Post-absorptive and insulin-mediated muscle protein metabolism in liver-transplanted patients.

Cirrhotic patients after liver transplantation show a near-normal glucose homeostasis when in stable condition. In contrast, the basal and insulin-mediated whole-body protein metabolism remain altered several years after the graft. To examine whether the persisting defect of protein metabolism was due to the muscle, 7 non-diabetic liver-transplanted patients in stable condition were studied by means of the catheterization of the brachial artery and the deep forearm vein (to measure the balance across the forearm) and the infusion of labelled leucine and phenylalanine associated with indirect calorimetry. Whole-body proteolysis (as determined by endogenous leucine flux, ELF), protein synthesis (from non-oxidative leucine disposal, NOLD) and leucine oxidation (LO) were reduced in comparison to previously obtained values in a normal population. Insulin infusion (while maintaining euglycemia) induced a not significant variation of forearm phenylalanine Ra (24.4-->16.5 micromol/100 ml forearm min(-1); proteolysis) and Rd (18.5-->19.7; protein synthesis). In contrast, the whole-body insulin-dependent inhibitions of ELF (31.5-->21.8 micromol/m(2) min) and NOLD (27.3-->18.4) were impaired with respect to a normal population. On the basis of the present results, we conclude that skeletal muscle is not responsible for the alterations of leucine metabolism persisting after liver transplantation. By exclusion, this points to the liver as the major determinant of the leucine metabolism defect.

Metabolic effects of restoring partial beta-cell function after islet allotransplantation in type 1 diabetic patients.

Successful intraportal islet transplantation normalizes glucose metabolism in diabetic humans. To date, full function is not routinely achieved after islet transplantation in humans, with most grafts being characterized by only partial function. Moreover, the duration of full function is variable and cannot be sufficiently predicted with available methods. In contrast, most grafts retain partial function for a long time. We hypothesized that partial function can restore normal protein and lipid metabolism in diabetic individuals. We studied 45 diabetic patients after islet transplantation. Labeled glucose and leucine were infused to assess whole-body glucose and protein turnover in 1) 6 type 1 diabetic patients with full function after intraportal islet transplantation (FF group; C-peptide > 0.6 nmol/l; daily insulin dosage 0.03 +/- 0.02 U x kg(-1) body wt x day(-1); fasting plasma glucose < 7.7 mmol/l; HbA1c < or = 6.5%), 2) 17 patients with partial function (PF group; C-peptide > 0.16 nmol/l; insulin dosage < 0.4 U x kg(-1) body wt x day(-1)), 3) 9 patients with no function (NF group; C-peptide < 0.16 nmol/l; insulin dosage > 0.4 U x kg(-1) body wt x day(-1)), and 4) 6 patients with chronic uveitis as control subjects (CU group). Hepatic albumin synthesis was assessed in an additional five PF and five healthy volunteers by means of a primed-continuous infusion of [3,3,3-2H3]leucine. The insulin requirement was 97% lower than pretransplant levels for the FF group and 57% lower than pretransplant levels for the PF group. In the basal state, the PF group had a plasma glucose concentration slightly higher than that of the FF (P = 0.249) and CU groups (P = 0.08), but was improved with respect to the NF group (P < 0.01). Plasma leucine (101.1 +/- 5.9 micromol/l) and branched-chain amino acids (337.6 +/- 16.6 micromol/l) were similar in the PF, FF, and CU groups, and significantly lower than in the NF group (P < 0.01). During insulin infusion, the metabolic clearance rate of glucose was defective in the NF group versus in the other groups (P < 0.01). Both the basal and insulin-stimulated proteolytic and proteosynthetic rates were comparable in the PF, FF, and CU groups, but significantly higher in the NF group (P = 0.05). In addition, the PF group had a normal hepatic albumin synthesis. Plasma free fatty acid concentrations in the PF and FF groups were similar to those of the CU group, but the NF group showed a reduced insulin-dependent suppression during the clamp. We concluded that the restoration of approximately 60% of endogenous insulin secretion is capable of normalizing the alterations of protein and lipid metabolism in type 1 diabetic kidney recipients, notwithstanding chronic immunosuppressive therapy. The results of the present study indicate that "success" of islet transplantation may be best defined by a number of metabolic criteria, not just glucose concentration/metabolism alone.

Effect of hypoglycemia on amino acid and protein metabolism in healthy humans.

