Helen M. Tepperman

ON THE RESPONSE OF HEPATIC GLUCOSE-6-PHOSPHATE DEHYDROGENASE ACTIVITY TO CHANGES IN DIET COMPOSITION AND FOOD INTAKE PATTERN.

1. n1.|Ethionine inhibition experiments suggest that the hepatic glucose-6-phosphate dehydrogenase increase seen on refeeding and after fructose feeding probably represents de novo protein synthesis. Some difficulties in interpreting experiments done with protein synthesis inhibiting maneuvers in whole animals are discussed. n n2. n2.|No increase in glucose-6-phosphate concentration of liver is detectable before or during periods of rapid dehydrogenase activity increase. n n3. n3.|Inhibition of lipogenesis by the isocaloric substitution of fat for protein in a high glucose diet is accompanied by failure of enhancement of glucose-6-phosphate dehydrogenase activity. When the enzyme level has been elevated as a result of carbohydrate feeding the addition of fat to the diet results in a lowering of enzyme activity but only after a 2 day latent period. n n4. n4.|The administration of 2 ml of corn oil by stomach tube to carbohydrate diet adapted rats has the following effects within 2 hr (a) decreased lipogenesis from glucose and acetate and (b) decreased appearance of glucose carbon 1 in CO2. These findings suggest that inhibition of lipogenesis results in a dimunition of glucose-6-phosphate oxidation by way of the pentose pathway. n n5. n5.|A number of treatments (methylene blue, menadione and phenobarbital administration) designed to increase cellular demand for NADPH2 were without effect on glucose-6-phosphate dehydrogenase activity. An attempt to increase tissue NADP concentration by nicotinamide feeding was similarly without effect.

ADAPTIVE HYPERLIPOGENESIS—LATE 1964 MODEL*

At a recent symposium we’ presented a 1963 model of the concept of adaptive hyperlipogenesis. Tha t essay was largely autobiographical, and no attempt will be made here to cover the same ground. We propose instead to show a couple of antique models of adaptive hyperlipogenesis and then to develop a conceptual scheme for a late 1964 model. For the construction of the latter we have shamelessly stolen component parts from many of our colleagues who, we hope, will forgive us for the uses to which we have pu t their data and ideas. The seeds of the idea of increased formation of fat from nonfat sources actually go back to the 1860 report of Lawes and Gilbert’ in which the first experimental proof of carbohydrate-to-fat conversion was presented. One of the earliest dynamic experiments which suggested very rapid fat formation was described in the 1901 report of B l e i b t r e ~ , ~ who demonstrated that geese fed a grain diet often showed Respiratory Quotients (R.Q.’s) far in excess of unity. For half a century the R.Q., sometimes used in conjunction with analytical techniques and iodine number determinations, was the methodologic standby for the demonstration of increased lipogenesis. A 1925 model (Wierzuchowski & Ling4) has about the same silhouette as Bleibtreu’s 1901 model had, although, in this case, the experimental animal was a hog instead of a goose. We can now turn our attention to the development of the late 1964 model. How many of the parts are interchangeable among the three major lipogenic cell types of the body-adipocyte, parenchymal liver cell and lactating mammary gland cell-we cannot say. At times we may make unjustifiable generalizations on the basis of observations made in only one tissue. However, model builders as a group tend to take the simple view. What we have called adaptive hyperlipogenesis occurs in various forms of experimental o b e ~ i t y ; ~ when low fat, high carbohydrate diets are fed after a period of starvation;6 when similar diets are fed after a period of feeding diets containing fat;’ in periodic overfeeding and starvatioqR when animals are force-fed diets containing appreciable amounts of ~ a r b o h y d r a t e ; ~ and when dietary carbohydrate consists largely of sucrose or fructose.” In all of these circumstances rates of lipogenesis from labeled precursors by surviving liver slices or f a t pads is markedly increased, as compared with rates obtained with tissues of chow fed animals with “normal” feeding patterns. Some of the dimensions of the problem of adaptive hyperlipogensis can be illustrated by an arbitrary division of the discussion into six general subjects

