate cyclase activity was assessed by measuring the ability of a GTP analogue, guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), to inhibit forskolin-stimulated adenylate cyclase activity; chronic exposure to GH prevented this effect of p[NH]ppG. Thus the attenuation of the inhibition of lipolysis by PIA by chronic exposure of adipocytes to GH appears to be due to an impairment in the interaction between adenylate cyclase and the alpha subunit of one or more isoforms of Gi.

Protein kinase C isoforms play differential roles in the regulation of adipocyte differentiation.

In this study we first established, by immunoblotting with specific antibodies, the temporal changes in cellular levels of protein kinase C (PKC) isoforms during differentiation of 3T3-F442A pre-adipocytes. Both pre-adipocyte and adipocyte 3T3-F442A cells were found to express PKC-alpha, -gamma, -delta, -epsilon, -zeta and -mu. However we were unable to detect PKC-beta, -eta or -theta. The same PKC isoform expression profile was found in rat adipocytes. The alpha, delta and gamma isoforms displayed similar temporal patterns of expression during differentiation of 3T3-F442A cells; all increased rapidly, peaking at day 2 of differentiation. Subsequently, the expression of these isoforms decreased, resulting in lower levels in fully differentiated adipocytes than in pre-adipocytes. The expression of PKC-epsilon increased steadily during differentiation, resulting in markedly elevated levels in adipocytes. Although expression of PKC-mu increased during differentiation, this was attributable to prolonged confluence rather than to the differentiation process itself. No change was observed in PKC-zeta levels during adipocyte development. Anti-sense oligodeoxynucleotides (ODNs) were used to deplete selectively the individual PKC subtypes. Each of the ODNs used effectively depleted the specific isoforms to undetectable levels and did not affect expression of the other PKC subtypes. This approach indicated that pre-adipocyte differentiation is not dependent upon PKC-zeta but that PKC-alpha,-delta and -mu each exert an inhibitory influence upon differentiation. Use of anti-sense ODNs to deplete PKC-epsilon and -gamma revealed that pre-adipocyte differentiation is dependent upon each of these isoforms. However, PKC-gamma, but not PKC-epsilon, appeared to be necessary for the clonal expansion of differentiating cells, suggesting that PKC-epsilon is required at a later phase in the differentiation process, when its expression is elevated, for the attainment and maintenance of the adipocyte phenotype.

Cyclic AMP potentiates growth hormone-dependent differentiation of 3T3-F442A preadipocytes: possible involvement of the transcription factor CREB.

We have examined the effects of cyclic AMP on the differentiation of 3T3-F442A preadipocytes. High concentrations of intracellular cyclic AMP potently inhibited differentiation whereas low concentrations of intracellular cyclic AMP, induced by a number of different agents, promoted differentiation. To analyse these effects of cyclic AMP more closely, we developed a two-phase protocol for the differentiation of 3T3-F442A cells. Growth hormone (GH) was necessary to prime confluent cells during the first phase, following which, the addition of insulin and other adipogenic agents then promoted terminal differentiation. Cyclic AMP potentiated the priming action of GH but exerted an inhibitory effect on terminal differentiation when added to cells which had previously been primed with GH showing that the effects of cyclic AMP on preadipocyte differentiation are stage-dependent. We analysed the stimulatory effects of cyclic AMP during GH priming and found that cyclic AMP induced phosphorylation of the cyclic AMP response element (CRE) binding protein CREB and activated transcription of a CRE-linked reporter gene. Furthermore, GH also stimulated CREB phosphorylation and activation and this effect was potentiated by cyclic AMP. These results suggest a mechanism for the synergistic priming of preadipocytes for terminal differentiation by cyclic AMP and GH via the activation of differentiation genes containing CREs.

Growth hormone and phorbol esters require specific protein kinase C isoforms to activate mitogen-activated protein kinases in 3T3-F442A cells.

Previous studies have shown that the activation of p44 and p42 mitogen-activated protein (MAP) kinases (ERK1 and ERK2) by growth hormone (GH) and phorbol esters, but not by epidermal growth factor, in 3T3-F442A preadipocytes is dependent on protein kinase C (PKC). In the present study two approaches have been taken to determine the PKC isoform dependence of MAP kinase activation in these cells. By immunoblotting with specific antibodies, the cells were found to express PKC-alpha, -gamma,-delta, -epsilon and -zeta. Treatment of cells with 500 nM PMA for 3 h led to the complete depletion of PKC-delta and the partial depletion of PKC-alpha but did not significantly affect the expression of the other PKC isoforms. In parallel, such treatment severely attenuated the ability of GH to activate MAP kinase. The degree of this attenuation was not increased by more prolonged PMA pretreatment, indicating that PKC-delta and perhaps PKC-alpha are important for MAP kinase activation by GH. These experiments further revealed that additional PKC isoforms were required for the full activation of MAP kinases by acute treatment with PMA. A second approach involved the use of anti-sense oligodeoxynucleotides (ODNs) to deplete the individual PKC isoforms selectively. Each of the ODNs used effectively depleted the relevant isoform to undetectable levels and did not affect the expression of the other PKC isoforms. Pretreatment of cells with PKC-delta anti-sense ODN, but not with anti-sense ODN to the other phorbol ester-sensitive isoforms, severely attenuated the activation of MAP kinases by GH. PKC-delta anti-sense ODN also blocked (by approx. 50%) the activation of MAP kinases by PMA. Furthermore a combination of PKC-delta and -epsilon anti-sense ODNs completely blocked the effect of PMA on MAP kinases. Collectively, these results indicate that the novel PKC-delta and -epsilon isoforms can couple to the MAP kinase pathway in 3T3-F442A cells but that the activation of MAP kinases by GH specifically involves PKC-delta.

