Insulin resistance: Review of the underlying molecular mechanisms

Most human cells utilize glucose as the primary substrate, cellular uptake requiring insulin. Insulin signaling is therefore critical for these tissues. However, decrease in insulin sensitivity due to the disruption of various molecular pathways causes insulin resistance (IR). IR underpins many metabolic disorders such as type 2 diabetes and metabolic syndrome, impairments in insulin signaling disrupting entry of glucose into the adipocytes, and skeletal muscle cells. Although the exact underlying cause of IR has not been fully elucidated, a number of major mechanisms, including oxidative stress, inflammation, insulin receptor mutations, endoplasmic reticulum stress, and mitochondrial dysfunction have been suggested. In this review, we consider the role these cellular mechanisms play in the development of IR.     

Roles of cAMP and free fatty acids on the activity of the hexose monophosphate shunt

Summary Basal glucose utilization by isolated rat adipocytes have been found to be increased ten times in the presence of certain preparations of albumin. In these conditions the effects of several adrenergic agonists and related compounds on glucose oxidation, lipolysis and triacylglycerol synthesis in isolated fat cells have been studied. Oxidation of D(1-¹⁴C) glucose in rat adipocytes was almost completely inhibited by norepinephrine and isoproterenol when added to incubated fat cells. Agents able to modify intracellular AMP cyclic levels by different mechanisms display a similar ability to imitate the effect of lipolytic agents. The inhibition of glucose oxidation due to norepinephrine and isoproterenol is partially reverted by propanolol. Under the same conditions in which norepinephrine and isoproterenol markedly reduced glucose conversion to ¹⁴CO₂, they stimulated lipolysis and triacylglycerol synthesis and in this case propanolol also reverted those actions. However, in these experimental conditions, norepinephrine and isoproterenol did not raise CAMP levels 10 min after hormone addition.It is concluded from these data that glucose oxidation through hexose monophosphate shunt, activation of lipolysis and triacylglycerol synthesis in isolated rat fat cells by lipolytic agents occurs by a mechanism(s) that depend(s) on intracellular free fatty acids levels.     

Molecular mechanisms beyond glucose transport in diabetes-related male infertility

Diabetes mellitus (DM) is one of the greatest public health threats in modern societies. Although during a few years it was suggested that DM had no significant effect in male reproductive function, this view has been challenged in recent years. The increasing incidence of DM worldwide will inevitably result in a higher prevalence of this pathology in men of reproductive age and subfertility or infertility associated with DM is expected to dramatically rise in upcoming years. From a clinical perspective, the evaluation of semen parameters, as well as spermatozoa deoxyribonucleic acid (DNA) integrity, are often studied due to their direct implications in natural and assisted conception. Nevertheless, recent studies based on the molecular mechanisms beyond glucose transport in testicular cells provide new insights in DM-induced alterations in male reproductive health. Testicular cells have their own glucose sensing machinery that react to hormonal fluctuations and have several mechanisms to counteract hyper- and hypoglycemic events. Moreover, the metabolic cooperation between testicular cells is crucial for normal spermatogenesis. Sertoli cells (SCs), which are the main components of blood–testis barrier, are not only responsible for the physical support of germ cells but also for lactate production that is then metabolized by the developing germ cells. Any alteration in this tied metabolic cooperation may have a dramatic consequence in male fertility potential. Therefore, we present an overview of the clinical significance of DM in the male reproductive health with emphasis on the molecular mechanisms beyond glucose fluctuation and transport in testicular cells.     

