The intestine expresses pancreatic triacylglycerol lipase: regulation by dietary lipid.

We identified the enzyme responsible for alkaline lipolysis in mucosa of rat small intestine. RT-PCR was used to amplify a transcript that, by cloning and sequencing, is identical to pancreatic triacylglycerol lipase. In rats fed normal laboratory chow, pancreatic triacylglycerol lipase mRNA was detected in all four quarters of the small intestine, with the first quarter expressing about three times as much of this transcript as was found in the more distal three-quarters combined. Both acutely and chronically administered dietary fat were shown to regulate pancreatic triacylglycerol lipase mRNA expression and lipase activity. The synthesis of pancreatic triacylglycerol lipase protein by the small intestine was demonstrated by in vivo radiolabeling experiments using [(35)S]methionine/cysteine followed by immunoprecipitation with an anti-pancreatic triacylglycerol lipase antibody. Immunohistochemical studies suggest that pancreatic triacylglycerol lipase protein expression is restricted to enterocytes throughout the small intestine. To our knowledge, this is the first report identifying rat small intestinal mucosa as a site of pancreatic triacylglycerol lipase synthesis and the first demonstration of its modulation in the mucosa by dietary fat. We propose that pancreatic triacylglycerol lipase is used by the intestine to hydrolyze the mucosal triacylglycerol that is not transported in chylomicrons.

Treatment of rat pancreatic islets with reactive PEG.

Covalent attachment of polymers to cells and tissues could be used to solve a variety of problems associated with cellular therapies. Insulin-dependent diabetes mellitus is a disease resulting from the autoimmune destruction of the beta cells of the islets of Langerhans in the pancreas. Transplantation of islets into diabetic patients would be an attractive form of treatment, provided that the islets could be protected from the host's immune system in order to prevent graft rejection. If reaction of polyethylene glycol (PEG) segments with the islet surface did not damage function, the immunogenicity and cell binding characteristics of the islet could be altered. To determine if this process damages islets, rat islets have been isolated and treated with protein-reactive PEG-isocyanate (MW 5000) under mild reaction conditions. An assessment of cell viability using a colorimetric mitochondrial activity assay showed that treatment of the islets with PEG-isocyanate did not reduce cell viability. Insulin release in response to secretagogue challenge was used to evaluate islet function after treatment with the polymer. The insulin response of the PEG-treated islets was not significantly different than untreated islets in a static incubation secretagogue challenge. In addition, PEG-isocyanate-treated islets responded in the same manner as untreated islets in a glucose perifusion assay. Finally, the presence of PEG on the surface of the islets after treatment with the amine-reactive N-hydroxysuccinimide-PEG-biotin (not PEG-isocyanate) was confirmed by indirect fluorescence staining. These results demonstrate the feasibility of treating pancreatic islets with reactive polymeric segments and provide the foundation for further investigation of this novel means of potential immunoisolation.

Pressor doses of angiotensin II increase hepatic glucose output and decrease insulin sensitivity in rats.

The metabolic effects of angiotensin II (AII) were studied under steady-state conditions of euglycaemic hyperinsulinaemia in anaesthetized rats. Pressor doses of AII (50 and 400 ng/kg per min) had dose-dependent hypertensive and hyperglycaemic effects during glucose clamp studies. Glucose turnover measurements showed that hepatic glucose output (HGO) increased equally at both pressor doses compared with either saline infusion or AII infusion at a dose without a pressor effect (20 ng/kg per min); however, glucose disposal increased significantly only at 50 ng/kg per min. Infusion of the AII receptor antagonist, saralasin, did not itself alter glucose output or disposal significantly, but it abolished the effects of a simultaneous infusion of AII. It is concluded that pressor doses of AII increase HGO by a receptor-mediated mechanism that is not related to the pressor response to the hormone. The hyperglycaemic reaction to this metabolic effect of AII is partially offset by increased glucose disposal at lower doses. The physiological significance of these metabolic actions of AII remains to be established, but they raise the possibility that AII could potentially play a role in glucose homeostasis in vivo.

Insulin resistance during euglycemic clamp studies in chronically undernourished rats with mild streptozocin diabetes.