The effect of age on Intestinal glucose transport in the chick (Gallus domesticus)

Abstract 1. 1. Intestinal 3-O methyl glucose transport was studied by the in vitro everted sac technique in chicks of various ages. 2. 2. Glucose transport was maximum in 1-week-old chicks and then decreased progressively with age. 3. 3. [U-14C]Glucose oxidation by isolated intestinal epithelial cells also decreased with age along with the DNA content of the cell preparations. 4. 4. Histological examination of the intestinal sections prepared from 1- and 8-week-old chicks showed that while the epithelial cell number decreased, thickness of the submucosal tissue increased with age. 5. 5. Oral 3-O-methyl glucose load given to 1- and 12-week-old chicks showed significantly greater intestinal absorption in the younger chicks. 6. 6. Our results suggest that glucose absorption in the chick decreases with age and that this is probably due to combined effects of decreased cellular performance, decreased cell number and an increase in submucosal tissue thickness.

Mechanisms of the fasting-induced dissociation of insulin binding from its action in isolated rat hepatocytes

SummaryFasting leads to an increase in insulin binding to isolated rat hepatocytes from 12 to 17%. This increase was accounted for by changes in the affinity of insulin receptors without alteration in their number. In contrast, the responsiveness of hepatocytes to insulin was markedly diminished in fasted rats. Both basal and insulin-stimulated rates of 14C-glucose incorporation into glycogen were significantly decreased in fasted animals. When insulin-induced 14C-glucose incorporation into glycogen was expressed as a percent above the basal rate, hepatocytes isolated both from control and fasted animals showed the same magnitude of maximal response (66 ± 13% in fed and 59 ± 12% in fasted animals, respectively). However, more insulin must be bound to hepatocytes isolated from fasted animals in order to elicit the same percent of insulins maximal effect.Incubation of ‘fed’ hepatocytes in the serum obtained from fasted rats significantly diminished their responsiveness to insulin. An addition of insulin (100 ng/ml), glucose (10 mM) and antibodies to glucagon (1:100) eliminated the inhibitory effect of ‘fasted’ serum on ‘fed’ hepatocytes.A 48-hour fast increased significantly the microviscosity (decreased fluidity) of hepatocyte plasma membranes and altered membrane phospholipid composition. These changes correlated with enhanced insulin binding to isolated membranes. Moreover, in response to insulin, plasma membranes isolated from ‘fasted’ hepatocytes generated only one half the amount of the second messenger (PDH activator) observed in membranes of fed animals. The amount of PDH activator generated by incubation of plasma membranes with insulin correlated inversely with both insulin binding and membrane microviscosity.We conclude that 1) fasting induces both coupling defect and post-receptor changes in insulins action; 2) both extracellular and intracellular factors contribute to fasting-induced dissociation of insulin binding from insulin action; 3) insulin/ glucagon ratio may influence hepatocyte responsiveness to insulin; 4) alterations in plasma membrane fluidity and phospholipid composition may alter insulin binding and contribute to its dissociation from the subsequent action; 5) membranes isolated from ‘fasted’ hepatocytes generate less mediator of insulin action than do membranes isolated from ‘fed’ hepatocytes.

Effects of cortisone and purified pituitary growth hormone on ketogenesis by surviving liver slices.

