Piyush C. Kothary

MAP Kinase Pathway is Involved in IGF-1-Stimulated Proliferation of Human Retinal Pigment Epithelial Cells (hRPE)

Purpose: To investigate the mitogenic activity of insulin-like growth factor-1 (IGF-1) on the proliferation of human retinal pigment epithelial cells (hRPE) and to elucidate the role of vascular endothelial growth factor (VEGF) and MAP kinase (MAPK) in the IGF-1 signaling cascade. Methods: Human RPE specimens were obtained from postmortem non-pathological eyes and cultured in vitro through several passages. Cellular proliferation in the presence of increasing concentrations of IGF-1 and IGF-1 + PD98059 (a known MAPK inhibitor) was measured by [3H]thymidine incorporation; trypan blue exclusion studies (T) verified cell viability. Under the same experimental conditions, synthesis of VEGF was measured utilizing [14C]methionine immunoprecipitation and immunocytochemical methods as well as Western blot analysis. Results: IGF-1 stimulated hRPE proliferation, as demonstrated by [3H]thymidine incorporation. There was also an IGF-1-induced increase in VEGF synthesis as measured quantitatively by [14C]methionine-VEGF immunoprecipitation. This was qualitatively confirmed by immunocytochemistry and Western blotting. PD98059 suppressed both IGF-1-induced cell proliferation as well as IGF-1-stimulated VEGF production. Conclusions: These studies suggest that IGF-1 is a mitogen for hRPE cells and also stimulates production of the angiogenic factor, VEGF. Additionally, PD98059 inhibits the production of VEGF, suggesting that the MAP kinase pathway is involved in IGF-1-mediated angiogenesis.

Treatment of cirrhotic rats with epidermal growth factor and insulin accelerates liver DNA synthesis after partial hepatectomy

Prevention of postoperative hepatic failure is important after hepatic resection. In patients with cirrhosis, impaired liver function and regenerative capacity after major hepatic resection are associated with increased morbidity and mortality. In this study, a combination of epidermal growth factor (EGF) and insulin were used as hepatotrophic factors in an attempt to stimulate DNA synthesis after 70% hepatectomy (HTX). Regenerative capacity was evaluated in normal and cirrhotic rat liver by measuring DNA synthesis in vivo. Micronodular liver cirrhosis was established by the simultaneous oral administration of CCl4 and phenobarbital. Epidermal growth factor plus insulin was injected subcutaneously immediately after and 12 h after HTX or sham operation was performed. Rats were killed 24 h after the operation and liver regeneration was estimated by [3H]‐thymidine incorporation into DNA as well as an autoradiographic nuclear labelling index. Hepatectomy increased [3H]‐thymidine incorporation significantly in both normal and cirrhotic rats. In cirrhotic rats, [3H]‐thymidine incorporation after HTX was significantly lower than in normal rats and administration of a combination of EGF and insulin after HTX enhanced [3H]‐thymidine incorporation. In conclusion, DNA synthesis 24 h after HTX is decreased in cirrhotic rats compared with normal rats and EGF supplementation with insulin accelerates DNA synthesis in hepatectomized cirrhotic rats. The data suggest that administration of combinations of exogenous hepatotrophic factors may play a useful role in the treatment of cirrhotic patients undergoing major hepatic resection.

Inhibition of DNA synthesis by somatostatin in rat hepatocytes stimulated by hepatocyte growth factor or epidermal growth factor

The antiproliferative effects of somatostatin on hepatocytes stimulated by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) were investigated using primary cultures of adult rat hepatocytes. Somatostatin inhibits HGF-induced (at a dose of 10 ng/mL) or EGF-induced (at a dose of 100 ng/mL) 3H-thymidine incorporation into hepatocytes in a dose-dependent manner (10(-10) to 10(-8) M). This inhibition was confirmed by autoradiography. The effect of somatostatin was nontoxic as judged by preserved albumin synthesis, a marker for differentiated hepatocyte function. In the presence or absence of somatostatin, neither HGF nor EGF significantly altered intracellular cyclic adenosine monophosphate (cAMP). We conclude that somatostatin is a potent inhibitor of HGF- or EGF-induced deoxyribonucleic acid synthesis in adult rat hepatocytes. The mechanism of this inhibition appears to be independent of cAMP. The significance of somatostatin in liver regeneration has yet to be assessed.

