Elevated plasma ammonia level in hepatic cirrhosis: role of glucagon.

Elevated plasma ammonia level in hepatic cirrhosis has been attributed to a lack of conversion of enteric ammonia into urea or to its entry into systemic circulation via portasystemic shunting, or to both. It is exaggerated by excessive protein intake. Because hyperglucagonemia is well documented in cirrhosis and a protein meal is an effective glucagon secretagogue, plasma glucose, insulin, glucagon, and ammonia levels were determined in 50 cirrhotic patients after an overnight fast. Effects of a protein meal were also assessed in 20 of these patients. Plasma glucose was normal and remained unaltered after a protein meal. Insulin, glucagon, and ammonia levels were elevated, but only in patients with advanced liver dysfunction. Ammonia levels correlated significantly with glucagon (r = 0.61, p less than 0.001), but not with insulin or glucose levels. Insulin and glucagon levels rose after a protein meal in all patients and controls; whereas a significant rise in the ammonia level occurred only in decompensated cirrhotics. Elevation of the ammonia level was significantly correlated with fasting glucagon (r = 0.54, p less than 0.05), as well as with glucagon response (r = 0.65, p less than 0.01), but not with basal insulin or insulin response. Furthermore, the rise in ammonia level occurred too early to be accounted for by enteric generation. Finally, direct effects of glucagon administration on plasma glucose and serum ammonia were examined in 15 cirrhotic patients. Glucose response was significantly blunted in cirrhotic patients as compared with normal subjects, whereas serum ammonia rose promptly but only in cirrhotics, with maximum rise being noted in cirrhotic patients with advanced liver dysfunction. This study, therefore, suggests that hyperglucagonemia may contribute significantly to hyperammonemia in hepatic cirrhosis.

Hyperglucagonemia in hepatic cirrhosis: its relation to hepatocellular dysfunction and normalization on recovery.

Plasma glucagon, insulin and glucose concentrations, and liver function tests were determined after an overnight fast in 24 normal subjects and 50 male cirrhotic patients. In cirrhotic patients with normal liver profiles, plasma glucagon remained within normal limits, irrespective of the presence of portasystemic anastomoses either pathological or surgical. Hyperglucagonemia was documented in presence of advanced liver dysfunction alone. Significant correlations were established between plasma glucagon and several liver function tests, i.e., serum bilirubin, albumin/globulin ratio, and prothrombin time. Moreover, hyperglucagonemia normalized on recovery from clinical manifestations and improvement in liver profile. Plasma insulin was raised primarily in the presence of a significant portasystemic shunting and maximum levels were observed in patients manifesting advanced liver dysfunction as well. However, no correlation was evident between plasma insulin and any of the liver function tests. Fasting plasma glucose was not altered in cirrhotic patients. Therefore, it is concluded that in hepatic cirrhosis, glucagon secretion by pancreatic alpha-cell may be dependent on the severity of the hepatocellular damage whereas portasystemic shunting may be responsible for hyperinsulinemia which may be further exaggerated in presence of advanced liver dysfunction.

Nutritional and metabolic effects of alcohol.

Alcohol is commonly used in almost all parts of the world, and its adverse effects on the liver, central nervous system, and other organs are widely recognized. Less well known are the deleterious actions of alcohol on nutrient metabolism. The purpose of this article is to call attention to the way in which alcohol intake influences the absorption and metabolism of carbohydrates, proteins, and lipids.

Dietary modulation of alpha-cell volume and function in strain 129/J mice.

Weanling female 129/J mice were maintained for 1, 2, 3, or 6 mo on either a control diet containing 60% sucrose and 23% protein or an isocaloric, high-protein, no-carbohydrate diet containing 83% protein and 0% sucrose. Mice were killed after each interval to assess the effect of diet on histological and physiological changes in the endocrine pancreas. Image analysis of islets stained immunocytochemically for alpha-cells, beta-cells, delta-cells, and PP cells was performed to quantify changes in islet structure. It was found that islet composition was strongly affected by diet. The volume density of the alpha-cells was significantly elevated in mice fed the high-protein diet (e.g., 35% vs. 16% in controls at 6 mo), whereas the volume density of beta-cells concomitantly decreased from 65 to 39%. Radioimmunoassay of the insulin and glucagon content of the pancreas and the plasma corroborated the morphometric findings. Pancreatic and plasma glucagon concentration in mice on the high-protein diet was elevated by an average of 2.5-fold above controls, whereas pancreatic insulin concentration was diminished by nearly half. The increase in alpha-cell volume density and pancreatic glucagon concentration appeared initially due to alpha-cell hypertrophy, although by 6 mo of high-protein feeding both hypertrophy and hyperplasia of the alpha-cells were evident. Presumably, these changes were compensatory responses to the increased functional demand on alpha-cells (i.e., glucagon biosynthesis and secretion) imposed by chronic high-protein feeding.

