Bert Binas

Preimplantation-stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning

Hematopoietic stem cells have been successfully employed for tolerance induction in a variety of rodent and large animal studies. However, clinical transplantation of fully allogeneic bone marrow or blood-borne stem cells is still associated with major obstacles, such as graft-versus-host disease or cytoreductive conditioning-related toxicity. Here we show that when rat embryonic stem cell-like cells of WKY origin are injected intraportally into fully MHC-mismatched DA rats, they engraft permanently (>150 days) without supplementary host conditioning. This deviation of a potentially alloreactive immune response sets the basis for long-term graft acceptance of second-set transplanted WKY cardiac allografts. Graft survival was strictly correlated with a state of mixed chimerism, which required functional thymic host competence. Our results provide a rationale for using preimplantation-stage stem cells as vehicles in gene therapy and for the induction of long-term graft acceptance.

Requirement for the heart-type fatty acid binding protein in cardiac fatty acid utilization

Nonenzymatic cytosolic fatty acid binding proteins (FABPs) are abundantly expressed in many animal tissues with high rates of fatty acid metabolism. No physiological role has been demonstrated for any FABP, although these proteins have been implicated in transport of free long‐chain fatty acids (LCFAs) and protection against LCFA toxicity. We report here that mice lacking heart‐type FABP (H‐FABP) exhibit a severe defect of peripheral (non‐hepatic, non‐fat) LCFA utilization. In these mice, the heart is unable to efficiently take up plasma LCFAs, which are normally its main fuel, and switches to glucose usage. Altered plasma levels of LCFAs, glucose, lactate and β‐hydroxybutyrate are consistent with depressed peripheral LCFA utilization, intensified carbohydrate usage, and increased hepatic LCFA oxidation; these changes are most pronounced under conditions favoring LCFA oxidation. H‐FABP deficiency is only incompletely compensated, however, causing acute exercise intolerance and, at old age, a localized cardiac hypertrophy. These data establish a requirement for H‐FABP in cardiac intracellular lipid transport and fuel selection and a major role in metabolic homeostasis. This new animal model should be particularly useful for investigating the significance of peripheral LCFA utilization for heart function, insulin sensitivity, and blood pressure.—Binas, B., Danneberg, H., McWhir, J., Mullins, L., Clark, A. J. Requirement for the heart‐type fatty acid binding protein in cardiac fatty acid utilization. FASEB J. 13, 805–812 (1999)

Impaired long-chain fatty acid utilization by cardiac myocytes isolated from mice lacking the heart-type fatty acid binding protein gene.

Heart-type fatty acid binding protein (H-FABP), abundantly expressed in cardiac myocytes, has been postulated to facilitate the cardiac uptake of long-chain fatty acids (LCFAs) and to promote their intracellular trafficking to sites of metabolic conversion. Mice with a disrupted H-FABP gene were recently shown to have elevated plasma LCFA levels, decreased cardiac deposition of a LCFA analogue, and increased cardiac deoxyglucose uptake, which qualitatively establishes a requirement for H-FABP in cardiac LCFA utilization. To study the underlying defect, we developed a method to isolate intact, electrically stimulatable cardiac myocytes from adult mice and then studied substrate utilization under defined conditions in quiescent and in contracting cells from wild-type and H-FABP(-/-) mice. Our results demonstrate that in resting and in contracting myocytes from H-FABP(-/-) mice, both uptake and oxidation of palmitate are markedly reduced (between -45% and -65%), whereas cellular octanoate uptake, and the capacities of heart homogenates for palmitate oxidation and for octanoate oxidation, and the cardiac levels of mRNAs encoding sarcolemmal FA transporters remain unaltered. In contrast, in resting H-FABP(-/-) cardiac myocytes, glucose oxidation is increased (+80%) to a level that would require electrical stimulation in wild-type cells. These findings provide a physiological demonstration of a crucial role of H-FABP in uptake and oxidation of LCFAs in cardiac muscle cells and indicate that in H-FABP(-/-) mice the diminished contribution of LCFAs to cardiac energy production is, at least in part, compensated for by an increase in glucose oxidation.

Uncoupling protein 3 as a mitochondrial fatty acid anion exporter

In contrast to UCP1, the primary function of UCP3 is not the dissipation of energy. Rather, several lines of evidence suggest that UCP3 is related to cellular long‐chain fatty acid homeostasis. If long‐chain fatty acids enter the mitochondrial matrix in their non‐esterified form, they cannot be metabolized and may exert deleterious effects. To test the feasibility that UCP3 exports fatty acid anions, we systematically interfered at distinct steps in the fatty acid metabolism pathway, thereby creating conditions in which the entry of (non‐esterified) fatty acids into the mitochondrial matrix is enhanced. First, reducing the cellular fatty acid binding capacity, known to increase cytosolic concentrations of non‐esterified fatty acids, up‐regulated UCP3 5.3‐fold. Second, inhibition of mitochondrial entry of esterified long‐chain fatty acids up‐ regulated UCP3 by 1.9‐fold. Third, high‐fat diets, to increase mitochondrial supply of non‐ esterified long‐chain fatty acids exceeding oxidative capacity, up‐regulated UCP3 twofold. However, feeding a similar amount of medium‐chain fatty acids, which can be oxidized inside the mitochondrial matrix and therefore do not need to be exported from the matrix, did not affect UCP3 protein levels. These data are compatible with a physiological function of UCP3 in facilitating outward transport of long‐chain fatty acid anions, which cannot be oxidized, from the mitochondrial matrix.

