Dietary and species influence on potential of plasma to stimulate differentiation and lipid accumulation in cultured adipocyte precursors

Sera and plasma from different species and from rats of various dietary statuses were compared with regard to effects on proliferation, glycerophosphate dehydrogenase (GPDH) activity, and lipid-filing of rat adipocyte precursors converting to adipocytes in primary cell culture. All of the tested sera and plasma samples were comparably supportive of cell multiplication, but their effects on elevation of GPDH activity (a key event in adipocyte differentiation) and lipid-filling varied greatly. Plasma supported a much greater increase in GPDH activity than serum, while serum from cats supported a much lower increase than serum from humans, calves, goats, or rats. Dietary status of rats did not affect the potential of plasma to support GPDH activity, but did affect plasma support of lipid-filing. A higher than normal degree of lipid-filling was promoted by plasma from rats fed a high-fat, high-sugar diet, while a lower than normal degree was promoted by plasma from fasted rats. Lipid-filling was also found to vary in response to changes in content of very low density lipoprotein (VLDL) in human plasma. This suggests that the influence of diet on the potential of plasma to promote adipocyte lipid-filling may be mediated by the effect of diet on plasma VLDL. The absence of a diet-dependent effect of plasma either on multiplication of adipocyte precursors or on degree of elevation of GPDH activity leaves unresolved the mechanism by which diet affects adipocyte production in animals.     

Serum hormone levels and tissue metabolism in pair-fed lean and obese Zucker rats.

Obese Zucker rats were either pair-fed to their lean litter-mates or fed ad lib, to determine the effect of hyperphagia on serum hormone levels and tissue metabolism as indicated by enzyme activities and in vitro metabolite flux. Hyperphagia was shown to be non-essential for the elevation in serum insulin and suppression in serum growth hormone and prolactin in the genetically obese rat. It was also shown that the increased liver cell lipogenic rate was not dependent on hyperphagia in the obese rat and that adipose cell lipogenesis was not significantly altered in the pair-fed obese rat. The utilization of alanine for glucose synthesis in vitro was similar for both lean and obese rats, but its utilization for fatty acid synthesis was higher in the obese rat. Data is presented which suggest that the inhibitory effect of glucagon on liver lipogenesis is blunted in the obese rat.     

Effect of estrogen on lipoprotein lipase activity and cytoplasmic progestin binding sites in lean and obese Zucker rats

Injections of 5 micrograms estradiol benzoate (EB) for 5 days resulted in decreases in the rate of body weight gain in both lean (Fafa) and obese (fafa) Zucker rats. EB administration also resulted in significant induction of cytoplasmic progestin binding sites in both hypothalamic-preoptic area (H-POA) and adipose tissues from rats of both genotypes. However, EB treatment significantly decreased lipoprotein lipase (LPL) activity in adipose tissue from lean, but not obese, Zucker rats and the same treatment increased LPL activity in the uteri from lean, but not obese, Zucker rats. The data are discussed in terms of the metabolic and reproductive dysfunctions observed in the genetically obese rat.     

Use of Glycated Hemoglobin to Assess Glycemic Control in Wistar Diabetic Fatty Rats and Zucker Fatty Rats

The Wistar Diabetic Fatty rat (WDF fafa) is a con-genic strain of the Wistar Kyoto rat. Studies using blood glucose reveal that only fatty male (not female) WDF rats spontaneously develop hyperglycemia when fed a stock diet Blood glucose values have not provided consistent results for evaluation of glycemic status in fatty male WDF rats. Zucker fatty (fafa) rats, while sharing the fa gene and the development of hyperinsulinemia and hyperlipemia, do not spontaneously become hyperglycemic. In order to examine strain differences and the effects of age on long-term average glycemic status in WDF and Zucker rats, glycated hemoglobin (GHb) was analyzed. Glycated hemoglobin was measured in male lean and obese WDF and Zucker rats at 2,3,6, and 12 months of age. Nonfasted plasma glucose was measured in male lean and obese WDF rats at 2, 3, 6, and 12 months of age and in lean and obese Zucker rats at 3, 6, and 12 months of age. Plasma insulin was measured in lean and obese WDF and Zucker rats at 3, 6, and 12 months of age. Obese WDF rats had significantly elevated GHb compared to lean controls at 3, 6, and 12 months of age. Glycated hemoglobin was substantially above the normal range (3.8-6.5%) at 3 months of age (14.1%). Glycated hemoglobin significantly declined in the obese WDF rats between 6 and 12 months of age. Nonfasted plasma glucose was significantly elevated in the obese WDF rats at 3 months (14.1 ± 2.1 mM/L) and 6 months of age (16.2 ± 2.3 mM/L) compared to lean controls. At 12 months of age there was no difference in plasma glucose between obese and lean WDF rats. Obese and lean Zucker rats had similar levels of GHb and plasma glucose at all ages. In conclusion, GHb provides more integrated data for classifying disease status of WDF rats and evaluation of potential long-term complications associated with hyperglycemia.     

