Phosphoinositide 3-kinase activity is necessary for insulin-dependent inhibition of apolipoprotein B secretion by rat hepatocytes and localizes to the endoplasmic reticulum.

Insulin inhibits apolipoprotein B (apoB) secretion by primary rat hepatocytes through activation of phosphoinositide 3-kinase (PI 3-K). Current studies demonstrate that the PI 3-K inhibitor wortmannin inhibits both basal and insulin-stimulated PI 3-K activities. Wortmannin and LY 294002, two structurally distinct PI 3-K inhibitors, prevent insulin-dependent inhibition of apoB secretion in a dose-dependent manner. To link PI 3-K activation to insulin action on apoB, we investigated whether insulin induced localization of activated PI 3-K to the endoplasmic reticulum (ER), where apoB biogenesis is initiated. Insulin action results in a significant redistribution of PI 3-K to a low density microsome (LDM) fraction containing apoB protein and apoB mRNA. Insulin stimulates a significant increase in PI 3-K activity associated with insulin receptor substrate-1 as well as an increase in insulin receptor substrate-1/PI 3-K mass in LDM. Subfractionation of LDM on sucrose density gradients shows that insulin significantly increases the amount of PI 3-K present in an ER fraction containing apoB. Insulin stimulates PI 3-K activity in smooth and rough microsomes isolated from rat hepatocytes, the latter of which contain rough ER as demonstrated by electron microscopy. Studies indicate that 1) PI 3-K activity is necessary for insulin-dependent inhibition of apoB secretion by rat hepatocytes; 2) insulin action leads to the activation and localization of PI 3-K in an ER fraction containing apoB; and 3) insulin stimulates PI 3-K activity in the rough ER.

Organ procurement: experience from a southern Italian region.

The authors report their experience of organ procurement during the last 5 years to evaluate a program that began in 1988 to improve organ retrieval in Calabria. In this region only two donations were reported up to 1988, one each in 1980 and 1985. Because of the large population on dialysis and the willingness of a group of surgeons and anesthesiologists, this program was undertaken in 1988 under the supervision of C.C.S.T. (Co-ordination of Centre and South Italy for Transplantation). This program was designed to act on two levels: to create a large group of people directly involved in health care (physicians and nurses) motivated in organ procurement and transplantation, and to diffuse the "culture" of organ donation among lay people. This was achieved by means of scientific meetings inside the hospital and with conventions and TV programs, supported by an Association of Volunteers, where ethical and scientific problems of organ donation and transplantation were discussed in simple language. Various meetings were also held with high school students. During these meetings a questionnaire was distributed among students. Results of this questionnaire show that the main obstacles to organ donation are the "unclear" concept of "brain death" and religious feelings, but after the concept of brain death was explained, a significant number of students showed a different attitude toward organ procurement and transplantation. Results of this program are extremely encouraging (23 organ donations during the last 3 years). We hope to improve our results in the near future, and we do believe that a further and significant increase to our preliminary good results could be achieved by the possibility of performing at least kidney transplantation in our institution.

[Results of the multicenter study (A.C.O.I. Stapler Study Group) on 420 cases of esophagojejunal and 544 cases of colorectal anastomoses]. / Risultati dello studio multicentrico (A.C.O.I. Stapler Study Group) su 420 anastomosi esofago-digiunali e 544 colorettali.

The introduction of staplers in general surgery allowed to perform gastrointestinal anastomoses easier and with better results. Because many series report an important incidence of complications even using stapling instruments, the A.C.O.I. (Italian Association of General Hospital Surgeons), with the support of Auto Suture Italy, promoted a collaborative trial in Italy to evaluate the real incidence of complications after stapled colorectal or esophago-jejunal anastomoses. The study has been carried out during the period April 1990-December 1991 and 58 Centres throughout Italy participated with 420 esophagojejunostomies after Total Gastrectomy and 544 Colorectal anastomoses after Anterior Resection of the Rectum. Many variables were evaluated such as patients general conditions, the pathology and the site of anastomosis, the instrument used, the technique of reconstruction, if it was an urgent or an elective procedure, the incidence of intraoperative problems and the surgical experience. The most frequent complications were bleeding, leakage and stenosis. The incidence of bleeding was low in both groups, it was an occasional event not related with any of the variables previously described. Furthermore it was never important and never required reoperation or caused death of the patient. Leakage was the most frequent and serious complication (12.4% in colorectal anastomoses and 5.5% in esophagojejunostomies) and was mainly related to the incidence of intraoperative technical problems (tearing of the stump or purse string failure), to urgent procedures, to low and ultra low colorectal anastomoses and to centres with less experience, particularly with an experience of less than 50 mechanical anastomoses. Late complications such stenoses, were particularly frequent (8.4%) after a leakage and in very low colorectal anastomoses. Perioperative Mortality was very low in birth groups (1.1% for colorectal anastomoses and 2.6% for esophagojejunostomies) one of the lowest described in the Literature. This study seem to confirm that staplers are effective, safe and easy to use, showing a low incidence of complications and they make possible anastomoses otherwise technically impossible.

