Petr Brauner

n-3 PUFA: bioavailability and modulation of adipose tissue function

Adipose tissue has a key role in the development of metabolic syndrome (MS), which includes obesity, type 2 diabetes, dyslipidaemia, hypertension and other disorders. Systemic insulin resistance represents a major factor contributing to the development of MS in obesity. The resistance is precipitated by impaired adipose tissue glucose and lipid metabolism, linked to a low-grade inflammation of adipose tissue and secretion of pro-inflammatory adipokines. Development of MS could be delayed by lifestyle modifications, while both dietary and pharmacological interventions are required for the successful therapy of MS. The n-3 long-chain (LC) PUFA, EPA and DHA, which are abundant in marine fish, act as hypolipidaemic factors, reduce cardiac events and decrease the progression of atherosclerosis. Thus, n-3 LC PUFA represent healthy constituents of diets for patients with MS. In rodents n-3 LC PUFA prevent the development of obesity and impaired glucose tolerance. The effects of n-3 LC PUFA are mediated transcriptionally by AMP-activated protein kinase and by other mechanisms. n-3 LC PUFA activate a metabolic switch toward lipid catabolism and suppression of lipogenesis, i.e. in the liver, adipose tissue and small intestine. This metabolic switch improves dyslipidaemia and reduces ectopic deposition of lipids, resulting in improved insulin signalling. Despite a relatively low accumulation of n-3 LC PUFA in adipose tissue lipids, adipose tissue is specifically linked to the beneficial effects of n-3 LC PUFA, as indicated by (1) the prevention of adipose tissue hyperplasia and hypertrophy, (2) the induction of mitochondrial biogenesis in adipocytes, (3) the induction of adiponectin and (4) the amelioration of adipose tissue inflammation by n-3 LC PUFA.

Role of energy charge and AMP-activated protein kinase in adipocytes in the control of body fat stores.

As indicated by in vitro studies, both lipogenesis and lipolysis in adipocytes depend on the cellular ATP levels. Ectopic expression of mitochondrial uncoupling protein 1 (UCP1) in the white adipose tissue of the aP2-Ucp1 transgenic mice reduced obesity induced by genetic or dietary manipulations. Furthermore, respiratory uncoupling lowered the cellular energy charge in adipocytes, while the synthesis of fatty acids (FA) was inhibited and their oxidation increased. Importantly, the complex metabolic changes triggered by ectopic UCP1 were associated with the activation of AMP-activated protein kinase (AMPK), a metabolic master switch, in adipocytes. Effects of several typical treatments that reduce adiposity, such as administration of leptin, β-adrenoceptor agonists, bezafibrate, dietary n-3 polyunsaturated FA or fasting, can be compared with a phenotype of the aP2-Ucp1 mice. These situations generally lead to the upregulation of mitochondrial UCPs and suppression of the cellular energy charge and FA synthesis in adipocytes. On the other hand, FA oxidation is increased. Moreover, it has been shown that AMPK in adipocytes can be activated by adipocyte-derived hormones leptin and adiponectin, and also by insulin-sensitizes thiazolidinediones. Thus, it is evident that metabolism of adipose tissue itself is important for the control of body fat content and that the cellular energy charge and AMPK are involved in the control of lipid metabolism in adipocytes. The reciprocal link between synthesis and oxidation of FA in adipocytes represents a prospective target for the new treatment strategies aimed at reducing obesity.

Induction of muscle thermogenesis by high-fat diet in mice: association with obesity-resistance

The obesogenic effect of a high-fat (HF) diet is counterbalanced by stimulation of energy expenditure and lipid oxidation in response to a meal. The aim of this study was to reveal whether muscle nonshivering thermogenesis could be stimulated by a HF diet, especially in obesity-resistant A/J compared with obesity-prone C57BL/6J (B/6J) mice. Experiments were performed on male mice born and maintained at 30 degrees C. Four-week-old mice were randomly weaned onto a low-fat (LF) or HF diet for 2 wk. In the A/J LF mice, cold exposure (4 degrees C) resulted in hypothermia, whereas the A/J HF, B/6J LF, and B/6J HF mice were cold tolerant. Cold sensitivity of the A/J LF mice was associated with a relatively low whole body energy expenditure under resting conditions, which was normalized by the HF diet. In both strains, the HF diet induced uncoupling protein-1-mediated thermogenesis, with a stronger induction in A/J mice. Only in A/J mice: 1) the HF diet augmented activation of whole body lipid oxidation by cold; and 2) at 30 degrees C, oxygen consumption, total content, and phosphorylation of AMP-activated protein kinase (AMPK), and AICAR-stimulated palmitate oxidation in soleus muscle was increased by the HF diet in parallel with significantly increased leptinemia. Gene expression data in soleus muscle of the A/J HF mice indicated a shift from carbohydrate to fatty acid oxidation. Our results suggest a role for muscle nonshivering thermogenesis and lipid oxidation in the obesity-resistant phenotype of A/J mice and indicate that a HF diet could induce thermogenesis in oxidative muscle, possibly via the leptin-AMPK axis.

