Targeted disruption of the c-src proto-oncogene in mice has shown that src expression is required for normal bone resorption, since the src-deficient mutants develop osteopetrosis. To evaluate the mechanisms by which src-deficiency affects osteoclast function, we treated src-deficient mice with the stimulants of bone resorption, IL-1, parathyroid hormone, and parathyroid hormone-related protein, and analyzed the effects by quantitative bone histomorphometry and electron microscopy. Increased numbers of multinucleated cells with the morphological characteristics of osteoclasts appeared on bone surfaces, but these cells did not form ruffled borders or normal resorption lacunae. To confirm these in vivo findings, we cultured src-mutant bone marrow cells on dentine slices in the presence of 1,25 dihydroxyvitamin D3. Increased numbers of multinucleated cells were formed, but unlike normal murine bone marrow cells, they did not form resorption pits. These results indicate that osteoclasts appear in the absence of pp6Or, but that pp6O C expression is required for mature osteoclasts to form ruffled borders and resorb bone. (J. Clin. Invest. 1992.

. Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment.

Osteopetrosis is a bone modeling disorder resulting in excessive accumulation of bone matrix due to defective function of osteoclasts, the cells that resorb bone. Mice carrying a targeted disruption of the gene Src that encodes pp6WC-src (Src), a nonreceptor protein tyrosine kinase, develop this phenotype but do not exhibit other overt defects despite the fact that the kinase is normally present in a broad variety ofcell types. Because Src is expressed in osteoblasts as well as in osteoclasts and both are required for normal bone resorption, the basic defect could occur in either cell type. In this study we have used in vitro approaches and fetal liver transplantation into irradiated Src-recipients to demonstrate that the inherent defect is with osteoclasts and autonomous of the bone marrow microenvironment. This result (i) identifies a cell type in which Src function is essential and cannot be replaced by other related kinases and (u) should allow the isolation of a substrate that is specific to Src.The bone disorder, osteopetrosis, is an informative disease for studying the biology of the osteoclast, the bone cell that is responsible for bone resorption. Different models of this disease have provided valuable insights into the cellular origin of the osteoclast and into some of the extracellular factors and intracellular molecules required for normal osteoclastic bone resorption. In some models of this disease, the defect is restricted to the osteoclast and in others, such as the op mice, it is in the bone marrow microenvironment (1-3). Recently, osteopetrosis has been described in mice deficient in expression of Src nonreceptor tyrosine kinase after targeted disruption of the gene Src in embryonic stem cells (4). This observation shows that normal expression of Src is required for osteoclastic bone resorption. Additional studies showed that the osteoclasts from Src-deficient (Src-) mice failed to form ruffled borders and resorption lacunae (5). However, these studies do not show whether expression of Src is required in the cellular microenvironment of the osteoclast or within the osteoclast lineage itself. In this report, we use both in vitro and in vivo approaches to show that the inherent defect in Src-osteopetrosis is in the osteoclast lineage and is autonomous of the bone marrow microenvironment. MATERIALS AND METHODSImmunohistochemistry. Calvarial bones were obtained from wild-type (wt) and Src-mutant mice that had been treated with interleukin-1 to increase the numbers and activity of osteoclasts and osteoblasts as described (5). Specimens were fixed in 10% phosphate-buffered formalin for 24-72 hr, decalcified in 14% EDTA, and embedded in paraffin. Sections of human bone from an osteoarthritic hip were incubated overnight with the primary monoclonal antibody (mAb) 327 directed against the SH3 domain of Src (6) at 1:250 dilution and were developed with a standard peroxidaseconjugated streptavidin-biotin detection system (7) with osmium-enhanced diaminobenzidine as chromogen. Sections of murine bon...

Glucagon-like-peptide 1 (GLP-1) is expressed not only in gut endocrine cells, but also in cells in the caudal brainstem and taste buds. To better understand the functions of central GLP-1, GLP-1 expression was immunohistochemically profiled in normal rat brain and its distribution correlated with FOS induction following systemic administration of a GLP-1 receptor agonist, exendin-4. In the present study, only a small number of GLP-1-immunoreactive cell bodies were observed in the nucleus of the solitary tract (NTS). However, these neurons send abundant projections to other regions of the brain, in particular the forebrain, including the paraventricular and dorsomedial nuclei of the hypothalamus, the central nucleus of the amygdala, the oval nucleus of the bed nuclei of the stria terminalis, and the paraventricular nucleus of the thalamus. Intraperitoneal administration of exendin-4 resulted in extensive FOS expression in areas of the forebrain and the hindbrain. In the forebrain, FOS expression was largely confined to regions where a high density of GLP-1-immunoreactive terminals was also localized. The majority of GLP-1-immunoreactive cells in the NTS were not FOS-positive. FOS-positive cells appeared to represent a different population from those expressing GLP-1. Thus, GLP-1-containing neurons in the brainstem may not be involved in receiving and relaying to other regions of the brain the physiological signals of prandial GLP-1 secreted by intestinal L-cells. Projections of GLP-1-containing neurons to the distinctive structures in the forebrain imply that central GLP-1 may play an important role in the behavioral and metabolic integration of autonomic control and arousal in the rat.

Since absence of expression of the c-src gene product in mice indicates that the pp60CSF" tyrosine kinase is required and essential for osteoclastic bone resorption, we tested the effects of the antibiotic herbimycin A, which is an inhibitor of pp60 " on osteoclastic bone resorption in vitro and on hypercalcemia in vivo. We examined the effects of herbimycin A on the formation of bone resorbing osteoclasts in mouse long-term marrow cultures, on isolated rodent osteoclasts and on bone resorption in organ cultures of fetal rat long bones stimulated by parathyroid hormone. We found that herbimycin A in concentrations of 1-100 ng/ml inhibited bone resorption in each of these systems. We determined the effects of herbimycin A ( 100 ng/ml) on src tyrosine kinase activity in mouse marrow cultures and found that it was decreased. Herbimycin A also decreased elevated blood calcium levels that were induced either by repeated subcutaneous injections of recombinant human interleukin-la or by a human tumor. There was no evidence for toxicity in any of these culture systems or in mice treated with herbimycin A. A different tyrosine kinase inhibitor that does not inhibit pp6j(f was used as a control and caused none of these effects. These data suggest that pp6O45w tyrosine kinase inhibitors may be useful pharmacologic inhibitors of osteoclastic bone resorption and hypercalcemia. (J. Clin. Invest. 1993.91:2791-2795