The increased risk of thromboembolism in people with diabetes mellitus is in part due to changes in the hemostatic mechanism including abnormal platelet function leading to platelet activation, increase in several coagulation factors, decrease in natural anticoagulants, and impaired fibrinolytic activity. Both microangiopathy and atherosclerosis in people with diabetes will enhance the thrombotic potential of these abnormal hemostatic changes. The recent recognition of a role of the components of the plasminogen-plasmin system in many biologic functions at the cellular level has led to studies showing that the angiopathic complications of diabetes may also be caused by impaired plasminogen activator function.

AGEs are nonenzymatically glycosylated adducts of proteins that accumulate in vascular tissues with aging and at an accelerated rate in people with diabetes; AGEs are closely linked to tissue damage due to their high reactivity in protein cross-linking. A macrophage-monocyte receptor system for AGE moieties is shown to mediate the uptake of AGE-modified proteins by a process that also induces cachectin-TNF, IL-1, IGF-I, and PDGF secretion. Thus, in addition to removing senescent glucose-modified proteins and cells, AGE-mediated release of growth-promoting factors may represent a mechanism by which macrophages signal mesenchymal cells the need for replacement of senescent proteins. The age of the macrophage correlates inversely with the binding and removal capacity of the AGE receptor, possibly preventing the clearance of cross-linked proteins and the compounding aging-related tissue damage. In addition to monocyte and macrophages, other cells express similar receptors for AGE-proteins, including endothelial cells, fibroblasts, and mesangial cells. Endothelial cell AGE-receptors mediate transcytosis of AGEs to the subendothelium, induce increased permeability, and enhance endothelium-dependent procoagulant activity. Renal mesangial AGE receptors mediate PDGF-dependent extracellular matrix protein production. Fibroblast AGE receptors may influence cellular proliferation by EGF and EGF-receptor regulation. These findings, in connection with the known abundance of AGEs in aged and diabetic tissues, indicate that AGE-ligand-receptor interactions are crucial for the development of age- and diabetes-related vascular tissue and renal pathology.

The syndromes of insulin resistance are a group of clinically diverse disorders, and our understanding of their molecular pathogenesis has advanced in parallel with our understanding of the structure of the insulin receptor and the mechanism of insulin action. The most straightforward progress has related to defining the role of both anti-receptor antibodies and mutations in the insulin receptor gene in causing these disorders. Despite this progress, the cause of severe target cell resistance in patients without defects in the receptor locus remains unknown, and we are limited in our ability to relate specific molecular defects in insulin signalling to in vivo phenotypes, such as those relating to growth and development and function of adipose tissue and muscle. Answers to these questions may ultimately be explained by the existence of multiple species of insulin receptors expressed in different tissues, brought about by alternative splicing and receptor hybrids, and by divergent pathways of insulin signalling with different consequences for specific tissues. The possibility that the insulin receptor and GLUT4 may be candidate genes for inherited insulin resistance in NIDDM has been addressed with the aid of genetic screening techniques such as SSCP. Currently, the loci have not been implicated in studies in most patients. Transgenic methodologies will be powerful tools for pursuit of unanswered questions in the field of insulin resistance in coming years.

