The postprandial rates of glycogen and lipid synthesis of lean and obese female Zucker rats

OBJECTIVE: To determine the relative rates of glycogenesis and lipogenesis following administration of a test meal in lean and obese Zucker rats. PROTOCOL: Nine-week-old lean and obese Zucker rats were fasted overnight, then tube-fed a test meal of balanced composition amounting to 16kJ (lean rats and one group of obese rats) or 24kJ (one group of obese rats) and containing 200 mg 1-(13)C glucose. Immediately after the meal the rats were injected intraperitoneally with 5 mCi of 3H2O and killed 1 h later. METHODS: Glycogenesis was calculated from the incorporation of 3H into liver glycogen divided by the specific activity of plasma water. Lipogenesis was calculated similarly from the incorporation of 3H into saponifiable lipids in liver and perirenal adipose tissue. The proportion of glycogen synthesized by the indirect pathway via pyruvate was determined from the ratio of 3H labelling at positions C6 and C2 in the glycogen glucose residues. Glycogen synthesis from glucose was determined from the ratio of 13C enrichment in liver glycogen to that in plasma glucose. RESULTS: The rate of synthesis of glycogen was considerably lower in the livers of obese rats than those of lean controls, with the larger meal causing a small but significant increase in glycogenesis. The proportion of glycogen synthesized via pyruvate showed a non-significant increase in the obese rats, while the amount of glycogen synthesized from glucose was significantly decreased. Hepatic lipogenic rates were about five times higher in both groups of obese rats than the lean controls. In adipose tissue, lipogenesis per g tissue was slightly reduced in the obese rats, although there was clearly an increase in adipose tissue lipogenic activity per whole animal. The larger meal caused a greater rise in plasma glucose and insulin concentrations but did not affect lipogenic rates, although it did cause a greater suppression of lipolysis, as indicated by a lower plasma glycerol concentration. CONCLUSION: Ingested carbohydrate is partitioned predominantly into hepatic fatty acid synthesis in obese Zucker rats. Hepatic glycogen synthesis is suppressed and comes mainly from precursors other than glucose. The suppression of hepatic glycogen synthesis may contribute to the increased energetic efficiency of obese Zucker rats.     

Fructose utilization by hepatocytes isolated from fed obese fa/fa Zucker rats. Metabolic fate of fructose in the absence of the modulator

The utilization of fructose by isolated hepatocytes was investigated in fed obese Zucker fa/fa rats compared with their lean littermates (Fa/?) and Sprague-Dawley rats. Hepatocytes were incubated during 3 h using U14C fructose (20 mM). Our results show: a significant increase of fructose consumption, glucose, lactate and pyruvate production and faster turnover of glycogen by fa/fa rats. In these animals, synthesis of acylglycerol was also significantly enhanced. Our results suggest that fructose in fa/fa rats was used preferentially as precursor for lipid synthesis not only by the liver but also by the adipose tissue after a prior transformation into glucose by hepatocytes. All these abnormalities result in an accumulation of acylglycerols maintaining an obesity state in fa/fa rats.     

Carbohydrate metabolism in lean and obese Zucker rats

Carbohydrate metabolism was evaluated in lean and obese Zucker rats. Plasma glucose concentration, renal and hepatic gluconeogenesis, and hepatic glycogen content and rates of synthesis were investigated in 2-mo and 8-mo-old animals. Mild hyperglycemia was observed in obese Zucker rats compared to lean rats and was more pronounced in males than in females. Rates of glucose disappearance were normal in both female and male rats, although there was a trend toward decreased clearance in the male. Total organ hepatic and kidney PEPCK activity and kidney glucose production were elevated in obese compared to lean rats. Total organ hepatic glycogen levels and rates of glycogen synthesis were increased significantly in obese compared to lean, the increase being greater in males than females. The mild hyperglycemia present in obese Zucker rats is not associated with delayed disappearance of intravenously administered glucose, but may be due to the increased production of glucose by whole kidney and liver.     

