👤 M E Hadley

The adipose tissue-derived hormone leptin can drive decreases in food intake while increasing energy expenditure. In diet-induced obesity, circulating leptin levels rise proportionally to adiposity. Despite this hyperleptinemia, rodents and humans with obesity maintain increased adiposity and are resistant to leptin's actions. Here we show that inhibitors of the cytosolic enzyme histone deacetylase 6 (HDAC6) act as potent leptin sensitizers and anti-obesity agents in diet-induced obese mice. Specifically, HDAC6 inhibitors, such as tubastatin A, reduce food intake, fat mass, hepatic steatosis and improve systemic glucose homeostasis in an HDAC6-dependent manner. Mechanistically, peripheral, but not central, inhibition of HDAC6 confers central leptin sensitivity. Additionally, the anti-obesity effect of tubastatin A is attenuated in animals with a defective central leptin-melanocortin circuitry, including db/db and MC4R knockout mice. Our results suggest the existence of an HDAC6-regulated adipokine that serves as a leptin-sensitizing agent and reveals HDAC6 as a potential target for the treatment of obesity. Show less

Genetic studies might provide new insights into the biological mechanisms underlying lipid metabolism and risk of CAD. We therefore conducted a genome-wide association study to identify novel genetic determinants of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides. We combined genome-wide association data from 8 studies, comprising up to 17 723 participants with information on circulating lipid concentrations. We did independent replication studies in up to 37 774 participants from 8 populations and also in a population of Indian Asian descent. We also assessed the association between single-nucleotide polymorphisms (SNPs) at lipid loci and risk of CAD in up to 9 633 cases and 38 684 controls. We identified 4 novel genetic loci that showed reproducible associations with lipids (probability values, 1.6×10(-8) to 3.1×10(-10)). These include a potentially functional SNP in the SLC39A8 gene for HDL-C, an SNP near the MYLIP/GMPR and PPP1R3B genes for LDL-C, and at the AFF1 gene for triglycerides. SNPs showing strong statistical association with 1 or more lipid traits at the CELSR2, APOB, APOE-C1-C4-C2 cluster, LPL, ZNF259-APOA5-A4-C3-A1 cluster and TRIB1 loci were also associated with CAD risk (probability values, 1.1×10(-3) to 1.2×10(-9)). We have identified 4 novel loci associated with circulating lipids. We also show that in addition to those that are largely associated with LDL-C, genetic loci mainly associated with circulating triglycerides and HDL-C are also associated with risk of CAD. These findings potentially provide new insights into the biological mechanisms underlying lipid metabolism and CAD risk. Show less

Basement membranes are thin extracellular matrices which contact epithelial cells and promote their adhesion, migration, differentiation, and morphogenesis. These matrices are composed of collagen IV, heparan sulfate proteoglycan, laminin, and entactin as well as other minor components. Sertoli cells, like most epithelial cells, are in contact at their basal surface with a basement membrane. When cultured within three-dimensional basement membrane gels (Matrigel), Sertoli cells reorganize into cords that resemble testicular seminiferous cords found in the in vivo differentiating testis. Anti-laminin and anti-entactin antisera inhibit this cord morphogenesis by Sertoli cells whereas antisera against type IV and type I collagen, heparan sulfate proteoglycan, fibronectin, and preimmune sera had no effect. The RGD (RGDS-NH2) sequence, found in the cell binding domain of the integrin family of cell adhesion molecules as well as in the A chain of laminin and in entactin, effectively inhibited Sertoli cell cord formation at a concentration of 1.0 mg/ml but was unable to prevent Sertoli cell attachment at concentrations as high as 2.0 mg/ml. A synthetic pentapeptide from a cell-binding domain of the B1 chain of laminin. YIGSR-NH2, inhibited cord formation at a concentration of 0.25 mg/ml, but Sertoli cells were still adherent to the basement membrane matrix. At concentrations greater than 0.50 mg/ml, Sertoli cells detached. Antiserum against the YIGSR-NH2-containing sequence was also effective in inhibiting cord formation by Sertoli cells. Ligand (YIGSR-NH2 peptide) blot analysis of Sertoli cell lysates revealed an interaction with a major band at 60 kDa and with minor bands at 39 and 127 kDa. Furthermore, in Western blot analysis the anti-67-kDa laminin-binding protein antibody recognized a 59- to 60-kDa protein in Sertoli cells. The data indicate that laminin is involved in both Sertoli cell attachment and migration during formation of histotypic cord structures by these cells in culture. Two separate laminin cell-binding domains appear to be involved in Sertoli cell cord morphogenesis in vitro and are likely to participate in the formation of seminiferous cords in vivo. Show less