Liver fatty acid-binding protein initiates budding of pre-chylomicron transport vesicles from intestinal endoplasmic reticulum

The rate-limiting step in the transit of absorbed dietary fat across the enterocyte is the generation of the pre-chylomicron transport vesicle (PCTV) from the endoplasmic reticulum (ER). This vesicle does not require coatomer-II (COPII) proteins for budding from the ER membrane and contains vesicle-associated membrane protein 7, found in intestinal ER, which is a unique intracellular location for this SNARE protein. We wished to identify the protein(s) responsible for budding this vesicle from ER membranes in the absence of the requirement for COPII proteins. We chromatographed rat intestinal cytosol on Sephacryl S-100 and found that PCTV budding activity appeared in the low molecular weight fractions. Additional chromatographic steps produced a single major and several minor bands on SDS-PAGE. By tandem mass spectroscopy, the bands contained both liver and intestinal fatty acid-binding proteins (L- and I-FABP) as well as four other proteins. Recombinant proteins for each of the six proteins identified were tested for PCTV budding activity; only L-FABP and I-FABP (23% the activity of L-FABP) were active. The vesicles generated by L-FABP were sealed, contained apolipoproteins B48 and AIV, were of the same size as PCTV on Sepharose CL-6B, and by electron microscopy, excluded calnexin and calreticulin but did not fuse with cis-Golgi nor did L-FABP generate COPII-dependent vesicles. Gene-disrupted L-FABP mouse cytosol had 60% the activity of wild type mouse cytosol. We conclude that L-FABP can select cargo for and bud PCTV from intestinal ER membranes.

Liver fatty acid binding protein is required for high rates of hepatic fatty acid oxidation but not for the action of PPARα in fasting mice

Liver fatty acid binding protein (L‐FABP) has been proposed to limit the availability of long‐chain fatty acids (LCFA) for oxidation and for peroxisome proliferator‐activated receptor α (PPAR‐α), a fatty acid binding transcription factor that determines the capacity of hepatic fatty acid oxidation. Here, we used L‐FABP null mice to test this hypothesis. Under fasting conditions, this mutation reduced β‐hydroxybutyrate (BHB) plasma levels as well as BHB release and palmitic acid oxidation by isolated hepatocytes. However, the capacity for ketogenesis was not reduced: BHB plasma levels were restored by octanoate injection; BHB production and palmitic acid oxidation were normal in liver homogenates; and hepatic expression of key PPAR‐α target (MCAD, mitochondrial HMG CoA synthase, ACO, CYP4A3) and other (CPT1, LCAD) genes of mitochondrial and extramitochondrial LCFA oxidation and ketogenesis remained at wild‐type levels. During standard diet, mitochondrial HMG CoA synthase mRNA was selectively reduced in L‐FABP null liver. These results suggest that under fasting conditions, hepatic L‐FABP contributes to hepatic LCFA oxidation and ketogenesis by a nontranscriptional mechanism, whereas L‐FABP can activate ketogenic gene expression in fed mice. Thus, the mechanisms whereby L‐FABP affects fatty acid oxidation may vary with physiological condition.

Mammary gland specific hEGF receptor transgene expression induces neoplasia and inhibits differentiation

The epidermal growth factor receptor (EGFR) is overexpressed in about 48% of human breast cancer tissues. To analyse the role of the EGFR in mammary tumor development we generated transgenic mice expressing the human EGFR under the control of either the MMTV-LTR (MHERc) or the β-lactoglobulin promoter (BLGHERc). The BLGHERc-transgene was expressed exclusively in the female mammary gland, whereas the MHERc transgene was expressed more promiscuously in other organs, such as ovary, salivary gland and testis. Female virgin and lactating transgenic mice of both strains have impaired mammary gland development. Virgin EGFR transgenic mice developed mammary epithelial hyperplasias, whereas in lactating animals progression to dysplasias and tubular adenocarcinomas was observed. In both strains the number of dysplasias increased after multiple pregnancies. The transgene expression pattern was heterogeneous, but generally restricted to regions of impaired mammary gland development. Highest EGFR transgene expression was observed in adenocarcinomas. By using a whole mount organ culture system to study the differentiation potential of the mammary epithelium, we observed a reduced number of fully developed alveoli and a decrease in whey acidic protein expression. Taken together, EGFR overexpression results in a dramatic effect of impaired mammary gland development in vitro as well as in vivo, reducing the differentiation potential of the mammary epithelium and inducing epithelial cell transformation.

