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Psammomys obesus (a desert gerbil, nicknamed the “sand rat”) with innate insulin resistance was transferred to a high-energy (HE) diet at a young (8 to 20 weeks) and older (38 to 45 weeks) age. The young Psammomys progressed to in vivo insulin resistance, followed by pronounced hyperglycemia and hyperinsulinemia, as described previously. Analysis of the time dependency of these changes in response to the HE diet showed that the increase in serum glucose preceded the increase in insulin and plateaued earlier, reverting to normal together with insulin in the older Psammomys . Implants releasing insulin 2 IU/24 h did not induce appreciable hypoglycemia, a decrease in free fatty acids (FFAs), or a suppression of hepatic phosphoenolpyruvate carboxykinase (PEPCK) activity in young animals after 5 hours, despite a markedly increased circulating insulin. However, in the older Psammomys , the exogenous hyperinsulinemia produced a significant decline in serum glucose and FFA and a suppression of hepatic PEPCK activity. A euglycemic-hyperinsulinemic clamp confirmed that hepatic glucose production (HGP) was lower in older Psammomys versus the young and was almost completely abolished by insulin (from 5.6 ± 0.6 to 0.2 ± 0.1 mg · min −1 · kg −1 v 10.9 ± 0.8 to 3.9 ± 0.5 mg · min −1 · kg −1 ). This indicates that HGP, rather than glucose underutilization, was the main contributor to the hyperglycemia and that the hepatic insulin resistance in Psammomys is attenuated with age. In relation to the human condition, these findings point out that while the type 2 diabetes prevalence in Western populations generally increases with age, the excessive nutritional intake in high-risk populations produces a pattern of diabetes prevalence that tapers off with age. As such, the nutritionally induced diabetes in Psammomys represents a similar model for a differing pattern of the age-related prevalence of diabetes.

Animal species with genetic or nutritionally induced insulin resistance, diabetes and obesity (diabesity) may be divided into two broad groups: those with resilient pancreatic beta-cells, e.g. ob/ob mice and fa/fa rats, capable of long-lasting compensatory insulin over-secretion, and those with labile beta-cells in which the secretion pressure leads to irreversible beta-cell degranulation, e.g. db/db mice, Macaca mulatta primates, ZDF diabetic rats. Prominent in this group is the Israeli desert gerbil Psammomys obesus (sand rat), which features low insulin receptor density in liver and muscle. On a diet of relatively high energy, the capacity of insulin to activate the receptor tyrosine kinase (TK) is reduced, in the face of hyperinsulinemia. With the following hyperglycemia, the rising insulin resistance imposes a vicious cycle of insulinemia and glycemia, accentuating the TK activation failure and the beta-cell failure. Among various factors affecting the insulin signaling pathway, multisite phosphorylation, including serine and threonine on the receptor beta-subunit, due to overexpression of certain protein kinase C isoforms, seems to be responsible for the inhibition of the critical step of TK phosphorylation activity. The compromised TK activation is reversible by diet restriction which restores to normal the glycemia and insulinemia. The beta-cell response to long-lasting stimulation and the receptor malfunction in diabesity have implications for a similar etiology in human insulin resistance syndrome and type 2 diabetes, particularly in populations emerging from a food scarce environment into nutritional affluence, inappropriate to the human metabolic capacity. It is suggested that the "thrifty gene" is characterized by a low threshold for insulin secretion and low capacity for insulin clearance. Thus, nutritionally-induced hyperinsulinemia is potentiated and becomes the primary phenotypic expression of the thrifty gene, linked to the insulin receptor signaling pathway malfunction.

Psammomys lapses into fully fledged diabetes when maintained on a high-energy diet. Progression to diabetes has been classified into stage A of normoglycemia and normoinsulinemia (

Lymph chylomicrons and plasma VLDL, 14 C-labelled in vivo, were isolated from normal and nephrotic rats and injected into normal or nephrotic recipients. In normal recipients, the half-life of chylomicrons of nephrotic vs. normal origin was significantly longer (5.2 ± 0.5 vs . 3.5 ± 0.4 min −1 ). The nephrotic chylomicrons were larger in size, deficient in apo-E and apo A-I, rich in triacylglycerol and cholesterol, but poor in phospholipids, indicating that factors related to composition affected their removal. The half-life of nephrotic vs. normal VLDL, given to normal recipients, was unexpectedly shorter, (4.5 ± 0.2 vs . 5.8 ± 0.2 min −1 ). The nephrotic VLDL were also triacylglycerol- and cholesterol-rich and phospholipid-poor, but had a large diameter spread and contained a dense fraction according to the zonal ultracentrifugation pattern, suggesting the presence of faster removable IDL-like particles. When nephrotic rats received normal particles, a pronounced removal delay was seen, paralleling the extent of plasma triacylglycerol elevation. The half-life of chylomicrons was 8.3 ± 1.4 and 15.2 ± 2.5 min −1 in moderately and severely nephrotic rats, respectively, that of VLDL was 11.72 ± 2.1 and 37.8 ± 7.1 min −1 correspondingly. The chylomicron-triacylglycerol uptake was reduced both by adipose tissues and muscles of normal or nephrotic recipients, with some increase in entry into lungs, kidneys and spleen. Tissue distribution patterns of VLDL-triacylglycerol was similar to that of chylomicrons, except that the liver took up approx. 90% of the label. The low share of triacylglycerol uptake by tissues rich in lipoprotein lipase indicates that the activity of this enzyme was unlikely to limit the rate of removal. Lipoprotein lipase activity in adipose tissue and heart was slightly decreased in moderately nephrotic rats and declined only by approx. 35% in severely nephrotic ones. These results indicate that the removal defect in nephrosis seems to be due, in part, to changes in the composition of triacylglycerol-rich particles, compromising their accessibility to lipolysis and, in part, to their abundance, saturating the lipolytic capacity.

