• Samples for blood, plasma and serum were collected in glass tubes (heparinized for plasma) kept on ice, and then centrifuged (4^oC, 10 min, 2,000 rpm) to obtain the plasma or serum fraction.

• Determined using GLUCO GOD/POD/PAP Test of Galenopharm according to Trinder for the determination of the glucose concentration of blood [10Trinder P. Determination of blood glucose using an oxidase-peroxidase system with a non-carcinogenic chromogen. J Clin Pathol 1969; 22(2): 158-61.
  [http://dx.doi.org/10.1136/jcp.22.2.158] [PMID: 5776547] ].

• Triglyceride concentrations of serum and liver homogenates were tested by the colorimetric assay of Biggs et al. [11Biggs HG, Erikson JM, Moorehead WR. A manual colormetric assay of triglycerides in serum. Clin Chem 1975; 21(3): 437-41.
  [PMID: 1112059] ]. Duncombe’s [12Duncombe WG. The colorimetric micro-determination of long-chain fatty acids. Biochem J 1963; 88(1): 7-10.
  [http://dx.doi.org/10.1042/bj0880007] [PMID: 16749030] ] colorimetric test was used for the determination of FFA contents of the same samples.

• Concentration of plasma was determined using Murphy’s Competitive Protein Binding Assay [13Murphy BEP. Some studies of the protein-binding of steroids and their application to the routine micro and ultramicro measurement of various steroids in body fluids by competitive protein-binding radioassay. J Clin Endocrinol Metab 1967; 27(7): 973-90.
  [http://dx.doi.org/10.1210/jcem-27-7-973] [PMID: 4291524] ], with an inter-assay coefficient of variance of 2%.

• The liver samples were stored in tightly wrapped aluminum foil, frozen immediately in liquid nitrogen and stored at -70^oC until analyzed. Prior to analysis the samples were thawed on ice, homogenized in 3.0 ml of cold Sorensen-phosphate buffer (pH 7.4, 0.15 mol).

• The protein contents of liver, pancreas homogenates and small intestinal content samples were determined by Lowry’s protein assay [14Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193(1): 265-75.
  [PMID: 14907713] ].

• Samples were assayed by the method of Good at al. [15Good CA, Kramer H, Somogyi M. The determination of glycogen. J Biol Chem 1933; 100: 485-91.], and by Trinder’s method [16Trinder P. Determination of blood glucose using an oxidase-peroxidase system with a non-carcinogenic chromogen. J Clin Pathol 1969; 22(2): 158-61.
  [http://dx.doi.org/10.1136/jcp.22.2.158] [PMID: 5776547] ].

• The Lower+upper Duodenal (DP) and Gastric+Splenic (GSP) Pancreatic lobes were separeted (Fig. 1) and homogenized with Potter-Elvehjem homogenizer in ice-cold saline. The homogenized samples were centrifuged (4 ^oC, 10 min, 2,000 rpm) and the supernatants were stored at -70^o C until further analysis.

Fig. (1) Diagrammatic presentation of the separation of the Duodenal (DP) and Gastric+Splenic (GSP) Pancreatic regions.

• Pancreatic tissue was excised from the GSP tissue near the spleen and immediately homogenized in a stop solution. Pancreatic RNA was prepared by standard guanidiumthiocyanate/phenol/ethanol extraction and proinsulin-1 mRNA was determined by Northern blotting [17Sambrook J, Frittsch EF, Maniatis T. Molecular Cloning. Laboratory Manual. Cold Spring Harbor Laboratory, 1989 New York].

• Plasma and pancreas tissue PP concentrations were determined as described by Gettys et al. [18Tom W. Gettys, Richard Garcia, Karin Savage, David C. Whitcomb, Shuji Kanayama, and Ian L. Taylor insulin-sparing effects of pancreatic polypeptide in congenitally obese rodents. PancreasVol 1991; 6(1): 46-53.
  [http://dx.doi.org/10.1097/00006676-199101000-00007] ].

Pancreatic insulin and glucagon concentrations were tested following acid ethanol extracts (0.18N HCl; 75% ethanol) of the homogenized pancreatic tissue.

The insulin concentrations of plasma and tissue extracts were determined by R-2 RIA Kit (MTA, Hungary) using rat insulin standard (Novo, Denmark). The sensitivity of the RIA procedure was 35±16pmol/l. The inter-assay coefficient of variance was 8.3%.

The glucagon concentrations of plasma and tissue extract were determined by RIA, using C-terminal specific antibodies [19 Phedebas Amylase Tests, Pharmacea Diagnostics AB, Upsala, Sweden]. The sensitivity of the assay was 3.6±1.2 pmol/l. The inter-assay coefficient of variance was 7.3%.