alpha-Melanotropin (alpha-melanocyte-stimulating hormone, alpha-MSH) is a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2. The minimal sequence of alpha-MSH required for agonism in the lizard (Anolis carolinensis) skin bioassay was determined to be Ac-His-Phe-Arg-Trp-NH2 (Ac-alpha-MSH6-9-NH2). Smaller fragments of this sequence (Ac-alpha-MSH6-8-NH2, Ac-alpha-MSH6-7-NH2, Ac-alpha-MSH7-9-NH2, and Ac-alpha-MSH7-8-NH2) were devoid of melanotropic activity. The tetrapeptide, Ac-alpha-MSH7-10-NH2, was also inactive, thus again demonstrating the importance of His at position 6 for minimal activity. The important potentiating amino acids were found to be Met-4, Lys-11, and Pro-12, since Ac-alpha-MSH4-10-NH2 was about 100 times more potent than Ac-alpha-MSH5-10-NH2, and Ac-[Nle4]-alpha-MSH4-11-NH2 was about 40 times more potent than Ac-alpha-MSH4-10-NH2 or Ac-[Nle4]-alpha-MSH4-10-NH2. Ac-alpha-MSH4-12-NH2 and Ac-[Nle4]-alpha-MSH4-12-NH2 were equipotent and about six times more potent than alpha-MSH. Since [Nle4]-alpha-MSH and Ac-[Nle4]-alpha-MSH4-13-NH2 were both equipotent but about sixfold less active than Ac-[Nle4]-alpha-MSH4-12-NH2, it is clear that valine at position 13 does not contribute to the potency of alpha-MSH, except possibly in a negative way. The minimal message sequence for equipotency to alpha-MSH appears to be Ac-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-NH2, since the analog, Ac-[Nle4]-alpha-MSH4-11-NH2, was as active as the native hormone. Ser-1, Tyr-2, Ser-3, Glu-5, and Val-13 are not important for melanotropic potency since Ac-alpha-MSH4-12-NH2 was more potent than alpha-MSH, and Ac-alpha-MSH5-10-NH2 and Ac-alpha-MSH6-10-NH2 were equipotent, being about 4,000 times less active than alpha-MSH. Show less

Primary cultures of Sertoli cells maintained in conventional cultures on plastic culture vessels do not retain many of the structural and functional properties of their in vivo counterparts. Sertoli cell phenotype is better maintained by incorporating certain environmental parameters, intrinsic to the testis, into the Sertoli cell culture system. These environmental parameters include a) high cell density, b) a unique extracellular matrix, c) a semipermeable support between the basal plasma membrane of the cells and blood-derived nutrients in the interstitium, d) chemically distinct microenvironments at the apical and basal surfaces of the cells, and e) cell-to-cell interactions among Sertoli cells and other testicular cell types. Using three variations of Sertoli cell culture we have demonstrated the importance of each of these environmental parameters in obtaining a better Sertoli cell culture model. Show less

The distribution of laminin, type IV collagen, heparan sulfate proteoglycan, and fibronectin was investigated in the rat testicular lamina propria by electron microscopic immunocytochemistry. Distinct patterns were observed for each antigen within the extracellular matrix (ECM) layers of the lamina propria. Laminin, type IV collagen, and heparan sulfate proteoglycan all localized to the seminiferous tubule basement membrane. Type IV collagen and heparan sulfate proteoglycan, but not laminin, localized to the seminiferous tubule side of the peritubular myoid cells. All four of the antigens were localized between the peritubular and lymphatic endothelial cells. Failure to localize fibronectin in the ECM layer between the Sertoli and peritubular myoid cells tends to support the concept that adult Sertoli cells do not produce this protein in vivo. Intracellular immunostaining was insufficient to allow unambiguous identification of the cellular source of any of the ECM molecules. Show less