Since Burn and Ling1 first described the ketonuric effect of crude pituitary extracts twenty-one years ago, many investigators have confirmed the fact that injected pituitary extracts produce ketonemia and ketonuria.2After it had been indicated by the work of Collip and his colleagues4 and by the studies of Mirsky6 that the liver is essential for the ketonemic response, the effects of crude pituitary extracts on the metabolism of surviving liver slices were studied by Shipley,* by Ennor: and by Campbell and Davidson? While there are certain points on which these authors do not agree, their collective.work stands in support of the view that the liver is a site of action of the ketogenic substance or group of substances that can be extracted from the pituitary gland. Shipleye was the only one of these investigators who described enhancement of ketogenesis by surviving liver slices when crude pituitary extract was added to the system in witro. This constituted the only available evidence that a ketogenically active material in crude extracts causes the liver to produce ketone bodies at an accelerated rate by stimulating the liver cells directly, and not merely by offering them an excess of lipid precursor mobilized from the fat depots. When purified pituitary hormones became available for physiologic study, Bennett and coworkersg described enhanced ketonemia in response to both growth hormone and ACTH. In fact, their data indicate a pronounced synergistic effect on the level of ketonemia between these two substances. When viewed in the light of the studies cited above, the report of Bondy and WilhelmilO appeared to contain a certain paradox. These authors, in the course of well-designed and carefully executed experiments, found a ketogenic defect in liver slices obtained from rats many months after hypophysectomy and described their efforts to repair this defect by injecting certain hormones. They found that, while ketogenesis by liver slices of hypophysectomized rats could be restored to essentially normal levels by thyroxin administration, the injection of pursed growth hormone in wivo was without effect. These results were clearly difficult to reconcile with those of earlier workers, who used crude extracts or purified hormones in intact animals. The fact that the rate of ketogenesis by the slices could be accelerated by thyroxin administrationlo did not appear to us to rule out the possibility that lack of other hormones may have contributed to the ketogenic defect in the hypophysectomized rat. Accordingly, the experi-

The Effects of Streptozotocin Diabetes on the Activities of Rat Liver GlycosyItransferases

The effects of streptozotocin diabetes on the activities of rat liver glycosy Itransferase enzymes have been investigated. Liver microsomal fractions were prepared from rats that had been injected with streptozotocin (65 mg/kg, i.v.) 3 wk to 2 mo earlier. Preparations from diabetic rats had decreased activities of N-acetylglucosaminyl transferase compared with those of agematched controls (0.98 ± 0.11 nmol transferred per mg protein in 30 min versus 3.19 ± 0.34 for controls, P < 0.001). Galactosyltransferase activity was also lower in diabetic rat livers (1.48 ± 0.26 nmol transferred per mg protein in 30 min versus 3.32 ± 0.56 for controls, P < 0.025). Sialytransferase activities were not significantly different between diabetic and control rat livers. There were no significant differences between the diabetic and control rat liver microsomes in the activities of UDP N-acetylglucosamine pyrophosphatase, UDP galactose pyrophosphatase, or CMP sialic acid phosphatase. The glycosidases, N-acetylglucosaminidase and galactosidase, had similar activities in the livers of both groups of rats. Sialidase activity could not be detected in microsomal preparations from either diabetic or control rat livers. These results are discussed in relation to our previously reported alterations in glycosyltransferase activities, and plasma membrane glycoprotein composition in the livers of rats made insulin-resistant by a carbohydrate-free, high-fat diet and to the observation of Carter and his colleagues (FEBS Lett. 1979; 104:389–92.) that streptozotocin diabetes alters the glycoprotein composition of rat liver plasma membranes.

Stimulation of insulin secretion in the rat by glucagon, secretin and pancreozymin: Effect of aminophylline

Abstract Male, 150 gram rats were injected i.p. twice; first with either saline or aminophylline and second, after 10 or 14 minutes with saline or one of the following hormones: glucagon, secretin or pancreozymin. The animals were bled by cardiac puncture after a short time. Serum immunoreactive insulin and serum glucose were measured. All three hormones exerted a serum insulin elevating effect which was apparently potentiated by the phosphodiesterase inhibitor, aminophylline. This potentiation produced indirect evidence that glucagon, secretin and pancreozymin stimulate the release of insulin from the pancreatic beta cell through the mediation of 3′, 5′ cyclic AMP. It was further shown that secretin and pancreozymin do not stimulate insulin release indirectly by increasing the blood glucose concentration.

