Danica Grujic

Uncoupling protein-2 negatively regulates insulin secretion and is a major link between obesity, beta cell dysfunction, and type 2 diabetes

Abstract β cells sense glucose through its metabolism and the resulting increase in ATP, which subsequently stimulates insulin secretion. Uncoupling protein-2 (UCP2) mediates mitochondrial proton leak, decreasing ATP production. In the present study, we assessed UCP2s role in regulating insulin secretion. UCP2-deficient mice had higher islet ATP levels and increased glucose-stimulated insulin secretion, establishing that UCP2 negatively regulates insulin secretion. Of pathophysiologic significance, UCP2 was markedly upregulated in islets of ob/ob mice, a model of obesity-induced diabetes. Importantly, ob/ob mice lacking UCP2 had restored first-phase insulin secretion, increased serum insulin levels, and greatly decreased levels of glycemia. These results establish UCP2 as a key component of β cell glucose sensing, and as a critical link between obesity, β cell dysfunction, and type 2 diabetes.

Energy Metabolism in Uncoupling Protein 3 Gene Knockout Mice

Uncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier superfamily. Based upon its high homology with UCP1 and its restricted tissue distribution to skeletal muscle and brown adipose tissue, UCP3 has been suggested to play important roles in regulating energy expenditure, body weight, and thermoregulation. Other postulated roles for UCP3 include regulation of fatty acid metabolism, adaptive responses to acute exercise and starvation, and prevention of reactive oxygen species (ROS) formation. To address these questions, we have generated mice lacking UCP3 (UCP3 knockout (KO) mice). Here, we provide evidence that skeletal muscle mitochondria lacking UCP3 are more coupled (i.e. increased state 3/state 4 ratio), indicating that UCP3 has uncoupling activity. In addition, production of ROS is increased in mitochondria lacking UCP3. This study demonstrates that UCP3 has uncoupling activity and that its absence may lead to increased production of ROS. Despite these effects on mitochondrial function, UCP3 does not seem to be required for body weight regulation, exercise tolerance, fatty acid oxidation, or cold-induced thermogenesis. The absence of such phenotypes in UCP3 KO mice could not be attributed to up-regulation of other UCP mRNAs. However, alternative compensatory mechanisms cannot be excluded. The consequence of increased mitochondrial coupling in UCP3 KO mice on metabolism and the possible role of yet unidentified compensatory mechanisms, remains to be determined.

β3-Adrenergic Receptors on White and Brown Adipocytes Mediate β3-Selective Agonist-induced Effects on Energy Expenditure, Insulin Secretion, and Food Intake A STUDY USING TRANSGENIC AND GENE KNOCKOUT MICE

β3-Adrenergic receptors (β3-ARs) are expressed predominantly on white and brown adipocytes, and acute treatment of mice with CL 316,243, a potent and highly selective β3-AR agonist, produces a 2-fold increase in energy expenditure, a 50–100-fold increase in insulin levels, and a 40–50% reduction in food intake. Recently, we generated gene knockout mice lacking functional β3-ARs and demonstrated that each of these responses were mediated exclusively by β3-ARs. However, the tissue site responsible for producing these actions is unknown. In the present study, genetically engineered mice were created in which β3-ARs are expressed exclusively in white and brown adipocytes (WAT+BAT-mice), or in brown adipocytes only (BAT-mice). This was accomplished by injecting tissue-specific β3-AR transgenic constructs into mouse zygotes homozygous for the β3-AR knockout allele. Control, knockout, WAT+BAT, and BAT-mice were then treated acutely with CL, and the effects on various parameters were assessed. As previously observed, all effects of CL were completely absent in gene knockout mice lacking β3-ARs. The effects on O2 consumption, insulin secretion, and food intake were completely rescued with transgenic re-expression of β3-ARs in white and brown adipocytes (WAT+BAT-mice), demonstrating that each of these responses is mediated exclusively by β3-ARs in white and/or brown adipocytes, and that β3-ARs in other tissue sites were not required. Importantly, transgenic re-expression of β3-ARs in brown adipocytes only (BAT-mice) failed to rescue, in any way, CL-mediated effects on insulin levels and food intake and only minimally restored effects on oxygen consumption, indicating that any effect on insulin secretion and food intake, and a full stimulation of oxygen consumption required the presence of β3-ARs in white adipocytes. The mechanisms by which β3-AR agonist stimulation of white adipocytes produces these responses are unknown but may involve novel mediators not previously known to effect these processes.

