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New results have brought to light the importance of the regulation of glucagon by β-cells in the development of diabetes. In this perspective, we examine the normal paracrinology of α- and β-cells in nondiabetic pancreatic islets. We propose a Sherringtonian model of coordinated reciprocal secretory responses of these juxtaposed cells that secrete glucagon and insulin, hormones with opposing actions on the liver. As insulin is a powerful inhibitor of glucagon, we propose that within-islet inhibition of α-cells by β-cells creates an insulin-to-glucagon ratio that maintains glycemic stability even in extremes of glucose influx or efflux. By contrast, in type 1 diabetes mellitus, α-cells lack constant action of high insulin levels from juxtaposed β-cells. Replacement with exogenous insulin does not approach paracrine levels of secreted insulin except with high doses that “overinsulinize” the peripheral insulin targets, thereby promoting glycemic volatility. Based on the stable normoglycemia of mice with type 1 diabetes during suppression of glucagon with leptin, we conclude that, in the absence of paracrine regulation of α-cells, tonic inhibition of α-cells improves the dysregulated glucose homeostasis. These results have considerable medical implications, as they suggest new approaches to normalize the extreme volatility of glycemia in diabetic patients.

Cortical endoplasmic reticulum (cER) is a permanent feature of yeast cells but occurs transiently in most animal cell types. Ist2p is a transmembrane protein that permanently localizes to the cER in yeast. When Ist2 is expressed in mammalian cells, it induces abundant cER containing Ist2. Ist2 cytoplasmic C-terminal peptide is necessary and sufficient to induce cER. This peptide sequence resembles classic coat protein complex I (COPI) coatomer protein-binding KKXX signals, and indeed the dimerized peptide binds COPI in vitro. Controlled dimerization of this peptide induces cER in cells. RNA interference experiments confirm that coatomer is required for cER induction in vivo, as are microtubules and the microtubule plus-end binding protein EB1. We suggest that Ist2 dimerization triggers coatomer binding and clustering of this protein into domains that traffic at the microtubule growing plus-end to generate the cER beneath the plasma membrane. Sequences similar to the Ist2 lysine-rich tail are found in mammalian STIM proteins that reversibly induce the formation of cER under calcium control.

Coat protein complex II (COPII) is a multi-subunit protein complex responsible for the formation of membrane vesicles at the endoplasmic reticulum. The assembly of this complex on the endoplasmic reticulum membrane needs to be tightly regulated to ensure efficient and specific incorporation of cargo proteins into nascent vesicles. Recent studies of a genetic disease affecting COPII function, and a structural analysis of COPII subunit interactions emphasize the central role of the Sec23 subunit in COPII coat assembly. Similarly, the demonstration that Sec23 interacts physically and functionally with proteins involved in both vesicle tethering and the transport along microtubules indicates that the Sec23 subunit is crucially important in linking COPII vesicle formation to anterograde transport events.

Monolayer culture of pancreatic cells from newborn rats has been shown to yield ultrastructurally normal endocrine cells, virtual absence of differentiated exocrine cells, and the maintenance of the capacity to synthesize immuno-reactive insulin (IRI) and glucagon (ERG). Such a preparation has potential advantages for the study of mechanisms of hormone synthesis and release. Therefore, a survey of factors influencing hormone content and release from cultured cells was undertaken. The following general features were demonstrated: (1) highly reproducible responses within a given preparation of cells despite (2) some variations of absolute hormone content and release between preparations, (3) suitability for preparing sufficient quantities of cells to permit the simultaneous comparison of several factors within a given preparation, and (4) persistence of physiological responses to known modulators of IRI and IRG release. Thus, IRI release was stimulated in concentration-related fashion by glucose. Amino acids, tolbutamide and glucagon augmented release, and 2-deoxy-D-glucose, mannoheptulose and diazoxide inhibited glucose-induced release. Epinephrine inhibited glucose-induced IRI release through stimulation of an α-adrenergic receptor mechanism, and the presence of probable β-receptor stimulation of release was also demonstrated. Agents affecting the microtubular-microfilamentous system exerted effecte similar to those demonstrated in other preparations. Ouabain, as well as the absence or augmented potassium in the medium increased IRI release in the presence of non-stimulatory 2.75 mM glucose, whereas absence of calcium inhibited the response to 11 mM glucose without affecting baseline, non-stimulated release. IRG release was shown to be inversely related to the glucose concentration in the medium. It was stimulated by epinephrine, arginine, alanine, lactate and pyruvate, and inhibited by β-hydroxybutyrate. Diazoxide alone had no effect on IRG release. The monolayer culture employed in these studies provides a convenient, reproducible system for the further study of adult-type IRI and IRG secretory behaviour. In addition to acute or short-term regulation it may be especially suited for the study of long-term modulating effects during the culture period.

