Vincent Poitout and R. Paul Robertson

Glucotoxicity, lipotoxicity, and glucolipotoxicity are secondary phenomena that are proposed to play a role in all forms of type 2 diabetes. The underlying concept is that once the primary pathogenesis of diabetes is established, probably involving both genetic and environmental forces, hyperglycemia and very commonly hyperlipidemia ensue and thereafter exert additional damaging or toxic effects on the β-cell. In addition to their contribution to the deterioration of β-cell function after the onset of the disease, elevations of plasma fatty acid levels that often accompany insulin resistance may, as glucose levels begin to rise outside of the normal range, also play a pathogenic role in the early stages of the disease. Because hyperglycemia is a prerequisite for lipotoxicity to occur, the term glucolipotoxicity, rather than lipotoxicity, is more appropriate to describe deleterious effects of lipids on β-cell function. In vitro and in vivo evidence supporting the concept of glucotoxicity is presented first, as well as a description of the underlying mechanisms with an emphasis on the role of oxidative stress. Second, we discuss the functional manifestations of glucolipotoxicity on insulin secretion, insulin gene expression, and β-cell death, and the role of glucose in the mechanisms of glucolipotoxicity. Finally, we attempt to define the role of these phenomena in the natural history of β-cell compensation, decompensation, and failure during the course of type 2 diabetes.

TYPE 2 DIABETES is considered to be a complex syndrome of polygenic nature (1). Several genome-wide scans recently identified risk loci for type 2 diabetes that include gene controlling β-cell development or function, supporting the concept that the genetic susceptibility of the β-cell determines the risk of developing the disease.

Glucotoxicity, lipotoxicity, and glucolipotoxicity are secondary phenomena that are proposed to play a role in all forms of type 2 diabetes. The underlying concept is that once the primary pathogenesis of diabetes is established, probably involving both genetic and environmental forces, hyperglycemia and very commonly hyperlipidemia ensue and thereafter exert additional damaging or toxic effects on the β-cell. In addition to their contribution to the deterioration of β-cell function after the onset of the disease, elevations of plasma fatty acid levels that often accompany insulin resistance may, as glucose levels begin to rise outside of the normal range, also play a pathogenic role in the early stages of the disease.

The words “glucotoxicity” and “lipotoxicity,” as well as their combination form, “glucolipotoxicity,” are best described as medical jargon rather than scientific terms that can be precisely defined. They represent currently popular concepts that connote adverse or toxic influences on pancreatic β-cell function caused by excessive glucose and/or lipids. In a sense, these are paradoxical concepts because physiological levels of glucose and lipids are not toxic but instead are essential to normal β-cell function. Thus, a spectrum exists going from normoglycemic and normolipidemic conditions to hyperglycemic and hyperlipidemic abnormalities that must be taken into account. The root of these words, “toxicity,” implies damage and leads to the consideration that in certain instances glucose and lipid levels entering or synthesized within tissues might become so elevated that they can cause pathology.

Unger and colleagues first introduced the concepts of glucotoxicity and lipotoxicity. In their initial glucose toxicity paper, they put forward the concept that continuous overstimulation of the β-cell by glucose could eventually lead to depletion of insulin stores, worsening of hyperglycemia, and finally deterioration of β-cell function. Exposure of the β-cell to excessive levels of lipids was also hypothesized to be a cause of worsening β-cell function. In this view, failure to correct hyperglycemia and hyperlipidemia dooms the β-cell to a continual onslaught of glucotoxicity and lipotoxicity that perpetuates the downward spiral to β-cell dysfunction and death.

The term glucolipotoxicity has a more recent origin and was coined in recognition of the fact that the alterations in intracellular lipid partitioning underlying the mechanisms of lipotoxicity are dependent upon elevated glucose levels. Consequently, it is our view that hyperglycemia is a prerequisite for lipotoxicity to occur, and that therefore the term glucolipotoxicity, rather than lipotoxicity, is more appropriate to describe deleterious effects of lipids on β-cell function.