Alpha 1- and beta-adrenergic receptors in brown adipose tissue of lean (Fa/?) and obese (fa/fa) Zucker rats. Effects of cold-acclimation,sucrose feeding and adrenalectomy.

1. The populations of alpha 1- and beta-adrenergic receptors in brown adipose tissue (BAT) of genetically obese Zucker rats (fa/fa) were studied with [3H]prazosin and [3H]CGP-12177 respectively. 2. The density of alpha 1-adrenergic receptors in BAT was significantly lower in obese than in lean Zucker rats, both at 2-4 months of age and at 6 weeks of age. The density of beta-adrenergic receptors was identical in BAT of lean and obese 6-week-old Zucker rats. 3. Cold-acclimation increased the alpha 1-receptor density significantly in BAT of both lean and obese Zucker rats, and the number of beta-receptors was also somewhat increased. 4. Sucrose feeding did not affect the density of alpha 1-receptors in BAT of lean or obese Zucker rats, but it increased beta-receptor density. 5. Adrenalectomy restored the density of alpha 1-adrenergic receptors in BAT of obese Zucker rats to the value observed in lean rats. 6. It is concluded that there is a direct correlation between alpha 1-receptor density and tissue recruitment, and that alpha 1-receptor density is thus positively correlated with sympathetic activity. beta-Receptor density is apparently better correlated with feeding conditions.     

Plasma B-cell tropin (ACTH22-39) concentration in lean and obese (ob/ob) mice and lean and obese (fa/fa) Zucker rats

A method has been developed for the measurement of plasma concentrations of Beta-cell tropin (BCT), which is a potent insulinotropic and lipogenic peptide secreted by the pituitary. The method was employed to compare plasma Beta-cell tropin concentrations between lean and genetically obese (ob/ob) mice and between lean and genetically obese (fa/fa) Zucker rats. The plasma concentration in lean mice was 0.17 +/- 0.02 (5)nmole/l (mean +/- SEM, n = 5), while that in obese (ob/ob) mice was significantly higher, being 2.88 +/- 1.13 (5)nmole/l. The plasma BCT concentration in Zucker rats was 0.14 +/- 0.02 (15)nmole/l, while that in obese Zucker (fa/fa) rats was significantly higher, being 1.69 +/- 0.72 (16)nmole/l. These results explain previously observed differences in the Beta-cell tropin-like biological activity in plasma from lean and obese animals, and support the hypothesis that the peptide has a role in the development of hyperinsulinaemia and obesity.     

The Male Obese Wistar Diabetic Fatty Rat Is a New Model of Extreme Insulin Resistance

The male obese Wistar Diabetic Fatty (WDF) rat is a genetic model of obesity and non-insulin dependent diabetes (NIDDM). The obese Zucker rat shares the same gene for obesity on a different genetic background but is not diabetic. This study evaluated the degree of insulin resistance in both obese strains by examining the binding and post binding effects of muscle insulin receptors in obese, rats exhibiting hyperinsulinemia and/or hyperglycemia. Insulin receptor binding and affinity and tyrosine kinase activity were measured in skeletal muscle from male WDF fa/fa (obese) and Fa/? (lean) and Zucker fa/fa (obese) and Fa/Fa (homozygous lean) rats. Rats were fed a high sucrose (68% of total Kcal) or Purina stock diet for 14 weeks. At 27 weeks of age, adipose depots were removed for adipose cellularity analysis and the biceps femoris muscle was removed for measurement of insulin binding and insulin-stimulated receptor kinase activity. Plasma glucose (13.9 vs. 8.4 mM) and insulin levels (14,754 vs. 7440 pmoI/L) were significantly higher in WDF obese than in Zucker obese rats. Insulin receptor number and affinity and TK activity were unaffected by diet. Insulin receptor number was significantly reduced in obese WDF rats (2.778 ± 0.617 pmol/mg protein), compared to obese Zucker rats (4.441 ± 0.913 pmol/mg potein). Both obese strains exhibited down regulation of the insulin receptor compared to their lean controls. Maximal tyrosine kinase (TK) activity was significantly reduced in obese WDF rats (505 ± 82 fmol/min/mg protein) compared to obese Zucker rats (1907 ± 610 fmol/min/mg protein). Only obese WDF rats displayed a decrease in TK activity per receptor. These observations establish the obese WDF rat as an excellent model for exploring mechanisms of extreme insulin resistance, particularly post-receptor tyrosine kinase-associated defects, in non-insulin dependent diabetes.     