SummaryProteins trafficking through the secretory pathway must first exit the endoplasmic reticulum (ER) through membrane vesicles created and regulated by the COPII coat protein complex. Cranio-lenticulo-sutural dysplasia (CLSD) was recently shown to be caused by a missense mutation in SEC23A, a gene encoding one of two paralogous COPII coat proteins. We now elucidate the molecular mechanism underlying this disease. In vitro assays reveal that the mutant form of SEC23A poorly recruits the Sec13-Sec31 complex, inhibiting vesicle formation. Surprisingly, this effect is modulated by the Sar1 GTPase paralog used in the reaction, indicating distinct affinities of the two human Sar1 paralogs for the Sec13-Sec31 complex. Patient cells accumulate numerous tubular cargo-containing ER exit sites devoid of observable membrane coat, likely representing an intermediate step in COPII vesicle formation. Our results indicate that the Sar1-Sec23-Sec24 prebudding complex is sufficient to form cargo-containing tubules in vivo, whereas the Sec13-Sec31 complex is required for membrane fission.

Until 60 years ago, fatty heart was an accepted clinical entity. Since then, its very existence has been questioned, despite the fact that 2 of 3 Americans are now obese or overweight and obesity has been shown to be correlated with cardiac functional abnormalities. In 2000, a syndrome of “lipotoxic cardiomyopathy” resembling earlier pathologic descriptions of fatty human hearts was described in rodents, and fatty infiltration of cardiomyocytes was subsequently reported in patients with congestive failure. Now, magnetic resonance spectroscopy has been adapted to permit routine noninvasive screening for fatty heart. The use of this technique in human volunteers indicates that cardiomyocyte fat correlates well with body mass index and is elevated in uncomplicated obesity. It is more severe when glucose tolerance becomes abnormal or diabetes is present. It is associated with impaired diastolic filling, even in seemingly asymptomatic obese volunteers. Because fatty heart can be readily prevented by lifestyle modification and pharmacologic interventions that reduce caloric intake and increase fatty acid oxidation, it seems important to recognize its existence so as to intervene as early as possible.

The objective of this study was to determine whether the late failure of β-cells in islets transplanted via the portal vein is caused by excess insulin-stimulated lipogenesis and lipotoxicity and, if so, whether the damage can be prevented by reducing lipogenesis surrounding the islets. Based on the premise that high portal vein levels of nutrients and incretins would stimulate hyperinsulinemia, thereby inducing intense lipogenesis in nearby hepatocytes, normal islets were transplanted into livers of syngeneic streptozotocin-induced diabetic recipients. Hydrolysis of the surrounding fat would flood the islet grafts with fatty acids that could damage and destroy the β-cells. Reducing lipogenesis by leptin or caloric restriction should prevent or reduce the destruction. After a rise after transplantation, insulin levels gradually declined and hyperglycemia increased. Four weeks after transplantation mRNA of the lipogenic transcription factor, sterol regulatory element–binding protein-1c (SREBP-1c) and its lipogenic target enzymes were elevated in livers of these recipients, as was triacylglycerol content. Positive oil red O staining for lipids and immunostaining for SREBP-1 were observed in hepatocytes surrounding islets with damaged β-cells. Leptin-induced lipopenia prevented and caloric restriction reduced steatosis, hyperglycemia, and apoptotic β-cell destruction. Excessive SREBP-1c–mediated lipogenesis, induced in hepatocytes by insulin hypersecretion, is followed by β-cell destruction in the grafts and reappearance of diabetes. Graft failure is prevented by blocking lipogenesis. The results suggest that strict antilipogenic intervention might improve outcomes after human islet transplantation.