细胞糖代谢方式改变与肿瘤转移的相关性

肿瘤转移是引起肿瘤相关死亡的主要原因,肿瘤细胞的代谢异常在肿瘤转移中扮演重要角色。肿瘤的糖代谢以“Warburg效应”为显著特征,即细胞在有氧条件下也以糖酵解为主要糖代谢途径提供能量。而这种现象在转移性肿瘤细胞中更为突出,表现为葡萄糖的大量摄取、高糖酵解速率和核酸合成速率等,这为肿瘤细胞的快速生长和增殖提供了重要的能量和物质基础。对于肿瘤转移过程中相关代谢改变的研究,将为最终揭示肿瘤转移的机制打下基础。本文综述肿瘤细胞糖代谢中糖酵解、线粒体有氧代谢及磷酸戊糖途径中的变化与肿瘤转移发生的相关性,其结果为进一步从调控肿瘤代谢角度发现新的肿瘤转移控制手段提供了启示。     

Critical Role of H2O2 Generated by NOX4 during Cellular Response under Glucose Deprivation

Glucose is the most efficient energy source, and various cancer cells depend on glycolysis for energy production. For maintenance of survival and proliferation, glucose sensing and adaptation to poor nutritional circumstances must be well organized in cancer cells. While the glucose sensing machinery has been well studied in yeasts, the molecular mechanism of glucose sensing in mammalian cells remains to be elucidated. We have reported glucose deprivation rapidly induces AKT phosphorylation through PI3K activation. We assumed that regulation of AKT is relevant to glucose sensing and further investigated the underlying mechanisms. In this study, AKT phosphorylation under glucose deprivation was inhibited by galactose and fructose, but induced by 2-deoxyglucose (2-DG). Both 2-DG treatment and glucose deprivation were found to induce AKT phosphorylation in HepG2 cells. These findings suggested that glucose transporter may not be involved in the sensing of glucose and induction of AKT phosphorylation, and that downstream metabolic events may have important roles. A variety of metabolic stresses reportedly induce the production of reactive oxygen species (ROS). In the present study, glucose deprivation was found to induce intracellular hydrogen peroxide (H₂O₂) production in HepG2 cells. N-acetylcysteine (NAC), an antioxidant reagent, reduced both the increase in cellular H₂O₂ levels and AKT phosphorylation induced by glucose deprivation. These results strongly suggest that the glucose deprivation-induced increase of H₂O₂ in the cells mediated the AKT phosphorylation. RNA interference of NOX4, but not of NOX5, completely suppressed the glucose deprivation-induced AKT phosphorylation as well as increase of the intracellular levels of ROS, whereas exogenous H₂O₂ could still induce AKT phosphorylation in the NOX4-knockdown cells. In this study, we demonstrated that the ROS generated by NOX4 are involved in the intracellular adaptive responses by recognizing metabolic flux.     

Moving the insulin-regulated glucose transporter GLUT4 into and out of storage

The glucose transporter isoform GLUT4 is unique among the glucose transporter family of proteins in that, in resting cells, it is sequestered very efficiently in a storage compartment. In insulin-sensitive cells, such as fat and muscle, insulin stimulation leads to release of GLUT4 from this reservoir and its translocation to the plasma membrane. This process is crucial for the control of blood and tissue glucose levels. Investigations of the composition and structure of the GLUT4 storage compartment, together with the targeting motifs that direct GLUT4 to this compartment, have been extensive but have been controversial. Recent findings have now provided a clearer consensus of opinion on the mechanisms involved in the formation of this storage compartment. However, another controversy has now emerged, which is unresolved. This concerns the issue of whether the insulin-regulated step occurs at the level of release of GLUT4 from the storage compartment or at the level at which released vesicles fuse with the plasma membrane.     

Mechanism of maltose uptake and glucose excretion in Lactobacillus sanfrancisco.

