Depressed liver regeneration after partial hepatectomy of germ-free, athymic and lipopolysaccharide-resistant mice.

A hypothesis has been proposed by this laboratory that endogenous gut-derived lipopolysaccharide is responsible for systemic endotoxemia in animals with acute liver injury particularly after partial (67%) hepatectomy. Systemic lipopolysaccharide and possibly fibrin aggregates or tissue debris then elicit release of cytokines from phagocytizing macrophages and/or monocytes that may be essential for normal liver regeneration. To test this hypothesis liver regeneration was assessed in germ-free euthymic mice that lack the gram-negative bacterial source of lipopolysaccharide, as well as being deficient in lymphoid tissue and relatively resistant to endotoxin. To complement the germ-free animals, conventional athymic nude BALB/c mice and conventional lipopolysaccharide-resistant C3H/HeJ mice were also examined. Liver regeneration, quantified by [3H] thymidine incorporation into hepatic DNA after partial hepatectomy was performed on mice anesthetized with ether, was significantly depressed in germ-free euthymic and conventional athymic BALB/c mice and delayed in conventional lipopolysaccharide-resistant C3H/HeJ mice, as compared with conventional control BALB/c and C3H/HeN animals. Pretreatment of conventional euthymic control mice with lipopolysaccharide 24 hr before surgery significantly stimulated hepatic DNA synthesis after 67% liver resection. Germ-free euthymic, conventional athymic, and conventional lipopolysaccharide-resistant mice pretreated with endotoxin did not manifest significant stimulation of liver regeneration. Evidence is reviewed that cytokine release in response to endotoxin was depressed in germ-free euthymic, conventional athymic, and conventional lipopolysaccharide-resistant mice as compared with conventional euthymic controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute phase responses after acute liver injury by partial hepatectomy in rats as indicators of cytokine release.

The purpose of this study was to support the hypothesis that cytokines such as interleukin-1, tumor necrosis factor and interleukin-6 are released by macrophages or monocytes within 1 to 2 hr of phagocytosis of circulating, gut-derived bacterial lipopolysaccharide translocated by acute liver injury. Time courses of fever, neutrophilia and low blood-zinc levels generally attributed to cytokines were quantified after partial (67%) hepatectomy of rats under ether anesthesia. These acute phase responses in hepatectomized rats were compared with those after intravenous injection of exogenous endotoxin and human natural interleukin-1. Fever commenced 30 min after interleukin-1 injection, 4 hr after exogenous lipopolysaccharide injection and 6 hr after 67% liver resection. Similarly, rectal temperatures were significantly elevated in recipient rats 30 min after intravenous administration of donor plasma from hepatectomized animals, indicating that cytokines, not lipopolysaccharide, elicited the febrile response. Neutrophilia was present 1, 2, and 4 hr after interleukin-1 injection, lipopolysaccharide injection and hepatectomy, respectively. Furthermore, the reduction in plasma zinc, which depends on cellular metallothionein synthesis, occurred 4 hr after interleukin-1 administration and 6 hr after lipopolysaccharide injection or partial hepatectomy. Donor plasma from hepatectomized rats also elicited neutrophilia at 1 hr and low blood-zinc levels 4 hr after injection in recipient animals. The timing of these responses, just as for the fever, implies that cytokines and not lipopolysaccharide in the donated plasma elicited the neutrophilia and hypozincemia. Evidence was reviewed that interleukin-1, tumor necrosis factor and interleukin-6 function as hepatotrophic factors and have been identified in the circulation of humans with liver damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Central interleukin 1-elicited hyperinsulinemia is mediated by prostaglandins but not autonomics.

