Tissue-specific regulation of mitochondrial and cytoplasmic protein synthesis rates by insulin.

In vivo studies have reported conflicting effects of insulin on mixed tissue protein synthesis rates. To test the hypothesis that insulin has differential effects on synthesis rates of various protein fractions in different organs, we infused miniature swine (n = 8 per group) with saline, insulin alone (at 0.7 mU/kg(-1). min(-1)), or insulin plus an amino acid mixture for 8 h. Fractional synthesis rate (FSR) of mitochondrial and cytoplasmic proteins in liver, heart, and skeletal muscle, as well as myosin heavy chain (MHC) in muscle, were measured using L-[1-(13)C]leucine as a tracer. The FSR of mitochondrial and cytoplasmic proteins were highest in liver, followed by heart and then muscle. Mitochondrial FSR in muscle was higher during insulin and insulin plus amino acid infusions than during saline. Insulin had no significant effect on FSR of MHC in muscle. In contrast, FSR of both mitochondrial and cytoplasmic proteins were not stimulated by insulin in liver. Insulin also did not increase FSR of mitochondrial in heart, whereas insulin and amino acid stimulated FSR of cytoplasmic protein. In conclusion, insulin stimulates the synthesis of muscle mitochondrial proteins, with no significant stimulatory effect on synthesis of sarcoplasmic and MHC. These results demonstrate that insulin has different effects on synthesis rates of specific protein fractions in the liver, heart, and skeletal muscle.

Insulin's effect on synthesis rates of liver proteins. A swine model comparing various precursors of protein synthesis.

Insulin's effect on the synthesis of liver proteins remains to be fully defined. Previous studies using various surrogate measures of amino acyl-tRNA have reported variable results of insulin's effect on liver protein synthesis. We determined the effect of insulin with or without amino acid supplementation on the synthesis rates of liver proteins (tissue, albumin, and fibrinogen) using L-[1-13C]Leu as a tracer in 24 male miniature swine. In addition, we compared the isotopic enrichment of different precursors of liver proteins with that of amino acyl-tRNA using L-[1-13C]Leu and L-[15N]Phe as tracers. Although liver tissue fluid enrichment of [13C]Leu and [15N]Phe and that of plasma [13C]ketoisocaproatic acid (KIC) were very similar to that of tRNA, plasma isotopic enrichment of both Leu and Phe were substantially higher (P < 0.01) and VLDL apolipoprotein-B100 enrichment was lower (P < 0.01) than the respective amino acyl-tRNA enrichment. Plasma KIC enrichment most accurately predicted leucyl-tRNA enrichment, whereas plasma Leu enrichment was best correlated with that of tRNA. Neither insulin alone nor insulin plus amino acid infusion had an effect on liver tissue protein synthesis. In contrast, insulin alone decreased the albumin synthesis rate, and insulin with amino acids maintained the albumin synthesis rate. Insulin with or without amino acids inhibited the fibrinogen synthesis rate. These results, based on synthetic rates using amino acyl-tRNA, were consistent with those obtained using KIC or tissue fluid Leu or Phe as precursor pools. These studies demonstrated that plasma KIC enrichment is a convenient and reliable surrogate measure of leucyl-tRNA in liver. We also concluded that insulin has differential effects on the synthesis rates of liver proteins. Whereas insulin with or without amino acid supplement has no acute effect on the synthesis of liver tissue protein, insulin has a substantial inhibitory effect on fibrinogen synthesis. In contrast, insulin administration along with amino supplement is necessary to maintain albumin synthesis rate.

The effect of insulin on human small intestinal mucosal protein synthesis.