Lactobacillus sanfrancisco LTH 2581 can use only glucose and maltose as sources of metabolic energy. In maltose-metabolizing cells of L. sanfrancisco, approximately half of the internally generated glucose appears in the medium. The mechanisms of maltose (and glucose) uptake and glucose excretion have been investigated in cells and in membrane vesicles of L. sanfrancisco in which beef heart cytochrome c oxidase had been incorporated as a proton-motive-force-generating system. In the presence of ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), and cytochrome c, the hybrid membranes facilitated maltose uptake against a concentration gradient, but accumulation of glucose could not be detected. Similarly, in intact cells of L. sanfrancisco, the nonmetabolizable glucose analog alpha-methylglucoside was taken up only to the equilibration level. Selective dissipation of the components of the proton and sodium motive force in the hybrid membranes indicated that maltose is transported by a proton symport mechanism. Internal [14C]maltose could be chased with external unlabeled maltose (homologous exchange), but heterologous maltose/glucose exchange could not be detected. Membrane vesicles of L. sanfrancisco also catalyzed glucose efflux and homologous glucose exchange. These activities could not be detected in membrane vesicles of glucose-grown cells. The results indicate that maltose-grown cells of L. sanfrancisco express a maltose-H+ symport and glucose uniport system. When maltose is the substrate, the formation of intracellular glucose can be more rapid than the subsequent metabolism, which leads to excretion of glucose via the uniport system.     

Glycated collagen and altered glucose increase endothelial cell adhesion strength

Cell adhesion strength is important to cell survival, proliferation, migration, and mechanotransduction, yet changes in endothelial cell adhesion strength have not yet been examined in diseases such as diabetes with high rates of cardiovascular complications. We therefore investigated porcine aortic endothelial cell adhesion strength on native and glycated collagen‐coated substrates and in low, normal, and high glucose culture using a spinning disc apparatus. Adhesion strength increased by 30 dynes/cm² in cells on glycated collagen as compared to native collagen. Attachment studies revealed that cells use higher adhesion strength α_vβ₃ integrins to bind to glycated collagen instead of the typical α₂β₁ integrins used to bind to native collagen. Similarly, endothelial cells cultured in low and high glucose had 15 dynes/cm² higher adhesion strength than cells in normal glucose after 2 days. Increased adhesion strength was due to elevated VEGF release and intracellular PKC in low and high glucose cells, respectively. Thus glucose increased endothelial cell adhesion strength via different underlying mechanisms. These adhesion strength changes could contribute to diabetic vascular disease, including accelerated atherosclerosis and disordered angiogenesis. J. Cell. Physiol. 228: 1727–1736, 2013. © 2013 Wiley Periodicals, Inc.     

Catabolite inactivation of the sugar transporters in Saccharomyces cerevisiae is inhibited by the presence of a nitrogen source

Saccharomyces cerevisiae uses glucose preferentially to any other carbon source and this preferential use is ensured by control mechanisms triggered by glucose. The consensus is that inactivation of sugar transporters other than glucose transporters is one of these mechanisms. This inactivation is called catabolite inactivation because of its apparent analogy with the catabolite inactivation of gluconeogenic enzymes. Recently, doubt has been cast on the role of the inactivation of the sugar transporters in controlling the use of glucose because this inactivation neither is specifically triggered by glucose nor specifically affects non-glucose sugar transporters. Based on the fact that this inactivation has been almost exclusively investigated using nitrogen-starved cells, it has been proposed that it might be due to the stimulation of the protein turnover that follows nitrogen starvation. The results obtained in this work support this possibility since they show that the presence of a nitrogen source in the medium strongly inhibited the inactivation. It is concluded that, in growing yeast cells, the contribution of the inactivation by glucose of the non-glucose sugar transporters to the preferential use of glucose is much lower than generally believed.     