The small intestinal contents were diluted with distilled water and the samples were centrifuged (4^oC, 10 min, 2,000 rpm) and the supernatants stored at -70^oC until the test of the hydrolases.

Amylase [19 Phedebas Amylase Tests, Pharmacea Diagnostics AB, Upsala, Sweden], lipase [20Schön H, Rassler B, Henning N. Über die untersuchung der exretorischen pancreasfunction. Methoden zur activatsbestimmung von Trypsin, chymotrypsin, carboxipeptidase, lipase und diastase. Klin Wochenschr 1961; 39: 217-22.
[PMID: 13748352] ] and trypsin [21Szabó J, Ribiczey P, Fodor E, Tenk I. Influence of environmental factors on digestive enzymes and plasma corticosterone in rats. I. Diet. Acta Vet Acad Sci Hung 1981; 29(4): 399-422.
[PMID: 7053015] ] activities were tested as previously described. For the trypsin test the pancreas proteolytic zymogens of the samples were activated by enterokinase (Sigma Chemical Co., Louis, MO, USA) at 37^oC for 60 min in Na-citrate buffer (pH 5.6) and the trypsin activity was determined photometrically using Boehringer colorimetric test-combination (Cat. No.125024).

The blood glucose levels (Table 3) of the following 3 groups were significantly different from each other: 1. CEBDF positive control (7.16±0.57 mM/l), 2. POLYCOSE® (6.27± 0.25 mM/l), and Casein diets (6.15±0.66 mM/l), 3. Safflower oil and the negative CEF control group (4,76±0.46 mM/l; 5,31±0.70 mM/l). All of these values were inside the normal clinical range for fasted and fed rats, e.g. “the average fasted and fed postprandial glucose level of normal Wistar rats was 3.95 ± 1.31 mM/l and 5.65 ± 1.63 mM/l; the 95% upper limit was 6.2 mM/L and 7.9 mM/L respectively” [31 Wang, Yang Y, Xiang X, Zhu Y, Men J, He M. Estimation of the normal range of blood glucose in rats. 2010. Wei Sheng Yan Jiu. 2010; 39(2):133-7, 142. [Article in Chinese]].

Table 3
Effects of macronutrients on blood Glucose, plasma triglycerides and serum free fatty acids.

According to Byers and Friedman [32Byers SO, Friedman M. Site of origin of plasma triglyceride. Am J Physiol 1960; 198: 629-31.
[http://dx.doi.org/10.1152/ajplegacy.1960.198.3.629] [PMID: 13806614] ] the liver is the major source of plasma triglyceride. In our CEF animal model fasting and the feeding of casein as a single nutrient, resulted in significantly smaller triglyceride levels then it was in the other treatment groups (Table 3). Higher triglyceride concentrations were detected in rats fed POLYCOSE®, safflower oil and the CEBDF diets.

Contrary to our results, Ettinger et al. [33Ettinger WH, Klinefelter HF, Kwiterovitch PO. Effect of short-term, low-dose corticosteroids on plasma lipoprotein lipids. Atherosclerosis 1987; 63(2-3): 167-72.
[http://dx.doi.org/10.1016/0021-9150(87)90117-1] [PMID: 3493784] ] reported that low-doses of corticosteroids markedly increased the LDL cholesterol, and triglycerides but not the HDL cholesterol level.

Since the triglyceride concentration of plasma is an important risk factor for cardiovascular disorders, our data show that casein as a single nutrient may have beneficial effects, however this effect also can be the result of the lack of lipids and carbohydrates from the diet of the casein fed group.

Serum FFAs (Table 3) are mobilized from the adipose and peripheral tissues and utilized by the liver, muscle, myocardium and other tissues [34Gordon RS Jr. Unesterified fatty acid in human blood plasma. II. The transport function of unesterified fatty acid. J Clin Invest 1957; 36(6 Part 1): 810-5.
[http://dx.doi.org/10.1172/JCI103486] [PMID: 13439020] ]. It is generally believed that elevated plasma FFAs play significant role in the pathogenesis of insulin resistance [35Roden M, Price TB, Perseghin G, et al. Mechanism of free fatty acid-induced insulin resistance in humans. J Clin Invest 1996; 97(12): 2859-65.
[http://dx.doi.org/10.1172/JCI118742] [PMID: 8675698] ].

In our CEF animal model, only the safflower oil increased significantly the serum FFA levels above the values found in the other treatment groups (Table 3).