BACKGROUND & AIMS: Insulin deficiency was recently shown to stimulate splanchnic protein synthesis in vivo, whereas insulin enhances small intestinal mucosal cell proliferation in vitro. Because insulin is a postprandial hormone, it was hypothesized that it has an important role in regulating small intestinal protein synthesis in humans. METHODS: Small intestinal mucosal protein synthesis was measured in C-peptide-negative patients with type 1 diabetes mellitus during insulin deprivation (n = 6) and during insulin treatment (n = 6) and in nondiabetic control subjects (n = 6). Mucosal protein synthesis was measured from the increment of [(13)C]leucine enrichment in endoscopically obtained duodenal mucosa samples during a primed continuous infusion of L-[1-(13)C]leucine. RESULTS: During insulin treatment, the rate of mucosal protein synthesis in patients with type 1 diabetes was similar (1.32% +/- 0.05%/h) to that of nondiabetic controls (1.33% +/- 0.06%/h). However, during insulin deprivation, the mucosal protein synthesis rate in patients with type 1 diabetes was significantly lower (1.15% +/- 0.33%/h) than during either insulin treatment (P = 0.01) or in nondiabetic controls (P = 0.04). CONCLUSIONS: These studies show that insulin is required for the maintenance of normal rates of protein synthesis in small intestinal mucosa. Because protein synthesis is an essential component of the remodeling process of this fast turning over tissue, the decline in the synthesis rate of small intestinal mucosa during insulin deprivation may be a contributing factor in the development of gastrointestinal complications that occur in poorly controlled type 1 diabetic patients.

The concentrations of free amino acids in human liver tissue obtained during laparoscopic surgery.

The concentrations of the free amino acids in individual tissues gives information concerning amino acid, energy and protein metabolism. In muscle and intestinal mucosa, different metabolic states are distinctly characterized by an altered free amino acid pattern. Furthermore, the patterns are quite different in individual tissues. So far, liver tissue has not been investigated systematically in this respect. The aims of this investigation were to establish a standardized sampling procedure for liver tissue during laparoscopic surgery and to characterize the free amino acid concentrations in human liver tissue. Aspartate was the most abundant amino acid in the liver, followed by taurine, glutamine, glutamate, glycine and alanine. These six, and most abundant, amino acids constitute 90% of the total hepatic amino acid concentration. In the future, liver tissue sampling during laparoscopic surgery may be used as a model for investigating the influence of nutrition and hormones on hepatic amino acid and protein metabolism in man.

Elective abdominal operations alter the free amino acid content of the human intestinal mucosa.

OBJECTIVE: To assess the impact of a standard moderately severe surgical operation on the mucosal amino acid content of the duodenum and the colon. DESIGN: Open study. SETTING: University hospital, Sweden. SUBJECTS: Nine patients who were to undergo elective open cholecystectomy. INTERVENTIONS: Endoscopically obtained biopsy specimens from the intestinal mucosa. MAIN OUTCOME MEASURES: Changes in the content of free amino acids in the duodenum and colon at three days postoperatively. RESULTS: The concentration of glutamine in the duodenum increased by 27% and that of glutamic acid by 34% after operation, whereas their content in colon remained unaltered. The concentration of branched chain amino acids increased by 26% in the duodenal mucosa after operation and by 24% in the colonic mucosa. The total concentration of amino acids (excluding taurine) increased by 9% in the duodenum, but remained unaltered in the colon. CONCLUSION: This study shows characteristic and consistent alterations in the free amino acid content of the intestinal tract after a moderately severe operation.

Intestinal amino acid content in critically ill patients.

BACKGROUND: The purpose of the study was to determine the concentrations of free amino acids and the total protein content of the human intestinal mucosa during critical illness. METHODS: The free amino acid and protein concentrations in endoscopically obtained biopsy specimens from the duodenum and the distal colonic segments were determined on 19 critically ill patients. The free amino acids were separated by ion exchange chromatography and detected by fluorescence, and the protein content was quantified by the method of Lowry. RESULTS: In general, the typical amino acid pattern of the intestinal mucosa was seen, with very high levels of taurine, aspartate and glutamic acid. The main difference, as compared to a reference series of healthy subjects, was the elevated glutamine concentration of the duodenal mucosa. This amino acid was unaltered in the descending colon and depressed in the rectum. At the same time, the glutamatic acid concentrations were unaltered, suggesting that the degradation of glutamine was not increased in the septic state of the majority of the patients studied. Phenylalanine and the two branched-chain amino acids, valine and leucine, were elevated in the duodenal mucosa, and in the colonic mucosa, methionine and phenylalanine were elevated; otherwise, all the other individual amino acids were unaltered or depressed. CONCLUSIONS: The alterations seen in mucosal free amino acid and protein concentrations in connection with critical illness are different in many respects and contrast with the findings seen after starvation or moderate surgical trauma.

Short-term starvation alters the free amino acid content of the human intestinal mucosa.