This article is divided into two parts. A retrospective overview summarizes some of the work that provided the framework and tools of the more recent studies. The five novel areas of research are related to the indirect effects of insulin. Regulation of plasma glucose is of central importance in health and diabetes. Understanding this precise regulation requires sensitive isotope dilution methods that can measure the rates at which glucose is produced by the liver and used by the tissues on a minute-to-minute basis. Validation studies indicated that the non-steady-state tracer method yields reasonable results when the specific activity of plasma glucose does not change abruptly. During hyperinsulinemic glucose clamps, the decrease in specific activity of glucose can be prevented by the MSTI. During exercise, the decrease of specific activity can be only in part ameliorated by step-tracer infusion. Depancreatized dogs are used extensively as a model of selective insulin deficiency, because dog stomach secretes physiological amounts of glucagon. This strategy can avoid injections of somatostatin, which can have other affects in addition to the suppression of insulin and glucagon. In human diabetes, in addition to an increase of glucose production, there is also an increase in glucose cycling in the liver. In animal models of diabetes, mild NIDDM, and in glucose intolerance, the percentage of increments of glucose cycling are much larger than those of glucose production. We hypothesize, therefore, that measurements of glucose cycling can be used as an early marker of glucose intolerance. Application of different tracer strategies and use of the depancreatized dog as a model of diabetes, we investigated the importance of the indirect effects of insulin in the pathogenesis of diabetes. 1) Because, in the treatment of IDDM, insulin is administered by the peripheral routes we compared the relative importance of hepatic and peripheral effects of insulin in regulating the rate of glucose production. Experiments were performed in depancreatized dogs that were initially maintained at moderate hyperglycemia (10 mM) with subbasal portal insulin infusion. During the experimental period, insulin was infused either peripherally or portally at 0.9 mU.kg-1.min-1. In addition, peripheral infusions were also given at 0.45 mU.kg-1.min-1. We concluded that when suprabasal insulin levels are provided to moderately hyperglycemic depancreatized dogs, the suppression of glucose production is more dependent on peripheral than portal insulin concentrations. This indirect effect of insulin may be mediated by limitation of the flow of precursors and energy substrates for gluconeogenesis and/or by suppressive effect of insulin on glucagon secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Of all the common diseases that have a genetic component, IDDM is probably the most tractable to the experimentalist. Large numbers of nuclear multiplex families are available, which can be stored as permanent cell lines; diagnosis is relatively unambiguous; and a mouse strain, the NOD, spontaneously develops autoimmune IDDM similar to the human disorder. In addition, the resolution and accessibility of the human genome map has been revolutionized by the discovery and widespread application of the PCR, particularly the amplification of short, tandemly repeated segments of DNA called microsatellites, which display high levels of allelic polymorphism. With these reagents, the stage is set for dissection of the genetic factors that control the pathophysiology of IDDM.

Cytokines have been implicated as immunological effector molecules that induce dysfunction and destruction of the pancreatic beta-cell. The mechanisms of cytokine action on the beta-cell are unknown; however, nitric oxide, resulting from cytokine-induced expression of nitric oxide synthase, has been implicated as the cellular effector molecule mediating beta-cell dysfunction. Nitric oxide is a free radical that targets intracellular iron-containing enzymes, which results in the loss of their function. The cytokine IL-1 beta induces the formation of nitric oxide in isolated rat islets and the insulinoma cell line, Rin-m5F. NMMA and NAME, both inhibitors of nitric oxide synthase, completely protect islets from the deleterious effects of IL-1 beta. These inhibitors are competitive in nature and inhibit both the cytokine-inducible and constitutive isoforms of nitric oxide synthase with nearly identical kinetics. This may preclude their use as therapeutic agents because of increases in blood pressure which result from the inhibition of constitutive nitric oxide synthase activity. Aminoguanidine, an inhibitor of nonenzymatic glycosylation of cellular and extracellular constituents associated with diabetic complications, recently has been reported to inhibit nitric oxide synthase. Aminoguanidine is approximately 40-fold more effective in inhibiting the inducible isoform of nitric oxide synthase, suggesting that aminoguanidine or analogues may serve as potential therapeutic agents to block diseases associated with nitric oxide production by the inducible isoform of nitric oxide synthase. In vivo administration of TNF IL-1 has been shown to induce anti-diabetogenic effects in the NOD mouse. This anti-diabetogenic effect of cytokines appears to conflict with evidence suggesting that cytokines mediate beta-cell dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)