It is interesting that the serum FFA level of CEF and CEBDF animals were practically identical. Because in the CEBDF the plasma insulin level was very high, we may assume that there was no FFA mobilization and catabolic amino acid disposal was minimal. This suggests that contrary to the fasted rats, the plasma FFA of CEBDF rats did not come from the lipid stores, but because the animals were in an absorptive metabolic state, FFA more likely originated from the safflower oil content of the diet and from FFA synthetized by the liver from the absorbed glucose. This assumption is supported by calculating the plasma FFA level of the dietary treatment groups. The control diet contained 75% polycose and 5% safflower oil. Adding up the 75% of the serum FFA (0.41 mM/l) of POLYCOSE® group (0.55 mM/l) and 5% of the serum FFA (0.04 mM/l) of safflower group (0.87 mM/l), we arrive at almost exactly the serum FFA (0.46 mM/l) concentration of CEBDF rats (0.47 mM/l).

We conclude that in the CEF animal model the blood glucose level remained in the clinically acceptable range. Among the tested 33 parameters the blood glucose showed correlation only with the insulin level of plasma (Fig. 2). The plasma triglyceride negatively correlated with plasma corticosterone and the lipase content of DP tissue. The significant correlations between the plasma triglyceride and corticosterone concentrations may have importance regarding the acute phase response during stress and is in accord with the observation of Chait et al. [36Chait A, Brunzell JD, Johnson DG, et al. Reduction of plasma triglyceride concentration by acute stress in man. Metabolism 1979; 28(5): 553-61.
[http://dx.doi.org/10.1016/0026-0495(79)90197-5] [PMID: 221783] ]; they found that different forms of stress resulted in acute reduction of plasma triglyceride concentrations. The serum FFA was significantly increased only by the safflower oil diet. The correlation analysis (Fig. 2) indicates significant negative relation of serum FFA to the trypsinogen content of the GSP (r=-0.878 p=0.050).

The clear understandings of the clinical significance of these correlations needs further investigation.

The highest liver protein concentration (Table 4) was in the fasted and the casein fed groups. The lowest level was in the polycose fed rats. The liver protein showed negative relationships with the I/G ration of plasma (r=-0.917 p=0.028), liver FFA (r=-0.910 p=0.032), proinsulin-1 mRNA in GSP (r=-0.900 p=0.032), glucagon in the DP tissue (r=-0.932 p=0.021) and amylase activity in the small intestinal content (r= 0.894 p=0.041) (Fig. 2).

Table 4
Effect of nutrients on liver (Li) protein, triglyceride, FFA, glucose and glycogen contents.

According to Delgado et al. [37Delgado TC, Pinheiro D, Caldeira M, et al. Sources of hepatic triglyceride accumulation during high-fat feeding in the healthy rat. NMR Biomed 2009; 22(3): 310-7.
[http://dx.doi.org/10.1002/nbm.1327] [PMID: 19012281] ], the Hepatic Triglyceride (HTG) concentrations are influenced by dietary lipid concentrations in healthy rats, although the de novo lipogenesis contribution to HTG content is suppressed by a high-fat diet. This effect is insufficient to prevent overall increases in HTG concentrations.

In our CEF animal model the safflower oil and the POLYCOSE® feeding resulted in significantly higher HTG concentrations (Table 4) than it was in the fasted, casein and CEBDF groups.

This effect of safflower oil is in accord with the result of Neschen et al. [38Neschen S, Moore I, Regittnig W, et al. Contrasting effects of fish oil and safflower oil on hepatic peroxisomal and tissue lipid content. Am J Physiol Endocrinol Metab 2002; 282(2): E395-401.
[http://dx.doi.org/10.1152/ajpendo.00414.2001] [PMID: 11788372] ]. In accordance with the high plasma insulin concentration, POLYCOSE® appears to have stimulated the de novo HTG synthesis. In the control group where safflower oil, POLYCOSE® and casein were included in the diet the HTG concentration was significantly lower (87.9±22.8 mg/g protein) than either in rats on the safflower oil or POLYCOSE® diets, suggesting that the lipid accumulation in the liver is suppressed by casein.

This suggests that the presence or absence of casein, a high quality protein source, may be an important factor in the lipid VLDL transport from the liver to the peripheral tissues, likely through changes in lipoprotein synthesis and transport. This speculation is in accordance with results of Pfueuffer and Barth [39Pfeuffer M, Barth CA. Modulation of very low-density lipoprotein secretion by dietary protein is age-dependent in rats. Ann Nutr Metab 1986; 30(5): 281-8.
[http://dx.doi.org/10.1159/000177205] [PMID: 3752927] ], that dietary casein may increase the influx of lipoprotein cholesterol and triglycerides into the plasma compartment. The triglyceride content of liver was in negative correlation with the trypsinogen content of DP (r=-0.902 p=0.036) and GSP (r=-0.963 p=0.009) tissues (Fig. 2). These relationships are unclear and need further investigation.