This review of the current literature and studies performed in our laboratories will first present experimental evidence in support of glucotoxicity and glucolipotoxicity. We will then propose the existence of a spectrum from glucolipoadaptation to glucolipotoxicity and attempt to define the role of these phenomena in the natural history of β-cell compensation, decompensation, and failure during the course of type 2 diabetes. In accordance with editorial guidelines for this Recent Progress in Hormone Research (RPHR) edition, this review mostly focuses on the work performed in our own laboratories. Although we have attempted to quote the relevant literature from other groups, we could not, due to space limitations, exhaustively cite the work of all other investigators who contributed to this field.

Most of the earliest work demonstrating the paradoxical ability of glucose to diminish β-cell function was reported from experiments using β-cell lines. A key design feature in these experiments was to expose cells to media containing high concentrations of glucose for a protracted period of time, as long as 3–12 months. For example, using HIT-T15 cells and glucose concentrations of 0.8 and 11.1 mm, we observed that prolonged culturing of cells in RPMI 1640 medium containing the lower glucose concentration maintained insulin mRNA levels, insulin content, and glucose-induced insulin secretion. On the other hand, insulin gene expression, insulin content, and glucose-induced insulin secretion were progressively and drastically compromised over time when the higher glucose concentration was used in the media. Subsequent observations revealed that culturing in media with high glucose concentrations also caused deterioration in insulin promoter activity as well as pancreas-duodenum homeobox-1 (PDX-1) and MafA binding activity. It is noteworthy that loss of MafA binding occurred much earlier than the loss of PDX-1 binding and that the decrease in insulin content correlated in time much closer to the loss of MafA binding. The decreased levels of insulin mRNA, insulin content, and insulin release were taken as evidence that chronic exposure to high glucose concentrations caused glucotoxic effects on the β-cells. In later experiments it was shown that the diminution in β-cell function observed in cultures containing high glucose could be reversed by switching to low glucose; however, the efficacy of this intervention was time-dependent. β-Cell function returned when the intervention was performed 5 or 10 wk after glucotoxic defects were present, but not if a longer period of time had elapsed. Differentiation of glucotoxic effects from β-cell exhaustion was provided by experiments in which HIT-T15 cells were cultured for long periods of time in media containing 0.8 or 11.1 mm glucose with and without somatostatin, an inhibitor of insulin secretion. The cells cultured with somatostatin had dramatically less insulin in the culture media, demonstrating β-cell rest. Yet, the cells exposed to high glucose and somatostatin still experienced glucotoxic effects on insulin gene expression, content, and glucose-stimulated insulin secretion, thereby eliminating β-cell exhaustion as a cause.

Another β-cell line, the INS-1 cell, was also used in similar experiments. In this case, however, the paradoxical effect of a high glucose concentration to decrease insulin mRNA was seen within 24 h and was quickly reversible after culturing the cells in a low glucose concentration. This abbreviated time course for establishing adverse effects of a high glucose concentration and their ready reversal is more reminiscent of a glucose desensitization phenomenon than glucose toxicity. More recent work using INS-1 cells suggests that glucotoxic β-cells may have additional, more distal defects in the exocytotic pathway. A third β-cell line, the βTC-6 cell, behaved very similarly to HIT-T15 cells when cultured under conditions of high and low glucose, with the exception that only MafA binding and not PDX-1 binding to DNA was affected. This finding presaged our later reports indicating that a decline in MafA alone is sufficient to cause loss of insulin gene expression in glucotoxic states.

Evidence that glucotoxicity might be related to oxidative stress can be found in early reports that N-acetylcysteine and aminoguanidine, both antioxidants, can protect HIT-T15 cells and isolated islets from the adverse effects of prolonged culturing with media containing high glucose concentrations. We asked whether the decreased PDX-1 binding and decreased insulin mRNA levels in HIT-T15 cells that we observed might also be related to oxidative stress. Using either N-acetylcysteine or aminoguanidine in cells cultured for many passages under high glucose conditions, we observed an antioxidant drug concentration-related preservation of insulin promoter activity, PDX-1 binding, and levels of insulin mRNA.