1. The effects of short-term starvation and refeeding on the free amino acid concentrations of the intestinal mucosa were characterized in male subjects (n = 6), using endoscopically obtained biopsy specimens from the duodenum and from all four segments of the colon. 2. The alterations in the amino acid concentrations in response to short-term starvation were overall uniform in both duodenal and colonic mucosa as well as in plasma. Most amino acids decreased, whereas branched-chain amino acids increased. 3. In the colon, glutamic acid and glutamine decreased during the starvation period, whereas they remained unaltered in the duodenum. This was the major difference in response to short-term starvation between the amino acid concentrations in the intestinal mucosa of the duodenum and colon. 4. Refeeding for 3 days normalized the amino acid concentrations except for glutamic acid, asparagine and histidine, which remained low in the colon, and threonine, which showed an overshoot in both parts of the intestine. 5. The changes in mucosal amino acid concentrations seen in response to starvation and refeeding were uniform in the four segments of the colon. This suggests that sampling from the rectum/sigmoid colon will give representative values for the free amino acid concentrations of the entire large intestine.

Free amino acids in biopsy specimens from the human colonic mucosa.

The content of free amino acids and total protein was determined in endoscopic biopsy specimens from the rectum, descending colon, transverse colon, and ascending colon in 10 patients. The amino acids were quantified by ion-exchange chromatography and were detected by fluorescence. The amino acid pattern and the rank order of the individual amino acids in the colon were different compared to those in plasma. Glutamate, taurine, aspartate, glutamine, glycine, alanine, serine, lysine, valine, and ornithine were the 10 most abundant amino acids in the colon. The rank order was the same in all of the different segments of the colon. The concentrations of the amino acids decreased the more aborally the biopsies were taken. The protein content in the rectum was significantly lower than that in the transverse colon, but there were no difference between the different segments otherwise. The study demonstrated the possibility of determining free amino acids from endoscopic biopsies of human colonic mucosa. Biopsy specimens from the descending colon and/or rectum/sigmoid colon may be considered representative of the entire large intestine. The technique may be used for repeated sampling in studies of the amino acid metabolism of the intestinal mucosa.

The content of free amino acids in the human duodenal mucosa.

The free amino acid concentrations and the total protein content of the duodenal mucosa were determined in biopsy specimens obtained during endoscopic examinations in 10 healthy subjects. The amino acids were separated and quantified by ion exchange chromatography using fluorescence detection. The protein content was analysed according to Lowry. The amino acid pattern found in the duodenal mucosa was quite different from that in the plasma. The total amounts of all individual free amino acids were considerably higher in the mucosa than in the plasma (16.2 +/- 0.6 mmol/kg biopsy weight compared to 2.4 +/- 0.1 mmol/l). Taurine, glutamate and aspartate constituted more then 65% of the total content of all amino acids in the mucosa. Glutamine, the most abundant amino acid in plasma (21%), ranked only as sixth in the duodenal mucosa (4%); still, the absolute concentrations were quite similar in the mucosa and plasma (0.60 +/- 0.05 mmol/kg vs. 0.53 +/- 0.02 mmol/l). This study demonstrates the possibility of determining free amino acids in endoscopic biopsy specimens from the human duodenum. The technique is recommended for repeated sampling in clinical studies on the amino acid metabolism of the intestinal mucosa.

A simple regimen for control of postoperative sepsis in colorectal surgery.

A preoperative schedule based on three principles: rigorous mechanical cleansing of the intestine, nutrition for four days with a formula diet (Biosorbin MCT), and short-time systemic prophylaxis with doxycycline has been tested in a prospective study of 252 patients who underwent elective colorectal surgery. There were 11 wound infections, most of them of minor importance (4.4 per cent). Intra-abdominal abscesses occurred in two patients, both successfully reoperated, giving an overall incidence of septic complications of 5.1 per cent. The schedule was easy for the staff to administer, well tolerated by the patients, an inexpensive. The complication rate is probably as low as could be anticipated with this type of surgery.

The effect of chemical and surgical sympathectomy on gastric secretion and innervation.

