Eve Reaven

Effect of Age and Diet on Insulin Secretion and Insulin Action in the Rat

The effects of aging on various aspects of insulin secretion and action were studied in male Sprague-Dawley rats, maintained from 1½ to 12 mo of age on conventional rat chow, sucrose-rich, or calorie-restricted diets. In chow-fed rats, islet volume increased as the animals grew from 1½ to 12 mo of age, but glucose-stimulated insulin secretion (per volume islet) declined over the same interval. In addition, in vivo insulin-stimulated glucose utilization fell in these rats. However, the plasma insulin response to an oral glucose challenge was sufficient to prevent frank decompensation of glucose tolerance (presumably due to an increase in total pancreatic endocrine cell mass). All these changes, with the exception of the decline in glucose-stimulated insulin secretion per volume islet, were accentuated by feeding sucrose. Thus, 12-mo-old sucrose-fed rats had larger islets and higher plasma insulin levels in response to an oral glucose challenge, and the rats were more insulin-resistant than chow-fed rats. However, glucose-stimulated insulin release per volume islet was similar in 12-mo-old chow-fed and sucrose-fed rats. In contrast, calorie restriction led to an amelioration in all but one of the age-related changes, i.e., islets from calorie-restricted rats were comparable in size to those of 2-mo-old rats, the animals had lower plasma insulin levels in response to an oral glucose load, and they were less insulin resistant than the other two groups of 12-mo-old rats. On the other hand, glucose-stimulated insulin secretion per volume islet was similar to that of the other 12-mo-old rats. These results suggest that aging leads to marked changes in both insulin secretion and insulin action. The decline in glucose-stimulated insulin secretion per unit endocrine pancreas appears to be an inevitable consequence of the aging process. In contrast, the age-related changes in islet size, insulin response to a glucose load, and in vivo insulin-stimulated glucose uptake are extremely responsive to variations in amount and kind of calories.

Vasopressin: Possible Role of Microtubules and Microfilaments in Its Action

Colchicine, vinblastine, podophyllotoxin, and cytochalasin B inhibit the action of vasopressin and cyclic adenosine monophosphate on osmotic water movement across the toad bladder. The findings suggest that microtubules, and possibly microfilaments, play a role in the action of vasopressin, perhaps through involvement in the mechanism of release of secretory material from the bladder epithelial cells.

Effect of age on glucose-stimulated insulin release by the beta-cell of the rat.

To assess the effect of age on beta-cell insulin release, collagenase-isolated islets of Langerhans were obtained from rats aged 2--18 mo and incubated with increasing concentrations of glucose. Similar islets were analyzed for insulin content or subjected to morphometric measurements to identify both the number of beta-cells and the volume of beta-granules per islet. In parallel studies, the islet content of intact pancreata was also determined. The results showed that beta-cell number increased from 2,300 t0 5,000 cells as rats aged from 2 to 18 mo and islet insulin content doubled. However, glucose-stimulated insulin release decreased progressively with age, and this was especially striking when considered in terms of the increase in number of beta-cells/islet; e.g., mean (+/- SEM) insulin secretion (nanounits per minute per beta-cell) of islets incubated with 450 mg/dl of glucose was 1.3 (+/- 0.02), 1.0 (+/- 0.1), 0.4 (+/- 0.05), and 0.3 (+/- 0.01), respectively for 2-, 6-, 12-, and 18-mo-old rats. Thus, insulin secretion per beta-cell was decreased, despite increased stores of insulin per cell. These findings demonstrate that the aging process leads to a profound defect in glucose-stimulated insulin release from the beta-cell. Whether this is a global secretory defect, or solely a failure of the beta-cell to respond to glucose, remains to be defined.

Morphological evidence that high density lipoproteins are not internalized by steroid-producing cells during in situ organ perfusion.

Although it is clear that high density lipoproteins (HDL) can support steroidogenesis in several rat cell systems, questions still arise as to how HDL are processed by cells. In particular, it is not yet clear whether HDL are internalized by a pathway similar to that used for low density lipoproteins. This issue was examined in the present study using the luteinized ovaries of hormone-primed immature rats in an in situ perfusion system. Ovaries were perfused for 2-120 min with 125I-labeled human or rat HDL and processed for autoradiographic studies at the light and electron microscopic level, or homogenized and used for isolation of subcellular membranes. The results show that the luteal cells of this tissue bind both human and rat HDL with great specificity. Moreover, the intact HDL particle does not appear to be internalized by the luteal cell during the period of perfusion: i.e., the protein moiety of the labeled HDL remains associated with the plasma membrane at all times. Evidence from the autoradiographs suggest, however, that with time, an increasing proportion of the plasma membrane-bound protein is associated with inverted microvilli, which are embedded within the cytoplasm and make close contact with structures of the interior of the cell. We speculate that HDL-cholesterol may be transferred at such sites.

