Effect of dietary carbohydrate and phenotype on sucrase, maltase, lactase, and alkaline phosphatase specific activity in SHR/N-cp rat.

The obese spontaneous hypertensive rat/NIH-corpulent (SHR/N-cp) rat exhibits some of the metabolic and pathologic alterations associated with non-insulin-dependent diabetes mellitus and hypertension. The current study was conducted to investigate the influence of phenotype (ob versus In) and source of dietary carbohydrate (sucrose versus starch) on intestinal sucrase, maltase, lactase, and alkaline phosphatase activity in SHR/N-cp rats. For 3 months, lean and obese male SHR/N-cp rats were fed isocaloric diets containing as the sole source of carbohydrate either 54% cooked corn starch or sucrose. Serum and urine markers for diabetes were observed in obese rats. Wet weight and length of intestines were significantly increased in obese rats compared with lean littermates. Among the intestinal enzymes measured, statistical tests confirmed that sucrase activity was significantly increased (P < 0.01) by both phenotype (ob > In) and feeding a sucrose diet. Diet alone (sucrose > starch) significantly increased (P < 0.05) maltase activity in obese rats, but had no effect on lean rats. Lactase activity was significantly higher (P < 0.05) in obese sucrose-fed rats compared with obese starch-fed and/or lean littermates. Statistical tests revealed that intestinal alkaline phosphatase activity was significantly altered (P < 0.05) by both phenotype and diet. Intestinal alkaline phosphatase was higher in starch-fed lean rats compared with lean littermates fed sucrose and to starch or sucrose-fed obese rats. These results are not indicative of a simple, nonspecific increase in intestinal enzyme activity, since the effects observed in intestinal alkaline phosphatase contrast the effects observed in intestinal sucrase, maltase, and lactase activity. These results indicate that both phenotype and diet alter structural and enzymatic intestinal activities of SHR/N-cp rats. Distinct variations in the observed intestinal enzymatic activities suggest that these enzymes are under the control of genetic, hormonal, and dietary factors. Rationale for these differences are discussed.

Adaptation in enzyme (metabolic) pathways to obesity, carbohydrate diet and to the occurrence of NIDDM in male and female SHR/N-cp rats.

Twenty-four male (12 obese and 12 lean) and 21 female (11 obese and 10 lean) SHR/N-cp rats were fed a diet containing either 54% sucrose or starch for periods of 3-4 months. Rats were killed after a 14-16 h fast and liver enzyme activities were determined in both sex groups. Liver glucose-6-phosphatase (G6Pase), fructose 1,6-bisphosphatase (FBPase), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME), phosphofructokinase (PFK), glucokinase (GK), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (per total liver capacity) were significantly affected by phenotype (obese > lean). Arginase and ornithine transcarbamylase levels were analysed only in male rats and were found to be elevated in obese rats as compared to lean littermates. Some of the above changes in enzyme levels were exaggerated by sucrose feeding but not the changes in FBPase, PEPCK, ME and GK (in both sexes) plus AST, arginase and arginine synthase activities in male rats and ALT levels in female rats. Results from SHR/N-cp rats published in this paper were compared to results obtained from LA/N-cp rats published previously. Comparison of the non-diabetic obese LA/N-cp with the diabetic obese SHR/N-cp male shows a greater excess in lipogenic capacity of the liver in the LA/N-cp male rat. The SHR/N-cp obese female also shows a greater liver lipogenic capacity as compared with the obese male SHR/N-cp rat. The results suggest that an adaptation of excessive lipogenesis in the liver of obese rats may be an anti-diabetogenic adaptation resulting in increased glucose conversion to lipids, thus reducing blood glucose levels.

Effect of dietary carbohydrate on liver and kidney enzyme activities and plasma amino acids in the LA/N-cp rat.