Malnutrition has been shown to impair insulin sensitivity, but it is not known whether this effect has any impact on coexisting diabetes. Insulin sensitivity was therefore studied using the glucose clamp technique in rats with chronic nutritional deprivation superimposed on mild streptozocin (STZ) diabetes mellitus. In pair-feeding experiments, 4-week-old littermate rats were either allowed ad libitum access to food or restricted to 50% of ad libitum intake for 8 weeks, and were injected with STZ 40 mg/kg intraperitoneally halfway through the experiment. Fasting plasma glucose (FPG) was similar in both groups of rats, but fasting plasma insulin (FPI) was lower in the undernourished group (P = .016). Undernourished rats were significantly more insulin resistant during euglycemic hyperinsulinemia of the same degree, with glucose disposal rate being impaired by 50% as compared with that in ad libitum-fed diabetic littermates (24.4 +/- 2.8 v 51.5 +/- 4.4 mumol/kg/min, P = .0008). The insulin sensitivity index was significantly lower in the undernourished group (3.03 +/- 0.32 v 5.67 +/- 0.6, P = .0057). The results show that chronic undernutrition markedly reduces insulin sensitivity in rats with mild STZ diabetes. This is further evidence that chronic undernutrition is a deleterious modifying influence on coexisting diabetes mellitus. It suggests that the insulin resistance of malnutrition-related diabetes mellitus (MRDM) could potentially be an acquired defect mediated by the coexistent undernutrition, rather than a "distinctive" feature that is intrinsically unique to this diabetic syndrome.

Adaptations in glucose homeostasis during chronic nutritional deprivation in rats: hepatic resistance to both insulin and glucagon.

The role of glucagon in glucose homeostasis during chronic malnutrition was studied in weanling-littermate rats either fed ad libitum or restricted to 60% of ad libitum intake for 8 weeks. Fasting glucose and insulin levels were lower in malnourished rats, and their response to glucagon (0.02 mg/kg intravenous [IV]) after a 16-hour fast was significantly less than in control littermates for both glucose (P = .039) and insulin (P = .008). During euglycemic glucose clamp studies at identical plasma glucose (PG) levels, insulin suppression of hepatic glucose production (HGP) was impaired in malnourished rats, indicating insulin resistance (mean +/- SE HGP: 48 +/- 5 v 32 +/- 10 mumol.kg-1.min-1 for controls, P = .028). Glucose disposal was not significantly different in the two groups. However, after IV glucagon, the increase in HGP was markedly impaired in malnourished rats (P = .0004), with the total amount of glucose produced by the liver over 15 minutes being 1,397 +/- 114 mumol/kg as compared with 2,031 +/- 118 in controls (P = .0047). The impaired response was not due to defective glycogenolysis, because the release of glucose from prelabeled glycogen in response to glucagon injection contributed only 6% to 8% of the overall increase in glucose output from the liver, and was not different in the two groups. Furthermore, liver glycogen stores were virtually exhausted after the 16-hour fast, without glucagon injection. Glucagon receptor affinity and number were not affected by malnutrition. It is concluded that (1) chronic malnutrition is associated with hepatic resistance to both insulin and glucagon, (2) the glucagon resistance is not due to impaired glycogenolysis, and (3) it is mediated by a postreceptor defect.(ABSTRACT TRUNCATED AT 250 WORDS)

Fasting glucose homeostasis in the adaptation to chronic nutritional deprivation in rats.

The hormonal basis for the metabolic paradox of relative hypoglycemia despite insulinopenia and insulin resistance in chronic nutritional deprivation was studied in weanling rats restricted to 60% of ad libitum intake over 8 wk (n = 12 each). Lower insulin and glucagon levels were observed in both peripheral and portal blood (P = 0.0016) in the malnourished rats on multivariate analysis of variance, indicating decreased islet secretion. Peripheral and portal hormone levels were proportionately similar, indicating that hepatic extraction was not altered. Despite relative hypoglycemia, glucose turnover rate, total glucose mass, and volume of distribution of glucose were not altered. This indicates that the sum total of the effects of malnutrition on the various hormonal influences controlling glucose turnover had resulted in the establishment of a new dynamic equilibrium associated with lower plasma glucose levels. It is concluded that fasting glucose levels are sustained at a lower level in chronic malnutrition by an adaptive process that includes insulin resistance and insulinopenia, counterbalanced not only by glucagon resistance, as shown earlier, but also by decreased glucagon secretion.