The liver FFA may originates from three sources: from the FFA content of the circulating blood, the chylomicron remnant uptake and the de novo lipogenesis in the liver [40Mashek DG. Hepatic fatty acid trafficking: multiple forks in the road. Adv Nutr 2013; 4(6): 697-710.
[http://dx.doi.org/10.3945/an.113.004648] [PMID: 24228201] ]. In addition to these three, intracellular pathways, triglyceride hydrolysis may also contribute to the FFA concentration in the liver.

Relative to the CEF negative control group, each diet increased the FFA content of the liver (Table 4). The POLYCOSE® doubled the liver FFA level, suggesting that the de novo lipid synthesis from absorbed glucose is significant.

The second most effective FFA stimulator was the safflower oil. This may be the result of increased liver chylomicron remnant uptake due to lymphatic transport of safflower oil into the systemic circulation. The increasing effect of casein on the liver FFA is only 19% suggesting that a smaller proportion of the absorbed amino acids were used for lipid synthesis. Compared to the CEBDF control, POLYCOSE® increased while casein and fasting decreased significantly the liver FFA concentration (Table 4, Fig. 3). There were negative correlations between the liver protein and liver FFA, and the trypsinogen contents of both pancreatic lobes (Fig. 2).

Fig. (3) Heatmap indicating diet effects on the parameters measured as very positive +1.5 to very negative -1.5. The two way claster analysis indicate the closeness of parameters (vertical) and the treatment groups (horizontal) to each others.

Between the FFA and triglyceride contents of liver a polynomial relationship can be detected (y= -0.0095x2 + 3.152x-81.51; R² = 0.968), where y represents the FFA and x is for triglyceride suggesting that the real relationship between the liver FFA and triglyceride levels is not linear (Fig. 4).

Fig. (4) Polynomial relationship between the triglyceride and FFA concentrations of liver.

Feeding POLYCOSE® and the CEBDF diets induced the highest liver glucose and glycogen concentrations (liver glucose: 208.0±20.1, 247.2±26.5; glycogen: 68.3±44.2, 88.1± 44.0 mg/g protein respectively) but there were no significant differences in liver glycogen among the casein, safflower oil and fasted (5.7± 3.2, 6.6±1.8, 4.0±0.7, 1.0±0.9 mg/g protein respectively) groups (Table 4). Among the tested 33 parameters the feed intake, energy intake, plasma I/G, small intestinal amylase activity and glucagon in the DP tissue correlated positively and significantly with the glucose and glycogen contents of liver (Fig. 2).

Compared to the negative CEF control animals, the CEBDF positive control, the casein and the POLYCOSE® diets significantly increased the plasma insulin concentration (Table 5, Fig. 4). The safflower oil depressed the plasma insulin level, but this difference (-29%) was not statistically significant.

Table 5
Macronutrient effects on plasma insulin, glucagon a pancreatic polypeptisde (PP).

Plasma insulin positively correlated with feed intake (r=0.899; p=0.038), bodyweight change (R=0.887; p=0.044), blood glucose (r=0.970; p=0.006) and negatively to plasma PP (r=-0.840; p=0.075) concentration (Fig. 2), however polynomial regression analysis shows Fig. (5) very close relation between plasma insulin and PP (R²=0.991), suggesting that plasma PP is the negative partner of insulin in the control mechanism/s of metabolism.

Fig. (5) There is a highly significant polynomial relationship between plasma insulin and PP.

It is interesting that the insulin and glucagon levels of plasma did not show significant relationship with each other. This result is in agreement with the suggestion of Gerich at al [41Gerich JE, Charles MA, Grodsky GM. Characterization of the effects of arginine and glucose on glucagon and insulin release from the perfused rat pancreas. J Clin Invest 1974; 54(4): 833-41.
[http://dx.doi.org/10.1172/JCI107823] [PMID: 4430717] ]. that ”endogenous insulin per se has little or no effect on Immunoreactive Glucagon (IRG) secretion and that endogenous glucagon does not appreciably affect pancreatic beta cell function”.

On the basis of these data, it is clear that both the casein and POLYCOSE® diets stimulated the insulin secretion significantly and safflower oil slightly but not significantly depressed it (-29%). These data are in agreement with the results of Gerich at al. [42Gerich JE, Charles MA, Grodsky GM. Characterization of the effects of arginine and glucose on glucagon and insulin release from the perfused rat pancreas. J Clin Invest 1974; 54(4): 833-41.
[http://dx.doi.org/10.1172/JCI107823] [PMID: 4430717] ], that both arginine and glucose alone and in combination with each other stimulated the insulin secretion of pancreas probably through different mechanisms. The 29% decrease (relative to CEF) of insulin concentration in the safflower oil fed group may be due to prolonged high plasma FFA concentrations, which may inhibit insulin secretion. This is in agreement with the result of Kashyap et al. [43Kashyap S, Belfort R, Gastaldelli A, et al. A sustained increase in plasma free fatty acids impairs insulin secretion in nondiabetic subjects genetically predisposed to develop type 2 diabetes. Diabetes 2003; 52(10): 2461-74.
[http://dx.doi.org/10.2337/diabetes.52.10.2461] [PMID: 14514628] ] that prolonged lipid exposure impairs beta cell function in both in vitro and in vivo animal studies.