Lipoprotein excretion was investigated in the urine of hyperlipidemic rats with nephrotic syndrome induced by aminonucleoside of puromycin. Incubation with phosphatidylcholine liposomes was employed to float apoproteins not bound to lipids, by ultracentrifugation at d = 1.21 g/ml. On ultracentrifugation of whole, untreated urine, the amount of protein floated was 6-fold greater in nephrotic vs. control rats and consisted mainly of HDL-like particles. Sodium dodecylsulfate-polyacrylamide gel electrophoresis showed that control urine contained apoproteins A-I, A-II, E and traces of C, whereas in the nephrotic urine apo-E and a large amount of apo-C was found. Addition of liposomes to the ultracentrifugal d = 1.21 g/ml infranate and reflotation at the same density resulted only in slight increment in the floated apoproteins, mainly C and A-I. Addition of liposomes to the whole urine and centrifugation at d = 1.21 g/ml also did not produce a greater yield in the floated apoproteins of control or nephrotic urine. These results indicated that the urine is virtually devoid of lipid-free apoproteins and those floated from both the nephrotic and control urine are complexed with lipids. The plasma VLDL + LDL fraction of nephrotic rats, though increased in quantity, did not differ markedly in composition from that of control rats. The HDL, approximately 3-fold elevated in nephrotic rats, were poorer in esterified cholesterol and richer in phospholipids. Relative to plasma HDL, the nephrotic urine HDL were protein-rich and phospholipid-poor and appeared to be larger in particle size as suggested by the lower estimated specific volume. The modified plasma HDL in nephrosis may have a pathophysiological implication.

Pairs of spiny mice (Acomys cahirinus), maintained on a 50% sucrose diet in two experiments lasting 18 or 8 months, gained less weight and exhibited a greater mortality rate of both parents, and pups, than mice kept on regular or fat-rich diets. According to the number of pups born and number of productive pairs, the sucrose-fed mice were also less fertile. The litter size and the number of pups born per productive pair, were slightly but not significantly lower. These findings are discussed against the background of metabolic and pathologic changes induced in rodents maintained on sucrose-rich diets.

Sand rats (Psammomys obesus) maintained on a diet providing a free choice between laboratory chow and salt bush (Atriplex halimus) were classified into four groups differing in extent of the diabetic syndrome: A, normoglycemic-normoinsulinemic; B, normoglycemichyperinsulinemic; C, hyperglycemic-hyperinsulinemic; or D, hyperglycemic with reduced insulin levels. The metabolic pattern of these groups was characterized by measuring 1) the uptake of fatty acid-labeled, very-lowdensity lipoprotein-borne triglycerides (VLDL-TG) and [3H]%-2-deoxyglucose (2-DOG) into muscle and adipose tissues; 2) incorporation of [14C]alanine into glycogen in vivo; 3) gluconeogenesis from lactate, pyruvate, and alanine in hepatocytes; 4) the effect of insulin on glycogen synthesis from glucose; 5) the oxidation of albumin-bound [1-14C]palmitate and [14C]glucose in strips of soleus muscle; 6) activities of muscle and adipose tissue lipoprotein lipase; and 7) activities of rate-limiting enzymes of glycolysis, gluconeogenesis, and fatty acid synthesis in liver. In group A, uptake of VLDL-TG and activity of lipoprotein lipase were higher in adipose tissue and lower in muscle than in albino rats. In the liver, gluconeogenesis and the activity of phosphoenolpyruvate carboxykinase, as well as lipid synthesis and the activity of NAOP-malate dehydrogenase,were higher than in albino rats, whereas activity of pyruvate kinase was lower. In group B, uptake of VLDL-TG by adipose tissue and muscle and lipoprotein lipase activity were similar or higher than in group A. Uptake of 2-DOG by muscle and adipose tissue and activity of liver phosphoenolpyruvate carboxykinase were lower than in group A. In groups C and D, uptake of VLDL-TG and lipoprotein lipase activity in muscle were further increased. In adipose tissue a progressive decrease in VLDL-TG uptake and lipoprotein lipase activity was found, and uptake of 2-DOG by muscle and adipose tissue was further reduced. In the liver, gluconeogenesis was increased, and activity of phosphoenolpyruvate carboxykinase reached a maximum in group D. These results suggest that in the hyperinsulinemic stage (group B), uptake of glucose by muscle and adipose tissue is reduced, but insulin suppresses gluconeogenesis and stimulates hepatic synthesis and adipose tissue uptake of TG. Hyperglycemia manifests itself when insulin resistance results in increased gluconeogenesis and a further reduction in peripheral glucose uptake. These characteristics can be regarded as a model for the development of type II diabetes in humans evoked by nutritional affluence.

Administration of triamcinolone or dexamethasone to rats led to a prompt, marked and persistent rise in liver acetyl-CoA carboxylase activity. The activity of fatty acid synthetase increased to a lesser extent and after a more prolonged glucocorticoid treatment, whereas the changes in that of NADP-malate dehydrogenase and ATP-citrate lyase were not appreciable. The overall channeling of [1-14-C]acetyl-CoA to fatty acids was enhanced. The triamcinolone effect on acetyl-CoA carboxylase activity appeared to be dependent on the coincident hyperinsulinemia since it was not obtained in alloxan-diabetic rats, whereas the alanine-aminotransferase-inducing effect of this hormone was additive to that of insulin deficiency. In adipose tissue triamcinolone treatment caused a reduction in the activity of all lipogenesis enzymes and blunted their response to insulin administration. The antagonism of glucocorticoids toward insulin, selectively modulating the responses of the insulin-sensitive enzymes in liver and adipose tissue is discussed. The rise in hepatic lipogenic capacity, through the retention of the ability of insulin to induce acetyl-CoA carboxylase, may be physiologically important in restraining the ketogenesis from acetyl-CoA despite the increased fat utilization during glucocorticoid excess.