Intense hyperleptinemia completely depletes adipocyte fat of normal rats within 14 days. To determine the mechanism, epididymal fat pads from normal wild-type (+/+) and obese (fa/fa) Zucker Diabetic Fatty (ZDF) donor rats were transplanted into normal +/+ and fa/fa ZDF recipients. Hyperleptinemia induced by adenovirus-leptin administration depleted all fat from native fat pads and from fat transplants from +/+ donors but not from transplants from ZDFfa/fa donors with defective leptin receptors. In both native and transplanted +/+ fat pads, large numbers of mitochondria were apparent, and genes involved in fatty acid oxidation were up-regulated. However, +/+ fat pads transplanted into fa/fa recipients did not respond to hyperleptinemia, suggesting lack of an essential leptin-stimulated cohormone(s). In +/+ but not in fa/fa rats, plasma catecholamine levels rose, and both P-STAT3 and P-CREB increased in adipose tissue, suggesting that both direct and indirect (hypothalamic) leptin receptor-mediated actions of hyperleptinemia are involved in depletion of adipocyte fat.

Hyperleptinemia rapidly depletes adipocyte fat in lean rats, whereas comparable hyperleptinemia produced by adipocytes in diet-induced obesity does not, implying a leptinergic blockade in adipocytes during overnutrition. Indeed, activated STAT-3 in white adipose tissue (WAT) of normal rats was less on a 60% high fat diet (HFD) than on 4% fat, despite a 10-fold higher plasma leptin. In 6 days of a HFD, mRNA of the postreceptor leptin inhibitor, suppressor of cytokine signaling-3, increased 22-fold in WAT, while leptin receptor (Lepr-b) mRNA gradually disappeared, implying leptinergic blockade at both postreceptor and receptor levels. Adipocyte-specific Lepr-b overexpression of a Lepr-b transgene completely prevented the adipocyte hypertrophy and hyperplasia and the increase in body fat induced in wild-type mice by HFD. Activated STAT-3 and AMP-activated protein kinase (AMPK), and the mRNA of lipooxidative enzymes, peroxisome proliferator-activated receptor-γ-coactivator-1α, and uncoupling protein-1 and -2 were increased in WAT. Body temperature was elevated in the transgenic mice, suggesting uncoupled fatty acid oxidation of surplus fatty acids. In conclusion, storage of surplus calories in WAT and the development of diet-induced obesity require the blockade of a latent leptin-stimulated caloric sump in white adipocytes.

Mutant forms of presenilin (PS) 1 and 2 and amyloid precursor protein (APP) lead to familial Alzheimer's disease. Several reports indicate that PS may modulate APP export from the endoplasmic reticulum (ER). To develop a test of this possibility, we reconstituted the capture of APP and PS1 in COPII (coat protein complex II) vesicles formed from ER membranes in permeabilized cultured cells. The recombinant forms of mammalian COPII proteins were active in a reaction that measures coat subunit assembly and coated vesicle budding on chemically defined synthetic liposomes. However, the recombinant COPII proteins were not active in cargo capture and vesicle budding from microsomal membranes. In contrast, rat liver cytosol was active in stimulating the sorting and packaging of APP, PS1, and p58 (an itinerant ER to Golgi marker protein) into transport vesicles from donor ER membranes. Budding was stimulated in dilute cytosol by the addition of recombinant COPII proteins. Fractionation of the cytosol suggested one or more additional proteins other than the COPII subunits may be essential for cargo selection or vesicle formation from the mammalian ER membrane. The recombinant Sec24C specifically recognized the APP C-terminal region for packaging. Titration of Sarla distinguished the packaging requirements of APP and PS1. Furthermore, APP packaging was not affected by deletion of PS1 or PS1 and 2, suggesting APP and PS1 trafficking from the ER are normally uncoupled.

Genetic and biochemical evidence has established that a SNARE complex consisting of syntaxin 5 (Sed5)-mYkt6 (Ykt6)-GOS28 (Gos1)-GS15 (Sft1) is required for transport of proteins across the Golgi stack in animals (yeast). We have utilized quantitative immunogold labeling to establish the cis-trans distribution of the v-SNARE GS15 and the t-SNARE subunits GOS28 and syntaxin 5. Whereas the distribution of the t-SNARE is nearly even across the Golgi stack from the cis to the trans side, the v-SNARE GS15 is present in a gradient of increasing concentration toward the trans face of the stack. This contrasts with a second distinct SNARE complex, also required for intra-Golgi transport, consisting of syntaxin 5 (Sed5)-membrin (Bos1)-ERS24 (Sec22)-rBet1 (Bet1), whose v-(rBet1) and t-SNARE subunits (membrin and ERS24), progressively decrease in concentration toward the trans face. Transport within the stack therefore appears to utilize countercurrent gradients of two Golgi SNAREpins and may involve a mechanism akin to homotypic fusion.

