Leptin receptors in human skeletal muscle

Abstract

Human skeletal muscle expresses leptin receptor mRNA; however, it remains unknown whether leptin receptors (OB-R) are also expressed at the protein level. Fourteen healthy men (age = 33.1 ± 2.0 yr, height = 175.9 ± 1.7 cm, body mass = 81.2 ± 3.8 kg, body fat = 22.5 ± 1.9%; means ± SE) participated in this investigation. The expression of OB-R protein was determined in skeletal muscle, subcutaneous adipose tissue, and hypothalamus using a polyclonal rabbit anti-human leptin receptor. Three bands with a molecular mass close to 170, 128, and 98 kDa were identified by Western blot with the anti-OB-R antibody. All three bands were identified in skeletal muscle: the 98-kDa and 170-kDa bands were detected in hypothalamus, and the 98-kDa and 128-kDa bands were detected in thigh subcutaneous adipose tissue. The 128-kDa isoform was not detected in four subjects, whereas in the rest its occurrence was fully explained by the presence of intermuscular adipose tissue, as demonstrated using an anti-perilipin A antibody. No relationship was observed between the basal concentration of leptin in serum and the 170-kDa band density. In conclusion, a long isoform of the leptin receptor with a molecular mass close to 170 kDa is expressed at the protein level in human skeletal muscle. The amount of 170-kDa protein appears to be independent of the basal concentration of leptin in serum.

leptin is a 16-kda hormone structurally related to cytokines (66) that plays a crucial role in the regulation of appetite and fat deposition (20, 38). This hormone is primarily released by white adipose tissue and acts on brain and peripheral receptors (19, 24, 45) that belong to the class I type cytokine receptor family (61, 65). There are at least six isoforms of leptin receptors (OB-Rs) generated by mRNA alternative splicing and/or proteolytic processing of the subsequent protein products (18, 33, 61). All of these receptors contain identical extracellular and transmembrane domains and differ in the length of the intracellular amino acid sequence (18, 33, 61). The long form of the leptin receptor (OB-Rb) has an intracellular domain, highly conserved in several species, that is critical for the effects of this hormone (18, 61, 65). Upon leptin binding, the OB-Rb is activated, leading to stimulation of the janus kinase/signal transducer and activator of transcription signaling pathway, like the other class I cytokine receptors (9, 12, 61). In the central nervous system, leptin/OB-Rb interaction leads to the activation of janus kinase-2 by transphosphorylation and subsequent phosphorylation of tyrosine residues (Tyr985 and Tyr1138) in the cytoplasmic part of OB-Rb (11, 27).

Expression of OB-R mRNA has also been found in nonneuronal tissues (32), such as bone, heart, liver, lung, adrenal glands, testes, spleen, small intestine, pancreatic islets, the placenta, adipose tissue, and skeletal muscle (1, 10, 21, 36, 43, 48). However, the presence of OB-R protein has not been shown in some human tissues in which mRNA for OB-R has been detected, such as skeletal muscle (17), cultures of primary skeletal muscle cells (55), subcutaneous adipose tissue (51), and hypothalamus (15).

In addition to its locomotive function, skeletal muscle accounts for the majority of the basal metabolic rate and is also the primary tissue responsible for whole body glucose and fatty acid metabolism (57). Animal experiments have shown that leptin has physiological effects in skeletal muscle (17, 23, 37); however, it remains unknown if human skeletal muscle is actually able to respond to circulating leptin (7). Plasma leptin concentration is directly proportional to adipose tissue mass. Increasing fat mass results in higher levels of circulating leptin (19, 24), while reducing the body fat stores through regular exercise and/or dieting results in lower plasma leptin concentrations (28, 46, 62). Human obesity is characterized by a high concentration of leptin in plasma associated with leptin resistance (8, 60). Obesity also causes insulin resistance in humans (30, 44), which has been associated with raised plasma leptin concentrations, independent of body fat mass (50, 56). Leptin resistance could be caused by a downregulation and/or desensitization of OB-Rs, among other mechanisms.

In this study, we planned to test two hypotheses: first, that leptin receptors are expressed at the protein level in human skeletal muscle; and second, that the amount of OB-R protein expression in skeletal muscles depends on the basal concentration of leptin. To test these hypotheses, we carried out Western blot analysis in protein extracts obtained from human muscle biopsies and from a human hypothalamus. The hypothalamus protein extract was used as a control to verify that any band identified as a potential OB-R in muscle is also present in the hypothalamic protein extract, since the hypothalamus is rich in OB-R protein content (53). To test the second hypothesis, we determined whether plasma leptin concentration correlates with the protein expression of OB-R in skeletal muscle.