In vivo lipogenesis and enzyme levels in adipose and liver tissues from pair-fed genetically obese and lean rats

Genetically obese Zucker rats pair-fed to lean controls were similar in body weight and food intake, however, epididymal fat pads were considerably larger than lean controls. $\text{In}$$\text{vivo}$ incorporation of acetate-¹⁴C into adipose tissue lipid was not significantly different, however, $\text{in}$$\text{vivo}$ liver lipogenesis was elevated in the obese rat. Characterization of enzyme profiles in both liver and adipose tissues revealed that enzymes normally associated with lipogenesis were elevated in liver tissue from obese rats. Malic enzyme and citrate cleavage enzyme were both depressed in adipose tissue of obese animals. From these data, it appears that the liver may be prominently involved in the development of excessive blood lipid and enlarged fat cells in the Zucker obese rat.     

Altered alkaline phosphatase activity in obese Zucker rats liver respect to lean Zucker and Wistar rats discussed in terms of all putative roles ascribed to the enzyme

Biliary complications often lead to acute and chronic liver injury after orthotopic liver transplantation (OLT). Bile composition and secretion depend on the integrated action of all the components of the biliary tree, starting from hepatocytes. Fatty livers are often discarded as grafts for OLT, since they are extremely vulnerable to conventional cold storage (CS). However, the insufficiency of donors has stimulated research to improve the usage of such marginal organs as well as grafts. Our group has recently developed a machine perfusion system at subnormothermic temperature (20°C; MP20) that allows a marked improvement in preservation of fatty and even of normal rat livers as compared with CS. We sought to evaluate the response of the biliary tree of fatty liver to MP20, and a suitable marker was essential to this purpose. Alkaline phosphatase (AlkP, EC 3.1.3.1), frequently used as marker of membrane transport in hepatocytes and bile ducts, was our first choice. Since no histochemical data were available on AlkP distribution and activity in fatty liver, we have first settled to investigate AlkP activity in the steatotic liver of fatty Zucker rats (fa/fa), using as controls lean Zucker (fa/+) and normal Wistar rats. The AlkP reaction in Wistar rats was in accordance with the existing data and, in particular, was present in bile canaliculi of hepatocytes in the periportal region and midzone, in the canals of Hering and in small bile ducts but not in large bile ducts. In lean ZR liver the AlkP reaction in Hering canals and small bile ducts was similar to Wistar rat liver but hepatocytes had lower canalicular activity and besides presented moderate basolateral reaction. The difference between lean Zucker and Wistar rats, both phenotypically normal animals, could be related to the fact that lean Zucker rats are genotypically heterozygous for a recessive mutated allele. In fatty liver, the activity in ductules and small bile ducts was unchanged, but most hepatocytes were devoid of AlkP activity with the exception of clusters of macrosteatotic hepatocytes in the mid-zone, where the reaction was intense in basolateral domains and in distorted canaliculi, a typical pattern of cholestasis. The interpretation of these data was hindered by the fact that the physiological role of AlkP is still under debate. In the present study, the various functions proposed for the role of the enzyme in bile canaliculi and in cholangiocytes are reviewed. Independently of the AlkP role, our data suggest that AlkP does not seem to be a reliable marker to study the initial step of bile production during OLT of fatty livers, but may still be used to investigate the behaviour of bile ductules and small bile ducts.     

Lipoprotein secretion in lean and obese Zucker female rats in vivo and in a single-pass-perfused liver preparation