Glycogen metabolism in rat heart muscle cultures after hypoxia

Elevated glycogen levels in heart have been shown to have cardioprotective effects against ischemic injury. We have therefore established a model for elevating glycogen content in primary rat cardiac cells grown in culture and examined potential mechanisms for the elevation (glycogen supercompensation). Glycogen was depleted by exposing the cells to hypoxia for 2 h in the absence of glucose in the medium. This was followed by incubating the cells with 28 mM glucose in normoxia for up to 120 h. Hypoxia decreased glycogen content to about 15% of control, oxygenated cells. This was followed by a continuous increase in glycogen in the hypoxia treated cells during the 120 h recovery period in normoxia. By 48 h after termination of hypoxia, the glycogen content had returned to baseline levels and by 120 h glycogen was about 150% of control. The increase in glycogen at 120 h was associated with comparable relative increases in glucose uptake (~ 180% of control) and the protein level of the glut-1 transporter (~ 170% of control), whereas the protein level of the glut-4 transporter was decreased to < 10% of control. By 120 h, the hypoxia-treated cells also exhibited marked increases in the total (~ 170% of control) and fractional activity of glycogen synthase (control, ~ 15%; hypoxia-treated, ~ 30%). Concomitantly, the hypoxia-treated cells also exhibited marked decreases in the total (~ 50% of control) and fractional activity of glycogen phosphorylase (control, ~ 50%; hypoxia-treated, - 25%). Thus, we have established a model of glycogen supercompensation in cultures of cardiac cells that is explained by concerted increases in glucose uptake and glycogen synthase activity and decreases in phosphorylase activity. This model should prove useful in studying the cardioprotective effects of glycogen.     

Carbohydrate Uptake and Utilization byClostridium beijerinckiiNCIMB 8052

The clostridia are a diverse group of obligately anaerobic bacteria with potential for the fermentative production of fuels, solvents and other chemicals. Several species exhibit a broad substrate range, but there have been few studies of the mechanisms involved in regulation of uptake and metabolism of fermentable carbohydrates.Clostridium beijerinckii(formerlyClostridium acetobutylicum) NCIMB 8052 exhibited transport activity for hexoses and hexitols. Glucose-grown cells transported glucose and fructose, but not galactose, glucitol (sorbitol) or mannitol, transport of which was induced by growth on the respective substrates. Phosphorylation of glucose, fructose, glucitol and mannitol by cell extracts was supported by phosphoenolpyruvate, indicating the involvement of a phosphotransferase system in uptake of these substrates. Fructose phosphorylation was also demonstrated by isolated membranes in the presence of fructose 1-phosphate, thus identifying this derivative as the product of the fructose phosphotransferase system. The presence of phosphotransferase activities in extracts prepared from cells grown on different carbon sources correlated with transport activities in whole cells, and the pattern of transport activities reflected the substrate preference of cells growing in the presence of glucose and another carbon source. Thus, glucose and fructose were co-metabolised, while utilization of glucitol was prevented by glucose, even in cells which were previously induced for glucitol metabolism. Of the substrates examined, only galactose appeared to be transported by a non-phosphotransferase mechanism, since a significant rate of phosphorylation of this sugar was supported by ATP rather than phosphoenolpyruvate.     

Mathematical modeling and analysis of glucose and glutamine utilization and regulation in cultures of continuous mammalian cells

A number of factors have been shown to affect the metabolism of glucose and glutamine in mammalian cells and their mechanisms have been partially elucidated. Despite these efforts, a quantitative knowledge of the significance of these factors, the regulation of glucose and glutamine utilization, and particularly the interactions of these two nutrients is still lacking. Controversies exist in the literature. To clarify some of these controversies, mathematical models are proposed in this work which enable to separate and identify the effects of individual factors. Experimental data from five cell lines obtained in batch, fed-batch, and continuous cultures, both under steady-state and transient conditions, were used to verify the model formulations. The resulting kinetic models successfully describe all these cultures. According to the models, the specific consumption rate of glucose (Q(Glc)) of continuous animal cells under normal culture conditions can be expressed as a sum of three parts: a part owing to cell growth; a part owing to glucose excess; and a part owing to glutamine regulation. The specific consumption rate of glutamine (q(Glc)7) can be expressed as a sum of only two parts: a part owing to cell growth; and a part owing to glutamine excess. Using the kinetic models the interaction and regulation of glucose and glutamine utilizations are quantitatively analyzed. The results indicate that, whereas q(Glc) is affected by glutamine, q(Gln) appears to be not or less significantly affected by glucose. It is also shown that the relative utilizations of glucose and glutamine by anabolism and catabolism are mainly affected by the residual concentrations of the respective compounds and are less sensitive to growth rate and the nature of growth limitation.(c) 1995 John Wiley & Sons, Inc.     