In response to hypoglycemia, healthy individuals rapidly antagonize insulin action on glucose and lipid metabolism, but the effects on protein metabolism are unclear. Because amino acids are an important substrate for gluconeogenesis and a fuel alternative to glucose for oxidation, we evaluated whether hypoglycemia antagonizes the hypoaminoacidemic and the antiproteolytic effects of insulin and changes the de novo synthesis of glutamine, a gluconeogenic amino acid. To this purpose, in 7 healthy subjects, we performed 2 studies, 3.5 h each, at similar insulin but different glucose concentrations (i.e., 4.9 +/- 0.1 mmol/l [euglycemic clamp] or 2.9 +/- 0.2 mmol/l [hypoglycemic clamp]). As expected, hypoglycemia antagonized the insulin suppression of glucose production achieved in euglycemia (from 21 +/- 15 to 116 +/- 12% of basal, P < 0.001), the stimulation of glucose uptake (from 207 +/- 28 to 103 +/- 7% of basal, P < 0.01) and the suppression of circulating free fatty acids (from 30 +/- 5 to 80 +/- 17% of basal, P < 0.001). In contrast, hypoglycemia increased the insulin suppression of circulating leucine (from 63 +/- 1 to 46 +/- 2% of basal, P < 0.001) and phenylalanine (from 79 +/- 3 to 64 +/- 3% of basal, P < 0.001) concentrations. Hypoglycemia did not change the insulin suppression of proteolysis (from 79 +/- 2 to 82 +/- 4% of basal, P < 0.001). However, hypoglycemia doubled the insulin suppression of the glutamine concentrations (from 84 +/- 3 to 63 +/- 3% of basal, P < 0.01) in the absence of significant changes in the glutamine rate of appearance, but it also caused an imbalance between glutamine uptake and release. This study demonstrates that successful counterregulation does not affect proteolysis. Moreover, it does not increase the availability of circulating amino acids by de novo synthesis. In contrast, despite the lower concentration of circulating amino acids, hypoglycemia increases the uptake of glutamine that can be used for gluconeogenesis and as a fuel alternative to glucose.

Postabsorptive muscle protein metabolism in type 1 diabetic patients after pancreas transplantation.

Insulin was shown to induce protein anabolism in vivo mainly by inhibiting proteolysis. Heterotopic pancreas transplantation in type 1 diabetes mellitus is characterized by peripheral hyperinsulinemia due to systemic rather than portal insulin delivery. Therefore, we studied the postabsorptive muscle protein metabolism in type 1 diabetic patients with or without pancreas transplantation. The forearm balance technique was performed in 9 type 1 diabetic patients on exogenous insulin treatment, in 4 type 1 diabetic patients following successful pancreas transplantation and in 6 healthy volunteers. Labelled leucine and phenylalanine were infused to quantify whole-body and muscle protein synthesis, respectively. In the postabsorptive state, whole-body protein synthesis (leucine kinetics) was similar in pancreas-transplanted patients and controls. In contrast, muscle protein synthesis tended to be less negative in pancreas-transplanted patients with respect to type 1 diabetic patients and healthy volunteers. The present data suggest that recipients with peripheral insulin delivery and chronic hyperinsulinemia are characterized by a preferential stimulation of protein synthesis in muscle rather than in the splanchnic district. When insulin was infused acutely, while maintaining euglycemia, the whole-body and muscle protein synthesis rates were approximately halved in type 1 diabetic patients with and without pancreas transplantation. We conclude that pancreas transplantation is able to normalize basal and insulin-stimulated protein metabolism. Chronic hyperinsulinemia counteract steroid-induced protein degradation by means of a mild, but persistent stimulation of muscle protein synthesis.

Prevention of cystic fibrosis in Italian families by DNA studies.

We performed a feasibility study of prenatal diagnosis in the Italian population by DNA analysis utilizing the probes: metD, metH, J3.11, KM.19 and XV-2c. With these probes, 118 out of 126 families (93.7%) were fully informative. We also tested part of the families with new additional tightly linked probes, E.9 and W3D1.4. With all the probes feasibility was complete in 44 out of 45 families (97.8%). In particular with a set of only 3 tightly linked probes, KM.19 + XV-2c + W3D1.4, 71% of the families were fully informative. We carried out 8 prenatal diagnoses, 4 of which with the polymerase chain reaction (PCR) for KM.19. These data show that these probes are highly informative and can be used for feasibility studies and prenatal diagnosis of CF in the Italian population.

New restriction fragment length polymorphism (probe E9) reveals the highest linkage disequilibrium in Italian CF patients.

We report that the allele distribution for RFLP's flanking the CF gene differs between patients with and without pancreatic insufficiency. The present study confirms this difference. In both classes the linkage disequilibrium (LD) is highest with the RFLP revealed by probe E9. The haplotype distribution identified by these RFLP's can be used for indirect carrier detection.

Haplotypes in cystic fibrosis patients with or without pancreatic insufficiency from four European populations.