The Human Uncoupling Protein-3 Gene GENOMIC STRUCTURE, CHROMOSOMAL LOCALIZATION, AND GENETIC BASIS FOR SHORT AND LONG FORM TRANSCRIPTS

Uncoupling protein-3 (UCP3) is a recently identified candidate mediator of adaptive thermogenesis in humans. Unlike UCP1 and UCP2, UCP3is expressed preferentially and at high levels in human skeletal muscle and exists as short and long form transcripts,UCP3 S and UCP3 L.UCP3 S is predicted to encode a protein which lacks the last 37 C-terminal residues of UCP3 L. In the present study, we have defined the intron-exon structure for the human UCP3 gene and determined thatUCP3 S is generated when a cleavage and polyadenylation signal (AATAAA) located in the last intron prematurely terminates message elongation. In addition we have mappedUCP3 to the distal segment of human chromosome 11q13 (between framework markers D11S916 and D11S911), adjacent toUCP2. Of note, UCP2 and UCP3 in both mice and humans colocalize in P1 and BAC genomic clones indicating that these two UCPs are located within 75–150 kilobases of each other and most likely resulted from a gene duplication event. Previous studies have noted that mouse UCP2 maps to a region of chromosome 7 which is coincident with three independently mapped quantitative trait loci for obesity. Our study shows thatUCP3 is also coincident with these quantitative trait loci raising the possibility that abnormalities in UCP3 are responsible for obesity in these models.

Differential regulation of uncoupling protein gene homologues in multiple tissues of hibernating ground squirrels

Nonshivering thermogenesis in brown adipose tissue (BAT) provides heat through activation of a mitochondrial uncoupling protein (UCP1), which causes futile electron transport cycles without the production of ATP. Recent discovery of two molecular homologues, UCP2, expressed in multiple tissues, and UCP3, expressed in muscle, has resulted in investigation of their roles in thermoregulatory physiology and energy balance. To determine the expression pattern of Ucp homologues in hibernating mammals, we compared relative mRNA levels of Ucp1, -2, and -3 in BAT, white adipose tissue (WAT), and skeletal muscle of arctic ground squirrels (Spermophilus parryii) hibernating at different ambient and body temperatures, with levels determined in tissues from ground squirrels not in hibernation. Here we report significant increases in mRNA levels for Ucp2 in WAT (1. 6-fold) and Ucp3 in skeletal muscle (3-fold) during hibernation. These results indicate the potential for a role of UCP2 and UCP3 in thermal homeostasis during hibernation and indicate that parallel mechanisms and multiple tissues could be important for nonshivering thermoregulation in mammals.Nonshivering thermogenesis in brown adipose tissue (BAT) provides heat through activation of a mitochondrial uncoupling protein (UCP1), which causes futile electron transport cycles without the production of ATP. Recent discovery of two molecular homologues, UCP2, expressed in multiple tissues, and UCP3, expressed in muscle, has resulted in investigation of their roles in thermoregulatory physiology and energy balance. To determine the expression pattern of Ucp homologues in hibernating mammals, we compared relative mRNA levels of Ucp1, -2, and -3 in BAT, white adipose tissue (WAT), and skeletal muscle of arctic ground squirrels ( Spermophilus parryii) hibernating at different ambient and body temperatures, with levels determined in tissues from ground squirrels not in hibernation. Here we report significant increases in mRNA levels for Ucp2 in WAT (1.6-fold) and Ucp3 in skeletal muscle (3-fold) during hibernation. These results indicate the potential for a role of UCP2 and UCP3 in thermal homeostasis during hibernation and indicate that parallel mechanisms and multiple tissues could be important for nonshivering thermoregulation in mammals.