The minimal sequence required for biological activity of alpha-MSH (alpha-melanotropin, alpha-melanocyte stimulating hormone) was determined in the frog (Rana pipiens) skin bioassay. The sequence required to elicit measurable biological activity was the central tetrapeptide sequence, Ac-His-Phe-Arg-Trp-NH2 (Ac-alpha-MSH6-9-NH2), which was about 6 orders of magnitude less potent than the native tridecapeptide. Smaller fragments of this sequence (Ac-His-Phe-NH2, Ac-Phe-Arg-NH2, Ac-His-Phe-Arg-NH2) were devoid of melanotropic activity at concentrations as high as 10(-4) M. We were unable to demonstrate biological activity for the tetrapeptide, Ac-Phe-Arg-Trp-Gly-NH2 (Ac-alpha-MSH7-10-NH2), and for several carboxy terminal analogues including Ac-Lys-Pro-Val-NH2 (Ac-alpha-MSH11-13-NH2). We prepared a series of fragment analogues of alpha-MSH in an attempt to determine the contribution of each individual amino acid to the biological activity of the native hormone. The minimal potency of Ac-alpha-MSH6-9-NH2 could be enhanced about a factor of 16 by the addition of glycine to the C-terminus, yielding Ac-alpha-MSH6-10-NH2 (Ac-His-Phe-Arg-Trp-Gly-NH2). Addition of glutamic acid to the N-terminus provided the peptide, Ac-alpha-MSH5-10-NH2, which was only slightly more potent than Ac-alpha-MSH6-10-NH2, indicating that position 5 contributes little to the biological potency of alpha-MSH in this assay. Addition of methionine to the N-terminus of Ac-alpha-MSH5-10-NH2 resulted in the heptapeptide, Ac-alpha-MSH4-10-NH2, which was only about 4-fold more potent than Ac-alpha-MSH5-10-NH2. Addition of lysine and proline to the C-terminal of the Ac-alpha-MSH4-10-NH2 sequence yielded the peptide, Ac-alpha-MSH4-12-NH2 with a 360-fold increase in potency relative to Ac-alpha-MSH4-10-NH2. This peptide was only about 6-fold less potent than alpha-MSH. A series of Nle-4-substituted analogues also were prepared. Ac-[Nle4]-alpha-MSH4-10-NH2 was about 4 times more potent than Ac-alpha-MSH4-10-NH2. Ac-[Nle4]-alpha-MSH4-11-NH2 also was about 4 times more potent than Ac-alpha-MSH4-10-NH2, demonstrating that lysine-11 contributes somewhat to the biological activity of alpha-MSH on the frog skin melanocyte receptor.(ABSTRACT TRUNCATED AT 250 WORDS) Show less

Sertoli cells in vivo are believed to secrete androgen-binding protein (ABP) as well as other proteins in a polarized manner, primarily into the adluminal (apical) compartment of the seminiferous tubules. It is now possible to examine polarized secretion by Sertoli cells in vitro by growing them in dual environment (bicameral) culture chambers such that there is a separation of the apical and basal compartments of the cells. Sertoli cells obtained from 17-day-old rats were grown in these chambers on Millipore filters coated with a reconstituted basement membrane matrix. Within 3 days a confluent epithelial sheet of Sertoli cells (30-40 microns in height) is established, and these cells are joined along their baso-lateral plasma membranes by typical Sertoli-Sertoli tight junctions. We have previously shown that this epithelial sheet of Sertoli cells establishes an electrical resistance, as well as a permeability barrier to small molecules, between the basal and apical compartments of the culture chamber. In the absence of Sertoli cells, proteins equilibrate within 8 h across the matrix-coated filter support. Between 12 and 48 h of culture the Sertoli cell monolayers secrete approximately 4- and 2-fold more ABP and transferrin (Tf), respectively, into the apical compartment than into the basal compartment when grown in serum-free defined medium. ABP secretion is diminished 10-fold by the removal of testosterone from the serum-free defined medium. In addition, the apical/basal ratio of ABP secretion decreases from 4.1 to 1.7 in the absence of testosterone. In contrast, neither the amount nor the direction of Tf secretion is altered by the removal of testosterone. These results demonstrate the bidirectional secretion of ABP and Tf by Sertoli cells grown in bicameral chambers. In addition, the polarity of secretion of these two proteins by Sertoli cells appears to be under differential regulation by testosterone. Show less