Twenty obese and 20 lean LA/N-cp male rats and 20 male Sprague-Dawley rats were fed a diet containing either 54 percent sucrose or starch for six weeks. After a 14-16 hour fast, rats were killed. Liver and kidney enzyme activities were determined in the LA/N-cp rats while plasma urea and selected amino acids were determined in all rats. Liver glucose-6-phosphatase (G6PASE), fructose-1,6-bisphosphatase (FBPASE), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME), glucokinase (GK), pyruvate kinase (PK), phosphofructokinase (PFK), glutamic-oxaloacetic-transaminase (GOT), glutamic-pyruvic transaminase (GPT), arginase (ARGASE), arginine-synthase (ARG-SYN) and ornithine transcarbamylase (OTC) levels were significantly affected by phenotype (obese greater than lean). All the above changes in enzyme levels were exaggerated by sucrose-feeding with the exception of PK, PFK, GOT, GPT, ARGASE and ARG-SYN. Kidney cortex G6PASE, PEPCK and ARGASE activities were higher in the obese rats as compared to the lean littermates. Sucrose feeding resulted in higher cortex G6PASE, FBPASE and PEPCK as compared to starch-fed rats. A phenotype effect was noted with plasma glutamate, urea, leucine, isoleucine and valine (obese greater than lean) and a diet effect was seen with aspartate, phenylalanine, leucine and valine (sucrose greater than starch) concentration. Sprague-Dawley rats had higher plasma urea and lower alanine than lean LA/N-cp males. Metabolic obesity in the LA/N-cp rat appears to involve an elevated capacity for pathways of glycolysis, gluconeogensis, lipogenesis and amino acid catabolism in the liver.

The effects of low-dose Bay-m-1099 (Miglitol) on serum lipids and liver enzyme activity of obese and obese-diabetic corpulent rats.

1. Groups of lean, obese, and obese-non-insulin-dependent diabetic LA/N-cp and SHR/N-cp rats were administered the a-glucosidase inhibitor Miglitol (150 mg/kg diet, ad libitum) from 8 until 15 weeks of age. 2. Phenotype effects (obese greater than lean) were present for weight gain, adiposity, serum glycemic and lipid parameters, and for liver glucokinase, glucose-6-phosphate dehydrogenase, and malic enzyme activity. Miglitol treatment was associated with improvements in glucokinase and malic enzyme in both strains, and in improvements in glycemic parameters in obese rats. 3. These results are consistent with variable improvements in glycemic control and insulin action following low dose Miglitol treatment, and indicate that indirect effects of the drug on insulin sensitivity in peripheral tissues and on glucoregulatory enzymes may contribute to the glycemic improvements observed with this drug, while greater dosages or longer treatment may be required to observe comparable improvements in adiposity or plasma lipid profiles.

Glucose-6-phosphate dehydrogenase. Characteristics revealed by the rat liver enzyme structure.

The primary structure of glucose-6-phosphate dehydrogenase from rat liver has been determined, showing the mature polypeptide to consist of 513 amino acid residues, with an acyl-blocked N-terminus. This structure is homologous to those of both other eutherian and marsupial mammals (human and opossum), thus characterizing a mammalian type enzyme to which the human form, notwithstanding its large number of genetic variants, conforms. The mammalian type differs from the fruit fly enzyme by about 50%. Known mutant forms exhibit further differences, widely distributed along the polypeptide chain. Structural patterns show glucose-6-phosphate dehydrogenases to consist of a few variable regions intermixed with relatively constant segments.

Lack of specificity of polyunsaturated fats in the inhibition of rat liver glucose-6-phosphate dehydrogenase.

The mechanism of the effect of polyunsaturated fatty acids (PUFA) on glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (G6PDH) was studied in young, male Wistar rats. Starvation-refeeding increased G6PDH level above that seen in ad libitum-fed animals (enzyme overshoot). A second episode of starvation-refeeding produced even higher levels of G6PDH activity (induction increment). Interposing a high fat diet (containing PUFA) between starvation and feeding the inducer diet abolished one-half to two-thirds of the overshoot. Feeding a high fat diet between the two starvations abolished the induction increment. Inhibitors of arachidonic acid metabolism were not able to reverse the PUFA effect. In another set of experiments it was shown that both linoleic and linolenic acid are equally effective in either reducing the overshoot or abolishing the induction increment. The evidence was interpreted as supporting a hypothesis that the PUFA effect does not require the formation of a specific end product of arachidonic metabolism in a direct way.