The plasma lipoprotein composition as well as lipoprotein synthesis and secretion were studied in vivo and in a single-pass-perfused liver preparation in lean and obese Zucker rats. Compared with their lean littermates the levels in the plasma of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) + low density lipoprotein (LDL) and high density lipoprotein (HDL) were increased 4-, 2- and 2.5 fold, respectively, in obese rats. In these rats both VLDL and IDL + LDL were enriched in triglycerides, while the HDL were enriched in cholesterol. Although the VLDL and IDL + LDL protein concentrations were the same in lean and obese rats, the HDL protein concentration was 3-fold greater in the obese rats. Both the lean and obese rats incorporated similar amounts of [14C]leucine into total liver protein. However, obese rats incorporated 2.5-fold and 6-fold more [14C]leucine into VLDL and HDL in vivo, 2.7-fold and 1.7 fold more [35S]methionine in VLDL and HDL present in the perfusate, than did lean rats. The perfusate [35S]S-labelled apoproteins (apo-B100, B48; apo-E, apo-AI, apo-AIV and apo-C) were separated by gel electrophoresis and identified by autoradiography. Incorporation of [3H]glycerol into liver, VLDL, IDL + LDL and HDL triglycerides was 2-, 48-, 13- and 1.5-fold higher in obese than in lean rats, respectively. The [3H]-labelled triglycerides in VLDL and IDL + LDL present in the perfusate was 5.4-fold and 4.4-fold more in obese rat. There was no difference in the incorporation of [3H]glycerol into triglycerides of perfusate HDL between the two genotypes of rats. Thus, the hypertriglyceridaemia observed in obese Zucker rats results from very high synthetic rates of both the lipid and protein moieties of plasma lipoproteins. Before this study, no report of the simultaneous triglycerides and protein synthesis in vivo and in a single-pass-perfused liver preparations had been reported.     

Expression of hepatic microsomal cholesterol 7 alpha-hydroxylase activity in lean and obese Zucker rats

The activity of hepatic microsomal cholesterol 7 alpha-hydroxylase was studied in genetically obese and lean Zucker rats. The liver microsomal cholesterol 7 alpha-hydroxylase activity in fatty Zucker rats (fa/fa) is about 50% to 70% lower than that of the lean (Fa/-) rats of the same sex, when animals were sacrificed at the middle of the dark cycle. When rats were sacrificed at the middle of the light cycle, cholesterol 7 alpha-hydroxylase activity was the same as in the dark cycle in obese rats of both sexes, but was 65% lower in lean rats. However, cholesterol 7 alpha-hydroxylase activity was stimulated by the treatment with cholestyramine in both obese and lean rats. Our results suggested that the diurnal regulation of cholesterol 7 alpha-hydroxylase activity is lost in obese rats but was present under cholestyramine treatment in the genetically obese strain of rats.     

Visfatin: gene expression in isolated adipocytes and sequence analysis in obese WOKW rats compared with lean control rats

Recently, Fukuhara et al. have shown that the novel visfatin is predominantly released from visceral adipocytes and shares metabolic functions with insulin. The authors suggested that there is a relationship between visfatin and the metabolic syndrome in humans. These findings prompted us to clarify the visfatin gene expression from visceral and subcutaneous adipocytes in WOKW rats, as an animal model for polygenically inherited metabolic syndrome, compared to lean Dark Agouti (DA) rats. Moreover, visfatin sequence analysis was performed in WOKW, DA rats, and disease-resistant Fisher 344, Lewis, Brown Norway, and Karlsburg wild rats. The relative gene expression of visfatin displays no significant changes in adipocytes from WOKW rats compared with DA and visfatin sequence analysis of the coding region was identical to the GenBank. But, we found length differences of two repeats, GT and GA, in intron 2 between the strains. In summary, the relative visfatin gene expression is not associated with the metabolic syndrome in WOKW rats.     

Development of gut microflora in obese and lean rats

The influence was evaluated of post-weaning normal nutrition and over-nutrition upon the development of the intestinal microbiota, the alkaline phosphatase activity (AP) and occurrence of obesity in male Sprague-Dawley rats (from days 21 to 40 the control rats were submitted to ad libitum intake of a standard laboratory diet whereas overfed rats received the same diet supplemented with milk-based high fat liquid diet). The jejunal numbers of two dominant divisions of bacteria, i.e. Firmicutes (Lactobacillus/ Enterococcus — LAB) and the Bacteroidetes (Bacteroides/Prevotella — BAC), were determined using the fluorescent in situ hybridization (FISH) method, and the jejunal AP activity was assayed histochemically. On day 40, the overfed rats in comparison with control animals displayed increased adiposity accompanied by enhanced AP activity, abundance of LAB, lower amounts of BAC and, thereafter, higher LAB/BAC ratio (L/B). The numbers of LAB and L/B index positively correlated with body fat, energy intake and AP activity, whereas numbers of BAC showed an opposite tendency. These results revealed the significance of nutritional imprint upon the post-weaning development of intestinal microbial and functional axis and contribute to better understanding of their involvement in energy-balance control and in adverse and/or positive regulation of adiposity.     

