Cellular mechanism of exocrine pancreatic insufficiency in diabetes mellitus
Diabetes mellitus (DM) is associated with the compromised digestion of carbohydrates. This complication is described as exocrine pancreatic insufficiency. Whilst this causes malnutrition in patients and contributes to diabetic morbidity, the physiology and molecular biology leading to this state is not well defined. This disease-induced inability to digest foodstuffs could have many levels of regulation. Obvious candidates are ligand proteins involved in stimulating secretion, receptor defects, intracellular ion levels and post-receptor signal transduction encompassing transcription and translation. To address these points, the current studies aimed to characterise the effects of experimental type I DM upon both physiological and molecular events. The first study investigated the effects of cholecystokinin-octapeptide (CCK-8) and exogenous insulin on exocrine pancreatic amylase secretion in streptozotocin (STZ)- induced diabetic rats compared to healthy age-matched controls in vivo and in vitro. Seven - eight weeks after the induction of diabetes, animals were either anaesthetised for the study of in vivo exocrine secretion or humanely killed and pancreatic acinar cells isolated for the measurement of intracellular free calcium and magnesium concentrations ([Ca 2+ ]1 and [Mg2+ ]1), total protein, and amylase output employing the Phadebas method. For rats in both in vivo and in vitro studies, fasting blood glucose in control and diabetic rats was 73.3 ± 3.4 mg dl-1 (n = 44) and 380.0 ± 25.9 mg dl-1 (n = 27), respectively. Basal pancreatic juice flow rate in STZ-diabetic rats was significantly increased (P<0.001) whereas protein and amylase outputs were significantly decreased (P<0.001) compared to control rats. CCK-8 infusion (150 pmol kg -1 h-1 for 100 mm) resulted in marked elevations in flow rate as well as in protein and amylase secretion in control animals (P<0.05 compared with the corresponding basals). In contrast, in diabetic rats, CCK-8 evoked a small increase in flow rate, which was not significant when compared to basal. In these animals, CCK-8 stimulated the secretion of amylase and protein output, but the secretory rates were dramatically lower compared with those in control rats. Administration of insulin (1 U, I.P.) in healthy rats significantly increased pancreatic flow rate, amylase secretion, protein output and blood glucose levels in vivo compared to basal (P<0.05). Infusion of CCK-8 together with insulin (1 U) in control rats markedly potentiated pancreatic juice flow and amylase secretion. Pretreatment with atropine (0.2 mg kg-1, I.P.) abolished the effects of insulin on secretory parameters despite a similar reduction in glycaemia. In diabetic rats, insulin (4 U, I.P.) did not modify exocrine pancreatic secretion either alone or in combination with CCK-8. In vitro experiments revealed that either (ACh (10-8 – 10-4 M) or CCK-8 (10" - 10-8 M)) can evoke total amylase release which was elevated in healthy control pancreatic acinar cells compared to diabetic acinar cells. In contrast, 10-6 M insulin produced a significant increase (Pc0.05) in the amount of total amylase output in control acinar cells compared to diabetic acinar cells. Combining insulin (10-8 – 10-6 M) with either ACh or CCK-8 had little or no effect on total amylase release in both control and diabetic acinar cells. There were no significant differences among the groups in unstimulated [Ca2+]j and [Mg2+]i. However, the peak [Ca 2+ ]i induced by 10-8 M CCK-8 was depressed (P<0.05) in diabetic cells (275.3 ± 11.5 nM n = 8) compared to (359.7 ± 27.5 nM, n = 6) control cells. Similarly, CCK-8 significantly decreased (P<0.05) [Mg2 ']1 in diabetic acinar cells compared to control. On a molecular level, the gene encoding amylase was under transcriptional dysregulation. Healthy control animals had a significantly lower (P>0.05) crossing point value (8.54 ± 0.131. n = 8) compared to STZ-induced diabetic animals (17.96 ± 0.272, n = 7), respectively. On a protein level, those mediators controlling translation such as p70 S6K and 4E-BPI were present at significantly lower (P>0.05) relative concentrations, suggesting an impaired capacity for protein synthesis. Interestingly, the actual activity of these proteins as measured by phosphorylation was slightly increased. It is suggested that this is a cellular mechanism to counteract loss in transcription and/or translation of mRNA encoding these proteins. Protein ubiquitination was also elevated suggesting increased protein breakdown which could be responsible for pancreatic atrophy and net protein loss. The NFkβ protein widely implicated in tissue atrophy was actually lower in STZ-induced DM, and therefore probably does not contribute to pancreatic wasting. To conclude, the results indicate that DM-induced exocrine pancreatic insufficiency is associated with decreased levels of total protein output and amylase secretion and these changes may be in part be associated with derangements in cellular Ca2+ and Mg2+ homeostasis. Furthermore, transcription of the α-amylase gene is reduced suggesting a reduced protein level and thus capacity for stimulus-secretion coupling. Finally, there appears impaired protein translation and elevated ATP-dependent protreasome mediated protein breakdown in STZ-induced DM.