Effect of systemic glucagon administration on ACTH secretion in anaesthetized rats.

The effect of glucagon on ACTH secretion was studied in anaesthetized rats injected with either saline (0.1 ml i.m.) or glucagon (0.02 mg/kg i.m.). For the first 90 min after glucagon injection, plasma ACTH fell by 50% from the basal value of 23 +/- 4 pmol/l (mean +/- S.E.M.) to 11 +/- 2 (P = 0.011), after which an abrupt return to baseline occurred (120 min value: 26 +/- 2 pmol/l). In saline injected rats, the baseline ACTH value was not significantly different from either the 90 min value or the 120 min value (27 +/- 3 vs 21 +/- 4 and 24 +/- 3 pmol/l respectively; P > 0.10). Plasma glucose after glucagon peaked at 11.6 +/- 1.1 mmol/l by 15 min but subsequently fell rapidly, attaining the baseline by 60 min. Insulin levels increased sharply after glucagon, from 381 +/- 78 pmol/l to 3172 +/- 668 pmol/l at 15 min, and plateaued at approximately 1000 pmol/l thereafter. No changes in glucose or insulin were seen in saline injected rats. The magnitude of suppression of ACTH after glucagon was not affected either by sustained hyperinsulinaemia (congruent to 1400 pmol/l), induced with continuous glucose infusion to maintain plasma glucose > 12 mmol/l, or by pretreatment with the long-acting somatostatin analogue octreotide (50 micrograms/kg s.c.). However, the return to baseline between 100 and 120 min was prevented both by hyperinsulinaemia induced with sustained hyperglycaemia, and by octreotide. It is postulated that glucagon may inhibit ACTH secretion either by a direct effect on the hypothalamus or indirectly through insulin, which is known to stimulate endogenous somatostatin release.

Bilateral massive adrenal hemorrhage.

A systematic review of the etiopathogenesis of adrenal hemorrhage reveals that the factors that determine the occurrence of this complication in a patient can be identified prospectively. Such prospective diagnostic suspicion allows the significance of the otherwise nonspecific clinical features heralding this catastrophe to be appreciated in the precrisis interval. Confirmation of the diagnosis is achieved by visual demonstration of bilaterally enlarged and hyperdense adrenal glands on a CT scan and by demonstration of adrenocortical failure on hormonal evaluation. Preemptive steroid therapy and diagnostic evaluation during the precrisis period prevent the occurrence of a potentially disastrous adrenal crisis in a patient who is seriously ill from other problems and who cannot afford a setback of this severity. Because steroid therapy delayed until the stage of crisis may be unsuccessful in salvaging such a patient, prospective recognition must be based on identifying the patient at risk for BMAH and recognizing the significance of the nonspecific clinical features that accompany it in the appropriate clinical context.

Tissue-specific regulation of the growth hormone receptor gene in streptozocin-induced diabetes in the rat.

Abnormalities of GH secretion and clearance are well-documented in poorly controlled insulin-dependent diabetes mellitus (IDDM), but the contribution of the receptor (GHR) and the GH-binding protein (GHBP) to these abnormalities has not been defined. We studied the expression of the GHR/GHBP gene in the livers, hearts and kidneys in streptozocin-induced diabetes (STZ-D) in the rat. GHR and GHBP mRNA levels were measured by Northern blot and ribonuclease protection assays. Whereas levels of GHR and GHBP mRNA were significantly decreased in liver and heart of STZ-D rats when compared with the control group (P < 0.01), GHR mRNA was significantly increased in the kidneys of STZ-D rats (P = 0.03). Six days of insulin treatment did not significantly alter the levels of GHR/GHBP mRNA in the liver or heart of STZ-D rats, but significantly decreased GHBP mRNA (P = 0.04) in the kidney. Circulating IGF-I was reduced, as was IGF-I mRNA in the liver and heart of STZ-D rats; only circulating IGF-I was restored by insulin treatment. Neither STZ-D nor insulin treatment affected IGF-I or IGF-I receptor mRNA concentrations in the kidney. We conclude that (1) STZ-D modulates the expression of the GHR/GHBP gene and (2) that these changes in GHR/GHBP mRNA concentrations are tissue-specific; STZ-D decreases GHR/GHBP mRNA in liver and heart tissue but increases GHR mRNA concentrations in the kidney. Our results indicate a role for decreased numbers of hepatic GHRs in the pathogenesis of resistance to GH's actions in terms of IGF-I generation and promotion of linear growth in IDDM. We postulate that increased GHR expression in the kidney may be involved in the renal complications of IDDM.