To be noted, when the plasma insulin level of CEF rats (0.41 ng/ml) is deducted from the sum of the plasma insulin in the single nutrient groups (casein 1.26 + POLYCOSE® 1.79 + safflower oil 0.29 = 3.34 ng/ml), we get almost exactly the insulin concentration (3.34 - 0.41 = 2.93) observed in rats on the CEBDF positive control diet (2.90), suggesting that the combined effect of casein (amino acids), POLYCOSE® (glucose) and safflower oil on insulin secretion of the pancreas is possibly cumulative but not synergistic.

Our better understanding of how nutrients affect glucagon secretion was brought to light in the 1970s when Unger et al. [44Unger RH, Eisentraut AM, McCall MS, Keller S, Lanz HC, Madison LL. Glucagon antibodies and their use for immunoassay for glucagon. Proc Soc Exp Biol Med 1959; 102(10): 621-3. 3
[http://dx.doi.org/10.3181/00379727-102-25338] ] developed a specific glucagon radioimmunoassay. It is generally accepted that glucose has a direct inhibitory effect on alpha cells [45Vieira E, Salehi A, Gylfe E. Glucose inhibits glucagon secretion by a direct effect on mouse pancreatic alpha cells. Diabetologia 2007; 50(2): 370-9.
[http://dx.doi.org/10.1007/s00125-006-0511-1] [PMID: 17136393] ] and protein (soybean and casein) rich diet increase the arginine stimulated glucagon secretion [46Noseda G, Fragiacomo C, Gatti E, Descovich G, Sirtori CR. Glucagon release following experimental diets in man: effects of soybean and casein enriched diets. Pharmacol Res Commun 1982; 14(9): 867-78.
[http://dx.doi.org/10.1016/S0031-6989(82)80011-8] [PMID: 6757973] ]. Further the type of protein may influence the molecular weight pattern of glucagon in plasma. The effect of lipids (free fatty acids) is unclear since both inhibitory and stimulatory effects have been reported under thermo-neutral conditions [47Edwards JC, Howell SL, Taylor KW. Fatty acids as regulators of glucagon secretion. Nature 1969; 224(5221): 808-9.
[http://dx.doi.org/10.1038/224808a0] [PMID: 4902928] , 48Gilon P, Rui C-X. Bao Khanh Lai, Hee-Young Chae, and Ana Gomez-Ruiz Physiological and pathophysiological control of glucagon secretion by pancreatic alpha cells. In: Islets of Langerhans, 2nd ed. 2014, pp 1-73. ].

Under cold stress the negative CEF control and the positive CEBDF control diet induced practically identical plasma glucagon levels. POLYCOSE® and safflower oil significantly decreased while casein significanlty increased the plasma glucagon concentration (Table 4, Fig. 3). The low blood glucose level is a stimulating factor for glucagon secretion. This stimulating effect of low blood glucose level was diminished in the safflower oil fed group. It is generally believed that the insulin/glucagon ratio are regulated in a yin-yang relationship. In our CEF animal model this antagonistic effect is not apparent. The plasma glucagon correlated only with the plasma corticosterone concentration (r=0.890; P=0.043) (Fig. 2) and no correlation was found between the plasma glucagon and plasma insulin or blood glucose concentrations. Our results suggest that glucagon may have role in increaseasing substrate availabilty for gluconeogenesis to assure the carbohydrate refill of liver in stress situations.

The I/G ratio (Table 5) was 2.7 in the CEF animals and it was not significantly different from the casein and the safflower oil groups; however casein induced higher (3.8) and the safflower oil fed group showed lower (2.0) I/G ratios. The feeding of POLYCOSE® or CEBDF diets resulted in very high I/G ratio (1028, 634 respectively). According to the correlation analysis the feed intake, the protein and glycogen contents of the liver, the proinsulin mRNA content of the GSP tissue, the glucagon content of the DP tissue and the amylase activity in the small intestinal content significantly correlated with the plasma I/G ratios (Fig. 2). The question as to whether which of these correlations are causal links, with similar signalling modes needs further investigation.

The effect of macronutrients on plasma PP concentrations (Table 5 and Fig. 2) were practically opposite to the plasma insulin concentrations. The linear correlation between these two parameters was negative (r =-0.840; p=0.074), and a close polynomial relation between these two parameters (R²=0.990) can be detected (Fig. 5).