Portal vein blood insulin and glucagon are increased in experimental hyperthyroidism.

Decreased glucose tolerance in hyperthyroidism has been long recognized; however, the mechanism(s) responsible for altered carbohydrate metabolism have not been elucidated. Studies of insulin secretion in hyperthyroidism have been reported by several investigators; however, the results are not conclusive since hormone secretion has been reported as increased, normal, or decreased. Few investigations of glucagon secretion in hyperthyroidism have been described; thus, the role of the alpha cell hormone in the glucose intolerance associated with thyrotoxicosis has not been established. To clarify these matters, experimental hyperthyroidism was induced in male rats by injecting T4 ip for 9 days. On the 10th day, blood was collected from the portal vein for measurement of insulin and glucagon. Animals injected with T4 (500 microgram/kg . day) became clearly thyrotoxic as evidenced by elevated plasma T4 and T3 and diminished weight gain. Rats receiving 750 micrograms T4/kg demonstrated marked enhancement of pancreatic insulin and glucagon release as evidence by elevated portal vein hormone values. Plasma insulin and glucagon in T4-treated rats were substantially elevated regardless of whether the animals were fed or were fasted for 20 h before blood samples were obtained. Insulin and glucagon in vena cava blood were far lower than in the portal vein in both control and hyperthyroid rats, and there was no significant difference between the two groups. We believe that portal vein hormone concentrations more accurately reflect insulin and glucagon secretion than do peripheral blood levels because the hormones and partially degraded in the liver. It is postulated that elevated T4 stimulates the alpha cell to release excess amounts of glucagon, which enhances hepatic glucose output. The possible increase in hepatic glucose output plus insulin resistance, which is characteristic of hyperthyroidism, may account for a secondary rise in insulin release.

Dietary control of pathogenesis in C57BL/KsJ db/db diabetes mice.

Weanling C57BL/KsJ homozygous diabetic (db/db) and normal littermate (+/+ or +/db) mice were maintained for 5 mon on isocaloric diets containing either 60% sucrose, 23% casein, 8% corn oil (diet C) or 0% sucrose, 83% casein, 8% corn oil (diet B). Diabetic homozygotes consumed more diet C than normals, but ate control amounts of diet B. Diabetic mice fed diet C exhibited 57% mortality between 4 or 5 mo of age. All diabetic mutants fed the carbohydrate-free diet B appeared healthy at 6 mo of age; mutant females were normoglycemic and mutant males were only moderately hyperglycemic. Histological examination of pancreatic islets confirmed the absence of islet degeneration. In diet B maintained mutants, increased carcass fat composition, plasma and pancreatic content of insulin and glucagon, and thymidine incorporation into islets, all established that the db gene was being fully expressed. These results indicate that dietary protein stimulates islet growth and function in db/db mice, while high levels of refined carbohydrate in the diet predispose islet beta cells to undefined changes that culminate in necrosis. Restricting mutants' intake of a carbohydrate-containing diet to one-half the caloric intake of normal mice failed to block onset of beta cell necrosis. Thus, dietary composition rather than total caloric intake appears to be critical in the induction of islet necrosis and atrophy in this animal model of genetically transmitted diabetes.

Impaired pancreatic alpha-cell response in hyperthyroidism.

Recently, we observed that in hyperthyroid patients, plasma glucagon was not adequately suppressed by an oral glucose load, suggesting altered pancreatic alpha-cell sensitivity. To further assess pancreatic alpha-cell function in hyperthyroidism, plasma glucose, glucagon, and insulin resonses to a protein meal were determined in normal subjects and hyperthyroid patients. Fasting plasma glucose was normal in hyperthyroid patients. A protein meal produced an increase in plasma glucose levels in hyperthyroid patients, whereas in normal subjects protein feeding was followed by a decline in blood glucose levels. Basal glucagon was markedly elevated in three of nine hyperthyroid patients, whereas in the remaining six, fasting plasma glucagon was unaltered. In both groups, protein feeding induced a glucagon rise; however, the increment was significantly smaller in hyperthyroid patients. In hyperthyroidism, fasting plasma insulin was raised and the insulin response to a protein meal was exaggerated. Furthermore, the insulin elevations were sustained and did not return to the basal level by 180 min as observed in normal subjects. We conclude that 1) the plasma glucagon response to a protein meal is blunted in hyperthyroidism, a finding which confirms our recent observation of decreased sensitivity of the pancreatic alpha-cell in hyperthyroidism; 2) fasting hyperinsulinemia with simultaneous euglycemia is consistent with the presence of insulin resistance in hyperthyroidism; and 3) the sustained and exaggerated plasma insulin rise after ingestion of a protein meal suggests hypersensitivity of the pancreatic beta-cell in hyperthyroidism.