Heart-Type Fatty Acid Binding Protein Regulates Dopamine D2 Receptor Function in Mouse Brain

Fatty acid binding proteins (FABPs) are essential for energy production and long-chain polyunsaturated fatty acid-related signaling in the brain and other tissues. Of various FABPs, heart-type fatty acid binding protein (H-FABP, FABP3) is highly expressed in neurons of mature brain and plays a role in arachidonic acid incorporation into brain and heart cells. However, the precise function of H-FABP in brain remains unclear. We previously demonstrated that H-FABP is associated with the dopamine D2 receptor long isoform (D2LR) in vitro. Here, we confirm that H-FABP binds to dopamine D2 receptor (D2R) in brain extracts and colocalizes immunohistochemically with D2R in the dorsal striatum. We show that H-FABP is highly expressed in acetylcholinergic interneurons and terminals of glutamatergic neurons in the dorsal striatum of mouse brain but absent in dopamine neuron terminals and spines in the same region. H-FABP knock-out (KO) mice showed lower responsiveness to methamphetamine-induced sensitization and enhanced haloperidol-induced catalepsy compared with wild-type mice, indicative of D2R dysfunction. Consistent with the latter, aberrant increased acetylcholine (ACh) release and depolarization-induced glutamate (Glu) release were observed in the dorsal striatum of H-FABP KO mice. Furthermore, phosphorylation of CaMKII (Ca2+/calmodulin-dependent protein kinase II) and ERK (extracellular signal-regulated kinase) was significantly increased in the dorsal striatum. We confirmed elevated ERK phosphorylation following quinpirole-mediated D2R stimulation in H-FABP-overexpressing SHSY-5Y human neuroblastoma cells. Together, H-FABP is highly expressed in ACh interneurons and glutamatergic terminals, thereby regulating dopamine D2R function in the striatum.

Long-Chain Fatty Acid Uptake by Skeletal Muscle Is Impaired in Homozygous, but Not Heterozygous, Heart-Type-FABP Null Mice

Previous studies with cardiac myocytes from homozygous heart-type fatty acid (FA)-binding protein (H-FABP)−/− mice have indicated that this intracellular: receptor protein for long-chain FA is involved in the cellular uptake of these substrates. Based on the knowledge that muscle FA uptake is a process highly sensitive to regulation by hormonal and mechanical stimuli, we studied whether H-FABP would play a role in this regulation. A suitable model system to answer this question is provided by H-FABP+/− mice, because in hindlimb muscles the content of H-FABP was measured to be 34% compared to wild-type mice. In these H-FABP+/− skeletal muscles, just as in H-FABP−/− muscles, contents of FA transporters, i.e., 43-kDa FABPpm and 88-kDa FAT/CD36, were similar compared to wild-type muscles, excluding possible compensatory mech-anisms at the sarcolemmal level. Palmitate uptake rates were measured in giant vesicles prepared from hindlimb muscles of H-FABP−/−, H-FABP+/− and H-FABP+/+ mice. For comparison, giant vesicles were isolated from liver, the tissue of which expresses a distinct type of FABP (i.e., L-FABP). Whereas in H-FABP−/− skeletal muscle FA uptake was reduced by 42–45%, FA uptake by H-FABP+/− skeletal muscle was not different from that in wild-type mice. In contrast, in liver from H-FABP−/− and from H-FABP+/− mice, FA uptake was not altered compared to wild-type animals, indicating that changes in FA uptake are restricted to H-FABP expressing tissues. It is concluded that H-FABP plays an important, yet merely permissive, role in FA uptake into muscle tissues.

Hormonal induction of functional differentiation and mammary-derived growth inhibitor expression in cultured mouse mammary gland explants

SummaryA method for the cultivation of organ explants from abdominal mammary glands of virgin mice has been established. In a serum-free medium containing aldosterone, prolactin, insulin, and cortisol (APIH medium) mammary gland development was documented by lobuloalveolar morphogenesis. The hormonal requirements for in vitro expression of beta-casein and of the mammary-derived growth inhibitor (MDGI) were tested. To this end, a full length cDNA coding for mouse MDGI was prepared displaying strong homologies to a mouse heart fatty acid binding protein, which is also expressed in the mammary gland. MDGI and beta-casein transcripts were found to be absent in the mammary tissue from primed virgin mice, and were induced upon culture of mammary explants in the APIH medium. An immunohistochemical analysis with specific antibodies against MDGI and casein revealed a different pattern of expression for the two proteins. In the APIH medium, MDGI was expressed mainly in differentiating alveolar cells of the lobuloalveolar structures, whereas beta-casein was present in both ductules and alveoli. The relationship between functional differentiation and MDGI expression was further studied in explants from glands of late-pregnant mice. At this stage of development, MDGI is found both in ducts and in alveoli. If explants were cultured with epidermal growth factor (EGF) and insulin, the lobuloalveolar structure was still present, whereas MDGI disappeared. Reinduction of MDGI expression was achieved by subsequent PIH treatment. Independent on developmental stage, EGF strongly inhibits MDGI mRNA expression. It is concluded that MDGI-expression is associated with functional differentiation in the normal gland.