Fatty acid oxidation and triacylglycerol hydrolysis are enhanced after chronic leptin treatment in rats

Leptin acutely increases fatty acid (FA) oxidation and triacylglycerol (TG) hydrolysis and decreases TG esterification in oxidative rodent muscle. However, the effects of chronic leptin administration on FA metabolism in skeletal muscle have not been examined. We hypothesized that chronic leptin treatment would enhance TG hydrolysis as well as the capacity to oxidize FA in soleus (SOL) muscle. Female Sprague-Dawley rats were infused for 2 wk with leptin (LEPT; 0.5 mg x kg(-1) x day(-1)) by use of subcutaneously implanted miniosmotic pumps. Control (AD-S) and pair-fed (PF-S) animals received saline-filled implants. Subsequently, FA metabolism was monitored for 45 min in isolated, resting, and contracting (20 tetani/min) SOL muscles by means of pulse-chase procedures. Food intake (-33 +/- 2%, P < 0.01) and body mass (-12.5 +/- 4%, P = 0.01) were reduced in both LEPT and PF-S animals. Leptin levels were elevated (+418 +/- 7%, P < 0.001) in treated animals but reduced in PF-S animals (-73 +/- 8%, P < 0.05) relative to controls. At rest, TG hydrolysis was increased in leptin-treated rats (1.8 +/- 2.2, AD-S vs. 23.5 +/- 8.1 nmol/g wet wt, LEPT; P < 0.001). In contracting SOL muscles, TG hydrolysis (1.5 +/- 0.6, AD-S vs. 3.6 +/- 1.0 micromol/g wet wt, LEPT; P = 0.02) and palmitate oxidation (18.3 +/- 6.7, AD-S vs. 45.7 +/- 9.9 nmol/g wet wt, LEPT; P < 0.05) were both significantly increased by leptin treatment. Chronic leptin treatment had no effect on TG esterification either at rest or during contraction. Markers of overall (citrate synthase) and FA (hydroxyacyl-CoA dehydrogenase) oxidative capacity were unchanged with leptin treatment. Protein expression of hormone-sensitive lipase (HSL) was also unaltered following leptin treatment. Thus leptin-induced increases in lipolysis are likely due to HSL activation (i.e., phosphorylation). Increased FA oxidation secondary to chronic leptin treatment is not due to an enhanced oxidative capacity and may be a result of enhanced flux into the mitochondrion (i.e., carnitine palmitoyltransferase I regulation) or electron transport uncoupling (i.e., uncoupling protein-3 expression).     

A Mycobacterium leprae-specific gene encoding an immunologically recognized 45 kDa protein

By screening a Mycobacterium leprae lambda gt11 expression library with a serum from an Ethiopian lepromatous leprosy (LL) patient a clone was isolated (LL4) belonging to hybridization group III of a panel of previously isolated M. leprae clones. Members of this hybridization group encode a serologically recognized 45 kDa protein. The complete DNA sequences of the partially overlapping clones LL4 and L1 (hybridization group III) are presented and these revealed the presence of an open reading frame (ORF) predicting a protein with a molecular size of 42 448 Da. Southern hybridizations on total genomic DNA of M. Ieprae, Mycobacterium tuberculosis and eight atypical mycobacteria showed that the LL4 DNA fragment is specific for M. Ieprae DNA even under low-stringency conditions. The M. Ieprae specificity of LL4 DNA was further confirmed by the polymerase chain reaction using four different sets of primers. Western blotting analyses showed that the M. Ieprae 45 kDa protein is frequently recognized by antibodies from leprosy patients and that this recognition is specific since no antibodies could be detected in sera of tuberculosis patients. T-cell proliferation assays also demonstrated T-cell recognition by leprosy patients and healthy contacts of the M. Ieprae 45 kDa protein. The specificity of the LL4 DNA region and the 45 kDa antigen that is encoded by hybridization group III could provide unique tools for the development of M. Ieprae-specific immunological and DNA reagents.     