Metabolic consequences of fasting in old lean and obese Zucker rats

The effects of fasting on lipid and carbohydrate metabolism and plasma insulin and glucagon levels were compared in lean and obese Zucker rats. Sixteen-month-old female and male rats were fasted for periods of 2, 4, 6 and 12 days. Fasting produced significant decreases in hepatic rates of lipid, cholesterol, and glycogen synthesis, as well as circulating levels of triglycerides, cholesterol, phospholipids, and insulin. Significant increases in hepatic lipid levels and serum free fatty acids were noted. When compared to lean rats, obese rats had elevated rates of hepatic lipid and glycogen synthesis, hepatic lipid and glycogen stores, serum triglycerides, cholesterol, phospholipids, and plasma insulin. Lean rats had higher plasma glucagon levels. Sex differences in several parameters were observed. Females demonstrated higher levels of lipid and cholesterol synthesis and serum free fatty acids, whereas serum cholesterol levels and hepatic glycogen stores were higher in males. Following a 12-day fast, carcass fat and protein content were decreased in both lean and obese rats, but the obese animals maintained an obese body composition. It is concluded that fasting results in qualitatively similar metabolic and hormonal changes in both lean and obese rats, but that abnormalities in carbohydrate and lipid metabolism persist in obese rats even after a 12-day fast.     

Oxidation and ketogenesis in hepatocytes of lean and obese Zucker rats

Ketone body production and oxidation of ¹⁴C fatty acids to CO₂ were measured in hepatocytes isolated from lean and obese Zucker rats. The oxidation of [1-¹⁴C]octanoate, [1-¹⁴C]palmitate and [1-¹⁴C]palmitoyl carnitine to ¹⁴CO₂ was 50%–70% less in obese than in lean rats. Although ketone body production in hepatocytes from both lean and obese rats was increased by fasting, there was a significantly lower rate of ketone body production in hepatocytes from obese rats. Ketone body production was reduced to a comparable extent by increasing the glucose concentration in the incubation media of hepatocytes from both lean and obese rats. Glucagon and carnitine increased ketogenesis and the effects were additive and similar in lean and obese rats. These data suggest that β-oxidation and ketogenesis are suppressed in the obese Zucker rat, and further that ketone bodies can be modulated similarly in hepatocytes from lean and obese rats by nutritional and hormonal intervention. It is postulated that the decreased β-oxidation and ketone body production may play a role in the development or maintenance of obesity in the Zucker rat.     

Brown adipose tissue metabolism in hypothalamic-obese rats

Studies were performed to evaluate the metabolic changes of brown adipose tissue (BAT) in rats with hypothalamic obesity (VMNL). In vitro 14C-palmitate oxidation and incorporation into triglycerides were similar in VMNL and control rats. However, protein and fatty acid content and incorporation of 14C-palmitate into phospholipid were significantly less in both hyperphagic and normophagic VMNL rats. In order to assess in vivo BAT lipogenesis, rats were injected with 3H2O. Plasma H2O incorporation into BAT lipids was significantly greater in VMNL rats. Likewise, BAT lipid content was higher in obese rats. In another experiment BAT was incubated with U-14C-glucose to evaluate glucose utilization by BAT. 14C-glucose was oxidized and incorporated into both lipids and glycogen more rapidly by obese than by normal rat BAT. Glycogen content was greater in VMNL rats. Tissues were also incubated with 1-14C-pyruvate and 2-14C pyruvate. Pyruvate incorporation into glyceride glycerol and oxidation of 2-14C pyruvate through the Krebs cycle were similar in both obese and control rats. However, the incorporation of pyruvate into glyceride fatty acids was increased in VMNL rats. The results indicate that both fatty acid and lipid synthesis are increased in BAT of obese rats whereas lactate production is decreased and Krebs cycle activity is normal. Some of these changes appear to be independent of the level of food intake.     