Molecular diversity of glucose-6-phosphate dehydrogenase: rat enzyme structure identifies NH2-terminal segment, shows initiation from sites nonequivalent in different organisms, and establishes otherwise extensive sequence conservation.

The NH2-terminal region of rat liver glucose-6-phosphate dehydrogenase (EC 1.1.1.49) is shown to differ radically from a reported amino acid sequence for the fruit fly enzyme and from one for the human enzyme. The results indicate considerable differences in the translational start point. However, a close relationship with another reported sequence for the human enzyme is established, now showing agreement between an indirectly deduced and a directly analyzed NH2-terminal structure of this enzyme type. The results provide evidence of one structural motif common to mammalian species but also suggest that genetic inconstancy 5' to, or at the start of, the region coding for the enzyme protein could be a source of intra- and interspecies diversity. This is of interest in relation to the large number of genetic variants of human glucose-6-phosphate dehydrogenase.

Dietary induction of hepatic glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and malic enzyme in lean and obese female Zucker rats.

Responses of the hepatic lipogenic enzymes, glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and malic enzyme (ME) to starvation refeeding and diet shifting were determined in lean and obese female Zucker rats. Rats were either fed nonpurified diet, starved 48 hr, and then refed nonpurified diet or one of the refined carbohydrate diets containing either glucose, fructose, cornstarch, or sucrose for 72 hr, or shifted from nonpurified diet directly to one of the refined carbohydrate diets for 72 hr. Initial activities were greater in obese than lean rats for all three enzymes studied. Similar to other strains of female rats, lean Zucker rats failed to demonstrate a starve-refeed response when refed nonpurified diet. Obese female littermates showed a statistically significant increase in enzymes when refed a nonpurified diet. Both lean and obese female Zucker rats demonstrated increases in enzyme activities above controls when starved and refed any of the refined carbohydrate diets. The greatest responses were observed when female rats were starved and refed sucrose; activities increased 2.6- to 3.5-fold in lean and 3.0- to 4.3-fold in obese Zuckers. In lean females 50-70% of the starve-refeed response observed with G6PDH and ME can be accounted for by simply shifting from a nonpurified diet to the respective refined carbohydrate diet, whereas in obese females only 33-55% of the increase could be attributed to diet shifting. Plasma testosterone/estrogen ratios were consistently 1.5 times higher in obese than in lean female rats. This phenotypic difference may potentiate the heightened starve-refeed overshoot response observed in obese rats.

Gender-linked differences in dietary induction of hepatic glucose-6 phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and malic enzyme in the rat.

The objective of these studies was to determine how alterations in dietary carbohydrate affect hepatic glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and malic enzyme (ME) activities in adult female rats. Rats were either starved 2 d and then refed a nonpurified diet or a purified 65% carbohydrate diet (glucose, sucrose, fructose or cornstarch) for 3 d, or switched from nonpurified to purified diets for 3 d. Liver G6PDH, 6PGDH and ME activities were determined. In males, enzyme activities were 8- to 12-fold and 3-fold higher when starved and refed purified diets and nonpurified diets, respectively, whereas in females, activities were 2- to 3-fold higher only when refed purified diets. Both genders had higher enzyme activities when shifted to purified diets. Females responded less dramatically than males. Of the higher enzyme activities observed during starvation-refeeding studies, in females 58-65% of the change is a function of switching rats from nonpurified to purified diets. In contrast, in males only 24-40% of the higher activities could be attributed to diet shifting. Results of these studies indicate that the effects of dietary carbohydrates on hepatic G6PDH, 6PGDH and ME activities are gender dependent.