Different susceptibility of liver grafts from lean and obese Zucker rats to preservation injury

We compared the susceptibility of liver grafts from lean and obese Zucker rats to preservation injury, using two organ-preservation techniques: conventional static preservation (SP) and machine perfusion (MP) preservation. SP: livers preserved by UW solution at 4, 8 or 20 °C for 6-h. MP: livers perfused for 6-h with an improved oxygenated Krebs–Henseleit solution (KH) at 4, 8 or 20 °C. Reperfusion with KH (2-h) was performed either with the SP or MP preserved livers. Fatty livers tolerate SP poorly at 4, 8 and 20 °C as compared with MP at the same temperatures. SP induced a decrease in the ATP/ADP ratio both at 8 and 20 °C in obese rats while an increase in energy status was found with MP at 8 and 20 °C. Nitrate/nitrite (NOx) concentration was higher and bile flow lower in livers preserved with SP than MP. In lean rats, no differences were observed between MP and SP as regards enzyme release, bile production and NOx levels except for SP at 20 °C in which high enzyme release and low bile flow were observed. In lean rats ATP/ADP was higher and NOx was lower with MP at 20 °C than with SP at 20 °C. To optimize steatotic liver preservation SP should be avoided because it is particularly detrimental as compared with MP.     

Kinetic parameters for plasma beta-endorphin in lean and obese Zucker rats

To determine plasma clearance kinetics for beta-endorphin (BE) by empirical compartmental analysis, a bolus of radioactive labeled 125I-BE was rapidly injected into a carotid artery catheter of unanesthetized lean (L) and obese (O) Zucker rats. The plasma disappearance of 125I was followed over a 3-h period. A 3-component exponential equation provided the best fit for plasma data. Plasma transit times were very short (10 s); however, plasma fractional catabolic rate was much slower. Plasma mean residence time was similar for both groups (50 min) as was recycle time (1.3 min). These data suggest that BE plasma disappearance kinetics are similar in L and O rats.     

Behavioral thermoregulation in obese and lean Zucker rats in a thermal gradient

Genetically obese Zucker (Z) rats have been reported to display a body core temperature (Tb) that is consistently below that of their lean littermates. We asked the question whether the lower Tb was a result of deficits in thermoregulation or a downward resetting of the set point for Tb. For a period of 45 consecutive hours, lean and obese Z rats were free to move within a thermal gradient with an ambient temperature (T(a)) range of 15-35 degrees C, while subjected to a 12:12-h light-dark cycle. Tb was measured using a miniature radio transmitter implanted within the peritoneal cavity. Oxygen consumption (VO2) was measured using an open flow technique. Movements and most frequently occupied position in the gradient (preferred T(a)) were recorded using a series of infrared phototransmitters. Obese Z rats were compared with lean Z rats matched for either age (A) or body mass (M). Our results show that obese Z rats have a lower Tb [37.1 +/- 0.1 degrees C (SD) vs. 37.3 +/- 0.1 degrees C, P < 0.001] and a lower VO2 (25.3 +/- 1.9 ml x kg(-1) x h(-1)) than lean controls [33.1 +/- 3.7 (A) and 33.9 +/- 3.9 (M) ml x kg(-1) x h(-1), P < 0.001]. Also, the obese Z rats consistently chose to occupy a cooler T(a) [20.9 +/- 0.6 degrees C vs. 22.7 +/- 0.6 degrees C (A) and 22.5 +/- 0.7 degrees C (M), P < 0.001] in the thermal gradient. This suggests a lower set point for Tb in the obese Z rat, as they refused the option to select a warmer T(a) that might allow them to counteract any thermoregulatory deficiency that could lead to a low Tb. Although all rats followed a definite circadian rhythm for both Tb and VO2, there was no discernible circadian pattern for preferred T(a) in either obese or lean rats. Obese Z rats tended to show a far less definite light-dark activity cycle compared with lean rats.     