Spiny mice (Acomys cahirinus) from the Geneva colony tend to develop diabetes, whereas those maintained in Jerusalem do not. The role of environmental factors in the development of glucose intolerance was investigated by diet exchanges in specimens from the two colonies. Spiny mice on the Geneva diet (laboratory chow supplemented by a seed mixture containing 15% fat by weight) developed massive obesity over 8–10 months; body lipid content increased threefold compared with albino mice and was twofold higher than in spiny mice maintained on the laboratory chow in Jerusalem or in spiny mice living in their natural habitat near the Dead Sea. Spiny mice from the Geneva colony, kept on the laboratory chow alone, were as lean as animals from the Jerusalem colony. Similarly, Jerusalem Acomys given pellets supplemented by seeds developed marked obesity. Liver and adipose tissue enzymes in spiny mice transferred to the seed-supplemented diet showed adaptation typical for fat feeding: decrease in the capacity of glycolysis, NADPH generation, and fatty acid synthesis. The obesity was associated with insulin resistance, evident from a negative correlation between the extent of hypoglycemia after i.v. insulin administration and body lipid content. The glucose disappearance rate (K value) was significantly reduced by obesity, but the insulin response to i.v. glucose increased only moderately. In all the Acomys groups studied, insulin response to i.v. glucose was markedly lower in comparison with the response in albino mice. The following conclusions are drawn: (1) Low insulin response to glucose is a species-characteristic of spiny mice whether the animals are bred in laboratories or live in freedom. (2) Given an appropriate diet, spiny mice develop obesity, accompanied by pronounced insulin resistance. (3) Obesity may be one of the causes of the marked islet hyperplasia in laboratory-kept spiny mice, but this does not result in increased insulin output. (4) The inability of spiny mice to respond with augmented insulin secretion when insulin efficiency is reduced may be responsible for the accelerated development of glucose intolerance in this species. Thus, the liability of Geneva spiny mice to develop diabetes may be caused by the obesity-inducing diet used in this colony rather than to a specific genetic characteristic. (5) The fact that insulin resistance in spiny mice occurs without the development of hyperinsulinemia suggests that similar mechanisms may operate in the development of glucose intolerance in human, low, insulin responders.

Protease activity of rat gastrocnemius myofibrillar fraction, optimally active at the alkaline pH range, was investigated. The activity was inhibited by soybean trypsin inhibitor, tranexamic acid and other inhibitors of proteases, as well as by 2- to 10-m m solutions of FeCl 2 , FeCl 3 , and ZnCl 2 . CaCl 2 , MgCl 2 , and MnCl 2 at the same concentration range did not affect the activity of the enzyme. Treating the myofibrillar fraction with 1 m KCNS or KCl solubilized the enzyme. The protease activity was increased after 4 days of fasting, and on Day 6 was five times higher than in nonfasted rats. This increase in specific activity was significant when expressed in terms of total protein, noncollagen protein, or muscle DNA content. Protease activity also increased in rats after glucocorticoid administration, in rats with alloxan diabetes, in aminonucleoside-nephrotic rats, and in rats with hypoproteinemia induced by plasmapheresis. These findings suggest that the activity of the myofibrillar protease is adaptive, and increases whenever catabolism of muscle proteins and mobilization of amino acids to the liver is required.

The effect of several endocrine and catabolic conditions on the activity of myofibril-bound alkaline protease in rat gastrocnemius, diaphragm and cardiac muscle in relation to changes in muscle mass was investigated. The catabolic conditions of prolonged fasting, streptozotocin diabetes, and massive protein loss due to nephrotic syndrome resulted in an increase of myofibrillar protease activity in the 3 muscles together with a decrease in muscle and total body weight. On treatment with triamcinolone acetonide or with other glycocorticoids the proteolytic activity rose in gastrocnemius and diaphragm but fell in the heart. Concordantly, the relative weight of the gastrocnemius muscle and diaphragm remained without change whereas that of the heart increased. Skeletal muscle differed from cardiac muscle also in sensitivity toward the glucocorticoid: the decrease in heart protease activity was obtained at smaller hormone dose compared to that required for the elevation of gastrocnemius protease activity. An analogous pattern of changes was obtained on triiodothyronine treatment. The activity of gastrocnemius and diaphragm protease became enhanced along with the weight loss of these muscles; in contrast, the activity of the heart protease fell together with retention of heart weight. Likewise to glucocorticoids, the heart protease was more sensitive than the skeletal muscle enzyme to the effect of triiodothyronine. Selective changes were also observed in other situations; glucagon treatment reduced cardiac protease activity without affecting that of the gastrocnemius enzyme and in muscular dystrophy the gastrocnemius but not the cardiac protease activity became increased. It is concluded that the myofibrillar protease is involved in the hormonal regulation of muscle protein turnover. The divergent adaptive behavior of this enzyme in the different muscles in response to various hormonal conditions reflects a tissue-specific mode of regulation of the enzyme.