Involvement of AMP-activated protein kinase in fat depot-specific metabolic changes during starvation

The mechanisms controlling fat depot‐specific metabolism are poorly understood. During starvation of mice, downregulation of lipogenic genes, suppression of fatty acid synthesis, and increases in lipid oxidation were all more pronounced in epididymal than in subcutaneous fat. In epididymal fat, relatively strong upregulation of uncoupling protein 2 and phosphoenolpyruvate carboxykinase genes was found. In mice maintained both at 20 and 30 °C, AMP‐activated protein kinase was activated in epididymal but did not change in subcutaneous fat. Our results suggest that AMPK may have a role in the different response of various fat depots to starvation.

Expression of mitochondrial uncoupling protein 3 and adenine nucleotide translocase 1 genes in developing rat heart: putative involvement in control of mitochondrial membrane potential

Postnatal maturation of the heart depends on the switch from glycolytic to oxidative metabolism and it is associated with decreasing tolerance to oxygen deprivation. Therefore, changes in composition and function of cardiac mitochondria during postnatal development require detailed characterization. Left-ventricular myocardium of prenatal, and 1-, 2-, 5-, 10-, 20-, 28-, 50-, 60-, and 90-d-old male Wistar rats was studied. The expression of uncoupling proteins (UCPs), adenine nucleotide translocase (ANT), and peroxisome proliferator-activated receptor alpha (PPARalpha) genes was characterized by northern blotting (UCP2), real-time quantitative RT-PCR (UCP2, UCP3, ANT1, ANT2, and PPARalpha), and by immunoblotting (UCP3). In isolated mitochondria, cytochromes a + a(3) were quantified by a spectrophotometry, and mitochondrial membrane potential (MMP) was measured using Rhodamine 123 (by spectrofluorimetry and flow cytometry). The specific content of cytochromes in mitochondria increased two-fold between birth and day 30, similarly, as the expression of ANT1 and PPARalpha genes. Postnatal activation in the expression of UCP2, UCP3, ANT1 and PPARalpha genes resulted in the expression maxima between days 20 and 30. The content/expression declined following day 20 (UCP2, UCP3, and PPARalpha) or 30 (cytochromes and ANT1), while expression of ANT2 declined continuously during the first month of life. In 1-d-old animals a single population of mitochondria with a relatively high MMP was observed; with increasing age, a second population of mitochondria with a significantly lower MMP appeared. The results support the view that mitochondrial energy conversion in heart changes during ontogeny and suggest the involvement of UCP3 and/or ANT1 in the control mechanism.

Modulation of Lipid Metabolism by Energy Status of Adipocytes Implications for Insulin Sensitivity

Abstract: It is becoming evident that insulin resistance of white adipose tissue is a major factor underlying the cardiovascular risk of obesity. Impaired fat storage rather than altered glucose metabolism in adipocytes probably contributes to development of insulin resistance in muscle and other tissues, in particular via increased delivery of nonesterified fatty acids into circulation. Lipid metabolism of adipose tissue is affected by the energy status of fat cells. In vitro experiments indicated the dependence of both lipogenesis and lipolysis on ATP levels in adipocytes. Thus, respiratory uncoupling in adipocytes that results in stimulation of energy dissipation and depression of ATP synthesis may contribute to the control of lipid metabolism, adiposity, and insulin sensitivity. This notion is supported by the expression of UCPs in adipocytes, for example, UCP2, UCP5, as well as some protonophoric anion transporters, and by induction of UCP1 and UCP3 in white fat by pharmacological treatments that reduce adiposity. A negative correlation between expression of UCPs in adipocytes and accumulation of white fat was also found. Expression of UCP1 from the adipose‐specific promoter in the aP2‐Ucp1 transgenic mice mitigated obesity induced by genetic or dietary factors. The obesity resistance, accompanied by respiratory uncoupling in adipocytes and increased energy expenditure, resulted from ectopic expression of UCP1 in white, but not brown fat. Probably due to depression of the ATP/ADP ratio, both fatty acid synthesis and lipolytic action of norepinephrine in adipocytes of transgenic mice were relatively low. Expression of regulatory G‐proteins, which are essential for both catecholamine and insulin signaling in adipocytes, was also altered by ectopic UCP1. These results support the role of protonophoric proteins in adipocytes in the control of adiposity and insulin sensitivity. Antidiabetic effects of thiazolidinediones, fibrates, β3‐adrenoreceptor agonists, dietary n‐3 PUFAs, and leptin may be explained at least partially by their effects on the energy and hence also the lipid metabolism of fat cells.