Impaired glycogen synthesis in hepatocytes from Zucker fatty fa/fa rats: the role of increased phosphorylase activity

Aims/hypothesis. The Zucker fatty fa/fa rat develops hyperinsulinaemia, insulin-resistance and severe obesity as a result of a homozygous mutation in the leptin receptor gene. The aim was to characterise the metabolic defect(s) in hepatocytes from fa/fa rats. Methods. Glucose metabolism and key regulatory enzymes were investigated in hepatocytes from fa/fa and Fa/? rats after short-term culture in the absence of insulin. Results. Hepatocytes from fa/fa rats have higher glucokinase activity and expression of the glucokinase regulatory protein and higher rates of glycolysis and lipogenesis, but lower rates of glycogen synthesis than hepatocytes from Fa/? controls. Insulin caused a similar stimulation of glycogen synthesis in hepatocytes from fa/fa rats as in controls ( > twofold) but did not restore the impaired glycogen synthesis in cells from fa/fa rats. Adenovirus-mediated glucokinase overexpression stimulated glycogen synthesis and glycolysis but aggravated rather than abolished the relative impairment of glycogen synthesis in cells from fa/fa rats. Inhibition of glycolysis with 2,5-anhydromannitol, an inhibitor of glycolysis and gluconeogenesis, increased glucose 6-phosphate concentrations and glycogen synthesis in hepatocytes from Fa/? and fa/fa rats but did not restore the impaired glycogen synthesis in cells from fa/fa rats. Hepatocytes from fa/fa rats had a higher activity of phosphorylase a in the basal state and after incubation with insulin or glucagon and higher total phosphorylase. Conclusion/interpretation. The increased activity of phosphorylase is a major contributing factor to the impaired glycogen synthesis in hepatocytes from fa/fa rats and could contribute to the lipogenic state by a glycogenolytic-glycolytic-lipogenic pathway. [Diabetologia (2000) 43: 589–597]     

Effect of caloric restriction on basal insulin levels and the in vivo lipogenesis and glycogen synthesis from glucose in the Koletsky obese rat

Fasting plasma immunoreactive insulin levels increased with age in hyperinsulinemic Koletsky obese rats, being almost four times as high as in lean siblings at 3 mo (40 ± 5 μU/ml) and rising steadily to 82 ± 4 μU/ml at 6 mo (about seven times higher than lean siblings). Restricting the food intake of the obese rats markedly reduced but did not normalize the hyperinsulinemia, which in these rats was accompanied by normal plasma glucose concentrations. The incorporation in vivo of D-U-¹⁴C-glucose into tissue lipids and glycogen was measured 1 hr after the intravenous injection of 1 g glucose (containing 100 μCi D-U-¹⁴C-glucose) per kg body weight in obese rats eating ad libitum, obese rats after 3 mo on a restricted food intake, and lean siblings. All tissues (heart, diaphragm, skeletal muscle, and adipose tissues and liver) of obese rats exhibited a significantly greater lipogenesis from glucose than those of lean siblings. Dietary restriction of the obese rats reduced the ¹⁴C incorporation into lipid to levels not significantly different from lean controls in all tissues except skeletal muscle and liver, where, although greatly reduced, lipogenesis was still significantly higher than in lean rats. Glycogen synthesis tended to be greater in all tissues of obese rats than in lean animals. Dietary restriction of obese rats did not greatly affect glycogen synthesis.     

Relationship between lipogenesis,ketogenesis, and malonyl-CoA content in isolated hepatocytes from the obese Zucker rat adapted to a high-fat diet

The relationship between lipogenesis and ketogenesis and the concentration of malonyl coenzyme A (CoA) was investigated in hepatocytes from adult obese Zucker rats and their lean littermates fed either a control low-fat diet or a high-fat diet (30% lard in weight). With the control diet, lipogenesis—although strongly inhibited in the presence of either 1 mmol/L oleate, 10^?6 mol/L glucagon or 0.1 mmol/L TOFA (a hypolipidemic drug)—remained about fifteen-fold higher in the obese rats than in the lean rats. In contrast, ketogenesis under some conditions (oleate + TOFA) was not significantly lower (30%) as compared with the lean rats. After adaptation to the high-fat diet, lipogenesis was depressed fourfold in the lean rats and ninefold in the obese ones; however its magnitude remained significantly higher in the latter, namely at a value close to that measured in control-fed lean rats. Ketogenesis was comparable in lean and obese rats and much higher in the presence of 1 mmol/L oleate than of 0.3 mmol/L oleate, whereas lipogenesis did not vary with increasing oleate concentration in the medium. Acetyl-CoA carboxylase activity measured in liver homogenates was higher in the obese group, but was stepwise inhibited by increasing concentrations of oleyl-CoA regardless of the diet for both lean and obese rats, thus showing no abnormality of in vitro responsiveness to this inhibitor. With the control diet, hepatocyte malonyl-CoA levels were significantly higher in the obese rats, both in the basal state and after inhibition of lipogenesis by oleate and TOFA. However, after the high-fat diet, there was no longer a significant difference between the genotypes. These results show that in the obese Zucker rats, ketogenesis is dependent on hepatocyte malonyl-CoA content in the sense that their ketogenic capacity becomes “normalized” when malonyl-CoA is decreased to the levels found in the lean littermates, as it is the case after fat-feeding. This normalization of malonyl-CoA levels in spite of higher lipogenesis in the obese rats may result from the activities of enzymes of its formation and utilization.     