The activity of rat liver lipogenic enzymes, acetyl-CoA carboxylase, fatty acid synthetase, ATP citrate lyase and NADP-malate dehydrogenase and the in vitro incorporation of [1- 14 C] acetyl-CoA to fatty acids became markedly reduced as a result of serial injections of aminonucleoside of puromycin, used to induce nephrosis. The activity of the lipogenic pathway returned to normal and even showed an adaptive increase in nephrotic rats 10–14 days after the termination of aminonucleoside injections. The initial decrease in the hepatic lipogenic capacity was attributed to the direct effect of aminonucleoside on enzyme synthesis since (1) it preceded the onset of proteinuria, (2) was observed in rats given doses of aminonucleoside insufficient to produce early nephrosis and (3) was also evident in adipose tissue. The rise in serum lipids closely followed the onset of nephrotic proteinuria and hypoproteinemia despite the coincident decrease in hepatic lipogenic capacity. The development of hyperlipidemia was associated with a marked increase in circulating free fatty acid levels which subsided later, coinciding with the rise in the activity of lipogenic enzymes. This observation, together with lack of change in liver triglyceride content, suggested an increased channeling of preformed free fatty acids into the lipoprotein-borne lipids. The findings indicate the need for careful dissociation of direct effects of aminonucleoside from those induced by nephrosis and demonstrate that the hepatic overproduction of apolipoproteins entails a complementary elaboration of lipid moieties either by de novo fatty acid synthesis or by the utilization of extrahepatic fatty acids.

Circadian rhythms of plasma lipids and lipoproteins, lipoprotein lipase activities and VLDL secretion rates were studied in fed and food-deprived (12 h) male rats after a light/dark synchronization of 14 days. In ad libitum fed rats, a circadian rhythm of plasma triacylglycerol, blood glucose and liver glycogen was clearly identified. A rhythm was also identified for plasma cholesterol, but not phospholipids. The peak of plasma triacylglycerol occurred 2 h after the beginning of the light period (7.00 a.m.), and the nadir, 2 h after the beginning of the dark period (7.00 p.m.). The differences of plasma triacylglycerol at these two circadian stages were even more pronounced in food-deprived rats and were confined to the very-low-density lipoprotein (VLDL) fraction. Plasma post-heparin and heart and muscle lipoprotein lipase activities were 50–100% higher at 7.00 p.m., the time when plasma triacylglycerol were lowest, as compared to 7.00 a.m. Plasma post-heparin hepatic lipase and adipose tissue lipoprotein lipase activities, in contrast, did not change. VLDL secretion rates were somewhat higher at 7.00 a.m. compared to 7.00 p.m., but this difference was not significant. It is concluded that physiological variation of heart and muscle lipoprotein lipase together with small differences of VLDL secretion rates are responsible for normal range oscillations of plasma VLDL triacylglycerol levels.

The activity of protease was investigated by cleavage of 14C-labelled globin by the isolated myofibrillar fraction of maternal and fetal muscles of nonpregnant, pregnant, and pregnant diabetic rats, on days 15 and 20 of pregnancy. Diabetes was produced by i.p. strep-tozotocin injection on day 5 or 12 of pregnancy. There was no change in muscle protease activity on day 15 of pregnancy in nondiabetic rats, compared to nonpregnant control rats, whereas on day 20 protease activity rose in the gastrocnemius by 25%, in the diaphragm by 30%, and only slightly in the heart. Diabetes, imposed during days 12–20 of pregnancy, induced a further increase in protease activity in the gastrocnemius (42 %), diaphragm (47 %), and heart (25%). In rats with diabetes lasting from day 5 to 20 of gestation there were only slight further increases in protease activity. In contrast, maternal diabetes did not induce changes in myofibrillar protease activity of fetal leg or heart muscles compared with the corresponding muscles of fetuses of nondiabetic rats. The increased protease activity on the maternal side occurred at low insulin levels and/or reduced insulin sensitivity, whereas the absence of changes in protease activity on the fetal side was associated with sustained or even increased presence of insulin. This suggests that the adaptive changes in protease activity, in both pregnancy and superimposed diabetes, are related to insulin availability and its effectiveness.

Age-related and muscle tissue-specific alterations in myofibrillar protease activity were observed in different muscles of the rat. Utilizing exogenous, denatured and 3 H-labelled hemoglobin as substrate, proteolytic activity of the myofibrillar enzyme was found to decrease with age in the gastrocnemius muscle while the same activity in the diaphragm and heart muscles increased with age. The extent of response of the enzymes to administration of the potent glucocorticoid triamcinolone was, however, found to be similar in young and old animals, and each muscle retained its specific mode of response to the exogenous glucocorticoid, for example enhancement of the activity in skeletal and diaphgragm muscles and diminution of the activity in the heart. Development was associated with a marked reduction in the number of glucocorticoid-specific binding sites in the cytosol of both gastrocnemius and heart muscles, with only negligible changes in the affinity of hormone binding. It is concluded that while the ability of the enzyme to response to exogenous, pharmacological doses of glucocorticoids is not affected by development, development does modify the myofibrillar protease activity in a tissue-specific manner.