Skeletal muscle mitochondrial FAT/CD36 content and palmitate oxidation are not decreased in obese women

A reduction in fatty acid oxidation has been associated with lipid accumulation and insulin resistance in the skeletal muscle of obese individuals. We examined whether this decrease in fatty acid oxidation was attributable to a reduction in muscle mitochondrial content and/or a dysfunction in fatty acid oxidation within mitochondria obtained from skeletal muscle of age-matched, lean [body mass index (BMI) = 23.3 +/- 0.7 kg/m2] and obese women (BMI = 37.6 +/- 2.2 kg/m2). The mitochondrial marker enzymes citrate synthase (-34%), beta-hydroxyacyl-CoA dehydrogenase (-17%), and cytochrome c oxidase (-32%) were reduced (P < 0.05) in obese participants, indicating that mitochondrial content was diminished. Obesity did not alter the ability of isolated mitochondria to oxidize palmitate; however, fatty acid oxidation was reduced at the whole muscle level by 28% (P < 0.05) in the obese. Mitochondrial fatty acid translocase (FAT/CD36) did not differ in lean and obese individuals, but mitochondrial FAT/CD36 was correlated with mitochondrial fatty acid oxidation (r = 0.67, P < 0.05). We conclude that the reduction in fatty acid oxidation in obese individuals is attributable to a decrease in mitochondrial content, not to an intrinsic defect in the mitochondria obtained from skeletal muscle of obese individuals. In addition, it appears that mitochondrial FAT/CD36 may be involved in regulating fatty acid oxidation in human skeletal muscle.     

Crucial role for LKB1 to AMPKα2 axis in the regulation of CD36-mediated long-chain fatty acid uptake into cardiomyocytes

Enhanced contractile activity increases cardiac long-chain fatty acid (LCFA) uptake via translocation of CD36 to the sarcolemma, similarly to increase in glucose uptake via GLUT4 translocation. AMP-activated protein kinase (AMPK) is assumed to mediate contraction-induced LCFA utilization. However, which catalytic isoform (AMPKα1 versus AMPKα2) is involved, is unknown. Furthermore, no studies have been performed on the role of LKB1, a kinase with AMPKK activity, on the regulation of cardiac LCFA utilization. Using different mouse models (AMPKα2-kinase-dead, AMPKα2-knockout and LKB1-knockout mice), we tested whether LKB1 and/or AMPK are required for stimulation of LCFA and glucose utilization upon treatment of cardiomyocytes with compounds (oligomycin/AICAR/dipyridamole) which induce CD36 translocation similar to that seen upon contraction. In AMPKα2- kinase-dead cardiomyocytes, the stimulating effects of oligomycin and AICAR on palmitate and deoxyglucose uptake and palmitate oxidation were almost completely lost. Moreover, in AMPKα2- and LKB1-knockout cardiomyocytes, oligomycin-induced LCFA and deoxyglucose uptake were completely abolished. However, the stimulatory effect of dipyridamole on palmitate uptake and oxidation was preserved in AMPKα2-kinase-dead cardiomyocytes. In conclusion, in the heart there is a signaling axis consisting of LKB1 and AMPKα2 which activation results in enhanced LCFA utilization, similarly to enhanced glucose uptake. In addition, an unknown dipyridamole-activated pathway can stimulate cardiac LCFA utilization by activating signaling components downstream of AMPK.     

Additive effects of insulin and muscle contraction on fatty acid transport and fatty acid transporters, FAT/CD36, FABPpm, FATP1, 4 and 6

Insulin and muscle contraction increase fatty acid transport into muscle by inducing the translocation of FAT/CD36. We examined (a) whether these effects are additive, and (b) whether other fatty acid transporters (FABPpm, FATP1, FATP4, and FATP6) are also induced to translocate. Insulin and muscle contraction increased glucose transport and plasmalemmal GLUT4 independently and additively (positive control). Palmitate transport was also stimulated independently and additively by insulin and by muscle contraction. Insulin and muscle contraction increased plasmalemmal FAT/CD36, FABPpm, FATP1, and FATP4, but not FATP6. Only FAT/CD36 and FATP1 were stimulated in an additive manner by insulin and by muscle contraction.     