A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.

The small G-protein Sar1 and the cytosolic complexes Sec23/24 and Sec13/31 associate sequentially on endoplasmic reticulum membranes to form a protein coat named COPII, which drives the formation of transport vesicles. Using dynamic light scattering, we show that Sec23/24 and Sec13/31 can self-assemble in a stoichiometric manner in solution to form particles with hydrodynamic radii in the range of 40-60 nm. Self-assembly is favoured by lowering the pH, the ionic strength and/or the temperature. Electron microscopy reveals the formation of spherical particles 60-120 nm in diameter with a tight, rough mesh on their surfaces. We suggest that these structures, which represent a minimal COPII cage, mimic the molecular organization of the membrane-associated COPII coat.

STIM proteins populate and expand cortical endoplasmic reticulum (ER) sheets to mediate store-operated Ca(2+) entry (SOCE) by trapping and gating Orai channels in ER-plasma membrane clusters. A longer splice variant, STIM1L, forms permanent ER-plasma membrane clusters and mediates rapid Ca(2+) influx in muscle. Here, we used electron microscopy, total internal reflection fluorescence (TIRF) microscopy and Ca(2+) imaging to establish the trafficking and signaling properties of the two STIM1 isoforms in Stim1(-/-)/Stim2(-/-) fibroblasts. Unlike STIM1, STIM1L was poorly recruited into ER-plasma membrane clusters and did not mediate store-dependent expansion of cortical ER cisternae. Removal of the STIM1 lysine-rich tail prevented store-dependent cluster enlargement, whereas inhibition of cytosolic Ca(2+) elevations or removal of the STIM1L actin-binding domain had no impact on cluster expansion. Finally, STIM1L restored robust but not accelerated SOCE and clustered with Orai1 channels more slowly than STIM1 following store depletion. These results indicate that STIM1L does not mediate rapid SOCE but can trap and gate Orai1 channels efficiently without remodeling cortical ER cisternae. The ability of STIM proteins to induce cortical ER formation is dispensable for SOCE and requires the lysine-rich tail of STIM1 involved in binding to phosphoinositides.

The balance between cell division and cell death determines the cell population of an organ. When cell death exceeds cell replacement in an organ, a functional deficit is created. A metabolic cause of programmed cell death, lipoapoptosis, has recently been identified to occur in obesity and aging. If nonadipose tissues are exposed to an excess of long-chain fatty acids, unless leptin action increases their oxidation sufficiently, unoxidized fatty acids enter nonoxidative pathways. While initially they are sequestered as harmless neutral fat, ultimately some will enter more toxic pathways. One of these, the de novo ceramide pathway, has been implicated in the lipoapoptosis of beta-cells and myocardiocytes of congenitally obese rats in which leptin action is defective. Here we review the mechanisms of lipoapoptosis and the diseases that result from this cause of a diminishing cell population of these organs. We suggest that some of the components of the metabolic syndrome of obese humans and the sarcopenia of aging may be result of failure of leptin liporegulation to prevent lipid overload of lean body mass and lipoapoptosis in certain organ systems.