The relative contribution of lipoproteins and free fatty acid to the cellular triacylglycerol content of cultured human fibroblasts was tested. Fibroblasts accumulated triacylglycerol in proportion to the molar ratio of free fatty acid (oleic acid) to albumin in the medium. Fibroblasts also accumulated triacylglycerol when exposed to medium containing human very low density liproprotein. This accumulation of triacylglycerol was apparently due to direct uptake of intact very low density lipoprotein particles initiated by binding of very low density lipoprotein to cell surface receptors. The amount of 125I-labeled very low density lipoprotein protein internalized and degraded by the cell saturated at the same very low density lipoprotein concentration that produced the maximum increase in cell triacylglycerol. Preincubations with lipoprotein-deficient serum, which enhanced the cell's ability to bind 125I-labeled very low density lipoprotein, increased the amount of 125I-labeled very low density lipoprotein internalized and degraded by the cell in parallel with increased levels of cellular triacylglycerol. Results suggest that the triacylglycerol that accumulates in the presence of very low density lipoprotein represents a lysosomal pool of partially degraded very low density lipoprotein. Measurements of lipase activity of fibroblast homogenates revealed three pH optima at (in descending order of magnitude of activity) pH 4, pH 6, and pH 8. The pH 8 lipase does not appear to represent lipoprotein lipase, since it is not activated by either serum or heparin. Exposure of the cells to medium with varying lipid composition had no effect on the lipase activities. The lipase activities of fibroblasts from donors with familial hypertriglyceridemia appear to be normal.

The possibility that free fatty acids (FFA) and their CoA esters may directly inhibit the activity of enzymes of glycolysis and lipogenesis was studied in liver and adipose tissue of acutely diabetic rats. Despite a marked elevation in tissue FFA, the activity of glucose-6phosphate dehydrogenase, phosphofructokinase, pyruvate kinase, α-glycerophosphate dehydrogenase, aldolase, citrate synthetase, and several other enzymes was not affected 3 or 6 h after anti-insulin serum injection. The activities of hepatic and adipose tissue acetyl-CoA carboxylase, hepatic glucokinase and adipose tissue hexokinase were decreased. The tissue FFA levels were compared to linoleate concentrations required for halfmaximal inhibition (Ki) of several enzymes in tissue fractions afterin vitro contact of 30 min at 37 °C. Linoleate irreversibly inactivated the enzymes to a varying degree but its effect was dependent on the protein content of the system. The Ki of linoleate increased linearly with cellular protein concentration; when extrapolated to the protein level in the intact cell it became unphysiologically high, markedly exceeding the liver or adipose tissue FFA concentration of acutely diabetic rats. The Ki linoleate and the actual FFA concentrations were particularly discrepant in the cytoplasmic compartment, comprising roost of the investigated enzymes, but only a small proportion of tissue FFA, as determined by measurements of intracellular FFA distribution. Similar discrepancy was found for the interaction of palmityl-CoA with glucose-6-phosphate dehydrogenase and acetyl-CoA carboxylase. Thus, the selective decrease in the activity of glucokinase and acetyl-CoA carboxylase in acute diabetes was attributed to factors other than FFA or their CoA esters. The nature of FFA-enzyme interaction, causing enzyme inactivation rather than specific inhibition is discussed, concluding that FFA do not act as direct homeostatic modifiers of enzymes regulating carbohydrate and fat metabolism.

Acetyl-CoA carboxylase in human skin fibroblasts, grown in a lipid-poor medium, was inducible by insulin. The activity, ranging from 200 to 500 pmol/ min per mg of cytoplasmic protein in various cell lines, rose up to 2-fold in the presence of 1 mU/ml insulin after 3 days of incubation. The rise in acetyl-CoA carboxylase activity which was independent of cell population growth was also stimulated in proportion to log insulin concentration. The induction of acetyl-CoA carboxylase by insulin was preventable by cycloheximide, indicating new protein synthesis, and was associated with increases in activity of other enzymes related to lipogenesis, NADP-malate dehydrogenase, ATP-citrate lyase and fatty acid synthetase. Enhanced lipogenesis was expressed by a 3-fold increase in the incorporation of 3 H 2 O label into fibroblast fatty acids. Addition to the incubation medium of very low density lipoproteins (VLDL) or albumin bound-free fatty acid resulted in a concentration-dependent decrease in cell population growth, cancelling the effect of insulin, and a marked accumulation of triacylglycerol. On a molar basis, the esterification of free fatty acid was more effective in increasing the cellular triacylglycerol content than the uptake of intact VLDL-triacylglycerol. The addition of VLDL or free fatty acid caused a decrease in fibroblast acetyl-CoA carboxylase activity which was, however, less pronounced in the presence of insulin. The effects of VLDL were caused in part by free fatty acid derived from VLDL due to residual activity of lipoprotein lipase inherent in most VLDL preparations. Binding of free fatty acid by the addition of free fatty acid-poor albumin to fibroblasts incubated with VLDL decreased the acetyl-CoA carboxylase inhibition and improved cell population growth. Incubation with insulin was without effect on lipolytic activity (pH 8.2) in acetone-extracted fibroblasts. This activity also did not exhibit other properties characteristic of lipoprotein lipase. Thus, the uptake of VLDL-triacylglycerol did not appear to proceed by lipolysis facilitated by insulin. The basal activity of acetyl-CoA carboxylase and the extent of its induction by insulin in fibroblasts from patients with various forms of familial hypertriglyceridemia were similar to those obtained from healthy subjects.