Effects of p-Chlorophenoxyisobutyric Acid (CPIB) on Adipose Tissue Triglycerides Synthesis, Lipogenesis, and Some Related Enzymes

Summary The addition of 0.25% CPIB to a glycerol-lard diet fed to rats resulted in increased adipose tissue activities of triglyceride synthesizing enzymes, hexose monophosphate shunt dehydrogenases, and NADP-malic enzyme. Incorporation of 14C-glucose and 3H from water into long chain fatty acids by fat pads in vitro was also greater in CPIB-treated rats. The drug had only small or no such effects when added to a fat-free high sucrose diet.

Effect of diet and insulin on the morphology and TPNH generating enzyme activities of rat adipose tissue.

Summary The activities of TPN malic enzyme and aggregate hexomonophosphate shunt dehydrogenases were determined in epididymal fat pads of rats subjected to the following treatment: (1) ad libitum chow fed; (2) fasted 72 hr; (3) refed sucrose diet 72 hr; (4) refed sucrose diet plus insulin; (5) refed high-fat diet; and (6) refed high-fat diet plus insulin. Refeeding the sucrose diet for 72 hr resulted in more than a six-fold increase in malic enzyme activity, and approximately a doubling of the activity of the shunt enzymes over the prefast levels. Refeeding the high-fat, carbohydrate-free diet resulted in restoration of enzyme activities to prefast levels. Administration of exogenous insulin resulted in no change beyond that produced by the respective diets alone. Fasting resulted in a decrease in nucleolar and cytoplasmic RNA staining and a reduction of the mean cell size. Refeeding sucrose induced a marked enlargement of nucleoli and increased cytoplasmic basophilia with essentially no changes in mean cell size. Refeeding the high-fat diet produced a striking hypertrophy of fat cells, whereas the nucleolar and cytoplasmic RNA staining was comparable to the prefast state. Exogenous insulin did not produce marked differences in either of the two diet groups. The authors are indebted to Elizabeth Zizzi and Patricia Hopkins for valuable technical assistance.

Effect of oral clofibrate administration on intestinal 3-O-methyl glucose absorption in rat and chick

Abstract 1. 1. These studies were undertaken to determine the effect of clofibrate ( p -chlorophenoxyisobutyrate, CPIB) on intestinal glucose absorption and the possible relationship to its hypolipidemic action in rat and chick. The effect of CPIB and diabetes on intestinal mucosal C-AMP concentration as well as the role of C-AMP in intestinal glucose absorption was also determined in rat. 2. 2. CPIB treatment decreased intestinal 3- O -methyl glucose absorption when expressed as absolute transport (1.37 ± 0.27 vs 3.47 ± 0.27 μ moles/g mucosa) or as serosal: mucosal ratio (1.28 ± 0.05 vs 1.62 ± 0.04) for the CPIB treated and control rats respectively. In the chick CPIB had no effect. 3. 3. In vitro addition of dibutyryl C-AMP significantly increased glucose transport in sacs prepared from control rats (1.96 ± 0.14 vs 3.23 ± 0.38 μ moles/g respectively) but not from CPIB treated rats. 4. 4. The mucosal C-AMP contents for the control, diabetic and CPIB treated rats were 248.2 ± 24.3, 428.5 ± 65.2 and 214.0 ± 23.9 pmole/g mucosa respectively. 5. 5. After an oral glucose load both serum glucose and insulin levels were higher during the first 20 min for the control rats compared to CPIB treated rats. 6. 6. Serum triglycerides were significantly lower in the CPIB treated rats compared to control rats and decreased in both groups following oral glucose load. 7. 7. Whereas CPIB increased liver weight in the rat (6.34 ± 0.20 vs 4.13 ± 0.09 g/100 g body wt.), it had no effect in the chick (2.57 ± 0.04 vs 2.56 ± 0.14 g/100 g body wt.) for the CPIB treated and controls respectively. 8. 8. Although CPIB decreased glucose transport only in the rat, it had a hypolipidemic effect in both the rat and the chick. 9. 9. Our results suggest that either intestinal glucose absorption is not involved in the hypolipidemic effect or there are species differences in the mechanism of action of CPIB.