Intracellular Events in the “Selective” Transport of Lipoprotein-derived Cholesteryl Esters

The current study utilizes human, apoE-free high density lipoprotein reconstituted with a highly specific fluorescent-cholesteryl ester probe to define the initial steps and regulatory sites associated with the “selective” uptake and intracellular itinerary of lipoprotein-derived cholesteryl esters. Bt2cAMP-stimulated ovarian granulosa cells were used as the experimental model, and both morphological and biochemical fluorescence data were obtained. The data show that cholesteryl ester provided through the selective pathway is a process which begins with a temperature-independent transfer of cholesteryl ester to the cells plasma membrane. Thereafter transfer of the lipid proceeds rapidly and accumulates prominently in a perinuclear region (presumed to be the Golgi/membrane sorting compartment) and in lipid storage droplets of the cells. The data suggest that lipid transfer proteins (or other small soluble proteins) are not required for the intracellular transport of the cholesteryl esters, nor is an intact Golgi complex or an intact cell cytoskeleton (although the transfer is less efficient in the presence of certain microtubule-disrupting agents). The intracellular transfer of the cholesteryl esters is also somewhat dependent on an energy source in that a glucose-deficient culture medium or a combination of metabolic inhibitors reduces the efficiency of the transfer. A protein-mediated event may be required for cholesteryl ester internalization from the plasma membrane, in that N-ethylmaleimide dramatically blocks the internalization phase of the selective uptake process. Taken together these data suggest that the selective pathway is a factor-dependent, energy-requiring cholesteryl ester transport system, in which lipoprotein-donated cholesteryl esters probably flow through vesicles or intracellular membrane sheets and their connections, rather than through the cell cytosol.

Mechanisms for Development of Diabetic Hypertriglyceridemia in Streptozotocin-Treated Rats EFFECT OF DIET AND DURATION OF INSULIN DEFICIENCY

A combined ultrastructural and functional approach was employed to define the effects of duration of diabetes and of diet on various aspects of lipid metabolism in rats with severe streptozotocin (SZ)-induced insulin deficiency. Plasma triglyceride (TG) levels rose to a mean of 479 mg/100 ml 24 h after SZ administration in rats eating a fat-free, high carbohydrate diet as compared to a mean of 324 mg/100 ml in rats eating a high fat diet. These changes were associated with a commensurate increase in hepatocyte Golgi very low density lipoprotein (VLDL) content, but only a small increase in estimates of VLDL-TG secretion rate (post-Triton WR 1339 increment in plasma TG level). Although these findings are consistent with the thesis that VLDL-TG synthesis and secretion are increased 24 h after administration of SZ, it seemed unlikely that the observed increase in VLDL-TG secretion could entirely account for the severity of the hypertriglyceridemia. Thus, although lipoprotein removal rate was not measured directly, it was necessary to postulate that a defect in VLDL-TG removal was also present at this stage. Hypertriglyceridemia was still present 7 days later, only in this instance plasma TG levels were higher in rats eating the high fat diet (a mean of 589 mg/100 ml, as compared to 263 mg/100 ml). Rats with diabetes of 7-day duration had a 50% decrease in both TG entry rate and hepatocyte Golgi complex VLDL content, irrespective of diet. Thus, there was no evidence of increased VLDL-TG secretion in chronic insulin deficiency. In this instance, although not assessed directly, it was necessary to postulate that the hypertriglyceridemia in chronically insulin-deficient rats is due entirely to a defect in lipoprotein removal, involving both dietary and endogenous fat.

Dimerization of the scavenger receptor class B type I: formation, function, and localization in diverse cells and tissues.

This study has examined the dimeric/oligomeric forms of scavenger receptor class B type I (SR-BI) and its alternatively spliced form, SR-BII, in a diverse group of cells and tissues: i.e., normal and hormonally altered tissues of mice and rats as well as tissues of transgenic animals and genetically altered steroidogenic and nonsteroidogenic cells overexpressing the SR-B proteins. Using both biochemical and morphological techniques, we have seen that these dimeric and higher order oligomeric forms of SR-BI expression are strongly associated with both functional and morphological expression of the selective HDL cholesteryl ester uptake pathway. Rats and mice show some species differences in expression of SR-BII dimeric forms; this difference does not extend to the use of SR-B cDNA types for transfection purposes. In a separate study, cotransfection of HEK293 cells with cMyc and V5 epitope-tagged SR-BI permitted coprecipitation and quantitative coimmunocytochemical measurements at the electron microscope level, suggesting that much of the newly expressed SR-BI protein in stimulated cells dimerizes and that the SR-BI dimers are localized to the cell surface and specifically to microvillar or double membraned intracellular channels. These combined data suggest that SR-BI self-association represents an integral step in the selective cholesteryl ester uptake process.