Mesenteric lymph was collected for 48 h from rats with aminonucleoside-induced nephrotic syndrome, receiving an intraduodenal infusion of a triacylglycerol emulsion. In nephrosis, the rates of lymph flow and triacylglycerol transport were approx. 2-fold higher, but the transport of total protein and of apoproteins A-I and E was 2- to 3-fold lower than that in control rats, resulting in chylomicrons with a 3-fold approx. elevated triacylglycerol/protein ratio. Supplementation of the triacylglycerol infusate with glucose and amino acids did not increase the protein or apoA-I and apoE transport. Production or transport of B and C apoproteins in nephrotic rats was also reduced, as indicated by tetramethylurea solubility, incorporation of intraduodenally infused [3H]leucine and staining of the chylomicron proteins on SDS-PAGE gels. Apoprotein A-IV was the only chylomicron component into which the leucine incorporation was elevated, but its relative content was not increased on SDS-PAGE gels. Lymph chylomicrons of nephrotic rats were larger in size (1498 +/- 37 vs. 1235 +/- 23 A), consistent with the higher triacylglycerol/protein ratio. The concentration of all lipoprotein classes was markedly elevated in the plasma of nephrotic rats, as was that of the total A-I and E apoproteins. Intravenous injection of 125I-labelled HDL, followed by tracing of the label in lymph chylomicrons, indicated a lower rate of transfer of HDL apoproteins from plasma to lymph in nephrotic rats. We conclude that the intestinal chylomicron formation in nephrosis is characterised by an enhanced triacylglycerol transport without the appropriate apoprotein complement. This is probably due to the limited capacity of enterocytes, in marked contrast to hepatocytes, to respond to the hypoproteinemia of nephrosis with increased production and/or transport of the apoproteins.

1. 1. The activity of hepatic fructokinase increased about 2-fold in desert-derived spiny mice (Acomys cahirinus) and laboratory bred albino mice and rats, maintained on a 50% sucrose diet for 3 months. 2. 2. The role of fructose as the specific inducer was apparent, as 25% fructose diet produced activity increases similar to those of sucrose in contrast to 25% glucose diet. 3. 3. The activity of hexokinase was not affected by the sucrose diet, that of glucokinase rose marginally but those of pyruvate kinase and NADP-malate dehydrogenase rose pronouncedly, especially in the spiny mice. 4. 4. Fructokinase activity increased significantly only after 2 weeks on the diet and continued to rise gradually. The activities of other glycolytic enzymes rose markedly already after 3 days and peaked at about 14 days. 5. 5. Fasting for 48 hr did not influence fructokinase activity while markedly reducing that of glucokinase, pyruvate kinase and NADP-malate dehydrogenase. 6. 6. Streptozotocin diabetes in rats resulted in a 40% reduction in fructokinase activity after 14 days which was restored after 6 days of insulin treatment. The activity increases of other glycolytic enzymes were more marked. However, the fructokinase induction on the sucrose diet was evident also in diabetic rats, suggesting that the insulin and substrate effects are independent. The preference of fructose over glucose phosphorylation capacity was clearly demonstrable in the non-diabetic and diabetic rats and became enhanced on sucrose feeding. 7. 7. The activity of triokinase also increased on the sucrose diet in the 3 rodent species, suggesting a coordinative substrate effect on the induction of these two rate-limiting fructolysis enzymes. 8. 8. It is concluded that the hepatic fructokinase, similarly to the intestinal fructokinase, should be regarded as an adaptive enzyme, responding to substrate availability and to endocrine changes.

The sections in this article are: 1 Fatty Acid Uptake and Release; Response to Hormones 2 Triglyceride (TG) Synthesis and Breakdown 3 Building of Fat Stores; Fatty Acid Synthesis and Triglyceride Uptake 4 Carbohydrate Metabolism 5 Composition and Distribution of Fat in Adipose Tissues 6 Concluding Comment

Epididymal adipose tissue of aminonucleoside-treated rats, investigated 3 to 6 days after induction of the nephrotic syndrome, had low glycogen levels and showed impaired esterification of free fatty acids and assimilation of lipoprotein triglyceride and markedly reduced liberation of lipoprotein lipase. These results were found to be influenced by the inadequate food intake of the acutely nephrotic animals and comparable to the values of control rats fasted for 2 days. On return to adequate nutrition, which occurred 12–20 days after aminonucleoside treatment, adipose tissue glycogen and free fatty acid assimilation returned toward normal levels but lipoprotein-lipase liberation remained below normal. In rats rendered nephrotic by antikidney serum, the assimilation of free fatty acids and lipoprotein-triglyceride by adipose tissue was impaired in spite of only minor reduction in food consumption. The results indicate that the defective metabolism of adipose tissue in nephrotic animals may be contributory to the nephrotic hypertriglyceridemia.

Activation of typsinogen and chymotrypsinogen was studied in pancreatic extracts from normal and aminonucleoside-treated rats with hypoproteinemia. Upon incubation of the pancreatic extracts, the usual 2–3 lag phase of the sigmoidal shaped activation curve was almost abolished in aminonucleoside-treated rats. The maximal activity of the two proteases obtained after complete activation was not affected. Evidence is presented that the early onset of autocatalytic appearance of tryptic or chymotryptic activity was due to the presence of preformed trypsin-like activity in the pancreatic extracts of aminonucleoside-treated rats. A short term treatment with aminonucleoside which did not lead to proteinuria and hypoproteinemia also resulted in an increased trypsin/trypsinogen ratio in pancreas and shortening of the lag period. A direct concentration-dependent effect of the drug was demonstrated in in vitro experiments showing a release of hydrolytic enzymes from isolated pancreatic zymogen granules. The possible physiologic implication of labilization of intracellular structures by aminonucleoside is discussed.