This finding is in agreement with the data of Park et al. [49Park HJ, Lee YL, Kwon HY. Effects of pancreatic polypeptide on insulin action in exocrine secretion of isolated rat pancreas. J Physiol 1993; 463: 421-9.
[http://dx.doi.org/10.1113/jphysiol.1993.sp019602] [PMID: 7504106] ], who showed that rat PP completely abolished the potentiating effect of exogenous insulin and glucose on the CCK stimulated flow rate and amylase output of the pancreas. They concluded that insulin is an important stimulatory hormone of pancreatic exocrine secretion, and PP exerts an inhibitory role on it by modulating insulin’s action. Fasting and safflower oil induced higher plasma PP concentration than in the positive control, POLYCOSE® and casein groups (Table 5).

Alphons et al. [50de Jong AJL, Hopman WPM, Jansen JBMJ, Lamers CBHW. Effect of medium-chain triglycerides and long-chain triglycerides on plasma pancreatic polypeptide secretion in man. Regul Pept 1985; 11(1): 77-81.
[http://dx.doi.org/10.1016/0167-0115(85)90033-3] [PMID: 4011957] ] found in human volunteers that the “Ingestion of LCT (long chain triglycerides) resulted in significant increases in plasma PP from 33 ± 7 to 55 ± 7 pmol/l (P < 0.01), whereas both MCT (medium chain triglycerides) and saline did not significantly increase plasma PP concentrations.” They concluded that the secretion of PP in response to triglycerides is dependent on the chain length of the fatty acids. These differences may also be due to the lymphatic transport of long chain fatty acids into the systemic circulation while MCT are transported directly to the liver via the portal vein, thereby undergoing very different metabolic pathways.

Based on these findings, we suggest that PP plays a significant indirect role in the control of blood glucose levels by inhibiting insulin synthesis and/or secretion from the GS pancreas. This assumption is supported by the negative correlations of plasma PP with plasma insulin (r = -0.840; p=0.074) and the insulin content of the GSP tissue (r = -0.940; p=0.015) (Fig. 2).

It has been shown that the timing of a meal [51English J, Hampton S, Dunne M, Marks V. The effect of timing of a standard meal on plasma cortisol levels in man. Horm Metab Res 1982; 14(2): 106.
[http://dx.doi.org/10.1055/s-2007-1018939] [PMID: 7200068] ] and the nutrient composition of the meal [52Szabo J, Fodor E, Koranyi L, Bruckner G. Effect of nutrients on plasma corticosteroid concentration in cold-stressed rats. Ann Nutr Metab 1993; 37(2): 53-61.
[http://dx.doi.org/10.1159/000177750] [PMID: 8517634] ] influence plasma corticosteroid concentrations. The physiological mechanism(s) responsible for these alteration is/are not clear. However Szabo and Bruckner [53Szabo J, Bruckner G. ATP potentioal of nutrients may regulate plasma corticosteroid concentration: a hypothesis. J Nutr Biochem 1995; 6: 12-20.
[http://dx.doi.org/10.1016/0955-2863(94)00002-4] ] proposed a hypothesis which suggests that the ”ATP Potential” of nutrients, by influencing the energy metabolism of the cells, is in a negative correlation with the plasma corticosteroid concentration”. The ATP Potential is defined as the net number of moles of ATP generated from a mole of nutrient per the number of TCA cycles used for its metabolism.

When compared to the CEF negative control, safflower oil, POLYCOSE® and the CEBDF positive control diets significantly decreased plasma corticosterone concentration, while casein increased it (Table 5, Fig. 3). The decreased plasma corticosteroid concentration in the POLYCOSE® and safflower oil fed group supports our ATP Potential hypothesis, but the high corticosteroid concentration induced by the casein diet suprisingly did not fit our model. The plasma corticosteron concentration negatively correlated with plasma triglyceride and positively to plasma glucagon and lipase content of DP tissue (Fig. 2).

These parameters were determined to study the effect(s) of single macro nutrient on the RNA and proinsulin-1 mRNA contents of the pancreas and to ascertain the relationships among the RNA, proinsulin-1 mRNA, pancreatic hormones (Table 6) and hydrolases (Table 7).

Table 6
Effect(s) of single macro nutrient on RNA, proinsulin-1mRNA and hormone contents of the pancreas.

Table 7
Effect of macronutrients on pancreatic hydrolases.

The lowest RNA and proinsulin-1 mRNA concentration was in the pancreas of the fasted animals. The different nutrients increased dramatically the concentrations of these parameters. The difference however between the negative and positive controls was ten to thirty times more (600 to 2962%) than Gidding et al. [54Giddings SJ, Chirgwin J, Permutt MA. The effects of fasting and feeding on preproinsulin messenger RNA in rats. J Clin Invest 1981; 67(4): 952-60.
[http://dx.doi.org/10.1172/JCI110145] [PMID: 6162861] ] reported (66%) in rats housed in a thermoneutral environment. This dramatic difference between the fed and fasted animals and among the different single nutrient fed rats suggests that in the cold exposed fasted animal model the metabolic effects of nutrients are magnified and this model is a useful tool for investigating nutrient metabolomic relationships.