Effects of angiotensin II on insulin sensitivity and fasting glucose metabolism in rats.

The hypothesis that angiotensin II (AII) might play a role in glucose homeostasis was studied in eight paired male littermate rats who had fasted for 16 h and were infused with either AII (50 ng/kg/min) or saline for 60 min. Mean arterial pressure (MAP) and plasma glucose (PG) did not change significantly in the saline-infused rats. In the AII-infused rats, however, MAP increased by 14.1 +/- 1.3 mm Hg and PG increased within 10 min to reach a new steady state that was approximately 10 mg/dL higher than the preinfusion PG (P < .001). Mean PG over 60 min was significantly higher during AII infusion compared to saline infusion (80.5 +/- 1 v 70.4 +/- 0.7 mg/dL, P = .0354 by repeated measures ANOVA), and plasma insulin was approximately 20 mu U/mL higher during AII infusion (66 +/- 6 v 46 +/- 2, P = .0023). Glucose turnover rate, determined isotopically from a single bolus injection of [3-(3)H]-glucose given after the new steady state was established, was no different in the two groups. However, insulin sensitivity declined by 33% in the AII-infused rats (0.116 +/- 0.012 v 0.152 +/- 0.016, P = .047). It is concluded that AII infusion at a dose that results in a physiologic increase in blood pressure acutely induces a new steady state of insulin resistance, from either reduced glucose utilization due to vasoconstriction or increased hepatic glucose production. The result of the insulin resistance is an increase in plasma glucose levels, and hyperinsulinemia, which overcomes insulin resistance, returning glucose turnover to normal despite continuing AII infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin resistance in spontaneously hypertensive rats. Difference in interpretation based on insulin infusion rate or on plasma insulin in glucose clamp studies.

Isotopic glucose turnover was measured during euglycemic glucose clamp studies in spontaneously hypertensive rats and normotensive Wistar-Kyoto control rats, under pentobarbital sodium anesthesia. At an insulin infusion rate of 4 mU.kg-1 x min-1, glucose disposal rate and hepatic glucose output were not significantly different in either group, at a plasma glucose of 6 mM. However, steady-state plasma insulin levels in spontaneously hypertensive rats were more than double those in Wistar-Kyoto rats (2.34 +/- 0.16 [mean +/- SE] vs. 6.78 +/- 0.58 nM, P < 0.00005, n = 7 in each group). Additional studies (n = 30 in spontaneously hypertensive rats and n = 32 in Wistar-Kyoto control rats) were conducted to match insulin levels in the two groups over a wide range, using infusion rates of 0.5-10 mU.kg-1 x min-1. When the responses of glucose disposal rate and hepatic glucose output to insulin were analyzed from the standpoint of plasma insulin levels, a highly significant difference was seen in the response to insulin between spontaneously hypertensive rats and Wistar-Kyoto rats, for both glucose disposal rate (P < 0.000005) and hepatic glucose output (P = 0.00007). The slope of the regression line for glucose disposal rate versus plasma insulin was lower in spontaneously hypertensive rats compared with Wistar-Kyoto rats (2.652 vs. 4.864, P < 0.001), indicating that insulin stimulation of glucose disposal rate was reduced by 50% in spontaneously hypertensive rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline lipase in rat intestinal mucosa: physiological parameters.