Ex vivo versus in situ resection of segmental liver grafts in pigs--a comparison in immediate and four-hour-stored grafts.

This study compared the function of reduced grafts prepared in situ or ex vivo and transplanted immediately or after 4 hr of cold storage. Measurements of acid/base balance, plasma electrolytes, albumin, and urea showed no differences between groups. There was no difference between the increase and decline of plasma AST in recipients of grafts transplanted immediately after either ex vivo or in situ reduction; the increase in plasma AST of recipients of stored grafts was up to 10-fold and persisted until the end of the study at 7 days, with some decline. Plasma fibrinogen decreased intraoperatively but levels were restored within 24 hr in all groups; plasma prothrombin and partial thromboplastin times were not significantly disturbed. The patterns of decline and return of tissue adenine nucleotides were similar in all groups. While the regenerative response measured by tissue thymidine kinase and mitotic figures was not different between the groups, comparison with results from a group of partially hepatectomized animals showed a 3-4-fold depression in response in reduced liver grafts. The contributions of the effects of ischemia, flushing, and preservation to the depressed regenerative response of reduced liver grafts need to be determined. The present studies suggest however, that with regard to functional assessment, results are not affected either by ex vivo or in situ reduction of the graft, or by cold storage for 4 hr.

Function of kidney grafts from brain-dead donor pigs. The influence of dopamine and triiodothyronine.

There are conflicting reports about the effects of administration of dopamine to brain-dead donors upon posttransplant organ function. This study compared the survival and serum creatinine levels in pigs that received renal grafts from untreated controls, from controls in which either the donor or donor and recipient received dopamine or from animals rendered brain dead for 16 hr by acute elevation of intracranial pressure, and given standard supportive treatment. In two additional groups, brain-dead donors were given dopamine or dopamine with triiodothyronine. Recipients of grafts from control animals or from brain-dead donors survived the 7-day period of study and showed minimal changes in serum creatinine. Recipients of grafts from brain-dead donors given dopamine however showed reduced survival and progressive increase in serum creatinine. This did not occur in the group given triiodothyronine concurrently with dopamine. It is suggested that if administration of dopamine is essential to treat donor hypotension, concurrent use of triiodothyronine may preserve posttransplant renal function.

Effects of nonketotic streptozotocin diabetes on apolipoprotein B synthesis and secretion by primary cultures of rat hepatocytes.

The effects of hypoinsulinemic nonketotic streptozotocin diabetes on hepatic apo B synthesis and secretion was studied in primary cultures of rat hepatocytes. Diabetic rats were characterized by their significantly elevated serum glucose, apo B, and triglyceride levels, while serum insulin levels were less than a third of normal. Serum transminase activities of diabetic rats were significantly elevated when compared with control rats, which was attributed to an increase in liver transaminase activity in diabetic rats. The pattern of enzyme activities of hepatocytes reflected that observed in livers of donor rats and the pattern was retained by primary cultures of hepatocytes over the culture period. Hepatocytes from diabetic rats secreted only one third of the apo B secreted by hepatocytes from control rats, which was determined by monoclonal immunoassay of rat total apo B. Decreases in secretion were confirmed by measurement of secretory [35S]methionine-labeled lipoprotein apo B radioactivity. The decreased apo B content of media of hepatocytes from diabetic rats was not due to increased apo B catabolism since hepatocytes from diabetic rats were shown to degrade less lipoprotein-apo B than hepatocytes from normal rats in control experiments. In addition, the apo B content of detergent-solubilized hepatocytes from diabetic rats was significantly less than that of hepatocytes from control rats. These results suggest that insulin is necessary for normal hepatic apo B synthesis and secretion and that the hyperlipidemia associated with hypoinsulinemia in vivo is primarily of intestinal origin.