The nutrients induced changes of proinsulin-1 mRNA (Fig. 3) correlated with the amylase in SIC (r=0.940; p=0.015), plasma I/G (r=0.940; p=0.017), liver glucose (r=0.900; p=0.038), glucagon in DP (r=0.900; p=0.036) and negatively with liver protein (r=-0.900; p=0.038) (Fig. 2).

The data depicted in Table 6, show that there were significant differences in the insulin concentrations of DP (lower + upper duodenal lobes) and GSP (gastric + splenic) lobes of pancreas. This is in concert with the histological structure of the different pancreatic regions reported by Elyat [55Elayat AA, el-Naggar MM, Tahir M. An immunocytochemical and morphometric study of the rat pancreatic islets. J Anat 1995; 186(Pt 3): 629-37.
[PMID: 7559135] ] “the lower and upper duodenal regions were significantly different from the gastric and splenic regions in the mean islet diameter and mean islet volume, whereas no difference was found either between the lower and upper duodenal regions or between gastric and splenic regions”. The same authors also observed that the volume density of B cells in the upper duodenal region were significantly lower than in the lower duodenal, gastric and splenic regions. Further, our data suggest that the insulin concentrations in the two investigated areas of the pancreas do not correlate with each other, indicating that the effect of nutrients, on the synthesis and the secretion of insulin, is not the same in the two-pancreatic regions. The changes of plasma insulin concentration were significantly and positively correlated with the insulin content of GSP tissue (r = 0.880, P=0.048), but not with the insulin contents of the DP lobe (Fig. 2). The relationship between the plasma insulin and DP insulin concentration was negative and was not significant.

In the CEBDF and safflower oil groups the insulin concentration of the DP pancreas (60.54±8.01; 68.55±17.05) was low and not different statistically from each other. The insulin concentrations of DP tissues in the POLYCOSE®, casein and CEF groups were identical and significantly increased compared to the control and safflower groups (99.03±31.94, 90.96±17.59, 94.97±8.20 respectively).

In the GSP lobes the insulin concentrations were similar in the safflower, the casein and the CEF animals; only the CEBDF control and POLYCOSE® diets increased the insulin level above the fasting value (Fig. 3).

There was no correlation between the glucagon contents of the two pancreatic sections of rats fed with different diets (Table 6), suggesting that the response of pancreatic alpha cells to the nutrients was different in the two sections. It should be pointed out, that the glucagon content of DP tissue showed significant positive correlations with the parameters of carbohydrate metabolism (plasma I/G, liver glucose and glycogen, proinsulin-1 mRNA and amylase activity in the small intestinal content) whereas the glucagon content of the GSP tissue showed negative correlation with the parameters of lipid and protein digestion (lipase content of DP tissue and trypsinogen contents of DP and GSP tissues) (Fig. 2).

The PP concentrations of the DP tissues were significantly higher than the PP content of GSP tissues (Table 6). This is aligned with the PP producing cell density of the two-pancreatic areas. In both pancreatic lobes the nutrient effect followed the same pattern in the CEF, casein, safflower oil and POLYCOSE® groups but not in the CEBDF group; however, the PP content of DP tissue decreased while PP in the GSP lobe was significantly increased over the CEF group.

In the DP lobes of POLYCOSE®, casein and fasting treatment groups the PP content was significantly higher than in the CEBDF and safflower groups. Contrary to this, the highest PP content was detected in the GSP tissues of the positive CEBDF animals. In both pancreatic lobes the safflower oil induced lower PP concentration.

We did not find correlation between the plasma PP concentration and hydrolase activity in the SIC, suggesting that in this in vivo system plasma PP did not influence the secretion of pancreatic enzymes. The DP PP correlated positively to the insulin content of DP tissue and negatively to the lipase activity in the small intestinal content (Fig. 2). The GSP tissue PP content showed a significant positive correlation with the plasma insulin level and the amylase contents of DP and GSP tissues. Further experiments may shed light on the physiological significance of these correlations.

Among the tested parameters, the feed intake, the energy intake and the body weight changes showed several significant correlations proving that the CEF model is a good tool to study nutrient induced hormonal and metabolic changes and the connections between or among different metabolomic parameters. For example the negative correlation between the plasma PP and feed intake, energy intake and body weight change, as well as between the plasma insulin and PP concentrations, suggests that in the regulation of appetite and body weight, insulin and PP have antagonistic functions. These correlations are in agreement with the finding of Bloom [56Batterham RL, Le Roux CW, Cohen MA, et al. Pancreatic polypeptide reduces appetite and food intake in humans. J Clin Endocrinol Metab 2003; 88(8): 3989-92.
[http://dx.doi.org/10.1210/jc.2003-030630] [PMID: 12915697] ], who demonstrated that PP reduces appetite and food intake in humans.