This laboratory previously reported that centrally administered interleukin 1 (IL-1) in fasted pentobarbital-anesthetized rats elicited significant hyperinsulinemic and febrile responses. In characterizing this putative central mechanism for the regulation of pancreatic insulin secretion, hyperinsulinemia and fever elicited by IL-1 injected intravenously (iv) or intracerebroventricularly (icv) was totally eliminated by prior cyclooxygenase inhibition with indomethacin, ibuprofen, or meclofenamate but not lipoxygenase inhibition with propyl gallate or leukotriene receptor antagonism with LY 171883. Furthermore, central administration of prostaglandin E2 at 10 and 100 ng doses consistently evoked hyperinsulinemic, hypercorticotropinemic, and febrile responses in anesthetized rats maintained on isothermal pads. beta-Adrenergic and vagus nerves to the pancreatic beta-cells seemed likely candidates to mediate the enhanced secretion of insulin elicited by IL-1 acting centrally. However, pretreatment of rats with hexamethonium, propanolol, atropine, or bilateral subdiaphragmatic vagotomy all failed to reduce hyperinsulinemia after IL-1 iv or icv. This evidence suggests that the central mechanism for enhanced pancreatic insulin secretion elicited by IL-1 may depend on a humoral rather than autonomic neural efferent pathway. Moreover, the hyperinsulinemia is mediated in part by prostaglandins just like the well-studied febrile response.

Hyperinsulinemia elicited by interleukin-1 and nonlethal endotoxemia in rats.

Nonlethal endotoxemia was produced in conscious fasted rats by the intravenous (i.v.) administration of Salmonella enteritidis lipopolysaccharide (LPS) at a dose of 30 micrograms/100 g together with the typical acute-phase response of fever at 4 hr post-LPS. Also at 4 hr post-LPS both hyperinsulinemia and hyperglucagonemia were manifested, the (insulin:glucagon) (I:G) molar ratio was not different from saline control animals, and normoglycemia was maintained. The monokine interleukin-1 (IL-1), which is synthesized de novo and then released by macrophages and monocytes following LPS phagocytosis, has been implicated in the typical responses to endotoxemia. Therefore, human natural IL-1 was injected i.v. at a dose of 50 U into conscious fasted rats. IL-1-induced fever occurred at 30 min postinjection. Hyperinsulinemia equal to two times the saline control value was also present at 30 min after monokine injection, with plasma insulin levels declining to below control values by 60 min and remaining depressed for up to 12 hr. In contrast, plasma glucagon concentrations were not significantly altered at any time between 15 min and 12 hr post-IL-1. Despite IL-1-elicited hyperinsulinemia with unchanged glucagon, which elevated the I:G molar ratio, normoglycemia was maintained after monokine administration. The coincident onset of fever and hyperinsulinemia at 30 min after i.v. administration of IL-1 suggests a common mediator for both responses.

Central administration of interleukin 1 elicits hyperinsulinemia in rats.

This laboratory previously demonstrated that human natural interleukin 1 (IL-1) administered intravenously at a dose of 50 U to conscious fasted male Holtzman rats elicited unstimulated as well as glucose- and arginine-stimulated hyperinsulinemia. The major question posed in the present investigation is whether the monokine IL-1 can act centrally on an area of the brain such as the preoptic area of the anterior hypothalamus, which participates in mediating the IL-1-induced febrile response, to elicit insulin secretion by the pancreas. Therefore, for this study, injections of IL-1 were administered intravenously (iv), intracerebroventricularly (icv), or into the preoptic area of the anterior hypothalamus (iPO) in pentobarbital-anesthetized fasted rats. Preliminary experiments determined that the hyperinsulinemic response was enhanced in anesthetized compared with conscious animals treated iv with IL-1. An icv administered 10-U dose of the monokine produced a delayed hyperinsulinemia equal to two times the saline control value starting near 20 min postinjection but declining quickly. When injected iPO, 5 U of IL-1 elicited a prompt substantial hyperinsulinemia equal to more than three times control beginning at 5 min postinjection and persisting through the 30-min experimental period. These findings indicate that an IL-1-induced signal for pancreatic insulin secretion can be generated within the central nervous system.

Endogenous gut-derived bacterial endotoxin tonically primes pancreatic secretion of insulin in normal rats.