Fast Decline of Hematopoiesis and Uncoupling Protein 2 Content in Human Liver after Birth: Location of the Protein in Kupffer Cells

Hepatic hematopoiesis is prominent during fetal life and ceases around birth. In rodent liver, the decline of the hepatic hematopoiesis starts abruptly at birth being accompanied by a decrease of mitochondrial uncoupling protein 2 (UCP2) expression in monocytes/macrophages, whereas hepatocytes may express UCP2 only under pathologic situations. The goals of this study were to characterize hepatic hematopoiesis in humans around birth, and to identify cells expressing UCP2. Hematopoiesis was evaluated histologically in the liver of 22 newborns (mostly very premature neonates), who died between 45 min and 140 d after birth, and one fetus. UCP2 expression was characterized by Northern blots, immunoblotting, immunohistochemistry, and by in situ hybridization. The number of hematopoietic cells started to decrease rapidly at birth, irrespectively of the gestational age (23–40 wk) of neonates. A similar decline was observed for UCP2 expression, which was relatively high in fetal liver. UCP2 was detected only in myeloid cells (mainly in Kupffer cells), but not in hepatocytes, although sepsis or other pathologies occurred in the critically ill newborns. Kupffer cells represent the major site of mitochondrial UCP2 expression in the human newborn. UCP2 may be essential for the differentiation and function of macrophages and serve as a marker for these cells in human liver during the perinatal period.

Expression of uncoupling protein 3 and GLUT4 gene in skeletal muscle of preterm newborns : Possible control by AMP-activated protein kinase

We seek to understand the mechanism for the delayed postnatal switch between glycolytic and oxidative metabolism in preterm newborns. Our previous study [Brauner et al. (Pediatr Res 53: 691-697, 2003)] suggested impaired postnatal recruitment of the gene for mitochondrial uncoupling protein 3 (UCP3) by nutritional lipids in skeletal muscle of neonates delivered before approximately 26 wk of gestation. UCP3 is linked to lipid oxidation and may be involved in the defective development of energy metabolism in skeletal muscles of very preterm newborns. In extension of our previous study, autopsy samples of musculus quadriceps femoris from 40 mostly preterm neonates and 5 fetuses were used for quantification of transcripts for UCP3, GLUT4, and their transcriptional regulator, AMP-activated protein kinase (AMPK). The new analysis confirmed the defect in the recruitment of the UCP3 gene expression by lipids in very preterm neonates. It also suggested involvement of AMPK in the control of expression of both metabolic genes, UCP3 and GLUT4, in the skeletal muscle of the newborns. Experiments on adult C57BL/6J mice confirmed the relationships between the transcripts and supported the involvement of AMPK in the control of UCP3 gene expression.

Induction of uncoupling protein 3 gene expression in skeletal muscle of preterm newborns.

Prematurity is associated with delayed postnatal activation of mitochondrial oxidative phosphorylation and impaired switch from glycolytic to oxidative metabolism. Fatty acids (FA), which represent a major energy substrate in mature muscle cells, are engaged in the postnatal activation of genes of energy metabolism and lipid oxidation. To understand the mechanism activating mitochondria in human newborns, expression of the genes for mitochondrial uncoupling proteins (UCP) was characterized in autopsy samples of skeletal (n = 28) and cardiac (n = 13) muscles of preterm neonates, who mostly died during the first postnatal month, and two aborted fetuses. Transcripts levels for UCP2, UCP3, and also for genes engaged in the transport of FA between cytoplasm and mitochondria were measured using real-time reverse transcriptase PCR. In accordance with studies in mice, our results document postnatal induction of UCP3 gene expression in skeletal muscle, involvement of nutritional FA in the induction, and a role of UCP3 in mitochondrial FA oxidation. They suggest impaired postnatal activation of UCP3 gene in neonates delivered before approximately 26 wk of gestation. Mean levels of the UCP3 transcript in skeletal muscle were by two orders of magnitude higher than in the heart. In contrast to UCP3, the UCP2 gene was active in fetuses, and its expression was not affected by nutrition. Our results support a role of UCP3 in postnatal activation of lipid oxidation in skeletal muscle and suggest the involvement of UCP3 in the delayed activation of mitochondrial energy conversion in very immature preterm neonates.