Conditioning Increases the Gain of Contraction-Induced Sarcolemmal Substrate Transport in Ultra-Endurance Racing Sled Dogs

Endurance exercise relies on transsarcolemmal flux of substrates in order to avoid depletion of intramuscular reserves. Previous studies of endurance trained sled dogs have shown a remarkable capacity of these dogs to adapt rapidly to endurance exercise by decreasing the utilization of intramuscular reserves. The current study tested the hypothesis that the dogs'' glycogen-sparing phenotype is due to increased sarcolemmal transport of glucose and fatty acids. Basal and exercise-induced transport of glucose and fatty acids into sarcolemmal vesicles was evaluated in racing sled dogs prior to and after 7 months of exercise conditioning. Sarcolemmal substrate transport capacity was measured using sarcolemmal vesicles and radiolabelled substrates, and transporter abundance was measured using Western blot quantification in whole muscle homogenates and the sarcolemmal vesicle preparations. Conditioning resulted in increased basal and exercise-induced transport of both glucose and palmitate. Neither acute exercise nor conditioning resulted in changes in muscle content of GLUT4 or FAT/CD36, but conditioning did result in decreased abundance of both transporters in the sarcolemmal vesicles used for the basal transport assays, and this decrease was further amplified in the vesicles used for the exercise-induced transport assays. These results demonstrate conditioning-induced increases in sarcolemmal transport of oxidizable substrates, as well as increased gain of exercise-induced sarcolemmal transport of these substrates. These results further indicate that increased sarcolemmal transport of oxidizable substrates may be due to either an increased intrinsic capacity of the existing transporters or to a different population of transporters from those investigated.     

Role of the inhibitory KIR ligand HLA-Bw4 and HLA-C expression levels in the recognition of leukemic cells by Natural Killer cells

Transplantation of acute myeloid leukemia (AML) patients with grafts from related haploidentical donors has been shown to result in a potent graft-versus-leukemia effect. This effect is mediated by NK cells because of the lack of activation of inhibitory killer cell immunoglobulin-like receptors (KIRs) which recognize HLA-Bw4 and HLA-C alleles. However, conflicting results have been reported about the impact of KIR ligand mismatching on the outcome of unrelated HLA-mismatched hematopoietic stem cells transplants (HSCT) to leukemic patients. The interpretation of these conflicting results is hampered by the scant information about the level of expression of HLA class I alleles on leukemic cells, although this variable may affect the activation of inhibitory KIRs. Therefore in the present study, utilizing a large panel of human monoclonal antibodies we have measured the level of expression of HLA-A, -B and -C alleles on 20 B-chronic lymphoid leukemic (B-CLL) cell preparations, on 16 B-acute lymphoid leukemic (B-ALL) cell preparations and on 19 AML cell preparations. Comparison of the level of HLA class I antigen expression on leukemic cells and autologous normal T cells identified selective downregulation of HLA-A and HLA-B alleles on 15 and 14 of the 20 B-CLL, on 2 and 5 of the 16 B-ALL and on 7 and 11 of the 19 AML patients tested, respectively. Most interestingly HLA-C alleles were markedly downregulated on all three types of leukemic cells; the downregulation was most pronounced on AML cells. The potential functional relevance of these abnormalities is suggested by the dose-dependent enhancement of NK cell activation caused by coating the HLA-HLA-Bw4 epitope with monoclonal antibodies on leukemic cells which express NK cell activating ligands. Our results suggest that besides the HLA and KIR genotype, expression levels of KIR ligands on leukemic cells should be included among the criteria used to select the donor-recipient combinations for HSCT.     

Closed-Loop Control of Active Sensing Movements Regulates Sensory Slip

1. Download high-res image (213KB)
2. Download full-size image

     

Cytological study of ischemic damage in rat kidney

Reparative regeneration in different periods following nephrectomy and the application of low-dose radiation has been studied by means of light and electron microscopy. Experimentally induced ischemia resulted in the destruction of renal corpuscles, the perishing of tubular epithelial cells, and the proliferation of connective tissue. Reparative regeneration is based on aseptic inflammation and its phase duration depends on the extent of organ injury. In nephrectomized rats, reparative regeneration is accompanied by necrosis and the deposition of calcium in the cortical substance. Calcium plays an important role in kidney metabolism and its increased content is characteristic of degenerative changes. The experiments showed that the use of low-dose radiation does not accelerate the process of reparative regeneration in rat kidney.