Evaluation of energy metabolism and calcium homeostasis in cells affected by Shwachman-Diamond syndrome.

Isomorphic mutation of the SBDS gene causes Shwachman-Diamond syndrome (SDS). SDS is a rare genetic bone marrow failure and cancer predisposition syndrome. SDS cells have ribosome biogenesis and their protein synthesis altered, which are two high-energy consuming cellular processes. The reported changes in reactive oxygen species production, endoplasmic reticulum stress response and reduced mitochondrial functionality suggest an energy production defect in SDS cells. In our work, we have demonstrated that SDS cells display a Complex IV activity impairment, which causes an oxidative phosphorylation metabolism defect, with a consequent decrease in ATP production. These data were confirmed by an increased glycolytic rate, which compensated for the energetic stress. Moreover, the signalling pathways involved in glycolysis activation also appeared more activated; i.e. we reported AMP-activated protein kinase hyper-phosphorylation. Notably, we also observed an increase in a mammalian target of rapamycin phosphorylation and high intracellular calcium concentration levels ([Ca(2+)]i), which probably represent new biochemical equilibrium modulation in SDS cells. Finally, the SDS cell response to leucine (Leu) was investigated, suggesting its possible use as a therapeutic adjuvant to be tested in clinical trials.

Pancreatic endocrine and exocrine function in children following near-total pancreatectomy for diffuse congenital hyperinsulinism.

CONTEXT: Congenital hyperinsulinism (CHI), the commonest cause of persistent hypoglycaemia, has two main histological subtypes: diffuse and focal. Diffuse CHI, if medically unresponsive, is managed with near-total pancreatectomy. Post-pancreatectomy, in addition to persistent hypoglycaemia, there is a very high risk of diabetes mellitus and pancreatic exocrine insufficiency. SETTING: International referral centre for the management of CHI. PATIENTS: Medically unresponsive diffuse CHI patients managed with near-total pancreatectomy between 1994 and 2012. INTERVENTION: Near-total pancreatectomy. MAIN OUTCOME MEASURES: Persistent hypoglycaemia post near-total pancreatectomy, insulin-dependent diabetes mellitus, clinical and biochemical (faecal elastase 1) pancreatic exocrine insufficiency. RESULTS: Of more than 300 patients with CHI managed during this time period, 45 children had medically unresponsive diffuse disease and were managed with near-total pancreatectomy. After near-total pancreatectomy, 60% of children had persistent hypoglycaemia requiring medical interventions. The incidence of insulin dependent diabetes mellitus was 96% at 11 years after surgery. Thirty-two patients (72%) had biochemical evidence of severe pancreatic exocrine insufficiency (Faecal elastase 1<100 µg/g). Clinical exocrine insufficiency was observed in 22 (49%) patients. No statistically significant difference in weight and height standard deviation score (SDS) was found between untreated subclinical pancreatic exocrine insufficiency patients and treated clinical pancreatic exocrine insufficiency patients. CONCLUSIONS: The outcome of diffuse CHI patients after near-total pancreatectomy is very unsatisfactory. The incidence of persistent hypoglycaemia and insulin-dependent diabetes mellitus is very high. The presence of clinical rather than biochemical pancreatic exocrine insufficiency should inform decisions about pancreatic enzyme supplementation.

An interfacial and comparative in vitro study of gastrointestinal lipases and Yarrowia lipolytica LIP2 lipase, a candidate for enzyme replacement therapy.