Role of sympathetic activity in controlling the expression of vascular endothelial growth factor in brown fat cells of lean and genetically obese rats

The thermogenic activity of brown adipose tissue (BAT) is heavily dependent on high perfusion, through its dense vascular system. Angiogenesis must go hand-in-hand with BAT functions, but little is known about the factors controlling it. In the present study we demonstrate that: (a) vascular endothelial growth factor (VEGF) is synthesised and released in brown adipocytes in culture; (b) VEGF mRNA isoforms and protein appear in dispersed mature brown adipocytes and whole tissue; (c) VEGF expression is increased in BAT from cold-exposed rats, and in cultured brown adipocytes exposed to noradrenaline and the β₃-adrenoceptor agonists; (e) BAT from genetically obese (fa/fa) rats exhibits reduced expression of VEGF as well as a change in the ratio of mRNA isoforms. It is concluded that sympathetic control of VEGF expression via noradrenaline acting on β₃-adrenoceptors plays a major role in developmental and adaptive angiogenesis, and defects in this contribute to the reduced thermogenic capacity of BAT in genetic obesity.     

Hormonal studies of young lean and obese Zucker rats

Plasma concentrations of insulin, corticosterone, T3, T4 and glucose were measured at 6 hour intervals throughout 24 hours in undisturbed, 34-day-old lean (Fa/?) and genetically obese (fa/fa) Zucker rats. fa/fa rats had higher plasma concentrations of insulin at all sampling times and higher plasma concentrations of corticosterone at 0300 and 0900 hours. Neither T3 nor T4 levels differed between phenotypes at any sampling time. Fasting for 24 hours at 34 days abolished the hyperinsulinaemia of fa/fa rats and raised the plasma corticosterone concentrations of both phenotypes. Before weaning there were no phenotypic differences in the plasma insulin or corticosterone concentrations measured at two sampling times in undisturbed rats. Following an intra-gastric glucose load however, fa/fa rats became hyper-insulinaemic compared with similarly treated Fa/? animals. Pancreatic insulin contents were higher in fa/fa rats at 34 days of age, but not before weaning. Somatostatin contents of the pancreas, hypothalamus and cerebral cortex did not differ between phenotypes at either 18 or 34 days of age. In conclusion, the elevated plasma concentrations of insulin and corticosterone in young fa/fa rats may contribute to their greater lipid deposition and lower protein deposition.     

Response of genetically obese Zucker rats to ciprofibrate,a hypolipidemic agent,with peroxisome proliferation activity as compared to Zucker lean and Sprague-Dawley rats

Summary— Genetically obese Zucker (fa/fa) rats were used as an experimental model to study the effects of hypolipidemic agents on peroxisome proliferation; comparison was made with Zucker lean phenotype (Fa/?) and Sprague-Dawley strain/phenotype. The pharmacokinetics of a single administration of ciprofibrate (1 or 3 mg/kg), appeared to be similar in all strains/phenotypes. After a 2-week oral administration at the same dosages, there were dosage-related increases in hepatocellular peroxisomal yield and in the hepatic enzymes' cyanide-insensitive acyl-CoA oxidase and catalase. The peroxisomal yield was less increased in Zucker than in Sprague-Dawley rats, while the enzyme activities were similarly increased. Although the absolute specific activity of microsomal ω-lauryl hydroxylase (cytochrome P4504A1) was lower in Zucker rats, it was increased more in this strain than in Sprague-Dawley rats in response to drug exposure. The hypolipidemic effect (cholesterol and triglyceride reduction) was more pronounced in Zucker obese rats. Based on biochemical and morphological results, no major differences between strains/phenotypes in terms of peroxisome proliferation were observed following a 2-week administration of ciprofibrate.     

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.     

