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Relatively high concentrations of leptin are present in plasma and it is thought to play a major role in lipid homeostasis. Leptin is reported to lower tissue triglyceride content by increasing intracellular oxidation of free fatty acids (FFA). However very little is known regarding the interaction between leptin and plasma FFA. We studied the interaction of FFA with leptin using a direct radiolabelled fatty acid binding assay, a fluorescence assay, electrophoretic mobility and autoradiobinding. All these data indicate that binding of FFA with leptin is reversible and shows a positive co-operativity. The binding of FFA to leptin produces a change in the pI value of the leptin and also increased the electrophoretic mobility of the protein in native polyacrylamide gels. The change in leptin's electrophoretic mobility depends on the chain length and the number of double bonds of the fatty acid, as stearic acid, 18:0, had no effect whereas oleic acid, 18:1n-9, linoleic acid, 18:2n-6, arachidonic acid, 20:4n-6, and docosahexaneoic acid, 22:6n-3, affected leptin's mobility to different degrees. The physiological implication of leptin-FFA interaction is not known, however the interaction may depend on the plasma FFA composition and concentration which are known to vary in different pathological/physiological conditions.

Hepatocytes situated at pericentral and periportal zones of the liver lobule show differences in the expression of several liver-specific genes, such as androgen-inducible alpha 2u globulin and androgen-repressible senescence marker protein-2 (SMP-2). A marked temporal difference in the expression of these two androgen-regulated genes has also been observed. The liver of the pre-pubertal male rat is insensitive to androgen, and during this period hepatocytes synthesize only SMP-2. During young adult life (greater than 40 days), the liver becomes androgen sensitive and concomitant synthesis of alpha 2u globulin and repression of SMP-2 occur. In the senescent male rat (greater than 750 days), the liver again becomes androgen insensitive when the decline in alpha 2u globulin is accompanied by an increase in SMP-2 synthesis. In this article we present results to show a correlation between the temporal and spatial (intralobular) changes in the expression of the androgen-inducible alpha 2u globulin and the androgen-repressible SMP-2 in rat hepatocytes. Results indicate that the temporal changes in hepatic androgen sensitivity are dictated by the intralobular location of the hepatocytes. Hepatocytes located around the central vein (pericentral/perivenous) may benefit from a paracrine advantage for the expression of a subset of genes, including the gene for the androgen receptor.

In vitro translation of total rat hepatic mRNAs has identified a 31 kilodalton senescence marker protein (SMP-2) which is present in higher amounts in prepubertal and senescent males than in the post-pubertal adult male (more than 10-fold). SMP-2 is an androgen-repressible protein. The negative regulation of the SMP-2 gene activity by androgen accounts for its increased expression during the androgen insensitive states of the prepubertal and senescent livers, and its constitutive expression in the female liver. A combination of separation procedures including salt fractionation, chromatofocusing, ion-exchange chromatography and preparative gel electrophoresis have led to the purification of SMP-2 to apparent homogeneity. The purified protein showed the same electrophoretic mobility as the sex- and age-specific in vitro translation product of hepatic mRNAs. The polyclonal antibody to SMP-2 was produced in the rabbit. The antibody selectively reacted with the 31 kDa sex- and age-specific translation product of hepatic mRNAs. Western blot analysis of the liver cytosol confirms monospecificity of the antiserum, as well as age- and sex-dependent changes in the tissue level of SMP-2. Histochemical staining of liver sections with the antiserum reveals a preferential periportal localization of SMP-2 in the hepatocytes. This finding is in marked contrast to the androgen-inducible alpha 2u globulin which is preferentially synthesized and localized in the pericentral hepatocytes. Thus, the zonal distribution of SMP-2 correlates with polarized androgen sensitivity of the hepatocytes within the liver lobule.

