Uncoupling Modifier Genes from Uncoupling Protein 2 in Pancreatic β-Cells

Intracellular levels of oxygen and glucose are higher in pancreatic insulin-secreting β-cells than any other mammalian cell. Indeed, among endocrine secretory cells, the pancreatic β-cell is unique in using glucose catabolism to meet its energy needs while relying on glucose metabolism to generate appropriate signals to maintain insulin secretion and glucose homeostasis. The unabated and successful execution of these two independent functions over the lifetime of the β-cell, requires fine coordination between several families of intracellular proteins. In addition to glucose, the β-cell also responds to other nutrients (fatty acids and amino acids), hormones, and nerve signals. Given this barrage of stimuli, it is logical to imagine that the β-cell would be armed with appropriate defense mechanisms to prevent excess stress that could lead to eventual cell dysfunction and/or death. On the contrary, the β-cell is particularly weak against the toxicity of free radicals including reactive oxygen species (ROS) and reactive nitrogen species (1,2) and, being a secretory cell, is also susceptible to endoplasmic reticulum (ER) stress (3,4). Thus, understanding the mechanisms and pathways that mediate the effects of oxidative or ER stress (3) is important to understand β-cell biology and to plan therapeutic approaches to prevent β-cell dysfunction and death.

Pancreatic β-cells sense glucose through its metabolism, and the resulting increase in the ATP/ADP ratio, plasma membrane depolarization, and Ca²⁺ influx leads to insulin secretion (5). In addition to membrane depolarization, the ATP/ADP ratio is also linked to the proton electron chemical gradient at the inner mitochondrial membrane (6). Uncoupling processes would reduce the mitochondrial membrane potential and attenuate mitochondria-derived ROS. Among the molecules that are involved in the proton leak into the mitochondrial matrix is the family of uncoupling proteins (UCPs), the prototype of which is UCP1, which plays a critical role in brown fat thermogenesis (7). UCP2, a member of the mitochondrial inner membrane carrier family, has high homology to UCP1 (7), is expressed widely, but at varying levels, in several tissues including the pancreatic β-cells where it mediates mitochondrial proton leak leading to reduced ATP production. Although UCP1 and UCP2 are highly homologous, the latter is not a classic UCP and does not contribute to adaptive thermogenesis (8).

Several studies have suggested that UCP2 may be able to decrease mitochondria-derived ROS (9). Given the susceptibility of β-cells to ROS, this raises the possibility that increasing UCP2 expression could promote β-cell survival and function. Consistent with this possibility, mice with global knockout (KO) of UCP2, on a mixed genetic background, exhibit higher islet ATP levels and increased glucose-stimulated insulin secretion (10). However, in this issue, Pi et al. (11) reveal a more complex picture. Although they too report that knockout of UCP2, on a mixed genetic background, improves β-cell function, knockout of UCP2 onto individual C57BL/6J (B6), 129 × 1/SvImJ (129), or A/J inbred strains actually worsens the phenotype. It is unclear whether the presentation of data on insulin secretion by Pi et al. (nanograms per milliliter per minute in studies on mixed background vs. percent insulin content in studies on individual strains) contributes significantly to the observed differences in glucose-stimulated insulin secretion.

These experiments by Pi et al. (11) provide several important lessons in the science of transgenics. First, genetic backgrounds can markedly alter the phenotype of a KO or transgene (for review see Ref. 12). Historically, it was recognized that culturing embryonic stem (ES) cells from B6 mice is difficult; therefore, most investigators used 129-derived ES cells (12), which were subsequently injected into B6 blastocysts for germline transmission. Naturally, the initial phenotyping was performed using mice on the mixed background and if necessary (and funds permitting) backcrossing the KOs onto individual strains with the B6 background being the most commonly used in metabolic studies. It should be noted that ES cells from B6 mice now exist (12,13,14). Several examples in the literature provide proof that phenotypes of KO mice on a mixed background alter when they are backcrossed to individual genetic strains (15,16,17,18,19). Among the most well recognized reasons that underlie the observation of alterations in phenotype when mice are maintained on a mixed genetic background vs. an inbred strain is the effect of genes flanking the target locus (12,20). Although a careful breeding strategy has been suggested to minimize alterations in phenotypes, it is impossible to completely rule out the impact of flanking genes (21). Second, the observations highlight innate differences in physiology between inbred strains of mice (22). The studies also raise the possibility that absence of UCP(s) in some human populations may be beneficial compared with others with consequent therapeutic implications for targeting UCPs.

Nevertheless, KO mice created on a mixed background and on individual strains each have their advantages. For example, notwithstanding the differences between mouse and man, and considering that virtually all humans are on a mixed genetic background, it should be informative to characterize phenotypes of mouse KOs created on more than a single background strain. For those genetically inclined, defining and comparing the phenotypes in mice on the mixed vs. one or more individual strains will allow identification of specific modifier genes that may contribute to the phenotype (23).

Although the phenotypes of the UCP2 KO mice either on the mixed background (10) or on the individual strains (11) are both compelling, the way forward will necessarily require additional studies. First, given the intriguing differences in phenotypes between the global UCP2 KO on a mixed background vs. that on single strains, it will be critical to create a β-cell-specific (preferably inducible) KO of UCP2 to establish the phenotype. This will also rule out potential secondary paracrine effects from neighboring α-cells, which express more UCP2 than β-cells (24), and effects from other islet cells, each of which can impact β-cell function within the islet in a global KO (Fig. 1). Second, it will be useful to examine whether modifier genes play a role when UCP2 is knocked out on different genetic backgrounds. Although this will be a tedious undertaking, the virtually opposing phenotypes of UCP2 KO mice on a mixed 129/B6 background vs. those on individual 129 or B6 backgrounds will likely unravel important modifier genes that regulate UCP2. Third, considering the variable effects of UCP2 in isolated islets from normal vs. Zucker diabetic fatty (ZDF) rats (25) and in ob/ob mice (10), which lack leptin and are hyperinsulinemic, it is worth examining the role of leptin and insulin signaling in the regulation of UCP2 in β-cells (26,27) (Fig. 1). Finally, it will be interesting to follow up on the findings of Pi and colleagues in regard to ROS as a potential second messenger in the modulation of insulin secretion (28,29).

Figure 1.

The schematic shows that knocking out UCP2 leads to enhanced oxidative stress that in turn blunts insulin secretion (depicted by dashed arrows and minus sign). The original observation of a knockout of UCP2 promoting insulin secretion is shown by the solid arrow and plus sign. Known inducers of oxidative and ER stress include hyperglycemia, hyperlipidemia, and cytokines, whereas the identities of potential modifier genes and the effects of insulin and leptin on UCP2 are largely unknown (shown by question mark). UCP2 is also expressed in α-cells, and it is unclear whether the concomitant absence of UCP2 in α-cells impacts the function of neighboring β-cells in a global UCP2 KO mouse. nuc, Nucleus.

As Robert Burns wrote in an ode “To a Mouse,”

But, Mousie, thou art no thy lane

In proving foresight may be vain:

The best laid schemes o’ mice an’ men

Gang aft a-gley,

An’ lea’e us nought but grief an’ pain,

For promised joy.

The essential experiments described above should be completed before one can begin to speculate about the potential for UCP2 as a drug target to modulate β-cell function and the promised joy.