Identification of gastrin molecular variants in gastrinoma syndrome

The molecular species of gastrin in the circulation and in tumor extracts were studied in two groups of patients: (1) with benign gastrinoma and (2) with gastrinoma with liver metastases. Radioimmunoassays (RIAs) and immunoaffinity chromatography for the amino (NH2)- and amidated COOH-terminus of gastrin-17 (antiserum G17) and the NH2-terminus of gastrin-34 (antiserum G34) were employed. In both benign and metastatic tumors the molecular forms of gastrin in boiling water extracts measured by the gastrin-17 NH2- and COOH-terminal assays were similar. In addition to a molecular component resembling the amidated gastrin-17, there were also significant amounts of larger molecular weight (mol. wt.) forms. The larger mol. wt. forms absorbed by the NH2-terminus of G17 antiserum corresponded to the COOH-terminus-extended forms of gastrin-17. Furthermore, larger mol. wt. gastrins immunopurified by antiserum to the NH2-terminus of gastrin-34 corresponded to gastrin-34 extended molecules. Sera of patients with liver metastases had higher concentrations of the NH2-terminal of gastrin-17 whereas sera of patients with benign gastrinoma contained predominantly gastrins detected by the COOH-terminal assay. These results suggest that: (a) there are differences in the molecular pattern of gastrin in the circulation of patients with benign and metastatic gastrinomas; (b) gastrins which are fully processed with carboxy-terminal amidation predominate in the circulation of patients with benign gastrinoma; and (c) gastrins containing the gastrin-17 and COOH-terminally extended gastrin-17 and gastrin-34 precursor molecules occur in high concentration in the circulation of gastrinoma patients with metastases to the liver.

Transforming growth factor-α (TGF-α) improves hepatic DNA synthesis after hepatectomy in cirrhotic rats

Impaired liver regeneration in cirrhosis complicates the surgical treatment of liver tumors which arise in this setting. We developed a rat model to investigate the regenerative response of cirrhotic liver after hepatectomy and studied the effect of exogenous transforming growth factor-α (TGF-α), a potent liver mitogen. Micronodular cirrhosis was established by the simultaneous administration of CCl4 and phenobarbital. Hepatic DNA synthesis ([3H]thymidine incorporation into DNA) 24 hr after partial hepatectomy in cirrhotic rats was 15.6 ± 3.4 cpm/μg DNA (means ± SEM), which was significantly lower than in normal rats (37.3 ± 3.4 cpm/μg DNA, P < 0.05). Exogenous TGF-α (30 nmole/kg, sc every 12 hr) significantly improved [3H]thymidine incorporation (35.6 ± 8.2 cpm/μg DNA, P < 0.05). An autoradiographic nuclear labeling index also confirmed increased DNA synthesis (6.7% vs 13.4%). TGF-α had no effect on normal regenerating liver (42.5 ± 8.8 cpm/μg DNA, NS). Although the significance of TGF-α-enhanced liver regeneration in cirrhosis has yet to be assessed, this model may be useful for the study of mechanisms which control hepatic proliferation.

Inhibitory effects of somatostatin on rat hepatocyte proliferation are mediated by cyclic AMP

Somatostatin (SS-14) is known as an antigrowth factor for a variety of cell types, including gastrointestinal mucosa, exocrine pancreas, lymphocytes, and some tumors. We have recently identified and biochemically characterized SS-14-binding protein on rat liver plasma membranes (S. E. Raper, P. C. Kothary, and J. DelValle, Gastroenterology 96: A408, 1989; P. C. Kothary et al., Digestion 46 (Suppl 1): 58, 1990). We hypothesized that SS-14 may affect liver growth as well and investigated cellular mechanisms of this phenomenon focusing on the second messenger cAMP. Freshly isolated rat hepatocytes were plated on tissue culture dishes coated with Matrigel (laminin, heparan sulfate, and type IV collagen). The medium was not supplemented with serum or hormones. Either dibutyryl-cAMP (1 mM) or isobutylmethylxanthine (IBMX, 0.1 mM) was added in the presence or absence of SS-14 (10 nM). DNA synthesis was estimated by the rate of [3H]thymidine incorporation into DNA and by the labeling index (an autoradiographic measurement of the number of labeled nuclei). SS-14 significantly inhibited both [3H]thymidine incorporation and labeling index of rat hepatocytes stimulated by dibutyryl-cAMP or IBMX. SS-14 also inhibited intracellular cAMP accumulation stimulated by IBMX. We conclude that SS-14 exerts at least part of its antiproliferative effects via the adenylate cyclase system. Further study using other signal transduction systems may yield more information about mechanisms of hepatocyte growth.