Expression of Human α2-Adrenergic Receptors in Adipose Tissue of β3-Adrenergic Receptor-deficient Mice Promotes Diet-induced Obesity

Catecholamines play an important role in controlling white adipose tissue function and development. β- and α2-adrenergic receptors (ARs) couple positively and negatively, respectively, to adenylyl cyclase and are co-expressed in human adipocytes. Previous studies have demonstrated increased adipocyte α2/β-AR balance in obesity, and it has been proposed that increased α2-ARs in adipose tissue with or without decreased β-ARs may contribute mechanistically to the development of increased fat mass. To critically test this hypothesis, adipocyte α2/β-AR balance was genetically manipulated in mice. Human α2A-ARs were transgenically expressed in the adipose tissue of mice that were either homozygous (−/−) or heterozygous (+/−) for a disrupted β3-AR allele. Mice expressing α2-ARs in fat, in the absence of β3-ARs (β3-AR −/− background), developed high fat diet-induced obesity. Strikingly, this effect was due entirely to adipocyte hyperplasia and required the presence of α2-ARs, the absence of β3-ARs, and a high fat diet. Of note, obese α2-transgenic, β3 −/− mice failed to develop insulin resistance, which may reflect the fact that expanded fat mass was due to adipocyte hyperplasia and not adipocyte hypertrophy. In summary, we have demonstrated that increased α2/β-AR balance in adipocytes promotes obesity by stimulating adipocyte hyperplasia. This study also demonstrates one way in which two genes (α2 and β3-AR) and diet interact to influence fat mass.

Regulation of skeletal muscle UCP-2 and UCP-3 gene expression by exercise and denervation

The factors that regulate gene expression of uncoupling proteins 2 and 3 (UCP-2 and UCP-3) in skeletal muscle are poorly understood, but both genes are clearly responsive to the metabolic state of the organism. Therefore, we tested the hypothesis that denervation and acute and/or chronic exercise (factors that profoundly affect metabolism) would alter UCP-2 and UCP-3 gene expression. For the denervation studies, the sciatic nerve of rat and mouse hindlimb was sectioned in one leg while the contralateral limb served as control. Northern blot analysis revealed that denervation was associated with a 331% increase (P < 0.001) in UCP-3 mRNA and a 200% increase (P < 0. 01) in UCP-2 mRNA levels in rat mixed gastrocnemius (MG) muscle. In contrast, denervation caused a 53% decrease (P < 0.001) in UCP-3 and a 63% increase (P < 0.01) in UCP-2 mRNA levels in mouse MG. After acute exercise (2-h treadmill running), rat UCP-3 mRNA levels were elevated (vs. sedentary control) 252% (P < 0.0001) in white gastrocnemius and 63% (P < 0.05) in red gastrocnemius muscles, whereas UCP-2 levels were unaffected. To a lesser extent, elevations in UCP-3 mRNA (22%; P < 0.01) and UCP-2 mRNA (55%; P < 0.01) levels were observed after acute exercise in the mouse MG. There were no changes in either UCP-2 or UCP-3 mRNA levels after chronic exercise (9 wk of wheel running). These results indicate that acute exercise and denervation regulate gene expression of skeletal muscle UCPs.The factors that regulate gene expression of uncoupling proteins 2 and 3 (UCP-2 and UCP-3) in skeletal muscle are poorly understood, but both genes are clearly responsive to the metabolic state of the organism. Therefore, we tested the hypothesis that denervation and acute and/or chronic exercise (factors that profoundly affect metabolism) would alter UCP-2 and UCP-3 gene expression. For the denervation studies, the sciatic nerve of rat and mouse hindlimb was sectioned in one leg while the contralateral limb served as control. Northern blot analysis revealed that denervation was associated with a 331% increase ( P < 0.001) in UCP-3 mRNA and a 200% increase ( P < 0.01) in UCP-2 mRNA levels in rat mixed gastrocnemius (MG) muscle. In contrast, denervation caused a 53% decrease ( P< 0.001) in UCP-3 and a 63% increase ( P < 0.01) in UCP-2 mRNA levels in mouse MG. After acute exercise (2-h treadmill running), rat UCP-3 mRNA levels were elevated (vs. sedentary control) 252% ( P < 0.0001) in white gastrocnemius and 63% ( P < 0.05) in red gastrocnemius muscles, whereas UCP-2 levels were unaffected. To a lesser extent, elevations in UCP-3 mRNA (22%; P < 0.01) and UCP-2 mRNA (55%; P < 0.01) levels were observed after acute exercise in the mouse MG. There were no changes in either UCP-2 or UCP-3 mRNA levels after chronic exercise (9 wk of wheel running). These results indicate that acute exercise and denervation regulate gene expression of skeletal muscle UCPs.