Effect of lactation on insulin signal transduction in sheep adipose tissue and skeletal muscle.

The molecular basis of the insulin resistance of adipocytes and skeletal muscle during lactation has been investigated in sheep. The number of insulin receptors per adipocyte or per unit membrane protein for skeletal muscle is unchanged by lactation. The ability of insulin to stimulate autophosphorylation of its beta-subunit was enhanced in adipocytes but not in skeletal muscle during lactation. This increased autophosphorylation was due, at least in part, to enhanced tyrosine phosphorylation and was found when both solubilised, immunoprecipitated insulin receptors and intact adipocytes were incubated with insulin. The ability of the insulin receptor kinase to phosphorylate other proteins did not appear to be altered by lactation; this was shown with lectin-purified insulin receptors using the artificial substrate, polyglutamyl tyrosine, and in intact adipocytes. Lactation had no effect on the ability of insulin to activate two key downstream kinases, mitogen-activated protein kinase and phosphatidyl inositol-3-kinase in adipocytes. The study thus shows that the insulin resistance of lactation in sheep is due to changes downstream of the receptor in both adipocytes and skeletal muscle.

Cyclic AMP stimulates the phosphorylation and activation of p42 and p44 mitogen-activated protein kinases in 3T3-F442A preadipocytes.

The effects of cyclic AMP on mitogen-activated protein (MAP) kinase activation were investigated in 3T3-F442A preadipocytes. Several agents, which raise intracellular cyclic AMP levels by distinct mechanisms, induced a transient activation of the p42 and p44 isoforms of MAP kinase and of a MAP kinase kinase. Activation of MAP kinase by cyclic AMP was prevented by two distinct inhibitors of cyclic AMP-dependent protein kinase and by PD 098059, a specific inhibitor of the activation of the MAP kinase kinase MEK 1. Therefore, in contrast to most cell types studied, cyclic AMP exerts a positive influence on the MAP kinase pathway in 3T3-F442A preadipocytes at a level upstream of the activation of MAP kinase kinase.

Requirement for phosphoinositide 3-OH kinase in growth hormone signalling to the mitogen-activated protein kinase and p70s6k pathways.

Pituitary growth hormone (GH) co-ordinately stimulates three distinct signalling pathways in 3T3-F442A preadipocytes, the STAT (signal transducer and activator of transcription) pathway, the mitogen-activated protein (MAP) kinase cascade and p70s6k. The mechanisms linking the GH receptor to these signals have not been fully identified. In this study we have examined the role of phosphoinositide 3-OH kinase (PI 3-kinase). Pretreatment of cells with wortmannin, a specific inhibitor of PI 3-kinase, prevented the activation of p70s6k and partially inhibited the activation of p42 and p44 MAP kinases by GH. In contrast, wortmannin failed to appreciably affect the GH-stimulated tyrosyl phosphorylation of JAK-2 or STAT-1. GH transiently increased the activity of PI 3-kinase recovered in antiphosphotyrosine immunoprecipitates. In addition, several tyrosyl-phosphorylated proteins were specifically adsorbed from lysates of cells exposed to GH by a glutathione S-transferase fusion protein containing the 85 kDa regulatory subunit of PI 3-kinase. GH also induced an increase in the PI 3-kinase activity associated with both JAK-2 and insulin receptor substrate-1 (IRS-1) immunoprecipitates. These results establish PI 3-kinase as an important mediator of GH signalling to the MAP kinase and p70s6k pathways and suggest that PI 3-kinase is activated by a mechanism involving JAK-2 and IRS-1.

Chronic effects of somatotropin treatment on response of subcutaneous adipose tissue lipolysis to acutely acting factors in vivo and in vitro.

The basis of the chronic lipolytic effect of somatotropin on adipose tissue was investigated in sheep. Lipolytic rate was assessed in subcutaneous adipose tissue both in vivo, by microdialysis, and in vitro. Somatotropin treatment resulted in a small increase in basal (unstimulated) lipolysis and also in the maximum lipolytic rate observed in the presence of catecholamines both in vivo and in vitro. There was a small increase in the number of beta-adrenergic receptors but no change in the amount of the two isoforms of the stimulatory GTP-binding protein, Gs. Treatment with somatotropin decreased the response to antilipolytic agents such as the adenosine analog N6-phenylisopropyladenosine and prostaglandin E1. There was, however, no change in the number of adenosine receptors or amounts of the inhibitory GTP-binding proteins (Gi-1 plus Gi-2). Somatotropin also decreased prostaglandin E2 production by subcutaneous adipose tissue in vivo. Somatotropin treatment thus alters lipolytic regulation in sheep and this is characterized by changes in a number of proteins involved in this process.