This laboratory has proposed that endogenous gut-derived bacterial endotoxin primes the pancreatic secretion of insulin in normal rats. Endogenous lipopolysaccharide (LPS) is continually absorbed from the gut into intestinal capillaries, and low-grade portal venous endotoxemia is the status quo. Under physiologic conditions, Kupffer cells of the liver totally phagocytize and degrade endotoxin from the portal circulation. Evidence from this and other laboratories indicates that administration of exogenous LPS to humans and rats enhances pancreatic secretion of both insulin and glucagon. Conversely, findings of the present study demonstrate that restriction of endogenous LPS in fasted rats depresses the basal and arginine-stimulated concentrations of plasma insulin. Techniques used to restrict gut-derived LPS availability included chronic daily gavage with neomycin and cefazolin for gut sterilization and with cholestyramine or lactulose to reduce endotoxin within the gut. In addition, induction of endotoxin tolerance was produced by progressively higher doses of LPS intraperitoneally (i.p.), and polymyxin B was administered subcutaneously (s.c.) daily to neutralize the lipid A portion of circulating LPS. Finally, isolator-reared, defined flora rats, which were gram-negative-bacteria-deficient, and, therefore, LPS-deficient, were compared with conventional counterparts. Basal plasma insulin but not glucagon levels were consistently and significantly reduced in endogenous LPS-restricted animals. Glucose-stimulated plasma insulin was decreased only after parenteral treatment by tolerance induction and polymyxin B administration. Both plasma insulin and glucagon were depressed in response to arginine challenge in most LPS-restricted rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Gut-derived endotoxin elicits hepatotrophic factor secretion for liver regeneration.

The influence of exogenous endotoxin pretreatment on liver regeneration after partial hepatectomy was evaluated. Partial hepatectomy was performed by 67% liver resection of ether-anesthetized rats with midline laparotomy and liver manipulation as the sham control. Animals were pretreated with endotoxin at a dose of 33 micrograms/100 g sc or iv 24 h before surgery and then fasted. Liver regeneration was quantified after partial hepatectomy by [3H]thymidine incorporation into hepatic DNA, and plasma levels of hepatotrophic factors were measured by radioimmunoassay or radioreceptor assay. Systemic endotoxemia occurred after exogenous endotoxin administration as well as after partial hepatectomy due to absorption of exogenous endotoxin from the gut into the portal circulation as determined by quantitative chromogenic lysate assay of perchloric acid-extracted plasma samples. Alterations in putative hepatotrophic factors, including insulin, glucagon, epidermal growth factor, vasopressin, and triiodothyronine, were remarkable similar in response to endotoxemia by exogenous endotoxin administration and by endogenous endotoxin absorption from the gut after partial hepatectomy. Our hypothesis purports that gut-derived systemic endotoxemia elicits hepatotrophic factor secretion for liver regeneration after partial hepatectomy and that endotoxin pretreatment expedites the hepatotrophic factor response, thus accelerating DNA synthesis in the proliferating liver after 67% resection.

Restriction of gut-derived endotoxin impairs DNA synthesis for liver regeneration.

The influence of restricting gut-derived endotoxin availability on liver regeneration after partial hepatectomy was evaluated. Partial hepatectomy was performed by 67% liver resection of ether-anesthetized rats. Liver regeneration was quantified after partial hepatectomy by [3H]thymidine incorporation into hepatic DNA; endotoxemia due to absorption of endogenous endotoxin from the gut into the portal circulation was determined by qualitative lysate assay of perchloric acid-extracted plasma samples, and plasma levels of the hepatotrophic factors insulin and glucagon were measured by radioimmunoassay. Treatments to restrict gut-derived endotoxin included chronic gavage with neomycin and cefazolin for gut sterilization, chronic gavage with cholestyramine to bind endotoxin within the gut, subcutaneous administration of polymyxin B to neutralize the lipid A portion of circulating endotoxin, intraperitoneal induction of endotoxin tolerance by progressively higher doses of endotoxin, and experimentation with isolator-reared defined flora Fisher rats that were Gram-negative bacteria deficient and therefore endotoxin deficient. All treatments to restrict endogenous endotoxin impaired DNA synthesis in regenerating livers particularly 21 h posthepatectomy when replication was increasing most rapidly in normal rats. We hypothesize that impairment of DNA synthesis after partial hepatectomy in endotoxin-restricted animals was due to the observed lack of normal systemic endotoxemic as well as hyperinsulinemic and hyperglucagonemic responses to 67% liver resection.