We hypothesized that pancreatic hormones (especially insulin) control the synthesis of pancreatic hydrolases (especially amylase). This hypothesis was based on the facts that in streptozotocin diabetic animals the amylase contents of the pancreatic tissue and activity in the small intestinal content were significantly lower than it was in the control or insulin supplemented diabetic rats [57Szabó J, Fodor E, Vetési F, Tenk I. Pancreatic and small-intestinal digestive enzyme activities in rats with streptozotocin-diabetes. Acta Vet Acad Sci Hung 1981; 29(4): 451-69.
[PMID: 7053017] ].

Because the size, the frequency and the distribution of Langerhans islands and also the hormone producing cells in the DP and GSP tissues are significantly different, we also hypothesized that the digestive enzyme concentrations in the two pancreatic lobes would also be significantly different. We also speculated that the hydrolase activity in SIC would correlate with the hydrolase contents of the pancreatic tissues.

Contrary to our hypothesis, we did not find significant difference between the amylase contents of the two-pancreatic lobes, however the amylase level was always lower in DP than GSP tissues. The nutrient induced changes (Table 7 and Fig. 3) showed identical tendencies in both lobes suggesting that the nutrient related control mechanism(s) of amylase synthesis in the DP and GSP tissues is/are identical and not influenced significantly by the different beta cell density in the two pancreatic areas. The amylase content of GSP tissue showed significant correlations with the PP content of GSP tissues (Fig. 2).

The second important finding is that the amylase activity in the small intestinal content did not follow the nutrient induced changes of amylase content of the pancreatic tissues, showing that the secretion of amylase is under different control mechanism(s) than the enzyme synthesis in the pancreas. The stimulatory effect of POLYCOSE® on amylase secretion (activity in the small intestinal content) was clearly apparent (Table 7).

Compared to the CEF control the casein and the safflower oil diet significantly decreased the amylase content of tissue homogenates in the DP lobe and only the CEBDF diet increased it significantly, however the amylase content of the two-pancreatic lobes was similar.

Only rats on the safflower oil diet showed significant decreases in amylase activity. It was surprising that the POLYCOSE® diet did not induce significant changes relative to the CEF control. Compared to the CEF group, the amylase activity in SIC did not change significantly in the casein and safflower groups; only the CEBDF and POLYCOSE® diets increased the amylase activity significantly (Table 7).

The correlation analysis (Fig. 2) showed that the blood glucose, the plasma insulin, the GSP PP, the DP amylase and the GSP lipase were in a significant linear relationship with the amylase content of GSP tissue.

On the basis of these data, we conclude, that the amylase content of the exocrine pancreatic tissue and amylase activity in the small intestinal content are influenced differently and most likely by differing mechanisms depending on the macronutrients fed.

However there are certain tendencies in the effects of macronutrients on the lipase contents of the DP and GSP tissues (Table 7) there is no statistically proven relationship between them.

As noted there were significant differences between the lipase contents of the two lobes in the CEF, the CEBDF, POLYCOSE® and safflower oil diets; the difference in the casein group was not significant.

The lipase content of DP tissues negatively correlated with the plasma triglyceride (r = -0.990; p=0.0007) and glucagon in GSP tissue (r = -0.893; p=0.041), while a positive correlations was detected regarding plasma corticosterone (r = 0.917; p=0.028) (Fig. 2).

The highest trypsinogen content was in the casein fed and CEF rats in both pancreatic lobes. Significantly lower trypsinogen concentrations were noted in the CEBDF and POLYCOSE® fed animals; however the lowest trypsinogen concentration was detected in both lobes of the safflower oil fed rats (Table 7).

The trypsinogen content was significantly different between the two-pancreatic regions in CEBDF, POLYCOSE®, safflower oil fed and fasted rats but not in rats on casein diet. The nutrient induced changes (Table 7, Fig. 3) followed the same pattern in both pancreatic lobes (r = 0.982; p=0.003).

The trypsin activity in the small intestinal contents did not correlate with the trypsinogen values of the two pancreatic lobes showing that the synthesis and the secretion of digestive enzymes are under different control mechanisms, as also noted for amylase and lipase.

In both pancreatic lobes the trypsinogen content correlated with liver triglyceride, liver FFA, glucagon in GSP tissue, lipase in DP tissue and trypsinogen in GSP tissue. The trypsin activity in SIC correlated with the blood glucose level of the rats (Fig. 2). More study is needed to understand the real significance of these correlations.