We examined the allele and haplotype frequencies of five polymorphic DNA markers in 355 European cystic fibrosis (CF) patients (from Belgium, the German Democratic Republic, Greece, and Italy) who were divided into two groups according to whether they were or not taking supplementary pancreatic enzymes. The level of linkage disequilibrium between each polymorphism and the CF mutation varied among the different populations; there was no significant association between KM.19 and CF in the Greek population. The distributions of alleles and haplotypes derived from the polymorphisms revealed by probes KM.19 and XV.2c were always different in patients with or without pancreatic insufficiency (PI) in all the populations studied. In particular, among 32 patients without PI, only 9 (or 28%) were homozygous for the KM.19-XV.2c = 2-1 haplotype (which was present in 73% of all the CF chromosomes in our sample) compared to 162 of 252 patients (or 64%) with PI. These findings are consistent with the hypothesis that pancreatic insufficiency or sufficiency may be determined by different mutations at the CF locus.

Prenatal diagnosis of carbamoyl-phosphate synthetase deficiency by fetal liver biopsy.

In a pregnancy at risk for carbamoyl-phosphate synthetase (CPS) deficiency, prenatal diagnosis was attempted by fetal liver biopsy, performed at 18 weeks of gestation. CPS activity was absent and the diagnosis was confirmed after termination of the pregnancy. The technique employed for fetal liver biopsy is described together with an evaluation of its possible role in prenatal diagnosis.

Bile acid malabsorption in cystic fibrosis with and without pancreatic insufficiency.

Serum conjugated cholic acid (CCA) and conjugated chenodeoxycholic acid (CCDCA) fasting levels were measured in 30 children with cystic fibrosis (CF) without liver involvement, and mean levels were not significantly different from control values. In seven children (four with partially corrected pancreatic insufficiency and three without pancreatic insufficiency) serum levels of both primary bile acids (BAs) were also measured after the ingestion of a standard liquid meal; the values were then compared with those for total and fractional fecal BA excretion. The CCA mean peak increase was significantly reduced in patients with pancreatic insufficiency (p less than 0.01), as well as in those without pancreatic insufficiency (p less than 0.05), as compared to controls. The CCDCA mean peak increase was reduced only in patients with pancreatic insufficiency (p less than 0.01). Fecal results confirmed serum data, showing a significantly increased excretion of cholic and deoxycholic acids in patients without pancreatic insufficiency as compared to controls (p less than 0.02), despite a similar total BA excretion. In patients with pancreatic insufficiency, total fecal BA levels were markedly increased compared to control values (p less than 0.001); the fecal percentage of chenodeoxycholic and lithocholic acids was greater than that recorded in patients without pancreatic insufficiency (p less than 0.05), in agreement with the different behaviour of serum CCDCA postprandial curves for the two groups of patients. The results are consistent with selective malabsorption of cholic acid in CF, independent of the presence of pancreatic insufficiency; they confirm the usefulness of serum BA postprandial determinations in measuring BA malabsorption.

Incidence of classic PKU in Italy estimated from consanguineous marriages and from neonatal screening.

As already described for cystic fibrosis and Friedreich ataxia, the incidence of PKU in Italy has been estimated by determining the increase of consanguineous marriages among 178 couples of PKU parents over the frequencies carefully established for the same marriages in the general Italian population for each of the 95 provinces during a 55-year period. The incidence estimated (between 1/15595 and 1/17815 according to two different formulas) is not very different from the incidence derived from screening programs (almost 1/12000). This indicates that the former method can be applied in Italy to the study of the incidence of other autosomal recessive disorders.

Evaluation of an oral ursodeoxycholic acid load in the assessment of bile acid malabsorption in cystic fibrosis.

Serum levels of ursodeoxycholic acid (UDCA) were measured by radioimmunoassay in 20 children with cystic fibrosis (CF) and in eight controls, who had received 300 mg of this bile acid orally. Area under the curve (AUC) after UDCA load was significantly reduced (25.24 +/- 9.54) in CF patients, as compared to controls (52.98 +/- 5.87 mean values +/- SD percent dose/liter X hr X kg body weight, P less than 0.001). AUC values were compared with daily fecal bile acids (BA) and fat excretion, and with serum fasting conjugated cholic (CCA) and chenodeoxycholic (CCDCA) acid levels. Total fecal BA were increased in CF patients (7.84 +/- 5.57 mg/kg/day) as compared to control values (2.7 +/- 1.1 mean +/- SD, P less than 00.5); they were inversely correlated with AUC values (r = 0.48) but not with steatorrhea (r = 0.32). UDCA load seems to be useful in detecting BA malabsorption in CF. Fasting CCA levels did not significantly differ in CF patients (0.87 +/- 0.61 mumol/liter) and in controls (0.60 +/- 0.26 mumol/liter) and did not correlate with fecal BA excretion. Fasting CCDCA mean levels were significantly increased (mainly in CF patients with liver damage), suggesting a lowered first-pass extraction in the liver: their determination may be useful in the follow-up of liver involvement in CF.