SUMMARY Treatment of normal rats with epinephrine in oil resulted in a rapid rise in plasma free fatty acid (FFA) levels. The return of FFA levels to normal coincided with the steeply increasing blood glucose concentration. The plasma FFA response to epinephrine was abolished by hypophysectomy or by adrenalectomy. The in vitro rate of release of FFA from the epididymal fat bodies of operated animals waa only about one-half that from fat bodies of normal animals. The in vitro rate of release of FFA from fat bodies removed 30 minutes after injection of epinephrine was two to three times as high as that in fat bodies taken from noninjected animals. The fat bodies taken from hypophysectomized and adrenalectomized animals showed stimulation by epinephrine, but the absolute rates of release were lower than those observed in fat bodies taken from intact rats. Normal rats receiving epinephrine showed highly significant elevations of serum cholesterol and phospholipid levels but no rise in triglyceride levels. The cholesterol and phospholipid responses to epinephrine were also abolished by hypophysectomy and substantially reduced by adrenalectomy. Attempts to mimic the action of epinephrine and induce an elevation of plasma lipoprotein levels by infusing sodium oleate intravenously were negative ; the amounts which could be safely infused, however, may have been inadequate. It was concluded that the pituitary and adrenal glands play an important role in the response to the lipid-mobilizing action of epinephrine, both in terms of FFA and lipoprotein responses.

The interaction of bilirubin, linoleate and human plasma albumin was investigated in an isolated model system, designed to evaluate the competitive effect of free fatty acids (FFA) on the binding of unconjugated bilirubin by albumin. Increasing amounts of linoleate were added to bilirubin-albumin solutions at a constant molar ratio, and the mixtures passed through Sephadex G-25 columns to absorb the displaced bilirubin, which was then eluted and determined. In solutions with molar ratio bilirubin/albumin of 0.5 or 1.0 no displacement was evident when the molar ratio of linoleate relative to albumin was below 5. Above molar ratio of linoleate/albumin of 6, free bilirubin in the system rose sharply. In solutions with molar ratio bilirubin/albumin of 1.8, bilirubin dissociated from albumin markedly when the molar ratio of linoleate relative to albumin approached 4. Thus, the displacement of bilirubin starts only after most of the FFA binding sites on albumin with high association constants become saturated, and the competition involves mainly multiple binding sites with low association constants. In two groups of infants without or with hyperbilirubinemia requiring exchange transfusion, the molar ratio FFA/albumin in plasma ranged from 1.07 to 2.37. These values are much lower than those initiating the displacement of bilirubin in the in vitro system. The significance of FFA in causing shifts of bilirubin between intra- and extravascular pools is briefly discussed. It is suggested that FFA in neonates, although elevated, do not seem to reach concentrations, relative to albumin, which are effective in bilirubin dissociation.

The electrophoretic pattern of the serum in the nephrotic syndrome is marked by decreased concentrations of albumin and elevated concentrations of alpha2- and beta-globulins. A large portion of the excess lipid in nephrotic serum migrates at pH 8.6 in the alpha2 region, whereas normally the major part of the lipid is found in the beta position. This abnormal finding has never been adequately explained. Thus, Swahn remarks that "the electrophoretic separation of alpha2- from beta-globulin in nephrotic sera is often poor and it is difficult to decide whether there is a pathological lipoprotein fraction migrating quicker than normal beta lipoprotein" (1). Unesterified fatty acids (UFA) have been recently demonstrated to be of considerable importance in lipid transport and metabolism (2). In normal serum these acids are generally considered to be predominantly carried by albumin. However, Gordon (3) and others (4-6) have shown that UFA can increase the electrophoretic mobility of serum lipoproteins when liberated in vivo by heparin-released lipoprotein lipase or when added directly to the serum in vitro. It was inferred by the above investigators that at these high levels, a significant fraction of the UFA became associated with the lipoproteins, thereby increasing their negative electrostatic surface charge. The present paper reports on direct measurements of the partition of UFA resulting from the competitive binding of UFA by the albumin and lipoprotein components of the serum. The distribution of UFA in normal sera was compared

SummaryActivity of liver phosphorylase in normal rats decreased after 4 days of fasting-by about two-thirds and recovered slowly on refeeding, whereas that of transglucosylase was not markedly affected. The glucose-6-phosphate-independent activity of transglucosylase was also only slightly influenced by fasting and feeding.In Aminonucleoside nephrotic rats, the activities of phosphorylase and transglucosylase and the content of liver glycogen exhibited a pattern of variations probably caused by a prolonged recovery from undernutrition following the injections or due to a direct effect of the drug. Absence of similar changes in animals rendered nephrotic by kidney anti-serum is not compatible with the theory that the metabolic adjustments of the nephrotic state are associated with changes in the activity of enzymes of glycogen metabolism.The authors are indebted to Mrs. Hanna Schramm and Mr. Eran Graff for skilled assistance.We gratefully acknowledge the supply of Aminonucleoside of Puromycin obtained thro...

Dogs receiving subcutaneous epinephrine in oil (1 mg. per kg.) showed a prompt but transient elevation in serum free fatty acids (FFA) and a delayed %-hour elevation of serum lipoproteins. On daily injections of epinephrine for 6 to 8 days the serum cholesterol rose to 91 per cent above control values and the phospholipid 53 per cent. The triglyceride response was smaller and quite variable. Little change was found in the d < 1.019 lipoprotein, a three to eightfold increase in the d = 1.019 to 1.063 fraction and a 15 to 41 per cent increase in the d = 1.063 to 121 fraction. Epinephrine prolonged alimentary lipidemia but did not inhibit disappearance of intravenously infused chylomicrons. By prior and concomitant administration of glucose the FFA elevation after epinephrine was prevented. Insulin alone also blocked the epinephrine-induced FFA response without parallel hyperglycemia, indicating that availability of glucose for effective tissue utilization rather than hyperglycemia per se controls the release of FFA. Despite the block in the FFA response to epinephrine by either glucose or insulin, there was a definite elevation of serum cholesterol and phospholipids at 24 hours, suggesting an at least partially independent lipoprotein mobilizing action of the hormone. The relation of these findings to stress-induced hypercholesterolemia is considered.