The purpose of this project was to evaluate the effects of chemical and surgical sympathectomy on gastric acid secretion (GAS) and mucosal innervation in the dog. Chemical sympathectomy was achieved with 6-OHDA and surgical sympathectomy was performed by excising the celiac and superior mesenteric ganglia. Gastric biopsies for fluorescence microscopy were taken before and after sympathectomy, as was pentagastrin (PPG) stimulated GAS. Each sympathectomy increased GAS in response to submaximal doses of PPG while simultaneously destroying adrenergic nerve terminals in the gastric mucosa. 6-OHDA caused a complete degeneration of nerve terminals at one week, but regeneration was complete by 9 weeks. The data support the concept that the sympathetic innervation of the stomach is an inhibitor of GAS.

A pharmacokinetic analysis of the vagal release of 5-hydroxytryptamine in the cat.

The kinetics of 5-hydroxytryptamine (5-HT) release into the portal system after efferent electrical vagal stimulation were studied in an in vivo model. Blood samples were drawn from cats before, during and after vagal nerve stimulation, and portal plasma levels of 5-HT were measured. Portal plasma concentration of 5-HT rose during stimulation. After termination of stimulation, the plasma levels returned to steady state values and were used to calculate half-life and elimination rate constant. When pharmacokinetic principles were applied, the plasma half-life (T1/2) was determined to be 1.32 min and the elimination rate constant (KE), 0.5246 min-1. Pharmacokinetic principles also were applied to data from earlier in vitro studies using exogenously administered 5-HT, and a half-life of 1.03 min and a KE of 0.67 min-1 were calculated. Therefore, it appears that exogenously administered 5-HT and neurogenically released endogenous 5-HT are metabolized at a similar rate, both having a short half-life.

Gastric acid secretion after chemical sympathectomy.

Adrenergic nerve terminals in several organs are selectively destroyed by 6-hydroxydopamine (6-OHDA) resulting in a chemical sympathectomy that is reversible. In this study the acute and chronic effects of 6-OHDA on gastric mucosa and acid secretion were evaluated. Four dogs were given 6-OHDA (40 mg/kg, intravenously). Gastric biopsies were taken before treatment and biweekly thereafter and were analyzed by fluorescence microscopy (Hillarp-Falck). Degeneration of adrenergic nerve terminals in the mucosa was complete at 1 week. Early regeneration was noted at 3 weeks and appeared to be complete at 9 weeks. In another group of seven dogs with a gastric fistula, dose-response curves to pnetagastrin (PPG, 0 to 5 microgram/kg/hr) were determined. Then 6-OHDA (40 mg/kg) was given to these dogs and secretory studies were repeated weekly thereafter for 8 weeks. After 6-OHDA administration, acid secretion increased in response to submaximal doses of PPG, whereas maximal secretion was unchanged. The peak increase occurred the second week; thereafter secretion gradually returned to control values. We conclude that chemical sympathectomy (6-OHDA) increases gastric acid secretion in response to submaximal PPG stimulation. This increase correlates well with the 6-OHDA--induced degeneration of adrenergic terminals in the mucosa. These data suggest that the adrenergic innervation of the stomach has an inhibitory effect on the control of acid secretion in the dog.

Origin of the adrenergic nerve fibers in the subdiaphragmatic vagus in the dog.

The vagi at the subdiaphragmatic level were studied by the Hillarp-Falck technic in combination with a nerve crush procedure in three normal dogs and in eight dogs that had undergone previous surgical excision of the superior cervical ganglion and/or the stellate ganglia. Unilateral ganglionectomies were performed so that the contralateral vagus served as a control. Based on these results, it is concluded that: (1) the subdiaphragmatic canine vagus contains numerous adrenergic nerve fibers; (2) the main portion of these vagal adrenergic fibers arises from the stellate ganglia; and (3) removal of both the stellate and the superior cervical ganglia results in nearly complete adrenergic denervation of the abdominal vagus.

Effects of celiacectomy and stellatectomy on gastric mucosal innervation and acid secretion in the dog.

The effect of surgical excision of the celiac and superior mesenteric ganglia and the stellate ganglia, respectively, on gastric acid secretion and adrenergic innervation of gastric mucosa was studied in five dogs. After removal of the abdominal ganglia, there was a significant increase in acid secretion while there was a marked decrease in the number of adrenergic nerve terminals in the mucosa. When, addition, the stellate ganglia were excised, acid secretion increased slightly in two dogs, while little change in mucosal innervation was noted. These data suggest that the adrenergic innervation of the stomach has an inhibitory role in the control of acid secretion in the dog. Vagal adrenergic nerve fibers seem to be of limited importance in gastric acid production.