Deposition of dietary fatty acids in young Zucker rats fed a cafeteria diet.

The content and accretion of fatty acids in 30, 45 and 60-day-old Zucker lean Fa/? and obese fa/fa rats fed either reference chow or a cafeteria diet has been studied, together with their actual fatty acid intake during each period. Diet had little overall effect on the pattern of deposition of fatty acids, but quantitatively the deposition of fat was much higher in cafeteria-fed rats. The fat-rich cafeteria diet allowed the direct incorporation of most fatty acids into the rat lipids, whilst chow feeding activated lipogenesis and the deposition of a shorter chain and more saturated pattern of fatty acids. Genetic, obesity induced a significant expansion of net lipogenesis when compared with lean controls. Cafeteria-fed obese rats accrued a high proportion of fatty acids, which was close to that ingested, but nevertheless showed a net de novo synthesis of fatty acids. It is postulated that the combined effects of genetic obesity and a fat-rich diet result in high rates of fat accretion with limited net lipogenesis. Lean Zucker rats show a progressive impairment of their delta 5-desaturase system, a situation also observed in obese rats fed a reference diet. In Zucker obese rats, cafeteria feeding resulted in an alteration of the conversion of C18:2 into C20:3. The cafeteria diet fully compensated for these drawbacks by supplying very high amounts of polyunsaturated fatty acids.     

Short-term influences of dichloroacetate on genetically hyperlipemic rats

The effects of short-term (7 days) administration of dichloroacetate (DCA) on carbohydrate and lipid metabolism in the Zucker obese and lean rat were investigated. Metabolic effects of the drug were more pronounced in the obese than in the lean rat. DCA decreased fasting blood glucose concentrations in both lean and obese rats, but more so in the fat animals, probably because of higher initial levels. The hypoglycemic action of DCA is likely attributable to a direct effect on liver and peripheral tissues and not to an indirect action caused by a decrease in the glucagon-to-insulin ratio because the drug induced just the opposite effect. DCA decreased plasma triglycerides (TG) and free fatty acids (FFA) in the hyperlipemic rats but not in lean rats. Intrahepatic triglyceride content diminished after drug treatment in fat rats, suggesting decreased hepatic TG synthesis. Hyperketonemia, induced in both lean and fat rats by DCA treatment, was also greater in the obese animal. This response was probably caused by accelerated hepatic ketone body production due to increased β-oxidation, and not to enhance FFA substrate supply. These data demonstrate that DCA is capable of correcting many of the underlying abnormalities in carbohydrate and fat metabolism in the obese Zucker rat.     

Changes in lipid metabolism in diet-induced obesity

Mature male Sprague-Dawley rats fed a powdered Purina Chow diet containing corn oil and condensed milk (CM) were compared to rats fed a Purina Chow diet (control). CM rats gained more weight and consumed more calories over a 73-day period than the control rats. The increased weight gain and body fat in CM rats was accompanied by increased cell number in retroperitoneal and inguinal but not epididymal fat pads while cell size was unchanged in all three pads. After obesity had developed there was an increase in insulin levels, lipolysis, hepatic fatty acid synthesis, and fatty acid oxidation. While CM rats demonstrated hyperinsulinemia and hyperglycerolemia, they maintained normal glucagon and glucose levels. They demonstrated higher rates of fatty acid synthesis in isolated hepatocytes but not in vivo, suggesting that a greater potential for fatty acid synthesis in CM rats was masked in vivo by the inhibitory action of dietary lipids. Beta-oxidation of (1-14C) palmitate in vivo and in vitro, and in vivo ketogenesis were greater in CM than in chow fed rats. These studies demonstrate that, after the development of obesity, CM rats, like genetically obese Zucker rats, are hyperinsulinemic and have elevated levels of fatty acid synthesis. However, unlike obese Zucker rats, CM rats displayed an increase in beta-oxidation. These studies suggest that increased insulin levels and hepatic fatty acid synthesis may contribute to dietary obesity (as they do to genetic obesity), whereas increased fatty acid oxidation in dietary obesity may be a compensatory response to maintain a lower body weight.     