Secretion of high and low molecular weight phosphorylated apolipoprotein B by hepatocytes from control and diabetic rats. Phosphorylation of APO BH and APO BL.

Apolipoprotein B (apo B) phosphorylation was examined in primary cultures of hepatocytes from control and nonketotic streptozotocin diabetic rats. Following a 5-h incubation with ortho[32P]phosphate, media lipoproteins (d less than 1.063 g/ml) were isolated, and delipidated apolipoproteins were separated by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis (SDS-PAGGE), and gels were heat fixed. Autoradiographic bands corresponding to high (apo BH) and low molecular weight apo B (apo BL) were observed in media lipoproteins isolated from control rats, and these bands were more prominent in media lipoproteins isolated from diabetic rats. Apo B-specific activity was estimated from aqueous alcohol-precipitated radioactivity and apo B monoclonal immunoassay of isolated media lipoproteins. In lipoproteins secreted by hepatocytes of diabetic rats, the calculated apo B specific activity was between 18- and 31-fold greater than that secreted by hepatocytes of control rats, consistent with the SDS-PAGGE gel data. The increase in secretory 32P-labeled apo B from hepatocytes of diabetic rats was due, at least in part, to an increase in labeled phospho-tyrosine as determined by phosphoamino acid analysis. These data demonstrate that apo BH may be secreted as a phosphorylated protein and that apo B phosphorylation occurs on tyrosine as well as serine residues.

Comparison of combined portal-arterial versus portal perfusion during liver procurement.

PAP of harvested livers is routinely used to minimize parenchymal anoxia during storage. PP is compared with PAP to evaluate the relative reliability of PAP. Sixty female Landrace pigs were used for 30 OLTs. Group 1 livers underwent PP, whereas group 2 livers were treated with PAP. The cold ischemic time was less than 120 minutes for both groups, with no warm ischemia. Intraoperative and 24-hour postoperative biochemical, coagulation, and histocytological data were analyzed. Morphological studies of cellular damage were based on the percentage of CVD and KP and classified as light, moderate, and severe damage. Data, at closing, were compared by using Fisher's test (group 1 v group 2,P = 0.003 for light damage and P = .04 for severe damage; first postoperative day for group 1 v group 2, P = .133 for light damage and P = .25 for severe damage. Blood samples at closing and 24 hours postoperatively showed significant differences between groups 1 and 2: At closing for groups 1 and 2, respectively: AST, 968.9 +/- 742.7 and 327.4 +/- 174.7 IU/L (P less than .001); ALT, 63.1 +/- 40.3 and 20.3 +/- 5.3 IU/L (P less than .001); AP, 292.2 +/- 107.1 and 139.5 +/- 45.3 IU/L (P less than .001); and 24 hours postoperatively for groups 1 and 2, respectively: AST, 1,664.9 +/- 917.8 and 419.3 +/- 230.9 IU/L (P less than .001): ALT. 180.4 +/- 28.9 and 66.4 +/- 17.5 IU/L (P less than .001); AP, 602.1 +/- 153.3 and 255.7 +/- 116.3 IU/L (P less than .01). Comprehensively, the results reflect a better perfusate distribution of the PAP livers compared with PP ones: uniform organ preservation, faster metabolic recovery, and reduced postoperative mortality.

Normal hepatic insulin receptor autophosphorylation in nonketotic diabetes mellitus.

Insulin receptor autophosphorylation is the earliest recognizable event in insulin action subsequent to insulin binding. To determine if the postbinding hepatic insulin resistance of nonketotic diabetes mellitus could reside in an inability of insulin to stimulate insulin receptor autophosphorylation, we evaluated the ability of insulin to stimulate 32P incorporation into the beta subunit of lectin-purified rat liver plasma membrane insulin receptors. The data indicate that both the absolute plasma membrane insulin receptor autophosphorylation in response to insulin as well as the insulin dose-response relationship for autophosphorylation are normal in diabetic animals when expressed per microgram of protein or per unit of binding activity. The previous data from our laboratory indicates that hepatic insulin resistance in non-ketotic streptozotocin-induced diabetes mellitus is present despite normal to increased insulin binding, is selective, is reversible with insulin treatment and involves an inability of insulin to stimulate the release of the putative mediator of insulin action. We conclude, therefore, that the hepatic insulin resistance of nonketotic diabetes mellitus resides distal to insulin receptor binding and autophosphorylation and is reflected in metabolic events at or near the plasma membrane which may include the generation or release of the putative mediator of insulin action.