All pancreatic beta-cells are identified by specific morphological characteristics. Similarity in microscopic features is not necessarily associated with identity in functional properties. In vitro studies on isolated rat beta-cells have indicated intercellular differences in the threshold for glucose-induced shifts in metabolic redox state. The cellular heterogeneity in glucose sensitivity results in a dose-dependent recruitment of glucose-exposed beta-cells into biosynthetic and secretory activities. The molecular basis of this diversity is not known. Indirect evidence supports the concept that the in situ pancreatic beta-cell population is also composed of functionally diverse subpopulations. The heterogeneity in glucose responsiveness is expected to create subpopulations of beta-cells with either constant, fluctuating, or occasional glucose-dependent functions; whether any subpopulation is preferentially responsive to other regulatory factors and/or committed to other activities is unknown. Morphological markers may help identify beta-cell subpopulations in situ and quantify their size in conditions known to affect total beta-cell mass or function. The concept of a functionally heterogeneous beta-cell population influences views on the role of pancreatic beta-cells in health and disease.

In the normal state, pancreatic secretion of insulin results in a portal/peripheral gradient with the highest concentrations of insulin in the liver. In diabetic patients with absent or insufficient pancreatic insulin secretion who require exogenous insulin, this normal gradient is lost, resulting in numerous abnormalities. This consideration led to interest in the intraperitoneal delivery of insulin, hoping to produce a therapeutic state more closely resembling normal physiology. The development of implantable insulin pumps, which can deliver insulin intraperitoneally, led to numerous studies on insulin-dependent diabetes mellitus (IDDM) patients, demonstrating that insulin delivered intraperitoneally is rapidly and predictably absorbed with most of it going into the portal system, resulting in hepatic delivery of insulin. Studies in IDDM patients have demonstrated that good glucose control can be achieved with intraperitoneal delivery of insulin from implantable pumps with lesser glycemic fluctuations and, therefore, fewer episodes of hypoglycemia. Furthermore, intraperitoneal insulin results in carbohydrate and particularly lipid metabolism that more closely mimics the normal physiological state than produced by injections of insulin. Thus, implantable insulin pumps are being studied for use in IDDM. Many non-insulin-dependent diabetes mellitus (NIDDM) patients have insufficient pancreatic secretion and require exogenous insulin. Because of alterations in hepatic sensitivity to insulin, increments in insulin delivery to the liver may be even more important in NIDDM than IDDM. Furthermore, insulin resistance, which is an integral part of NIDDM, results in higher physiological levels of insulin, which are required for glucose control, and thus significant peripheral hyperinsulinemia occurs in patients receiving exogenous insulin.(ABSTRACT TRUNCATED AT 250 WORDS)

The brain is not usually thought to be a target of chronic diabetes complications. Nonetheless, substantial evidence, summarized herein, suggests that diabetes causes brain damage. Clinical syndromes of diabetes-related brain abnormalities are discussed along with possible causes. Various physiological effects of diabetes are reviewed, and questions are raised about gaps in our knowledge. Appropriate directions for future research are suggested.

During our search for a marker for non-insulin-dependent diabetes mellitus (NIDDM) in a large multigenerational family with a form of NIDDM termed maturity-onset diabetes of the young (MODY), we learned a great deal that may serve to streamline the search for diabetes-susceptibility genes in other families. We describe here our experience and suggest strategies that may enhance the search for markers for other diabetes susceptibility genes with genetic linkage approaches.