Subchronic treatment with metformin produces anorectic effect and reduces hyperinsulinemia in genetically obese Zucker rats.

The effect of subchronic metformin treatment on food intake, weight gain and plasma and tissue hormone levels was investigated in genetically obese male Zucker rats and in their lean controls. Metformin hydrochloride (320 mg/kg/day for 14 days in the drinking water) significantly reduced 24 hour food intake both after one and two weeks treatment in obese rats. In contrast, metformin had only a transient effect on food intake in lean animals. The reduced food intake was associated with body weight decrease, particularly in obese rats. Metformin markedly reduced also the hyperinsulinemia of the obese animals without altering their plasma glucose or pancreatic insulin content which may reflect an improved insulin sensitivity after metformin treatment. Metformin did not change plasma corticosterone levels or insulin and somatostatin concentrations in the pancreas. Metformin reduced pyloric region somatostatin content in lean rats. It is concluded that metformin has an anorectic effect and reduces body weight and hyperinsulinemia in genetically obese Zucker rat.     

Effect of dietary Platycodon grandiflorum on the improvement of insulin resistance in obese Zucker rats

The effect of dietary Platycodon grandiflorum on the improvement of insulin resistance and lipid profile was investigated in lean (Fa/-) and obese (fa/fa) Zucker rats, a model for noninsulin dependent diabetes mellitus. Dietary Platycodon grandiflorum feeding for 4 weeks resulted in a significant decrease in the concentration of plasma triglyceride in both lean and obese Zucker rats. Furthermore, dietary Platycodon grandiflorum markedly decreased both plasma cholesterol and fasting plasma insulin levels, and significantly decreased the postprandial glucose level at 30 min during oral glucose tolerance test in obese Zucker rats. Although there was no statistical significance, the crude glucose transporter 4 protein level of obese rats fed Platycodon grandiflorum tended to increase when compared with that of obese control rats. Therefore, the present results suggested that dietary Platycodon grandiflorum may be useful in prevention and improvement of metabolic disorders characterized by hyperinsulinemia states such as noninsulin dependent diabetes mellitus, syndrome X, and coronary artery disease.     

Effects of Irbesartan on the Growth and Differentiation of Adipocytes in Obese Zucker Rats

Objective: The aim of this study was to evaluate the effects of the selective angiotensin receptor 1 antagonist irbesartan on the growth and differentiation of the adipocytes in obese Zucker fa/fa rats. Research Methods and Procedures: Obese Zucker fa/fa rats were treated by oral route for 3 weeks with irbesartan at doses of 3–10‐30 mg/kg per day. The adipocyte differentiation was evaluated by analyzing tissue samples of white (retroperitoneal) or brown (interscapular) adipose tissue for the presence of peroxisome proliferator activated receptor γ, leptin, and the activity of glycerol‐3‐phosphate dehydrogenase. Results: This study showed that the treatment of obese Zucker fa/fa with irbesartan effectively reduced the differentiation of adipocytes within brown (interscapular) and white (retroperitoneal) adipose tissue. In fact, irbesartan significantly (p < 0.01) and dose‐dependently reduced the tissue levels of leptin, peroxisome proliferator activated receptor γ, and the activity of the enzyme glycerol‐3‐phoshate dehydrogenase accepted markers of adipocyte differentiation. None of the tested doses of irbesartan affected these markers in non‐obese rats. Discussion: The antagonism of the angiotensin receptor 1 receptors with irbesartan reduces the adipogenic activity of angiotensin II in obese Zucker rats, with the endpoint being reduction of the growth and differentiation of the adipocytes within the adipose tissue.     

Lipoprotein lipase and cholesterol transfer activities of lean and obese Zucker rats.

Adult female lean and obese Zucker rats maintained under standard conditions were used for the estimation of plasma, liver and white adipose tissue (WAT) activity of lipoprotein lipase, plasma and liver hepatic lipase and plasma lecithin-cholesterol acyltransferase. No differences in plasma or tissue levels of lipoprotein lipase between lean and obese rats were detected, but the larger WAT size of the obese rats resulted in higher lipase activity per unit of rat weight. Hepatic lipase levels in plasma were higher in the obese, but in liver, the higher activity was found in lean rats. No significant differences were found for lecithin-cholesterol acyltransferase activity, except when the levels in the HDL fraction were expressed per unit of protein weight, showing lower activity in the obese rats. In conclusion, the essentially maintained enzyme activities in obese rat tissues suggest that they cannot explain the deficient lipoproteins processing of obese rats, and, consequently their dislipidaemia.     