Insulin binding in the hypothalamus of lean and genetically obese Zucker rats

Recent reports have suggested that the obesity and hyperphagia of the genetically obese Zucker rat may be related to defective insulin action or binding in the hypothalamus. We used quantitative autoradiography to determine if insulin binding is altered in specific hypothalamic nuclei associated with food intake. Insulin binding was measured in the arcuate (ARC), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei of 3–4-month-old lean (Fa/Fa) and genetically obese (fa/fa) Zucker rats. A consistently reproducible 15% increase in the total specific binding of 0.1 nM [¹²⁵I]-insulin was found in the ARC of the obese genotype. A slight increase in insulin binding in the DMN was also found. No difference in specific insulin binding was found between genotypes in the VMN. Nonlinear least squares analysis of competitive binding studies showed that the K_d of the ARC insulin binding site was 33% higher in the lean rats than in the obese rats, indicating an increased affinity for insulin. No difference in site number (B_max) was found in the ARC, DMN or VMN, and no evidence was found for reduced insulin binding in the hypothalamus of the obese (fa/fa) genotype. The results suggest that hyperphagia and obesity of the obese (fa/fa) Zucker rat genotype may be associated with increased insulin binding in the arcuate nucleus.     

Tissue and serum somatostatin-like immunoreactivity in lean and obese Zucker rats

Tissue and serum somatostatin levels were measured in genetically lean and obese Zucker rats. Immunoreactive somatostatin content was decreased in three central nervous system regions (hypothalamus, septum and preoptic area and thalamus) of obese rats but was increased in cerebral cortex. No differences were observed in antral or colonic somatostatin content but obese animals had significantly elevated pancreatic levels. Portal vein somatostatin-like immunoreactivity in contrast was significantly lower in obese rats. The widespread alterations in tissue and serum somatostatin-like immunoreactivity suggest either a diffuse abnormality of somatostatin physiology or a response to a generalised feature of the obese hyperinsulinaemic state.     

Effect of the estrous cycle and surgical ovariectomy on energy balance, fuel utilization, and physical activity in lean and obese female rats

This study presents an in-depth analysis of the effects of obesity on energy balance (EB) and fuel utilization in adult female rats, over the estrous cycle and immediately after surgical ovariectomy (OVX), to model pre- and postmenopausal states, respectively. Female Wistar rats were fed a high-fat (46%) diet for 16 wk to produce mature lean and obese animals. Stage of estrous was identified by daily vaginal lavage, while energy intake (EI), total energy expenditure (TEE), and fuel utilization were monitored in a multichamber indirect calorimeter and activity was monitored by infrared beam breaks. Metabolic monitoring studies were repeated during the 3-wk period of rapid OVX-induced weight gain. Component analysis of TEE was performed to determine the nonresting and resting portions of energy expenditure. Obesity was associated with a greater fluctuation in EB across the estrous cycle. Cycling obese rats were less active, expended more energy per movement, and oxidized more carbohydrate than lean rats. The changes in EB over the cycle in lean and obese rats were driven by changes in EI. Finally, OVX induced a large positive energy imbalance in obese and lean rats. This resulted primarily from an increase in EI in both groups, with little change in TEE following OVX. These observations reveal a dominant effect of obesity on EB, fuel utilization, and activity levels in cycling rats, which has implications for studies focused on obesity and EB in female rodents.     

Hepatic biotransformation in lean and obese Wistar Kyoto rats: comparison to that in streptozotocin-pretreated Sprague-Dawley rats

1. Phase I and phase II biotransformation was compared in streptozotocin-induced hypoinsulinemic (STZ) and genetic hyperinsulinemic (WKY-fatty) rats. 2. Total cytochrome P-450 concentrations were reduced in both STZ and WKY, whereas styrene oxide hydrolase and benzphetamine N-demethylase activities were normal in STZ and reduced in WKY. 3. UDP-glucuronosyltransferase activity was decreased toward testosterone and 1-naphthol in STZ and WKY, and was increased toward estrone in the obese female WKY. 4. Glutathione S-transferase activity was decreased in STZ toward 1-chloro-2,4-dinitrobenzene, ethacrynic acid and sulfobromophthalein, but was similar to that in normal rats for WKY.