The present studies describe an alkaline active lipase in the rat intestinal mucosa. Alkaline lipase activity was determined using a glyceryl tri[14C]oleate emulsion at pH 8.0. Subcellular fractions were prepared from mucosal homogenates by differential centrifugation. Cells from villus to crypt were sequentially released using citrate and EDTA buffer. The enzyme was found to be most active in the proximal quarter of the intestine and in villus tips. Most of the activity was found in the cytosolic fraction. Bile salts stimulated the enzyme activity threefold. The presence of both Ca2+ and taurocholate was essential for optimal activity. Mucosal activity was greatly reduced on intraduodenal glyceryl trioleate infusion. Activity was restored when phosphatidylcholine was added to the glyceryl trioleate infusion. The fact that mucosal lipase has its greatest activity in the villus tips of the proximal intestine and in cytosol suggests that it may play an important role in mucosal glyceryltrioleate metabolism.

Chronic malnutrition impairs insulin sensitivity through both receptor and postreceptor defects in rats with mild streptozocin diabetes.

The effect of coexisting chronic malnutrition on insulin sensitivity in mild diabetes was studied in rats. Food intake was restricted over an 8-week period to 50% of ad libitum intake by pair-feeding 4-week-old littermate rats injected with streptozocin ([STZ] 40 mg/kg intraperitoneally) at 8 weeks. Significantly greater glucose intolerance and hypoinsulinemia were seen in response to a glucose load in the malnourished diabetic group (P < .00005), suggesting that chronic malnutrition significantly accentuated beta-cell dysfunction from STZ. Significantly greater insulin resistance was also seen, with the percentage rate constant for glucose disappearance after a bolus of insulin being markedly impaired (P < .0001) in the malnourished diabetic group (1.5% +/- 0.2% x min-1 [SE]) compared with ad libitum-fed diabetic and normal rats (3.6% +/- 0.6% x min-1 and 4.7% +/- 0.7% x min-1, respectively; P < .01). Insulin binding to receptor in liver plasma membranes was altered by malnutrition (P < .00005), with the affinity of binding being significantly reduced compared with that of ad libitum-fed diabetic controls (P < .0001) at both the high-affinity site (3.9 +/- 0.2 v 13.7 +/- 3.3 x 10(-7) x mol/L-1) and low-affinity site (2.7 +/- 0.4 v 20.1 +/- 3.8 x 10(-5) x mol/L-1). During a constant glucose and insulin infusion (1.67 mU/kg/min), glucose clearance was significantly lower in malnourished rats (5.3 +/- 0.7 v 8.6 +/- 1.7 mL/gk/min, P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in insulin sensitivity from stress during repetitive sampling in anesthetized rats.

The effect of repetitive sampling on insulin sensitivity was studied in anesthetized rats. During glucose clamp studies, glucose disposal decreased from 9.3 +/- 0.9 (SE) to 6.5 +/- 1.1 mg.kg-1.min-1 (P less than 0.05), and hepatic glucose output (HGO) increased from 1.2 +/- 0.8 to 2.4 +/- 1.1 mg.kg-1.min-1 (P less than 0.05) after a cumulative blood loss of 9 ml/kg. After a loss of 15 ml/kg, HGO rose further to 4.7 +/- 1.6 mg.kg-1.min-1 (P less than 0.05). During repetitive sampling under identical conditions, plasma adrenocorticotropic hormone (ACTH) increased, despite simultaneous saline infusion, from 68 +/- 11 to 102 +/- 15 pg/ml (P less than 0.05) with a loss of 8 ml/kg, while plasma insulin increased from 39 +/- 7 to 124 +/- 20 mU/l (P less than 0.01) with a loss of 10 ml/kg. Thereafter, ACTH and insulin rose progressively. Plasma corticosterone closely followed the pattern of the ACTH response, indicating that the stress of cumulative blood loss had a significant effect on adrenal steroid production. Increases in ACTH were retarded by reduced volume loss and accelerated by increased loss. It is concluded that stress from blood loss greater than 7 ml/kg may be a source of error in the evaluation of glucose turnover and insulin sensitivity during clamp experiments in rats.

Effect of chronic undernutrition on hepatocyte insulin receptors in rats.