Biochemical pathways through which elevated levels of glucose can form excessive levels of reactive oxygen species (ROS), which cause oxidative stress and lead to β-cell dysfunction. Under normoglycemic conditions, glucose metabolites flow primarily (more ...)

Chronically culturing isolated pancreatic islets is a major laboratory challenge. Nonetheless, Briaud et al. were successful in culturing rat islets for up to 6 wk in media containing either 5.6 or 16.7 mm glucose and found at 6 wk that insulin mRNA levels were 50% decreased by the higher glucose concentration. In contrast, no changes were observed in mRNA levels for glucose transporter-2 or glucokinase. Adverse effects of chronically elevated levels of glucose were also studied earlier by Montana et al. who transplanted isolated C57BL/6 rat islets under the kidney capsule of syngeneic recipients previously rendered diabetic by streptozotocin. Their strategy was to transplant 150 islets, a number that was insufficient for normalization of glycemia, so that they would be exposed to hyperglycemic conditions after transplant. Control animals were transplanted with 300 islets, which restored and maintained normoglycemia. β-Cell mass in the grafts decreased in the former group but not the latter group. Other related work by this laboratory regarding the adverse effects of elevated glucose levels on β-cell mass and function has been extensively reviewed by Leahy et al.

Reports that β-cells have very low levels of antioxidant enzymes compared with other tissues suggest that the β-cell is particularly at risk for oxidative stress. This observation led to many efforts to determine whether overexpression of antioxidant enzymes would protect β-cells against oxidative stress in rodents. Tanaka et al. in our group performed studies to determine whether enhancing β-cell antioxidant levels would protect against ribose-induced oxidative stress. Ribose is a much stronger oxidant than glucose and provides a strategy to examine consequences of oxidative stress on islets that circumvents the requirement of culturing islets over many months under high glucose conditions. Adenovirally induced overexpression of glutathione peroxidase, a critical antioxidant enzyme, was used in the experimental approach. Although other antioxidant enzymes, such as superoxide dismutases and catalase, are present in the islet (but also in low concentrations), superoxide dismutase forms hydrogen peroxide whereas catalase, although capable of metabolizing hydrogen peroxide, does not catabolize lipid peroxides. Glutathione peroxidase, on the other hand, catabolizes both hydrogen peroxide and lipid peroxides to form the two nontoxic substances, oxygen and water. Adenoviral overexpression of glutathione peroxidase increased islet levels of this enzyme 6-fold or approximately to the levels found normally in liver. This overexpression protected islets from ribose-induced losses in insulin mRNA, insulin content, and glucose-stimulated insulin secretion. Tran et al. in similar studies examined the protective effect of overexpressing the glutamylcysteine ligase catalytic subunit, which regulates the use of cysteine as the rate-limiting substrate to form glutathione, the primary endogenous antioxidant substance. Adenoviral overexpression of this enzyme augmented the level of reduced glutathione in islets and prevented the adverse effects of IL-1-β on glucose-induced insulin secretion. Kaneto et al. observed that adenoviral overexpression of a dominant-negative (DN) mutant of c-Jun N-terminal kinase (JNK) preserved PDX-1 DNA binding in islets exposed to H2O2-induced oxidative stress. They also reported that rat islets infected with DN-JNK-expressing adenovirus and transplanted under renal capsules of streptozotocin-induced diabetic nude mice maintained insulin gene expression in the grafts. However, despite the repeated observation that overexpression of antioxidant enzymes in rodent models protects against oxidative stress, to date no one has reported whether overexpression of antioxidant enzymes is protective against type 2 diabetes mellitus in an animal model. Counter to the concept that oxygen radicals are damaging to islets, Pi et al. performed studies with INS-1 cells and isolated mouse islets and reported evidence that physiological levels of reactive oxygen species (ROS) may be required to support physiological β-cell function. This supports the concept that ROS in low levels are contributory to, but high levels may be harmful for, β-cell function.