Absence of a generalized disaccharide effect in adult female rats.

Adult female Sprague-Dawley rats were either prefed ground nonpurified diet, starved 48 h, then refed a purified carbohydrate diet for 72 h or shifted from ground nonpurified diet directly to a purified carbohydrate diet for 72 h. Diets were formulated to contain 65% carbohydrate either as the disaccharides maltose or sucrose or as their respective monosaccharide equivalents glucose and invert sugar (glucose: fructose, 1:1). Alternations in hepatic glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and malic enzyme (ME) activities, relative liver size and food efficiency were determined. Rats starved and refed invert sugar had higher levels of G6PDH and ME than those red glucose, indicating a positive fructose effect. The greatest changes in hepatic enzyme activities were observed in rats consuming diets containing sucrose. Positive fructose and disaccharide effects were obtained with sucrose for all enzymes studied in both dietary shift and starve-refeed studies. No disaccharide effect was observed with maltose. In conclusion, females did not display a generalized disaccharide effect with either dietary shifting or starvation refeeding.

Effects of dietary carbohydrate on hepatic gluconeogenesis in BHE rats.

The effects of feeding different carbohydrates on hepatic gluconeogenesis in BHE rats was studied. Three experiments were conducted that differed only in the aspects of gluconeogenesis examined. In experiment 1, gluconeogenesis and ketogenesis by hepatocytes isolated from 48-h starved rats were determined. In experiment 2, the activities of selected gluconeogenic enzymes were determined in starved and nonstarved rats. In experiment 3, the levels of the various metabolites of glycolysis and the citric acid cycle were determined in nonstarved rats. Rats were fed diets containing starch, maltose, glucose, sucrose or an equimolar mixture of glucose and fructose. In starved rats, gluconeogenesis was less in starch-fed rats than in rats fed any of the sugar diets. These same diet differences, with few exceptions, were also observed in ketogenesis. Glucose 6-phosphatase activity was lower in nonstarved rats fed starch and maltose than in rats fed glucose, sucrose, or glucose and fructose. These same nonstarved rats also had lower phosphoenolpyruvate carboxykinase and higher glutamate pyruvate transaminase activities than rats fed the other diets. In the starved rats, the diet differences were erased. Starved rats had lower activities of these gluconeogenic enzymes than nonstarved rats. Diet differences in the levels of the different metabolites in nonstarved rats were observed. The results show that dietary carbohydrate can influence gluconeogenesis. However, the mechanism of their effect is as yet unknown.

Preliminary crystallographic study of glucose-6-phosphate dehydrogenase from rat liver.

Crystals of D-glucose-6-phosphate: NADP+ oxidoreductase were obtained with the hanging drop, vapor diffusion and batch methods from ammonium sulfate-containing solutions. X-ray diffraction photographs indicate that the crystals belong to the orthorhombic space groups I222 or I2(1)2(1)2(1) with unit cell dimensions of a = 66.0 A, b = 140.8 A and c = 177.8 A. These data, together with results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crystal density experiments, indicate that there is one 116,000 Mr dimer per asymmetric unit. The crystals diffract to at least 2.2 A and are suitable for X-ray crystallographic structure determination.

The in vitro stability of rat liver glucose-6-phosphate dehydrogenase as a function of diet.

Rat liver glucose-6-phosphate dehydrogenase (G6PD) activity was studied in starved-refed rats given diets containing three levels of fat (35%, high-fat; 5%, low-fat; 0%, fat-free). Elution characteristics from DEAE-cellulose, Km for glucose-6-phosphate, pH optimum, and molecular weight appeared to be similar. During storage or heat denaturation, stability apparently was lowest of G6PD of livers from rats refed the high-fat diet. Heat stability was enhanced by the addition of NADP, but some differences due to diet persisted. Titration of a constant amount of enzyme with heating gave inconsistent results: in two of four experiments rats refed the high-fat diet had an equivalence point twice that of rats refed the fat-free diet. This difference disappeared if the antibody titration was carried out in the cold. The diet-induced instability of the G6PD, as measured in vitro, was reversible by changing the diet of the rats.