Three hypoglycemia-associated clinical syndromes in people with insulin-dependent diabetes mellitus (IDDM)--defective glucose counterregulation, hypoglycemia unawareness, and elevated glycemic thresholds for symptoms and activation of counterregulatory systems during effective intensive therapy--have much in common. They segregate together, are associated with increased frequency of severe iatrogenic hypoglycemia, and share several pathophysiological features, including reduced autonomic nervous system responses to a given degree of hypoglycemia. In the setting of reduced glucagon responses, the reduced adrenomedullary epinephrine responses play a key role in the pathogenesis of iatrogenic hypoglycemia in affected patients. Thus, these syndromes are examples of hypoglycemia-associated autonomic failure in IDDM, a disorder distinct from classical diabetic autonomic neuropathy. The pathogenesis of hypoglycemia-associated autonomic failure is not known, need not be the same in all three syndromes, and could be multifactorial even in a given syndrome. The recent finding that short-term antecedent hypoglycemia results in reduced symptomatic and autonomic (including adrenomedullary) responses to subsequent hypoglycemia in nondiabetic humans leads logically to the following hypothesis concerning one potential pathogenetic mechanism: recent antecedent iatrogenic hypoglycemia is a major cause of hypoglycemia-associated autonomic failure in IDDM, and hypoglycemia-associated autonomic failure, by reducing both symptoms of and defenses against developing hypoglycemia, results in recurrent severe hypoglycemia, thus creating a vicious cycle. If this hypothesis is confirmed, it will suggest strategies to reduce the frequency of iatrogenic hypoglycemia in people with IDDM.

This article presents a model for the HLA effect in insulin-dependent diabetes mellitus (IDDM) that is almost the mirror image of a model suggested by Nepom. In the Nepom model, the products of certain HLA alleles are associated with IDDM because they bind and present a specific peptide or peptides so as to induce an immune response to pancreatic beta-cells; certain other alleles can protect against IDDM if they compete strongly for binding of the diabetogenic peptide. My model focuses instead on the failure of the immune system to maintain tolerance to pancreatic beta-cells. I suggest that the HLA alleles negatively associated with IDDM (e.g., DR2 and DQw1) produce products with high affinity for certain beta-cell peptide or peptides needed to establish and maintain tolerance to beta-cells, whereas the alleles that are common in IDDM (e.g., DR3, DR4, and DQw8) produce products that have low affinity for the tolerogenic peptide or peptides or that bind the peptide or peptides in the wrong orientation or configuration for establishing tolerance. I also discuss the multiplicity of HLA loci, alleles, and amino acids contributing to IDDM and the fact that the associations of specific loci, alleles, and even genotypes with IDDM depend not only on their intrinsic properties but also on various population parameters.

The placenta is a specialized organ of exchange that provides nutrients to and excretes waste products from the fetus. The exchange of nutrients between placenta and fetus involves three major mechanisms: 1) direct transfer of nutrients from the maternal to the fetal plasma, 2) placental consumption of nutrients, and 3) placental conversion of nutrients to alternate substrate forms. Although direct transfer has been considered the primary means by which placental-fetal exchange controls the supply of nutrients to the fetus and thereby fetal metabolism and growth, the considerable metabolic activity of the placenta provides a large and fundamentally important contribution to both the quality and quantity of nutrient substrates supplied to the fetus; e.g., placental O2 and glucose consumption rates approach or even exceed those of brain and tumor tissue. Other placental metabolic activities include glycolysis, gluconeogenesis, glycogenesis, oxidation, protein synthesis, amino acid interconversion, triglyceride synthesis, and chain lengthening or shortening of individual fatty acids. Thus, consideration of the metabolism of the placenta is essential for a more complete understanding of how the placenta regulates nutrient transfer to the fetus, fetal energy balance, and fetal growth.