The liver-specific expression of the senescence marker protein 2 (SMP-2) in the male rat is markedly reduced during the androgen-sensitive state of young adulthood, whereas it is up-regulated during the androgen-insensitive phases of prepuberty and senescence. Nuclear runoff studies show that the age-dependent changes in SMP-2 expression are due to transcriptional regulation of the gene. In order to explore the mechanism of the regulatory process, we have cloned the upstream flanking regions of two distinct SMP-2 genes (SMP-2A and SMP-2B) and established their nucleotide sequence. These clones contain approximately 2.2 kb of the 5'-flanking sequence, exons 1 and 2, the first intron, and a portion of the second intron. The SMP-2 genes, as well as the upstream sequences, contain the sequence motifs for a number of cis-acting regulatory elements, such as the hepatocyte-specific element (HP1) and the androgen response element (ARE). S1 nuclease and primer extension analyses have established the transcription initiation sites for these genes. For functional analysis of the upstream sequences, we have constructed a hybrid plasmid containing the SMP-2A gene sequence (-1970 to +38 bases) fused to the structural gene for chloramphenicol acetyl-transferase (CAT). Upon transfection into rat hepatoma cells (FT02B), this construct was able to drive expression of the CAT gene. The same construct, however, failed to function in fibroblast-derived L cells, indicating tissue-specific regulation of the construct promoter.

Changes in the mRNAs coding for specific hepatic proteins in male rats during aging were examined by in vitro translation of the liver mRNA in the rabbit reticulocyte lysate. Characterization of the [35S]methionine-labeled translation products by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by autoradiography showed major age-dependent changes in the hepatic concentrations of three mRNA species. The translation products of these three mRNAs were found to have Mr = 28,500, 26,300, and 19,500 and are called senescence marker proteins (SMP) 1, 2, 3, respectively. On the basis of its immunochemical reactivity, SMP-3 (Mr = 19,500) is identified as alpha 2u-globulin while the mRNAs for SMP-1 (Mr = 28,500) and SMP-2 (Mr = 26,300) code for two yet uncharacterized proteins. The liver of the prepubertal male rats was found to contain the mRNA for SMP-2 and showed almost complete absence of the mRNAs for SMP-1 and SMP-3. However, the mRNAs for both SMP-1 and SMP-3 were present in the postpubertal young adults while the mRNA for SMP-2 was absent. Finally, when the animals reached senescence, the mRNAs for SMP-1 and SMP-3 disappeared from the liver with the reappearance of the SMP-2 mRNA. Age-dependent regulation of the mRNAs for these three senescence marker proteins can serve as an important model for the study of differential gene expression during aging.

SMP-2 is a rat liver protein whose synthesis is influenced by both androgens and aging. The steady-state level of its mRNA is repressed by the androgen. Compared to the adult male, SMP-2 mRNA is found in higher amounts in the prepubertal and senescent male rat livers which show relative androgen insensitivity. A cDNA library in the plasmid pBR322 was constructed from the female rat liver which contains a high level of SMP-2 mRNAs. Recombinant plasmids were screened by differential colony hybridization to 32P-labeled single-stranded cDNAs from adult female and adult male hepatic poly(A)+ RNAs. From a total of 3500 recombinant clones, 11 highly female specific clones were identified. From these female specific colonies the SMP-2 cDNA-containing plasmid (pSP11) was identified by its ability to select an mRNA species whose translation product is immunochemically and electrophoretically indistinguishable from SMP-2. This insert represents a 571-base pair portion of the SMP-2 cDNA. Rescreening of the library at a high colony density using the 32P-labeled cDNA insert of pSP11 identified several positive clones with larger inserts. Hybrid-selected mRNA translation again confirmed these clones to carry SMP-2 cDNA sequences. The plasmid pSP4a containing a 1040-base pair cDNA insert of SMP-2 was characterized by DNA sequence analysis. The size of the cDNA insert of pSP4a is close to the estimated size of the SMP-2 mRNA. The cDNA sequence provides an open reading frame of 282 amino acid residues. A comparison of the translated amino acid sequence with the protein sequences of NBRF-PIR, PSQNEW, and LOSALA data bases did not establish any sequence homology with known proteins. Northern blot analysis using the 32P-labeled cDNA insert of pSP4a confirms the androgenic repression of the SMP-2 mRNA.