Differential regulation of GLUT1 activity in human corneal limbal epithelial cells and fibroblasts

The corneal epithelial tissue is a layer of rapidly growing cells that are highly glycolytic and express GLUT1 as the major glucose transporter. It has been shown that GLUT1 in L929 fibroblast cells and other cell lines can be acutely activated by a variety agents. However, the acute regulation of glucose uptake in corneal cells has not been systematically investigated. Therefore, we examined glucose uptake in an immortalized human corneal–limbal epithelial (HCLE) cell line and compared it to glucose uptake in L929 fibroblast cells, a cell line where glucose uptake has been well characterized. We report that the expression of GLUT1 in HCLE cells is 6.6-fold higher than in L929 fibroblast cells, but the HCLE cells have a 25-fold higher basal rate of glucose uptake. Treatment with agents that interfere with mitochondrial metabolism, such as sodium azide and berberine, activate glucose uptake in L929 cells over 3-fold, but have no effect on glucose uptake HCLE cells. Also, agents known to react with thiols, such cinnamaldehyde, phenyarsine oxide and nitroxyl stimulate glucose uptake in L929 cells 3–4-fold, but actually inhibit glucose uptake in HCLE cells. These data suggest that in the fast growing HCLE cells, GLUT1 is expressed at a higher concentration and is already highly activated at basal conditions. These data support a model for the acute activation of GLUT1 that suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond within GLUT1 itself.     

Molecular mechanism of interaction of Mycobacterium tuberculosis with host macrophages under high glucose conditions

Mycobacterium tuberculosis has the potential to escape various cellular defense mechanisms for its survival which include various oxidative stress responses, inhibition of phagosome-lysosomes fusion and alterations in cell death mechanisms of host macrophages that are crucial for its infectivity and dissemination. Diabetic patients are more susceptible to developing tuberculosis because of impairement of innate immunity and prevailing higher glucose levels. Our earlier observations have demonstrated alterations in the protein profile of M. tuberculosis exposed to concurrent high glucose and tuberculosis conditions suggesting a crosstalk between host and pathogen under high glucose conditions. Since high glucose environment plays crucial role in the interaction of mycobacterium with host macrophages which provide a niche for the survival of M. tuberculosis, it is important to understand various interactive mechanisms under such conditions. Initial phagocytosis and containment of M. tuberculosis by macrophages, mode of macrophage cell death, respiratory burst responses, Mycobacterium and lysosomal co-localization were studied in M. tuberculosis H37Rv infected cells in the presence of varied concentrations of glucose in order to mimic diabetes like conditions. It was observed that initial attachment, phagocytosis and later containment were less effective under high glucose conditions in comparison to normal glucose. Mycobacterium infected cells showed more necrosis than apoptosis as cell death mechanism during the course of infection under high glucose concentrations. Co-localization and respiratory burst assay also indicated evasion strategies adopted by M. tuberculosis under such conditions. This study by using THP1 macrophage model of tuberculosis and high glucose conditions showed immune evasion strategies adapted during co-pathogenesis of tuberculosis and diabetes.     

Involvement of ATP-sensitive potassium (K(ATP)) channels in the loss of beta-cell function induced by human islet amyloid polypeptide