Somatostatin-14 blocks the hepatotrophic effects of insulin in the rat

UNLABELLED We hypothesized that somatostatin-14 (SS-14) might inhibit insulin-stimulated hepatic growth. Rat hepatocytes were isolated by a two-step collagenase perfusion technique and cultured on Matrigel. Differentiated hepatocyte function was documented by albumin synthesis. Hepatocytes were incubated with insulin in the presence or absence of SS-14. Hepatocyte proliferation was assessed by tritiated thymidine ([3H]thy) incorporation into DNA. [3H]thy incorporation was increased by 230% in the presence of insulin and was essentially abolished by the addition of SS-14. Insulin-stimulated cyclic-AMP accumulation was also decreased from 190 to 108% of control levels (P less than 0.05) by the addition of SS-14. Pretreatment with pertussis toxin, which inactivates the inhibitory G-protein, Gi, blocked the effect of SS-14. CONCLUSIONS (i) In the rat, SS-14 effectively blocks insulin-stimulated [3H]thy incorporation into DNA, possibly by blocking intracellular cAMP accumulation. (ii) Pertussis toxin blocks the growth inhibitory effects of SS-14, suggesting that inhibitory G proteins are involved in the mechanism of SS-14 action. Somatostatin may be useful in studying the role of second messengers in cell growth.

NH2-Terminal of gastrin-17 in duodenal ulcer disease: Identification of progastrin-17

Serum gastrin concentrations were measured using antisera with specificity for the carboxyl and amino terminus of gastrin-17 in 50 healthy subjects and 18 patients with active duodenal ulcer disease (DU). The amino terminal of gastrin-17 immunoreactivity was significantly higher in DU patients than in healthy subjects. NH2-terminus of gastrin-17 immunopurified material from serum of DU patients was subjected to Sephadex G50 column chromatography and eluates were monitored by an additional antiserum EG10 that recognizes COOH-terminally extended gastrin. Besides the NH2 terminal tridecapeptide of gastrin-17, COOH-terminally extended progastrin was found. This may reflect abnormal processing of gastrin in patients with active duodenal ulcer disease.

The effects of transforming growth factor alpha and somatostatin on regenerating hepatocytes in the rat.

Transforming growth factor alpha (TGF alpha) stimulates DNA synthesis in adult rat hepatocytes, and plays a physiological role after partial hepatectomy by an autocrine mechanism. Somatostatin (SS-14) is a potent inhibitor of gastrointestinal function and inhibits proliferation in various cell types. We examined the proliferative effect of TGF alpha and the inhibitory effect of SS-14 on hepatocytes isolated at various times after partial hepatectomy. To study the mechanism of SS-14 further, we treated rats with the long acting SS-14 analog, octreotide, before or after 70% hepatectomy to determine whether or not a differential effect could be seen. We confirmed the proliferative effects of TGF alpha, and the inhibitory action of SS-14 in the early phase of liver regeneration in vitro. Regenerating hepatocytes isolated from hepatectomized livers respond to TGF alpha only at early time points (2 h) but do not respond to SS-14. In addition, the long acting SS-14 analog, octreotide, inhibited hepatic regeneration only when administered prior to hepatectomy. We conclude that exogenous peptide stimulation is effective only in the early phase of the hepatic proliferative response. After the initial changes brought about by hepatectomy, subsequent steps of the regenerative process appear refractory to external stimuli.

The liver plays an important role in the regulation of somatostatin-14 in the rat☆

Since little is known about the in vivo disposition of circulating somatostatin-14 (SRIF-14), we examined hepatic processing of SRIF-14 in the rat. Three minutes after the intraportal injection of iodine 125 (125I)-labeled SRIF-14, 16.0 +/- 2.0% of the injected dose is localized to the liver. In the presence of unlabeled SRIF-14, hepatic uptake can be decreased by 68%. Five minutes after the intraportal injection of 125I-SRIF-14, 9.5 +/- 1.4% of the tracer is localized to the liver, more than any other organ tested. Serial collections of bile reveal peak radioactivity at between 10 and 20 minutes. Simultaneous administration of unlabeled SRIF-14 decreases biliary radioactivity by 40%. HPLC analysis of radioactive bile reveals a chromatographic profile similar to that of intact SRIF and is 73% immunoprecipitable by an anti-SRIF antibody. Pretreatment with chloroquine, a lysosomal enzyme inhibitor, does not significantly decrease biliary radioactivity. We conclude that the data are consistent with saturable hepatic uptake and predominantly nonlysosomal transcellular transport.