Increased synthase phosphatase activity is responsible for the super-activation of glycogen synthase in hepatocytes from fasted obese Zucker rats

Addition of 60 mM glucose caused a similar partial activation of glycogen synthase in hepatocytes isolated from overnight fasted Wistar rats and from fasted lean Zucker (Fa/fa?) rats. In contrast, the activation went rapidly to completion in cells from fasted obese (fa/fa) rats. Subsequent addition of 4 microM microcystin, a potent inhibitor of type 1 and type 2A protein phosphatases, induced a rapid inactivation of glycogen synthase, which occurred at a similar rate in all three types of hepatocytes. This suggests that the super-activation of glycogen synthase in hepatocytes from fasted obese rats is not due to a lower synthase kinase activity. Glycogen synthase phosphatase was quantitatively assayed in broken-cell preparations from the same livers, with exogenous synthase b as substrate. The synthase phosphatase activity in the fa/fa livers was 3-fold higher than that in the livers from both lean Zucker rats and Wistar rats. This difference has to be attributed to an increased synthase phosphatase activity of the glycogen-bound protein phosphatase-1 in livers of fasted obese rats. The results suggest that in the latter animals the available insulin exceeds the insulin resistance of the liver. The resulting overexpression of the insulin-dependent synthase-phosphatase-1G activity may explain the super-activation of glycogen synthase in response to a glucose challenge.     

Lipogenesis in situ in the genetically obese Zucker fatty rat (fa/fa): Role of hyperphagia and hyperinsulinaemia

Summary In situ fatty acid synthesis has been measured with ³H₂O in anaesthetised lean and obese Zucker (fa/fa) rats. The accumulation of fatty acids was increased in both the liver and adipose tissue of young fa/fa rats as a result of both an increased rate of lipogenesis and an increase in tissue mass. Whereas total hepatic lipogenesis increased with age, total adipose tissue lipogenesis decreased in older fa/fa rats. Experiments with hepatectomized rats showed that the liver was the major site of the excess fatty acid synthesis in fa/fa rats. The enhanced rate of lipogenesis in fa/fa rats was abolished by either pairfeeding or streptozotocin treatment. The results suggest that the increased fatty acid synthesis in fa/fa rats is secondary to the hyperphagia, hpyerinsulinaemia, and increased mass of hepatic and adipose tissues.The authors are grateful to the M. R. C. for award of a project grant.     

Cholesterol homeostasis in lean and obese male Zucker rats

Studies were performed in male Zucker rats to determine the metabolic effect of genetic obesity on whole body cholesterol homeostasis. Lean and obese mature Zucker rats were studied during intake of either a chow diet or a semisynthetic diet containing 10% corn oil; in addition growing animals were studied during constant body weight gain on a chow diet. Under all conditions the obese Zucker rats had significantly higher levels of total plasma cholesterol and triglyceride; however, measurements of the specific activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and of the rate of whole body cholesterol synthesis by sterol balance techniques demonstrated that the lean and obese animals did not differ in their endogenous rates of cholesterol synthesis. When sterol balance data were calculated per kilogram body weight, lean male Zucker rats synthesized a greater amount of cholesterol per day than obese animals. These studies demonstrate that the obese male Zucker rat, in many ways a model of human obesity, does not overproduce cholesterol and thus fails to exhibit one of major characteristics of the obese human.     