Induction of rat liver glucose-6-phosphate dehydrogenase and malic enzyme during blockage of glycogen accumulation.

Liver glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) and malic enzyme (ME, EC 1.1.1.40) activities were measured in rats that were starved for 2 days and then fed a high-glucose, adequate-protein diet for 3 days. During refeeding the rats were injected with thyroxine to block glycogen accumulations preceeding the enzyme "overshoot". The G6PD and ME "overshoot" at the end of refeeding was still evident in spite of a 90% reduction in the glycogen peak. The results showed that the glycogen accumulation prior to the enzyme "overshoot" was not obligatory to the subsequent rise in enzyme activity. The sequential accumulation/breakdown of liver glycogen (day 1 refeeding) followed by the accumulation of liver fat (day 2 of refeeding) are probably the result of the changes in enzymatic pathways available to deal with the inflow of excess glucose. Such dissociation of glycogen accumulation and G6PD and ME "overshoot" during starvation-refeeding makes it highly unlikely that either glucose or glucose-6-phosphate derived from glycogen would be the direct, primary inducer of G6PD or ME in rat liver.

Regulation of glucose-6-phosphate dehydrogenase and malic enzyme in liver and adipose tissue: effect of dietary trilinolein level in starved-refed and ad libitum-fed rats.

The responses of glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) and malic enzyme (ME) (EC 1.1.1.40) were studied in liver and adipose tissue of rats fed for 2 days a high glucose diet containing levels of synthetic trilinolein ranging from 0 to 25% (w/w) of the diet (trilinolein was substituted for glucose). One group of rats was starved for 2 days before the trilinolein-containing diets were fed (starved-refed); a second group of rats was fed a fat-free diet for 7 days before the trilinolein-containing diets were fed (ad libitum). Liver G6PD activity decreased exponentially and liver ME activity decreased linearly with increasing dietary trilinolein in starved-refed rats, but did not decrease significantly in ad libitum fed rats. Total liver lipid decreased exponentially with increasing trilinolein in starved-refed rats, but increased exponentially in ad libitum fed rats. Adipose tissue G6PD and ME activities decreased slightly with increasing trilinolein in starved-refed rats, but did not decrease in ad libitum fed rats. When the data were adjusted by analysis of covariance for differences in glucose intake, the liver responses in starved-refed rats were still significant but the adipose tissue responses were not, indicating that the responses of adipose tissue (but not of liver) may have resulted from decreased glucose intake rather than from increased trilinolein intake. The results suggest that dietary trilinolein inhibits the characteristic increase in liver G6PD, ME and total lipids upon starvation-refeeding. However, after the levels of these parameters have been increased by feeding a fat-free diet they cannot be decreased by dietary trilinolein in 2 days.

Independence of glycogen accumulation and glucose-6-phosphate dehydrogenase induction in rat liver.

The responses of liver glycogen and glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) to a high glucose, adequate protein diet were compared between rats previously starved 2 days, then refed a high protein, carbohydrate-free diet for 2 days, and rats previously fed the high protein diet for 4 days. Glycogen levels increased dramatically during the first day the high carbohydrate diet was fed, then decreased gradually on the second day. The response was the same regardless of whether the rats had been starved more before the high protein diet was fed. Liver G6PD activity also increased when the high carbohydrate diet was fed, and continued to increase on the second day. The increase in G6PD, however, was significantly greater in the rats which had been starved before the high protein diet was fed. It is suggested that some process occurs during starvation that predisposes the induction of G6PD upon refeeding a high carbohydrate diet, over and above any effect of glycogen accumulation and breakdown. Glucose or glucose-6-phosphate derived from glycogen does not appear to be the primary inducer of G6PD in rat liver.