A new mutation (db3J) at the diabetes locus in strain 129/J mice. I. Physiological and histological characterization.

A spontaneous recessive mutation appearing in strain 129/J mice at the diabetes (db) locus on Chromosome 4 has been characterized. The new allele, designated db3J, produced hyperphagia and severe obesity. Mutants weighed in excess of 70 g by 6 months of age, compared to 22-28 g for lean littermates. Although the disease was similar to the mild hyperglycaemia-severe obesity syndrome exhibited by db gene presentation on the C57BL/6J inbred background, the syndrome in 129/J mice reduced lifespan, with mutants exhibiting sudden weight loss, hypoglycaemia, and a 67% mortality between 6 and 14 months of age. Mutant males, but not females, were transiently hyperglycaemic between 2 to 4 months of age, attaining a maximum mean blood sugar of 196 +/- 27 (SEM) mg/dl. Thereafter glucose levels declined to normoglycaemic values (80-100 mg/dl), and with increasing age, mutants of both sexes became hypoglycaemic (60 mg/dl at 9 months). Mutants of both sexes were extremely hyperinsulinaemic at the earlier ages, with mean plasma insulin at months 5 reflecting 30-fold elevations above normal for males and 18-fold for females. These levels diminished with age, the decline being more marked in males. Plasma glucagon levels were 3-fold elevated in the younger mutants of both sexes (86 pg/ml versus 28 pg/ml in normal mice), mean levels increasing to almost 5-fold above mean control vaues in the older age group (198 pg/ml versus 41 pg/ml in normal mice). Histopathological findings were limited to pancreas. Increasing necrosis of the exocrine, but not endocrine, pancreas was noted in aging mutants. Aldehyde fushsin staining of the mutant pancreas revealed hyperplastic islets filled with heavily granulated B-cells. B-cell hyperplasia was accompanied by a 30-fold increase over controls in pancreatic insulin content in the 8 month old mutants, whereas pancreatic glucagon content was only doubled. Morphometric analysis showed less than a 2-fold increase in the mean number of A-cells per islet. Thus, an interesting feature of expression of the diabetes gene in the 129/J strain is the persisting hyperglucagonaemia in the face of moderating hyperinsulinaemia.

A new mutation (db3J) at the diabetes locus in strain 129/J mice. II. Studies of pancreatic alpha cell function in culture.

Monolayer cell cultures from pancreatic islets of aging 129/J strain diabetes (db3J/db3J) and lean littermate control mice were tested for differences in glucagon and insulin secretion in either serum-free Eagle's minimal essential medium (MEM) or Dulbecco's modified minimal essential medium (DMEM). There was a highly significant (p less than 0.0001) main effect of genotype and type of culture medium on glucagon secretion with time. Thus, although numbers of A-cells were not demonstrably increased in db3J/db3J cultures in DMEM, mean medium glucagon levels increased 2.7-, 18-, and 32-fold above littermate normal culture levels at days 4, 6, and 8 respectively. In MEM, the two populations could not be discriminated on the basis of glucagon secretion. By contrast, insulin secretion over culture days showed a highly significant (p less than 0.0001) dependence on genotype, but not type of medium, with the B-cell enriched db3J/db3J preparations secreting between 20 and 30 times as much insulin as controls in both medida. Analysis revealed that the heightened secretory responsiveness of mutatn A-cells in DMEM as compared to MEM was primarily elicited by the elevated DMEM amino acid concentration and specifically lysine (0.8 mmol/l in DMEM versus 0.4 mmol/l in MEM). In pulse-chase experiments using 14 day db3J/db3J cultures, incorporation of 3H-tryptophan into protein that eluted from Biogel P-10 columns in the native glucagon peak indicates that DMEM stimulated glucagon biosynthesis as well as secretion. This study reveals an augmented sensitivity of db3J/db3J A-cells to stimulation by basic amino acids in long-term culture.

Increased glucagon secretion in protein-fed rats: lack of relationship to plasma amino acids.