The effect of moderate chronic undernutrition on insulin receptors was studied in male rats, pair-fed 60% of the daily food intake of ad libitum-fed littermates, for 8 weeks. Body weights of undernourished rats were consistently found to be 35% to 40% less than control littermates, with no period of growth arrest at any point in the 8-week study. The binding-displacement curves of labeled insulin to hepatocyte receptors in the two groups in the presence of unlabeled insulin were significantly different (P = .0258 after repeated measures ANOVA). Significantly lower binding was observed in hepatocytes from the undernourished group (P less than .01) at all unlabeled insulin concentrations less than 20 nmol/L. In the absence of any unlabeled insulin, specific binding was reduced from 8.8% +/- 0.7%, (mean +/- SE) in controls, to 7.4% +/- 0.3% in undernourished rats (P less than .01). Half-maximal specific hormone binding to hepatocytes was achieved at a free insulin concentration of 362 nmol/L in the control group, compared with 447 nmol/L in the undernourished group, reflecting an increase of approximately 20%. The hypoglycemic response to intravenous insulin (0.1 U/kg body weight) was tested in a parallel experiment involving seven paired littermate rats, and found to be significantly impaired in the undernourished group (P = .0041 by repeated measures ANOVA).(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic undernutrition accentuates insulin deficiency in rats with mild streptozocin-induced diabetes.

The effect of chronic undernutrition on coexisting diabetes mellitus was studied in pair-fed littermate rats with mild streptozocin-induced diabetes. They were either fed ad libitum or 50% food restricted for 9 wk. Undernourished diabetic rats, in which weight gain was reduced by 40%, had significantly higher glucose intolerance (mean +/- SE, fractional rate of glucose disappearance during glucose tolerance test [Kgtt] 1.77 +/- 0.16) than diabetic littermates fed ad libitum (2.33 +/- 0.21, P less than 0.05) or nondiabetic controls (3.8 +/- 0.7, P less than 0.01). The total area under the insulin response curve was significantly reduced in both groups of diabetic rats, but the undernourished group showed a 45% greater reduction in overall secretion than normally nourished diabetic littermates (21.3 +/- 2.7 vs. 39.4 +/- 6.3 nM.min in the diabetic group, P less than 0.01, and 65.7 +/- 6.1 nM.min in controls). There was also a marked reduction in first-phase insulin secretion in the undernourished rats (4.75 +/- 0.24 vs. 9.84 +/- 1.36 nM, P less than 0.05, and 14.3 +/- 1.8 nM, P less than 0.01, respectively, in normally nourished diabetic littermates and controls). After refeeding, a significant improvement occurred in Kgtt (to 2.67 +/- 0.24, P less than 0.01) and first-phase insulin secretion (to 9.69 +/- 1.65 nM, P less than 0.05). The postrefeeding values were not different from those in the normally nourished diabetic littermates, indicating that the effect was fully reversible and solely attributable to undernutrition rather than to enhanced beta-cell cytotoxicity from streptozocin.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and partial characterization of intestinal acid lipase.

Intestinal acid lipase is an enzyme whose greatest specific activity is localized to the villus tips of the proximal intestine (Rao, R.H. and Mansbach, C.M. (1990) Biochim. Biophys. Acta 1043, 273-280). This suggests that it plays a role in the processing of dietary lipids. We purified the enzyme in order to better characterize it. Acid lipase was isolated from intestinal mucosa of rats by a combination of ammonium sulfate precipitation, butanol extraction and chromatography on DEAE Bio-Gel, CM Bio-Gel and Sephadex G-75. This resulted in a single protein of Mr 53,700 on SDS-polyacrylamide gel electrophoresis. The isolation scheme produced a 3344-fold purification resulting in an enzyme whose specific activity was 801 mumols/min per mg protein. The yield was 50%. The purified enzyme was stimulated (20-fold) by the addition of tauro- or glycocholate but no other conjugated bile acid. A sharp peak in activity occurred at pH 5.6. The pI of the enzyme was 6.2. The reaction products produced under prolonged incubation suggested that monoacylglycerol was not hydrolyzed since an overabundance of monoacylglycerol was found with respect to the amount of fatty acid produced. These results suggested that intestinal acid lipase is potentially important in the metabolism of dietary lipids. Its proportionate role awaits further documentation.