Preadipocyte stimulating factor in rat serum: evidence for a discrete 63 kDa protein that promotes cell differentiation of rat preadipocytes in primary cultures

In primary cultures of rat preadipocytes (PA) isolated from epididymal or perirenal depots, rat serum is more effective than other animal sera (fetal calf, newborn calf, human, horse, rabbit, cat, sheep, goat, dog, pig) in promoting adipogenic conversion, biochemical differentiation, and mitogenesis. Only mouse serum is comparable to rat serum. This activity is attributable to a specific growth factor (preadipocyte stimulating factor, PSF). An assay for PSF in rat serum was devised using PA from perirenal fat of 3-month-old Fischer 344 rats grown first to confluence in FCS for 8 days and then for the next 3 days in test serum, followed by measurement of triglyceride (TG) and glycerol-3-phosphate dehydrogenase (GPDH). Rat serum induces dose-dependent rapid cell division, which coincides with accumulation of TG and increase of GPDH; for routine quantitation, TG is assayed. The biochemical characteristics of PSF in serum are as follows: stable at 4 degrees C for up to 1 year; inactivated at 100 degrees C (80% loss, 30 min) but stable at 56 degrees C for 1 hr; stable at pH 2-12; non-dialyzable; completely resistant to pepsin, trypsin, and chymotrypsin but destroyed by pronase and subtilisn; stable to DTT and periodate; and m.w. between 68 kDa (Sephacryl-300) and 58 kDa (Sephacryl-300 in 5 M urea). PSF activity is greater in serum from Wistar than from Fischer 344 rats, while activity of serum from Zucker obese (fa/fa) rats is at least as great as that from Wistar rats and, like serum of rats made obese by feeding a high-fat, high-carbohydrate diet, is not suppressed. PSF activity is not due to insulin, insulin-like growth factor-1 (IGF-1), growth hormone, glucocorticoids, or combinations of these hormones. PSF activity was not seen with a number of growth factors including colony-stimulating factor (CSF-1), GM-CSF, interleukins 1, 2, and 3, neuroleukin, tumor necrosis factor, and others. PSF is distinct from the low molecular weight (4-8 kDa) differentiation factor present in rat serum, FCS, and human serum that promotes the adipogenic conversion and cellular differentiation of 3T3-L1, 3T3-F442A, and Ob17 cells. PSF appears to be a new differentiation factor for rat preadipocytes, has properties suggestive of a highly glycosylated protein, and may be highly species specific.     

Genotype and diet effects in lean and obese Zucker rats fed either safflower or coconut oil diets

Previously we reported that suckling lean heterozygous (FA/fa) Zucker rats had a number of adipose tissue measurements intermediate between those of homozygous lean (FA/FA) and obese (fa/fa) rats. However, in young adult male rats maintained on a low-fat diet, these differences were no longer apparent (i.e., values for the two lean genotypes were similar). In the present study we determined whether the heterozygous effect of the "fa" gene was dependent on the consumption of a high-fat diet. Mother rats were fed high-fat diets containing either safflower (SOD) or coconut (COD) oil throughout mating and lactation. Homozygous lean male and female rats were bred, as well as obese male and lean heterozygous female rats. Suckling rats were studied at 17 days of age. Additional male rats were maintained on the same diet as their mothers until 11-12 weeks of age. Obese suckling rats had higher body weights than lean pups. Inguinal fat pad weights and pad-to-body weight ratios followed the pattern of obese greater than lean (FA/fa) pups that were greater than lean (FA/FA) pups. A similar relationship was found for adipose tissue lipogenic enzyme activities. At 11-12 weeks of age, measurements followed the general pattern of obese rats having greater values than lean rats (i.e., FA/fa = FA/FA). SOD-fa/fa rats had higher hepatic lipogenic enzyme activities than COD-fa/fa rats. In addition, SOD rats had higher fat cell numbers than COD rats. These results suggest that specific fatty acids can alter adipocyte proliferation and/or differentiation in vivo. In addition, there appears to be a defect of fatty acid regulation in livers of genetically obese rats. The heterozygous effect of the "fa" gene in suckling Zucker rats was confirmed. However, high-fat feeding did not result in a heterozygous effect in young adult lean male rats. We will next evaluate the role of sex on this effect.     