The transferrin-mediated transcellular transport of 59Fe across confluent epithelial sheets of Sertoli cells grown on Millipore filters was investigated. These filters had been impregnated with reconstituted basement membrane and suspended in bicameral (two houses) culture chambers. After five days of culture, Sertoli cells from 10-day-old rats formed basally-located tight junctional complexes. Concomitantly, there was an increase in electrical resistance and the epithelial sheet became impermeable to lanthanum nitrate. The rate of passage of [3H]inulin across the epithelial sheet was considerably less than passage across a filter alone, a filter impregnated with reconstituted basement membrane or an epithelial sheet pretreated with 2 mM EGTA. We conclude from these permeability studies that the tight junctional complexes between Sertoli cells formed an effective transepithelial permeability barrier. Following addition of human serum [59Fe]transferrin to media bathing the basal cytoplasm of the cells, rat testicular [59Fe]transferrin was immunoprecipitated from apical media overlying the Sertoli cells. Cross-reactivity of the rabbit anti-rat transferrin antibody with human serum transferrin was less than 0.001%. Substitution of the primary antibody with normal rabbit serum reduced the amount of immunoprecipitable rat testicular [59Fe]transferrin to 20% of normal levels. Prior fixation of the Sertoli cell epithelial sheet in 2.5% glutaraldehyde, addition of a 100-fold excess of holotransferrin to the basal media, and incubation of the Sertoli cell epithelial sheet at 4 C all reduced the immunoprecipitable rat testicular [59Fe]transferrin in apical media to levels below that for the non-specific binding of the primary antibody. From these studies we conclude that 59Fe is shuttled across Sertoli cells by two different forms of transferrin. Serum transferrin delivers the 59Fe to the basal cytoplasm of the Sertoli cells. The 59Fe dissociates from the serum transferrin, is delivered to testicular transferrin, and is subsequently secreted from the apical surface of the epithelial sheet of Sertoli cells as testicular [59Fe]transferrin. Show less

Epididymal epithelial cells isolated from mature rats and Sertoli cells isolated from 10-day-old rats were cultured in serum-free defined media on extracellular matrix impregnated filters maintained in dual environment culture chambers. Epididymal epithelial cells had a polarized appearance only when plated at high density (greater than 1 X 10(6) cells/cm2). Confluent monolayers of these cells formed a permeability barrier to inulin. Sertoli cells were columnar and highly polarized when grown on extracellular matrix-impregnated filters, cuboidal when grown on filters alone, and squamous when grown on plastic. Confluent polarized monolayers of these cells excluded the electron-dense tracer lanthanum nitrate by way of basal-tight junctions. Therefore, polarized monolayers of epididymal epithelial cells and Sertoli cells can be obtained by growing the cells at high density on extracellular matrix-impregnated permeable supports. By maintaining the monolayers in specially constructed culture chambers, the cells can develop a permeability barrier, and are able to achieve the separation of apical from basal compartments so important for their function in vivo. Show less

Sertoli cell preparations isolated from 10-day-old rats were cultured on three different substrates: plastic, a matrix deposited by co-culture of Sertoli and peritubular myoid cells, and a reconstituted basement membrane gel from the EHS tumor. When grown on plastic, Sertoli cells formed a squamous monolayer that did not retain contaminating germ cells. Grown on the matrix deposited by Sertoli-myoid cell co-cultures, Sertoli cells were more cuboidal and supported some germ cells but did not allow them to differentiate. After 3 wk however, the Sertoli cells flattened to resemble those grown on plastic. In contrast, the Sertoli cells grown on top of the reconstituted basement membrane formed polarized monolayers virtually identical to Sertoli cells in vivo. They were columnar with an elaborate cytoskeleton. In addition, they had characteristic basally located tight junctions and maintained germ cells for at least 5 wk in the basal aspect of the monolayer. However, germ cells did not differentiate. Total protein, androgen binding protein, transferrin, and type I collagen secretion were markedly greater when Sertoli cells were grown on the extracellular matrices than when they were grown on plastic. When Sertoli cells were cultured within rather than on top of reconstituted basement membrane gels they reorganized into cords. After one week, tight junctional complexes formed between adjacent Sertoli cells, functionally compartmentalizing the cords into central (adluminal) and peripheral (basal) compartments. Germ cells within the cords continued to differentiate. Thus, Sertoli cells cultured on top of extracellular matrix components assume a phenotype and morphology more characteristic of the in vivo, differentiated cells. Growing Sertoli cells within reconstituted basement membrane gels induces a morphogenesis of the cells into cords, which closely resemble the organ from which the cells were dissociated and which provide an environment permissive for germ cell differentiation. Show less

The seminiferous epithelium in mature vasectomized Macaca fascicularis was examined quantitatively to assess spermatogenesis. Monkeys were bilaterally vasectomized and controls were bilaterally sham operated. At postoperative periods of 10 and 18 months, groups of monkeys were castrated and their testes prepared for morphologic analysis. Diameters were measured in 100 cross sections of seminiferous tubules from each animal. Numbers of spermatogonia (Ad and Ap), preleptotene spermatocytes, pachytene spermatocytes, and step 7 spermatids, relative 10 Sertoli cell nucleoli, were counted in stage VII tubules. Tubule diameter and germ cell numbers per Sertoli cell nucleoli were not altered by vasectomy. Our study demonstrates quantitatively that spermatogenesis in the monkey is not inhibited up to 18 months following vasectomy. Show less