Role of the liver in endotoxin-induced hyperinsulinemia and hyperglucagonemia in rats.

The intravenous administration of bacterial endotoxin to fasted rats elicited basal portal and systemic venous hyperinsulinemia and hyperglucagonemia. Enhanced pancreatic secretion of insulin and glucagon was implied by the elevated portal venous hormonal levels. Elevated insulin and glucagon levels were present at 4 hr after a 33 micrograms/100 gm intravenous endotoxin dose despite no fluctuation of the plasma glucose concentration. The role of the liver in the pancreatic hormonal response to endotoxin was investigated by infusing lipopolysaccharide slowly into the portal vein or systemic inferior vena cava. At doses of 33 and 100 micrograms per 100 gm, endotoxin administered via the systemic route stimulated significantly greater insulin and glucagon responses than did portal administration. Furthermore, rats with acute liver injury induced by partial (67%) hepatectomy, which depressed Kupffer cell phagocytosis, did respond to the 33 micrograms per 100 gm intraportal endotoxin dose with significantly greater hyperinsulinemia and hyperglucagonemia. These data suggest that hepatic Kupffer cells normally function to remove lipopolysaccharide from the portal venous blood and that at least at low pharmacological doses the pancreatic hormonal response to endotoxin is mediated by an unknown systemic mechanism.

Modulation of hepatic reticuloendothelial system phagocytosis by pancreatic hormones.

Experiments were conducted to determine the influence of the pancreatic hormones insulin, glucagon, and somatostatin on reticuloendothelial system (RES) phagocytosis both in vivo and in the isolated perfused livers of rats. Chronic pancreatic hormonal treatment consisted of twice daily injections SC of NPH insulin with doses ranging from 0.75 U on day 1 to 9.0 U on day 13 and unchanged doses of glucagon (200 micrograms) and somatostatin (50 micrograms). Chronic treatment with insulin significantly depressed by 48% intravascular phagocytosis of colloidal carbon administered IV at a dose of 8 mg/100 g, while glucagon and somatostatin stimulated macrophage endocytic function by 32% and 26%, respectively, compared to the control value. Acute treatment with the three pancreatic hormones at 30 min prior to carbon administration similarly produced insulin depression as well as glucagon and somatostatin stimulation of RES phagocytosis. Addition of the three hormones at near physiologic concentrations (20 ng/ml for insulin, 10 ng/ml for glucagon, and 5 ng/ml for somatostatin) to the recirculating perfusate of isolated perfused rat livers simultaneous with 24 mg of colloidal carbon likewise resulted in phagocytic reduction after insulin and enhancement after glucagon and somatostatin. Experiments involving insulin in vitro with isolated perfused livers as well as glucose replacement therapy concomitant with insulin in vivo demonstrated that hypoglycemia is not necessary for phagocytic depression by insulin while severe hypoglycemia in the perfusion medium is sufficient to depress carbon uptake by isolated perfused livers independent of insulin. Both pancreatic hormones and the level of glycemia seem to be important in modulating hepatic reticuloendothelial system phagocytosis.

Reticuloendothelial hyperphagocytosis occurs in streptozotocin-diabetic rats. Studies with colloidal carbon, albumin microaggregates, and soluble fibrin monomers.