Islet amyloid polypeptide (IAPP) is a major component of amyloid deposition in pancreatic islets of patients with type 2 diabetes. It is known that IAPP can inhibit glucose-stimulated insulin secretion; however, the mechanisms of action have not yet been established. In the present work, using a rat pancreatic beta-cell line, INS1E, we have created an in vitro model that stably expressed human IAPP gene (hIAPP cells). These cells showed intracellular oligomers and a strong alteration of glucose-stimulated insulin and IAPP secretion. Taking advantage of this model, we investigated the mechanism by which IAPP altered beta-cell secretory response and contributed to the development of type 2 diabetes. We have measured the intracellular Ca(2+) mobilization in response to different secretagogues as well as mitochondrial metabolism. The study of calcium signals in hIAPP cells demonstrated an absence of response to glucose and also to tolbutamide, indicating a defect in ATP-sensitive potassium (K(ATP)) channels. Interestingly, hIAPP showed a greater maximal respiratory capacity than control cells. These data were confirmed by an increased mitochondrial membrane potential in hIAPP cells under glucose stimulation, leading to an elevated reactive oxygen species level as compared with control cells. We concluded that the hIAPP overexpression inhibits insulin and IAPP secretion in response to glucose affecting the activity of K(ATP) channels and that the increased mitochondrial metabolism is a compensatory response to counteract the secretory defect of beta-cells.     

Endothelial cells' biophysical,biochemical, and chromosomal aberrancies in high‐glucose condition within the diabetic range

To date, many studies have been conducted to find out the underlying mechanisms of hyperglycemia‐induced complications in diabetes mellitus, attributed to the cellular pathologies of different cells—especially endothelial cells. However, there are still many ambiguities and unresolved issues to be clarified. Here, we investigated the alteration in biophysical and biochemical properties in human umbilical vein endothelial cells exposed to a high‐glucose concentration (30mM), comparable to glucose content in type 2 diabetes mellitus, over a course of 120 hours. In addition to a reduction in the rate of cell viability and induction of oxidative stress orchestrated by the high‐glucose condition, the dynamic of the fatty acid profile—including polyunsaturated, monounsaturated, and saturated fatty acids—was also altered in favor of saturated fatty acids. Genetic imbalances were also detected at chromosomal level in the cells exposed to the abnormal concentration of glucose after 120 hours. Moreover, the number of tip cells (CD31⁺/CD34⁺) and in vitro tubulogenesis capability negatively diminished in comparison to parallel control groups. We found that diabetic hyperglycemia was associated with a decrease in the cell‐cell tight junction and upregulation in vascular endothelial cadherin and zonula occludens (ZO)‐1 molecules after 72 and 120 hours of exposure to the abnormal glucose concentration, which resulted in a profound reduction in transendothelial electrical resistance. The surface plasmon resonance analysis of the human umbilical vein endothelial cells immobilized on gold‐coated sensor chips confirmed the loosening of the cell to cell intercellular junction as well as stable attachment of each cell to the basal surface. Our findings highlighted the disturbing effects of a diabetic hyperglycemia on either biochemical or biophysical properties of endothelial cells.     

Pig Reticulocytes. III. Glucose permeability in naturally occurring reticulocytes and red cells from newborn piglets

The loss of facilitated glucose transport of red cells occurring in the newborn pig was monitored in 11 density-separated cells from birth to a 4 wk of age. At birth there was a threefold increase in glucose permeability from the lightest cells to the most dense, suggesting that cells having progressively less glucose permeability are released into the circulation as gestation proceeds. Because of extraordinary stimulation of erythropoietic activity, the uppermost top fraction constituting 2-3 percent of the total cells is composed purely of reticulocytes in the growing animal. The glucose permeability of these reticulocytes which at birth has a slow but significant rate of 3.7 μmol/ml cell x min at 25 degrees C is rapidly decreased within 3-4 days to the level of reticulocytes produced in the adult in response to phenylhydrazine assault. Moreover, reticulocytes themselves discard their membrane permeability to glucose in the course of maturation to red cells. Thus, even though reticulocytes at birth are permeable to glucose, they will become red cells practically impervious to glucose within a few days. These findings suggest that the transition from a glucose- permeable fetal state to a glucose-impermeable postnatal state is brought about by two mechanisms: (a) dilution of fetal cells by glucose-impervious cells produced coincidentally with or shortly after birth; and (b) elimination of fetal cells, which have a shorter half-life, from the circulation.