This investigation was designed to test the hypothesis that protein feeding stimulated glucagon secretion because amino acids liberated during protein digestion function as glucagon secretagogues. Rats were fed high-protein (HP) or control diets for 9--10 days and blood taken from the aorta or portal vein (PV) at 0800, 1300, 1700, 1900, 2100, and 2300 for determination of amino acids, glucose, insulin, and glucagon. Glucose, insulin, and glucagon of control rats showed little change. In HP rats, PV glucose rose during fasting (0800-1700) and declined during feeding (1700-0800), changes that reflected alterations of glucagon and insulin secretion. PV glucagon in HP rats that was elevated 2--4 times rose during fasting, whereas PV and arterial amino acids declined. HP feeding caused enhanced glucagon release that was associated with increased amino acids in PV and arterial plasma, especially the branched-chain group. Although these findings suggest that protein feeding promotes glucagon release because branched-chain amino acids are elevated, these amino acids are known to have little effect on alpha cell function. Thus, we conclude that protein feeding influences glucagon secretion through some mechanism other than increased blood amino acid levels.

Decreased plasma insulin but normal glucagon in rats fed low protein diets.

Plasma glucagon and insulin were determined in rats fed three diets, one control and two low protein (LP 1 & LP 2). In LP 1, the protein omitted was replaced by carbohydrate while in LP 2, fat and alpha-cellulose replaced the omitted protein. Among rats fed LP diets ad libitum, food consumption decreased and body weight loss occurred. In order to separate the effects of reduced food intake and weight loss from the effects of LP diet alone, paired feeding and paired weight experiments were conducted. In another experiment, ingestion of a LP diet for 8 to 10 days was followed by refeeding the control diet for 5 days. The results demonstrate that plasma insulin was reduced in LP rats compared to the full-fed controls and pair-fed controls, the lowest levels being observed in rats fed LP 2 diet. Furthermore, the paired feeding experiment revealed that the diminished food consumption plays no significant role in lowering plasma insulin in LP rats. The refeeding experiment showed that the decline in plasma insulin in LP rats is a transient phenomenon and the plasma insulin promptly reverts to normal upon resumption of feeding the control diet. Plasma glucagon was unaltered throughout these dietary manipulations.

Glucagon, insulin, and gluconeogenesis in fasted odd carbon fatty acid-enriched rats.

Forty-eight male rats were fed a nutritionally complete diet containing 30% of dietary energy as fat. For 24 animals (control) the fat source was corn oil, for the remaining 24 rats (experimental) the fat source was a triundecanoin-corn oil mixture (7:3, wt/wt). After 6 wk, groups of control and experimental rats were killed after 0, 24, and 48 h of fasting. In the experimental group, adipose tissue fatty acids contained, on average, 280 mmol undecanoate/mol fatty acid. In the control group, no odd-numbered fatty acids were present. During fasting, the experimental groups had higher plasma glucose and alanine levels, higher plasma insulin-to-glucagon ration, and lower liver phosphenol pyruvate caboxykinase. The results suggest that the terminal propionate residues generated when odd carbon fatty acids are oxidized become gluconeogenic precursors and cause a reduced need for gluconeogenesis from protein.

A nonsuppressible increase of glucagon secretion by isolated islets of high-protein-fed rats.

We recently demonstrated increased plasma glucagon but nomal insulin in rats fed a high-protein, carbohydrate-free (HP) diet; however, other investigators have reported that both plasma glucagon and insulin are increased after protein feeding. For this reason, we have investigated the ffects of an HP diet on pancreatic secretion of insulin and glucagon. Male rats were fed an HP or control diet for one, three, or five days, and, at the end of the feeding period, blood was taken for glucose, insulin, and glucagon determinations. Additional animals fed the HP and control diets for up to 10 days were sacrificed, the pancreases removed, and islets of Langerhans isolated. Islets were incubated for 30 minutes in media with glucose concentration of1.7, 8.3, 16.7, or 33.4 mM. Insulin and glucagon secreted into the media were determined by radioimmunoassay. Plasma insulin was markedly reduced after one day of HP feeding but gradually returned to normal by the fifth day. Plasma glucagon was not altered on day 1 but was significantly increased after three days of HP feeding. The I/G molar ratio, which declined precipitously on day 1, increased thereafter but, as shown previously, remained at a level that promotes gluconeogenesis for up to 10 days. Insulin secretion by isolated islets of control and HP rats increased more than 10-fold as medium glucose was raised from 1.7 to 16.7 mM. There was no difference in insulin release by the two groups of islets. Glucagon secretion by HP islest at low medium glucose remained normal during the first five days; however, beginning on day 3 there was gradual loss of the suppressive effect of high medium glucose on glucagon secretion. After one week of HP feeding, glucagon secretion at low medium glucose was doubled and there was complete lack of suppression of the elevated hormone production by high medium glucose. The alterations of alpha-cell function induced by HP feeding are similar to those found in human and experimental diabetes.