In contrast to previous studies of diabetic humans and animals, which reported unchanged or depressed function, reticuloendothelial system (RES) hyperphagocytosis of colloidal carbon, 125I-albumin microaggregates, and 125I-fibrin monomers were observed in rats as early as 14 days after the induction of diabetes with streptozotocin (STZ). The fact that enhanced phagocytosis by RE macrophages was prevented by chronic insulin replacement therapy indicates that the diabetic internal environment of hyperglycemia and hypoinsulinemia was perhaps responsible for the observed changes. Experiments involving organ localization of intravenously administered particles, perfusion of isolated livers, and microscopic examination of the liver all suggested that increased Kupffer cell activity was the primary event in RES hyperphagocytosis by STZ-diabetic rats. Both hypertrophy and hyperplasia of Kupffer cells were apparent in livers of STZ-diabetic animals as evidenced by photomicrographs and hepatic cell quantification. Plasma fibronectin, which binds fibrin monomers to RE macrophages before phagocytosis, was significantly decreased in the circulation of STZ-diabetic rats, but the level of cell-associated fibronectin was not measured. Renal localization of urea-soluble 125I-fibrin monomers exceeded splenic and pulmonary uptake in normal control rats and was enhanced in animals with STZ-diabetes. Changes in fibronectin levels, fibrin monomer localization, and Kupffer cell size and numbers in experimental diabetes in rats may have implications for the pathogenesis of vascular disease involving phagocytic mesangial and foam cells in diabetic humans.

Endotoxin-induced hyperinsulinemia and hyperglucagonemia after experimental liver injury.

Basal portal and systemic venous hyperinsulinemia and hyperglucagonemia were present in fasted rats at 24 h after experimental liver injury by partial (67%) hepatectomy, carbon tetrachloride gavage, and intravenous D-galactosamine administration. Both enhanced pancreatic hormone secretion and depressed hepatic hormone extraction were likely responsible for the insulin and glucagon oversupply. Endogenous gut-derived endotoxin is proposed as the causative factor for the exaggerated hormonal response because intravenous exogenous endotoxin elicited an identical elevation of insulin and glucagon. Systemic endotoxemia at 24 h after liver injury was indicated by marked (78-100%) lethality in lead-sensitized rats and positive Limulus lysate gelation tests of plasma samples. Furthermore, antiendotoxin treatments, including endotoxin tolerance, polymyxin B, and gut sterilization, significantly reduced both lead-sensitized lethality and hyperinsulinemic and hyperglucagonemic responses at 24 h in most liver-injury groups. Portal versus systemic venous administration of endotoxin at a low dose implied that normal endotoxin phagocytosis by the liver suppressed the pancreatic endocrine response. A physiological negative-feedback control system involving gut-derived systemic endotoxemia after liver damage with insulin and glucagon hypersecretion by the pancreas for stimulation of hepatic regeneration is hypothesized.

Hepatocyte and Kupffer cell functions during liver regeneration in streptozotocin-diabetic rats.

The insulinoprivic influence of acute severe streptozotocin diabetes on liver regeneration in rats was evaluated by determining liver weights as well as hepatocyte and Kupffer cell functional capacities. Functional capacities were assessed by bromosulfophthalein uptake for hepatocytes and carbon phagocytosis for Kupffer cells. Evaluation immediately after partial hepatectomy revealed a 66% reduction of liver mass, a 63% decrease in hepatocyte bromosulfophthalein removal, and a 65% decline in Kupffer cell carbon phagocytosis. Per cent recovery at 48-hr posthepatectomy was considerably greater for carbon phagocytosis than for bromosulfophthalein removal by regenerating livers. This apparent difference in functional recovery was likely due in part to enhanced non-Kupffer cell carbon phagocytosis. No significant differences of the three regeneration indices were noted for untreated streptozotocin-diabetic rats compared to nondiabetic animals. However, insulin administration to fasted streptozotocin diabetics significantly stimulated liver regeneration above that of untreated fasted rats and almost equivalent to that of pair-fed animals. Fasted rats had in general slower liver regeneration than pair-fed animals as expected. Furthermore, insulin administration to fasted nondiabetic rats after partial hepatectomy caused severe hypoglycemia and resulted in a further depression of liver regeneration.

RES hyperphagocytosis by rats with streptozotocin-induced diabetes mellitus.