At least three types of phospholipase exist in the beta-cells of the pancreatic islet. Data regarding their physiological activation are incomplete but suggest that glucose (or its metabolite glyceraldehyde) either activates or potentiates the activation of several phospholipases. At least seven phospholipid hydrolysis by-products (diacylglycerol, myo-inositol 1,4,5-trisphosphate, lysophospholipids, arachidonic acid and its cyclooxygenase- and lipoxygenase-derived metabolites, phosphatidate) have been demonstrated to have effects compatible with their postulated roles as mediators or modulators of islet function. Presumptive mechanisms of action have been tentatively identified for these metabolites. However, key studies in the puzzle are missing, and current methodologies have important limitations. Shortcomings include the paucity of measurements of the mass of metabolites; the frequent use of static incubations rather than perfusions; a lack of complete time- and agonist concentration-dependence curves; the equation of metabolite accumulation with rates of metabolite generation (which ignores metabolite removal as a key variable); the use of nonspecific, insensitive, or ambiguous phospholipase assays; and the need for more studies directly correlating lipid metabolism and insulin secretion in physiologically functioning preparations. Like Rubik's Cube, the pancreatic islet is a dynamic puzzle comprised of many interrelated components requiring proper alignment and integration. Phospholipid turnover is one "panel" in the islet; however, an obligate role for phospholipase activation in glucose-induced insulin secretion is not yet rigorously established, despite tantalizing, inferential evidence. It may be that glucose serves principally to potentiate the phospholipase and secretory responses to other signals that act by initiating phospholipid hydrolysis.

More than a decade ago, Norbert Freinkel postulated that alterations in the maternal metabolic milieu at any time during gestation can influence intrauterine development and also may have long-term consequences for certain tissues such as adipocytes, myocytes, pancreatic beta-cells, and neurons. This review illustrates that metabolic alterations early in gestation, such as those that occur in diabetes mellitus, may impair growth of the embryo and increase the risk of dysmorphogenesis. Such delayed growth of the embryo may in turn influence size at birth. In midgestation, metabolic perturbations may accelerate functional maturation of fetal pancreatic beta-cells. Fetal beta-cell development is very sensitive to alterations in the nutrient milieu and may be enhanced in gestational diabetes mellitus (GDM) with only minimal elevations of plasma glucose and minor alterations in other nutrient fuels, including insulinogenic amino acids. Data are reviewed that suggest that the ensuing fetal hyperinsulinemia may promote the development of macrosomia even if metabolic control is satisfactory during late gestation. The overall potential influences of metabolic alterations on intrauterine growth are different in pregnancies complicated by diabetes mellitus throughout gestation (pregestational) and GDM. However, the implications in an individual pregnancy may be defined by the degree of metabolic control at the specific stages of gestation when growth of the embryo, development of fetal beta-cell function, and growth of insulin-sensitive tissues are most critically influenced by the metabolic milieu.

Diabetic pregnancy is associated with an increased risk for fetal maldevelopment for a largely unknown reason. A decade ago, Norbert Freinkel suggested that the altered fuel mixture offered to the growing conceptus may be the key to most of the changes in the embryogenesis of diabetic pregnancy. He coined the term fuel-mediated teratogenesis. During early pregnancy, periods of maternal hyper- and hypoglycemia may cause marked changes in the availability of glucose to the conceptus. Also, increased concentrations of lipids, notably ketone bodies, and branched-chain amino acids in the maternal circulation contribute to a changed fuel mixture for the embryo. In a recent experimental study of diabetic rats, it was found that the maternal metabolism of all three major classes of nutrients and maternal somatic growth during gestation covaried with the development of the embryo. Consequently, the maintenance of normal concentrations of metabolites from all nutrient classes may be important for prevention of adverse fetal outcome in diabetic pregnancy. In vitro, a high glucose concentration causes embryonic dysmorphogenesis by generation of free oxygen radicals. An enhanced production of such radicals in embryonic tissues may be directly related to an increased risk of congenital malformations in diabetic pregnancy. Thus, the notion that alterations in the net transfer of cellular fuels from the diabetic mother to her offspring may cause embryonic dysmorphogenesis, which suggests that combustion of the fuel may produce compounds that impair embryonic development, has obtained experimental support. If this is also true for human diabetic pregnancy, it has therapeutic implications.