Lipolytic activities of Yarrowia lipolytica LIP2 lipase (YLLIP2), human pancreatic (HPL) and dog gastric (DGL) lipases were first compared using lecithin-stabilized triacylglycerol (TAG) emulsions (Intralipid) at various pH and bile salt concentrations. Like DGL, YLLIP2 was able to hydrolyze TAG droplets covered by a lecithin monolayer, while HPL was not directly active on that substrate. These results were in good agreement with the respective kinetics of adsorption on phosphatidylcholine (PC) monomolecular films of the same three lipases, YLLIP2 being the most tensioactive lipase. YLLIP2 adsorption onto a PC monolayer spread at the air/water interface was influenced by pH-dependent changes in the enzyme/lipid interfacial association constant (KAds) which was optimum at pH 6.0 on long-chain egg PC monolayer, and at pH 5.0 on medium chain dilauroylphosphatidylcholine film. Using substrate monolayers (1,2-dicaprin, trioctanoin), YLLIP2 displayed the highest lipolytic activities on both substrates in the 25-35 mN m(-1) surface pressure range. YLLIP2 was active in a large pH range and displayed a pH-dependent activity profile combining DGL and HPL features at pH values found in the stomach (pH 3-5) and in the intestine (pH 6-7), respectively. The apparent maximum activity of YLLIP2 was observed at acidic pH 4-6 and was therefore well correlated with an efficient interfacial binding at these pH levels, whatever the type of interfaces (Intralipid emulsions, substrate or PC monolayers). All these findings support the use of YLLIP2 in enzyme replacement therapy for the treatment of pancreatic exocrine insufficiency, a pathological situation in which an acidification of intestinal contents occurs.

The growing pancreatic duct ligated pig as a model for exocrine pancreatic insufficiency in children: investigations to achieve sufficient vitamin A and vitamin E supply.

Human patients suffering from exocrine pancreatic insufficiency (EPI) are susceptible to deficiencies in fat-soluble vitamins. In children with cystic fibrosis, EPI is common and aspects of sufficient vitamin supply are of special interest. The aim of this study was to determine the best application form to maintain vitamin A and E levels in the physiological range in growing pigs with EPI (induced by pancreatic duct ligation) as a model for children. The pancreatic duct was ligated (PL) in twelve 8-wk-old pigs; 4 sham-operated pigs served as controls (Con). Pigs (n = 16) were individually housed and fed a diet containing 13,393 IU vitamin A and 122 mg vitamin E/kg DM. The PL pigs (n = 12) were divided into 3 groups (n = 4) 2 wk after surgery: PL-0, without extra vitamin supply; PL+ORAL, 90,000 IU vitamin A and 600 mg vitamin E/kg of DM plus emulsifier E 484 added to the diet; and PL+IM, intramuscular injection of vitamin A (5,250 IU) and vitamin E [aqueous; 3.15 mg/(kg BW · wk)] plus 700 mg vitamin E (oily)/(animal · wk). All PL pigs were supplemented with the pancrelipase Creon (19.8 g = 1,048,727 IU lipase/kg feed) beginning 2 wk after ligation of the pancreatic duct. Pigs were euthanized at 16 wk of age. Tocopherol levels (mg/kg DM) in liver were reduced (P ≤ 0.005) in PL-0 and PL+IM (6.91 and 8.61, respectively) whereas PL+ORAL did not differ from Con (27.4 and 25.8, respectively; P ≥ 0.77). Compared to control pigs (241 ± 14.1 mg vitamin A/kg DM of liver), the concentration of vitamin A (mg/kg DM) in liver was lower (P < 0.003) in PL-0 (136 ± 18.5) but higher (P < 0.003) in PL+ORAL (375 ± 50.0). In the group PL+IM a high individual variation was observed (288 ± 142 mg vitamin A/kg DM of liver). Extra dietary supply of high doses of vitamin A and E with an efficient emulsifier was adequate to maintain vitamin A and E in liver tissue within reference values. The present data underline the need for extra supplementation of vitamin A and E in juvenile patients with EPI and indicate that oral application is suitable.

Serum vitamin A concentration in dogs with experimentally induced exocrine pancreatic insufficiency.

Serum concentration of Vitamin A was determined in dogs with experimentally induced exocrine pancreatic insufficiency following oral administration of vitamin A, or pancreatic enzyme and vitamin A. In dogs receiving vitamin A alone, serum vitamin A concentration was significantly lower than that of dogs supplemented with pancreatic enzyme and vitamin A. In dogs with exocrine pancreatic insufficiency, serum vitamin A concentration was lower than in healthy dogs. In healthy dogs, the normal range of serum vitamin A concentration is high in comparison with other species, and humans. It is concluded that in dogs with exocrine pancreatic insufficiency, additional supplementation of vitamin A may be needed.