In contrast to previous studies of neutrophils from diabetic animals and humans in vitro and of macrophages from diabetic humans in vivo, which reported phagocytic depression, reticuloendothelial system (RES) hyperphagocytosis of colloidal carbon was observed in rats at 14 and 28 days after diabetes induction with streptozotocin (STZ). Carbon clearance half times were significantly enhanced to 6.3 +/- 0.79 and 8.1 +/- 1.04 min at 14 and 28 days post-STZ, respectively, compared with the nondiabetic value (12.7 +/- 0.98 min). The severity of uncontrolled STZ-induced diabetes in rats was confirmed by significant hypoinsulinemia, hyperglucagonemia, hyperglycemia, and hyperlipidemia. Although body weights of STZ-diabetic animals declined progressively, liver weights as a percent of body weight increased above the control value at 14 and 28 days post-STZ. In fact, expression of carbon phagocytosis as the corrected phagocytic index, which accounts for changes in liver and spleen weights relative to body weight, eliminated the significant difference between STZ-diabetic and nondiabetic animals. Antibiotic treatment of diabetic rats failed to alter the hyperphagocytosis, implying that a chronic bacterial infection was not the cause of phagocytic stimulation. Daily insulin replacements, but not a single large insulin dose to 14-day post-STZ rats, reversed the enhanced phagocytosis of colloidal carbon.

Hyperinsulinemia and hyperglucagonemia in fasted rats during liver regeneration.

The two pancreatic hormones, insulin and glucagon, have been implicated as hepatotrophic factors for the regenerating liver after partial hepatectomy. Similar to previous studies, hyperglucagonemia and hypoinsulinemia were noted following major liver resection of initially well-fed rats. In contrast to well-fed animals, rats fasted for 24 h prior to surgery to deplete hepatic glycogen and establish low basal insulin levels developed both portal venous hyperglucagonemia and hyperinsulinemia after 67% hepatectomy. Elevation of basal portal venous levels of the two hormones suggested enhanced pancreatic secretion of insulin and glucagon in response to acute liver injury in fasted animals. Because polypeptide hormones must bind to specific hepatic cell membrane receptors to exert their hepatotrophic influences, basal portal-systemic venous differences of insulin and glucagon and glucose-elicited portal-hepatic venous insulin differences were evaluated. The evidence implies that hepatic extraction and, therefore, cell membrane receptor binding of both insulin and glucagon by regenerating livers were enhanced even though portal venous levels of the two hormones were elevated.

Mechanisms of acute hyperinsulinemia after Kupffer cell phagocytosis.

Blockade of hepatic Kupffer cells by prior phagocytosis of a variety of particulate materials caused acute hyperinsulinemia in glucose-stimulated fasted rats under pentobarbital anesthesia. At 4-h postblockade a 125-250% increase in peripheral plasma insulin levels occurred due to a combination of enhanced pancreatic insulin secretion and depressed hepatic insulin extraction. Enhanced pancreatic insulin secretion was confirmed by a 36-54% elevation of portal venous insulin levels. Depressed hepatic insulin extraction was indicated by a 37-47% reduction in insulin uptake by in situ perfused livers as well as alterations in portal-hepatic venous insulin differences and intravenous insulin tolerance tests in vivo. All parameters began to return toward control values at 24 and 48 h postblockade. Return was slow after inert carbon phagocytosis and rapid after degradable bacteria phagocytosis. Peripheral plasma insulin levels were very highly correlated with glucose clearance rates in all groups both control and experimental. Mechanisms are proposed to explain these findings based on the release of lysosomal enzymes and endogenous pyrogens by phagocytizing Kupffer cells as well as the presence of insulin receptors on hepatocytes and Kupffer cells.

Degradation of particulate lipid by the large-granule fraction of rat liver (38555).

Radiolabeled particulate lipid administered intravenously was cleared from the circulation and localized primarily with in the liver. The phagocytized 131I-triglyceride was degraded with the subsequent release of free 131I-triglyceride emulsion in vitro with different hepatic subcellular fractions revealed that the lysosome rich large-granule fraction contained the highest specific deiodinative activity. This observation coupled with the findings on latency of activation and an acid findings on latency of activation and an acid pH optimum, support the concept that the lysosomes are critically involved in the degradation of the particulate phagocytized lipid in the liver.