We review some key aspects of the maturation of stimulus-secretion coupling and the regulation of DNA replication in the fetal beta-cell. During fetal life, the beta-cell shows a poor insulin response to glucose, although it responds to several other nonnutrient stimuli. However, chronic exposure to glucose in excess of basal levels can induce maturation of the stimulus-secretion coupling. Studies of glucose metabolism and the transmembrane flow of K+ and Ca2+ indicate that the attenuated glucose-stimulated insulin release is due to an immature glucose metabolism resulting in impaired regulation of ATP-sensitive K+ channels in the plasma membrane of the fetal beta-cell. In late fetal life, glucose is also a strong stimulus to beta-cell replication, and metabolism of glucose is a prerequisite for this process. Glucose stimulates proliferation by recruiting beta-cells from a resting state into a proliferative compartment composed of cells in an active cell cycle. The proliferative compartment comprises less than 10% of the total islet cell population even at maximal stimulation. The proliferation of fetal beta-cells is also regulated by several peptide growth factors such as growth hormone, insulinlike growth factor I, and platelet-derived growth factor. The observation that glucose can both induce precocious maturation of the stimulus-secretion coupling and stimulate proliferation of the fetal beta-cell explains the intrauterine hyperinsulinemia and beta-cell hyperplasia of the offspring of diabetic mothers with relatively mild hyperglycemia. However, severe hyperglycemia, at least when induced in rats, seems to retard rather than stimulate beta-cell growth.

During the first half of gestation in the rat, maternal net body weight increases rapidly, whereas in the second half of gestation, the mass of maternal structures declines, coincident with the rate of maternal fat accumulation. Enhanced maternal food intake, extrahepatic tissue lipoprotein lipase (LPL) activity, and adipose tissue lipogenesis are responsible for the progressive accumulation of maternal fat. However, during late gestation, decreased fat synthesis in maternal adipose tissue, enhanced lipolytic activity, and decreased LPL activity deplete maternal fat depots. These changes, plus enhanced endogenous production of triglyceride-rich lipoproteins, are also responsible for maternal hypertriglyceridemia. This condition benefits the offspring in two ways: 1) enhanced LPL activity in maternal liver when fasting increases triglyceride consumption for ketone body synthesis, giving the basis for accelerated starvation; and 2) induction of LPL activity in the mammary gland before parturition diverts maternal circulating triglycerides to milk synthesis in preparation for lactation. The magnitude of the maternal-fetal glucose transfer was higher than that of any of the other substrates studied, including alanine, and despite actions to spare glucose, this transfer causes maternal hypoglycemia, which is especially intense in the fasting condition. This increases sympathoadrenal activity in the mother, which may contribute to her active gluconeogenesis. Glycerol was a more efficient glucose precursor than alanine and pyruvate, and whereas glycerol placental transfer is very small, it is proposed that the fetus benefits from this product of adipose tissue lipolysis when it is previously converted into glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoglycemia continues to be found in infants of diabetic mothers, although the significance of the observation remains to be defined in terms of long-term neuro-development and behavior. Most follow-up studies no longer identify CNS problems. The criterion, i.e., the level of blood glucose at which the diagnosis should be made, has evolved and drifted toward the adult level. No single criterion can be used dependably to make this clinical and chemical diagnosis.

Several maternal plasma fuel abnormalities have been described in gestational diabetes mellitus (GDM), and all may contribute to the development of fetal macrosomia, generally because of the surfeit of calories they provide. Elevated maternal plasma glucose and amino acid concentrations represent key disturbances, because they are also well-known fetal pancreatic beta-cell secretagogues. Fetal hyperinsulinemia contributes to macrosomia in a special way by selectively accelerating fuel utilization and storage in insulin-sensitive fetal tissues. Maternal obesity intensifies the insulin resistance already present in late pregnancy and probably exaggerates the metabolic abnormalities attending GDM that impact on fetal growth and development. However, the means by which maternal obesity per se promotes the development of heavy babies in nondiabetic pregnancies remains poorly defined. Significant correlations exist between newborn birth weight and the levels of maternal plasma glucose, amino acids, free fatty acids, and triglycerides in diabetic pregnancies. However, the relative influence of each disturbance on fetal birth weight remains controversial and requires more detailed investigation.