Comparative hormonal profile of two newly developed obese congenic rat strains

1. The effect of feeding diets containing either 54% sucrose or cooked corn starch for 12 weeks on levels of fasting plasma insulin, corticosterone, growth hormone and glucagon were compared in two newly developed genetically obese rat strains--the normoglycemic LA/N-cp and the diabetic SHR/N-cp. 2. In corpulent rats of either strain, levels of plasma insulin and corticosterone were greater when compared to the lean littermates. Corpulent LA/N-cp rats had lower levels of plasma glucagon and higher levels of plasma growth hormone than did lean LA/N-cp rats. 3. SHR/N-cp rats fed sucrose had greater levels of corticosterone and glucagon than did SHR/N-cp rats fed starch.     

Unexpected regulation of hypothalamic neuropeptide Y by food deprivation and refeeding in the Zucker rat.

Neuropeptide Y strongly stimulates food intake when it is injected in the hypothalamic paraventricular (PVN) and ventromedian (VMN) nuclei. In Sprague-Dawley (SD) rats, NPY synthesis in the arcuate nucleus (ARC) is increased by food deprivation and is normalized by refeeding. We have previously shown that the obese hyperphagic Zucker rat is characterized by higher NPY concentrations in this nucleus. NPY might therefore play an important role in the development of hyperphagia. The aim of the present study was to determine if the regulation by the feeding state works in the obese Zucker rat. For this purpose, 10 weeks-old male lean (n = 30) and obese (n = 30) Zucker rats were either fed ad libitum, either food-deprived (FD) for 48 hours or food-deprived for 48 h and refed (RF) for 6 hours. NPY was measured in several microdissected brain areas involved in the regulation of feeding behavior. NPY concentrations in the ARC was about 50% greater in obese rats than in lean rats (p less than 0.02) whatever the feeding state. In the VMN, NPY concentrations were higher in the lean FD rats than in the obese FD rat (p less than 0.001). Food deprivation or refeeding did not modify NPY in the ARC, in the VMN or in the dorsomedian nucleus whatever the genotype considered. On the other hand, food deprivation induced a significant decrease in NPY concentrations in the PVN of lean rats. This decrease was localized in the parvocellular part of this nucleus (43.0 +/- 1.9 (FD) vs 54.2 +/- 2.1 (Ad lib) ng/mg protein; p less than 0.005). Ad lib levels were restored by 6 hours of refeeding. These variations were not observed in the obese rat. The regulation of NPY by the feeding state in the Zucker rat was therefore very different from that described in the SD rats. Strain or age of the animals used might explain these differences. High NPY levels and absence of regulation in obese Zucker rats could contribute to the abnormal feeding behavior of these rats.     

Comparison of the lipolytic activity of adipocytes isolated from white adipose tissue in Zucker and Wistar rats. In vitro effects of 3,5,3'-triiodo-L-thyronine

The in vitro effects of 3,5,3'-triiodo-L-thyronine (L-T3) on (-) epinephrine stimulated lipolytic activity were studied in adipocytes isolated from white adipose tissue of Zucker and Wistar male rats. Basic lipolytic activity was small and nearly insensitive to epinephrine in Zucker genetically obese rats. On the other hand, lipolytic activity was stimulated by epinephrine in Zucker lean rats and Wistar rats in the same way. There was no synergistic interaction between epinephrine and L-T3 on lipolytic activity, since the released glycerol levels were nearly the same. These data corroborate the hypothyroid status of Zucker genetically obese rats.     

Early development of adipose cell lipogenesis and glycerol utilization in Zucker obese rats.

A study of adipose cell metabolism was made at ages 5, 7, 10, and 14 wk of age in genetically obese Zucker rats. Adipose samples were surgically removed and used for in vitro adipose cell incubations and for characterization of enzyme patterns. Lipogenic capacity from glucose and enzymes normally associated with lipogenesis (malic enzyme, citrate cleavage enzyme and glucose-6-PO4 dehydrogenase) followed the same pattern of development. At 5 wk of age, the adipose cells of obese animals had a greater capacity for fat synthesis than the lean rats. At all other ages lipogenic activity and enzyme levels were either similar or less than the pair-fed lean littermates. Glycerol utilization by isolated fat cells was similar; however, adipose tissue glycerokinase was elevated in obese rats at 14 wk of age. It was concluded that there was no apparent change in specific lipogenic capacity of fat cells from the obese rat when compared to its lean littermate. It was also concluded that increased adipose glycerokinase activity in obese rats represented a secondary shift in metabolism.     

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.