In normal subjects the decrease of serum FFA levels after an oral glucose load was prompt and persistent with a drop to below 200 μEq./L. at one hour. Among patients with mild diabetes about 40 per cent showed FFA response similar to normal despite abnormal GT. Another 40 per cent diabetic patients had a delayed FFA decrease with a drop to minimum levels only after two hours and about 15 per cent of the patients had a deficient response with only a small decrease in FFA after glucose ingestion. In the two latter groups the slopes of FFA decrease (—log [FFA]/t) were less steep, indicating that the failure to reach the minimum level of normal subjects was not related to the higher initial serum FFA concentration. The occurrence of normal pattern of FFA decrease among a large proportion of diabetics was taken as an indication of normal function of adipose tissue. The tissue remained sensitive to ILA even when available in diminished amounts as assessed by comparisons of rises in the glucose uptake of rat diaphragm. A delayed FFA decrease appeared associated with late appearance of ILA, whereas the inability of adipose tissue to arrest the outflow of FFA was consistent with scarcity of ILA. The differential effectiveness of insulin as a result of selective antagonism toward its action on tissues is discussed.

Three cases of thrombocytopathic purpura with partial platelet dysfunction are reported. The platelets were defective in clot retraction, but normal in prothrombin conversion.

The distribution of free fatty acids of chain length of 16–24 carbon atoms between the albumin and lipoproteins of human serum was investigated by ultracentrifugal partition and electrophoretic separation. With elongation of the carbon chain less fatty acid was bound to albumin. More than 90% of the added palmitate remained with albumin, while the corresponding value for lignocerate was only 10–30%. The balance remained distributed with the lipoproteins or remained unbound. Presence of one or more double bonds resulted in an increased proportion of free fatty acids bound to albumin. The possible role of lipoproteins as carriers of free fatty acids of 20 or more carbon atoms is discussed.

Two patients had hypoglycemia associated with large and diffuse mesenchymal abdominal tumors. In these, the pattern of changes in blood glucose and free fatty acids after a glucose load or epinephrine injection, high plasma lactate levels, low serum insulin and insulin-like activity and excessive glucose uptake by tumor tissue in vitro indicated that the primary cause of hypoglycemia was enhanced glucose dissimilation by the large mass of the tumor. Deficient compensatory glucogenesis might have been a contributing factor. Divergent patterns of the response of serum free fatty acids to epinephrine injection were related to insulin-dependent and independent hypoglycemia.

Incorporation of [2-14C]pyruvate into adipose tissue triglycerides was measured in fasted, diabetic and triamcinolone treated rats and related to the activity of enzymes active in the elaboration of glyceride glycerol. Incorporation of pyruvate into glyceride glycerol was increased in fasting and diabetes. The activity of phosphoenolpyruvate carboxykinase increased in parallel with the enhanced glyceroneogenesis, whereas the activity of pyruvate carboxylase changed in the opposite direction. After triamcinolone treatment, the activity of both enzymes was decreased as was the incorporation of [2-14C]pyruvate into glyceride glycerol. In all experimental animals glutamate-pyruvate transaminase activity in adipose tissue decreased, whereas the activity of glutamate-oxaloacetate transaminase increased slightly. These results were interpreted as implying that the enhanced glyceroneogenesis in diabetes and fasting is related to the increase in phosphoenolpyruvate carboxykinase activity, whereas pyruvate carboxylase activity seems associated with changes in fatty acid synthesis. In contrast to adipose tissue, in the liver the activity of both enzymes of the dicarboxylic acid shuttle and of transaminases increased in the situations associated with enhanced gluconeogenesis. This finding is discussed in the light of possible differences in the availability of precursors for gluco- and glyceroneogenesis in the respective tissues. The role of glyceroneogenesis is pointed out, as one of the factors maintaining the balance between synthesis and breakdown of triglycerides in adipose tissue, particularly in conditions associated with rapid lipolysis.

The effect of triiodothyronine treatment on fatty acid synthesis was investigated in liver and adipose tissue of fed and 48-h fasted rats. In the liver, the thyrotoxic state was accompanied by an increased incorporation of [1-14C]acetyl-CoA to fatty acids and a rise in the activity of acetyl-CoA carboxylase and fatty acid synthetase, both in the fed and fasted rats. The activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, NADP-malate dehydro-genase and ATP citrate lyase was also enhanced by triiodothyronine treatment. Liver malate and citrate content was not changed in fed thyrotoxic rats, but was increased in fasted triiodo-thyronine-treated animals. Palmitate oxidation was increased in the liver of the same animals, either fed or fasted. The rise in acetyl-CoA carboxylase activity due to triiodothyronine administration was apparent when assayed both before and after citrate activation in vitro. Maximal activity of this enzyme in the presence of citrate was reached earlier in the liver of triiodothyronine-treated rats. In adipose tissue, triiodothyronine treatment induced an increase in the activity of acetyl-CoA carboxylase, fatty acid synthetase and in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The activity of NADP-malate dehydrogenase and ATP citrate lyase was slightly but insignificantly increased. Malate and citrate levels in adipose tissue were unchanged by triiodothyronine treatment. It is suggested that the triiodothyronine-induced simultaneous stimulation of opposing pathways (the synthesis of fatty acids and their oxidation) creates a futile metabolic cycle which contributes to the general energy waste and thermogenesis characteristic of thyrotoxicosis.