Uptake and utilization of lipoprotein cholesteryl esters by rat granulosa cells

Earlier studies have shown that rat granulosa cells grown in serum-free medium are exquisitely responsive to exogenously provided lipoprotein cholesterol. In this study we compare the amount of cholesterol (cholesteryl ester) actually delivered from various homologous and heterologous cholesterol-rich lipoproteins and examine the intracellular pathways used in the delivery system. Granulosa cells were incubated for 5 or 24 h with 125I-labeled human (h) HDL3, rat (r) HDL or hLDL equipped with non-releasable apoprotein and cholesteryl ether tags which accumulate within cells, even after degradation. We show that all the tested lipoproteins were similarly efficient in cholesteryl ester delivery; i.e., based on cholesterol: protein ratios of the starting ligands, each delivered approximately the same cholesteryl ester mass and evoked a similar progestin response. However, each lipoprotein was processed quite differently by the granulosa cells: hHDL3-cholesteryl ester was taken up almost exclusively by an non-endocytic pathway, hLDL-cholesteryl ester almost exclusively by an endocytic pathway and rHDL-cholesteryl ester by both pathways. In general, there was no correlation between the total amount of lipoprotein bound or apoprotein internalized and/or degraded by the cells with the amount of cholesteryl ester received or the level of the progestin response. Hormone stimulation upregulated the preferred pathway for each lipoprotein.

Age, glucose intolerance, and non-insulin-dependent diabetes mellitus.

Although insulin-dependent diabetes mellitus (IDDM) can occur in older patients, non-insulindependent diabetes mellitus (NIDDM) is by far the most common form of diabetes present in this age group. The primary goal of this article is to develop a hypothesis that explains why the incidence of NIDDM increases with age. To do so, it will be necessary to review available information pertaining to the effect of age on insulin secretion and insulin action and to present current views concerning the pathogenesis of NIDDM. In addition, attention will be directed to the health-related implications of the changes in insulin secretion and action that occur with aging. The benefit of this presentation to the reader will, it is hoped, be the acquisition of some new insights on an old issue. The benefit to us is considerable and stems from the challenge to prepare a coherent paper on NIDDM in the geriatric population. Since the rostrum is ours, it is only fair that certain prejudices be declared at the outset. Most important, we do not believe that one can develop a thoughtful view concerning the impact of NIDDM in the elderly by considering only the changes in carbohydrate metabolism associated with senescence. To be more specific, events that occur in middle life (excessive weight gain, for example) can have profound clinical effects 20 years later. Limiting discussion to studies of older subjects may be considered “pure” by the professional gerontologist, but we believe that this leads to a distorted view that can adversely affect our ability to minimize the deleterious effects of NIDDM in the older patient. In addition, we think it helpful to integrate information gained from animal studies with the results of clinical investigation. Specifically, questions that are difficult or iinpossible to answer in research on humans can sometimes be answered in animal studies, and the information thus accrued can be extremely useful in a variety

The Effect of Experimental Diabetes Mellitus and Insulin Replacement on Hepatic Ultrastructure and Protein Synthesis

The following study was conducted in order to define the specific alterations in hepatic ultrastructure responsible for the decrease in hepatic protein synthesis associated with experimental diabetes. Rats received intravenous alloxan (70 mg/kg) and 48 h later were either sacrificed or given insulin for 1, 2, 4, 6, or 24 h. Specimens for electron microscopic evaluation and morphometric analysis were taken from the same livers used to isolate ribosomes for measurement of in vitro protein synthesis. Our results show that hepatocytes from animals with untreated alloxan diabetes show varying degrees of disorganization and loss of rough endoplasmic reticulum (RER) which is directly related to the severity of the alloxan diabetes. A significant correlation existed between the severity of ultrastructural changes as judged by the loss of both membrane and polysome components of the RER and degree of inhibition of protein synthesis (P < 0.001). Abnormalities of hepatic ultrastructure and protein synthesis were reversed within 24 h of insulin administration. The data are consistent with the view that it is the relative decrease in hepatic polysomes that results from the loss of RER in alloxan diabetes that is responsible for the decrease in hepatic protein synthesis.