alpha 2u-Globulin, a protein of hepatic origin found in the urine of male rats, is accumulated in the kidney cortex during exposure to unleaded gasoline and has been implicated in the development of fuel hydrocarbon-induced nephropathy and renal neoplasia. The principal morphological feature of gasoline-induced nephropathy is accumulation of hyaline droplets (enlarged secondary lysosomes or phagolysosomes) in epithelial cells of the proximal convoluted tubule S1 and S2 segments. Inhibition of cathepsin B (a major lysosomal peptidase) by treatment of male rats with leupeptin causes rapid accumulation of phagolysosomes and alpha 2u-globulin in the kidney very similar to gasoline exposure. Further, the renal cortical subcellular distribution of alpha 2u-globulin, determined with an electron microscopic immunochemical method, is almost totally confined to phagolysosomes following administration of either gasoline or leupeptin. These results, taken together, indicate that the mechanism of nephrotoxicity of gasoline involves inhibition of renal phagolysosomal proteolysis.

Extensive peroxisomal proliferation in the hepatic parenchymal cells was observed when male rats were given a diet containing 0.1% Wy-14,643 [( 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid), a potent lipid-decreasing drug. This drug also caused a marked increase in the concentrations of the mRNA species coding for four proteins with Mr 77000, 61000, 43000 and 31000, and a similar decrease in the concentrations of three mRNA species coding for proteins of Mr 25000, 24000 and 19000. Specific immunoprecipitation studies identified the proteins of Mr 19000, 43000 and 77000 as alpha 2u-globulin, 3-ketoacyl-CoA thiolase (EC 2.3.1.16) and enoyl-CoA hydratase (EC 4.2.1.17) respectively. Comparisons of the Mr values suggest that the 61000- and 31000-Mr proteins may be equivalent to two additional peroxisomal enzymes, namely catalase (Mr 61000) and uricase (Mr 31000). The identity of the mRNA species coding for the 25000- and 24000-Mr proteins is at present unknown.

That the liver in oviparous females supplies the major part of the egg yolk proteins requires a marked degree of sexual dimorphism of this organ. In addition to vitellogenin, several minor components (e.g. vitamin binding proteins) are supplied by the liver to the oocyte in oviparous animals and to the developing embryo in viviparous females. Other metabolic adjustments to maintain reproductive competency of the female (e.g. increased lipid synthesis, detoxification of the waste products of the developing embryo, and reproductively sensible steroid metabolism) are some of the physiological bases for the differences between the female and male liver. Sex-differences in several other hepatic proteins, enzymes, and hormone receptors have also been established. alpha 2mu Globulin, Bond's protein, and carbonic anhydrase are clear examples of the sex specificity of rat liver. Differential expression of the genes for the male- and female-specific proteins in the liver is brought about by the androgenic and estrogenic hormones. The hepatic receptors for these hormones also show a marked degree of sexual dimorphism. During development and aging, these receptors seem to appear when the need for these hormones is most critical. The timely appearance of the hepatic estrogen and androgen receptor and the facilitated action of these hormones are mediated through "pre- and neonatal imprinting" by the sex hormones, especially androgen. Exploration of the physiological and molecular basis of this "imprinting" mechanism remains an exciting area of contemporary endocrinology.

We have shown that restricted calorie intake retards age-associated loss in androgen responsiveness of the rat liver. Sustained androgen receptivity delays age-dependent decline in the synthesis of the androgen-inducible alpha 2u globulin and derepression of the androgen-repressible senescence marker protein (SMP-2). Quantitation of mRNAs for alpha 2u globulin and SMP-2 in the liver of animals of various ages maintained on either ad libitum or restricted diets revealed that, although the 27-month-old ad libitum-fed rat had only 5% as much alpha 2u mRNA as the 6-month-old rat, the mRNA level was as high as 45% in the 27-month-old food-restricted rat. Conversely, the 27-month-old food-restricted rat had a much reduced amount (45%) of SMP-2 mRNA compared to the age-matched control that was allowed unlimited access to food. Furthermore, we have correlated the effect of dietary restriction on age-dependent changes in specific gene expression with the hepatic level of the immunoreactive cytoplasmic androgen-binding (CAB) protein. We observed that senescence in the male causes a substantial decrease in the circulating level of testosterone. However, dietary restriction does not retard the rate of decline in the plasma level of the male hormone during aging. These results indicate that age-dependent changes in the expression of androgen-responsive genes (alpha 2u globulin and SMP-2) reflect changing androgen sensitivity and that food restriction may directly influence the androgen receptivity of the liver.