Metabolic effects of high-protein diets in Zucker rats

The effects of dietary protein on the metabolism of proteins, carbohydrates, and especially, lipids were investigated in genetically obese Zucker rats and their lean siblings. For 40 days the rats received diets containing 15%, 64%, or 82% protein, included at the expense of cornstarch. In the obese animals, the high-protein diets led to decreased food intake and weight gain. While these diets decreased the activities of lipogenic enzymes along with the lipid gain, they did not decrease the final body-fat content. The increase protein intake stimulated hepatic ureogenesis and gluconeogenesis. Lipolysis was stimulated, as demonstrated by an accumulation of ketone bodies in the liver. Blood levels of triacylglycerols, free glycerol, and nonesterified fatty acids were concomitantly decreased, which suggests an accelerated turnover of lipids. Whatever the composition of the diet, total energy retention of the lean rats was always less than that of the obese rats. The changes observed on high-protein diets were essentially the same for the two groups, except that the final body-content of lipids in the lean rats was significantly lower. In the absence of exogenous carbohydrate, the lean rats were barely able to retain nitrogen and to maintain hepatic lipogenesis. Unlike the rats from other strains, the lean Zucker rats could not adapt to a low-carbohydrate diet; this failure may be due to a metabolic disorder.     

Glucose metabolism in isolated adipocytes from lean and obese Zucker rats following treatment with dehydroepiandrosterone

Previous work has demonstrated that chronic administration of dehydroepiandrosterone (DHEA) to obese Zucker rats reduces the severity of hyperinsulinemia that is usually present. There were also significant decreases in body weight, fat depot weight, and adipose tissue cellularity. It was hypothesized that the decreased serum insulin was a reflection of improved tissue responsiveness to insulin. The purpose of the present study was to evaluate this hypothesis by examining the insulin response in isolated adipocytes of DHEA-treated rats. Glucose incorporation into CO2, fatty acids, and glyceride-glycerol was measured in isolated parametrial and retroperitoneal adipocytes. Cells from control and DHEA-treated lean rats and control and DHEA-treated obese rats were used, as well as cells from a group of obese rats pair-fed to the DHEA-obese rats. Increased basal and insulin-stimulated rates of incorporation of glucose into CO2 and fatty acids were found in adipocytes from DHEA-lean rats compared to control, lean rats. In contrast, cells from DHEA-treated obese rats tended to incorporate less glucose into CO2 and fatty acids than either the control or pair-fed obese rats. These data indicate that the decrease in serum insulin levels seen in DHEA-treated obese rats is not due to an improvement of adipose tissue responsiveness.     

Different types of postinsulin receptor defects contribute to insulin resistance in hearts of obese Zucker rats

The influence of obesity on myocardial function and metabolism was studied in obese (fa/fa) and thin (Fa/Fa) Zucker rats using the isolated perfused heart as model. Cardiac performance of obese Zucker rats was not impaired. Instead, left ventricular pressure and contractility were increased as compared to controls. In agreement with these findings, creatine phosphate and the ratios of ATP/ADP and creatine phosphate creatine were elevated. The uptake and the conversion of glucose by hearts of obese Zucker rats were impaired. Insulin stimulated the uptake and oxidation of glucose. However, the responsiveness of these processes to insulin was diminished. Lipolysis of endogenous lipids was accelerated severalfold in obesity. Inhibition of fatty acid oxidation by a specific carnitine palmitoyl-transferase inhibitor, phenylalkyloxirane carboxylic acid (POCA), led to a slow rate of lipolysis, and to an acceleration of glucose oxidation and of the basal, noninsulin-dependent uptake of glucose. In the presence of POCA, insulin had, however, no additional stimulatory effect on the glucose uptake by hearts of obese rats. In contrast to hearts of ketotic, acutely diabetic rats where POCA fully restored myocardial responsiveness of glucose uptake and conversion to insulin, in hearts of obese rats only a shift in the glucose pathway from glycolytic formation of lactate and pyruvate to oxidation to CO2 was observed. Thus, POCA can be used as a tool to distinguish different forms of insulin resistance in obesity: 1) a lipid metabolism-dependent defect--presumably an inhibition of phosphofructokinase and pyruvate dehydrogenase by metabolites of fatty acid oxidation, influenced by inhibition of carnitine palmitoyltransferasei, and 2) a lipid metabolism-independent defect in the activation of uptake of glucose and glycogen synthesis by insulin not affected by POCA.