SEC16 encodes a 240-kD hydrophilic protein that is required for transport vesicle budding from the ER in Saccharomyces cerevisiae. Sec16p is tightly and peripherally bound to ER membranes, hence it is not one of the cytosolic proteins required to reconstitute transport vesicle budding in a cell-free reaction. However, Sec16p is removed from the membrane by salt washes, and using such membranes we have reconstituted a vesicle budding reaction dependent on the addition of COPII proteins and pure Sec16p. Although COPII vesicle budding is promoted by GTP or a nonhydrolyzable analogue, guanylimide diphosphate (GMP-PNP), Sec16p stimulation is dependent on GTP in the reaction. Details of coat protein assembly and Sec16p-stimulated vesicle budding were explored with synthetic liposomes composed of a mixture of lipids, including acidic phospholipids (major–minor mix), or a simple binary mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Sec16p binds to major–minor mix liposomes and facilitates the recruitment of COPII proteins and vesicle budding in a reaction that is stimulated by Sar1p and GMP-PNP. Thin-section electron microscopy confirms a stimulation of budding profiles produced by incubation of liposomes with COPII and Sec16p. Whereas acidic phospholipids in the major–minor mix are required to recruit pure Sec16p to liposomes, PC/PE liposomes bind Sar1p-GTP, which stimulates the association of Sec16p and Sec23/24p. We propose that Sec16p nucleates a Sar1-GTP–dependent initiation of COPII assembly and serves to stabilize the coat to premature disassembly after Sar1p hydrolyzes GTP.

The neuroendocrine processing endoproteases PC2 and PC1/3 are expressed in the beta cells of the islets of Langerhans and participate in the processing of proinsulin to insulin and C-peptide. We have previously shown that disruption of PC2 (SPC2) expression significantly impairs proinsulin processing. Here we report that disruption of the expression of PC1/3 (SPC3) produces a much more severe block in proinsulin conversion. In nulls, pancreatic and circulating proinsulin-like components comprise 87% and 91%, respectively, of total insulin-related immunoreactivity. Heterozygotes also show a more than 2-fold elevation in proinsulin levels to approximately 12%. Immunocytochemical and ultrastructural studies of the beta cells reveal the nearly complete absence of mature insulin immunoreactivity and its replacement by that of proinsulin in abundant immature-appearing secretory granules. In contrast, alpha cell morphology and glucagon processing are normal, and there is also no defect in somatostatin-14 generation. Pulse-chase labeling studies confirm the existence of a major block in proinsulin processing in PC1/3 nulls with prolongation of half-times of conversion by 7- and 10-fold for proinsulins I and II, respectively. Lack of PC1/3 also results in increased levels of des-64,65 proinsulin intermediates generated by PC2, in contrast to PC2 nulls, in which des- 31,32 proinsulin intermediates predominate. These results confirm that PC1/3 plays a major role in processing proinsulin, but that its coordinated action with PC2 is necessary for the most efficient and complete processing of this prohormone.

The spatial arrangement of COPII coat protein subunits was analyzed by crosslinking to an artificial membrane surface and by electron microscopy of coat proteins and coated vesicle surfaces. The efficiency of COPII subunit crosslinking to phospholipids declined in order of protein recruitment to the coat: Sar1p > Sec23/24p ≫ Sec13/31p. Deep-etch rotary shadowing and electron microscopy were used to explore the COPII subunit structure with isolated proteins and coated vesicles. Sec23/24 resembles a bow tie, and Sec13/31p contains terminal bilobed globular structures bordering a central rod. The surface structure of COPII vesicles revealed a coat built with polygonal units. The length of the side of the hexagonal/pentagonal units is close to the dimension of the central rod-like segment of Sec13/31. Partially uncoated profiles revealed strands of Sec13/31p stripped from the vesicle surface. We conclude that the coat subunits form layers displaced from the membrane surface in reverse order of addition to the coat.

Mice homozygous for a deletion in the gene encoding prohormone convertase 2 (PC2) are generally healthy but have mild hypoglycemia and flat glucose-tolerance curves. Their islets show marked alpha (A)-cell hyperplasia, suggesting a possible defect in glucagon processing (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J., Carroll, R., Ravazzola, M., Orci, L., Furuta, H., and Steiner, D. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646–6651). In this report we have examined the biosynthesis and processing of proglucagon in isolated islets from these mice via pulse-chase labeling and find that proglucagon undergoes essentially no processing in chase periods up to 8 h in duration. Only a small percent of cleavage at the sensitive interdomain site (residues 71 and 72) appears to occur. These observations thus conclusively demonstrate the essentiality of PC2 for the production of glucagon in the islet A-cells. Ultrastructural and immunocytochemical studies indicate the presence of large amounts of proglucagon in atypical appearing secretory granules in the hyperplastic and hypertrophic A-cells, along with morphological evidence of high rates of proglucagon secretion in PC2 null islets. These findings provide strong evidence that active glucagon is required to maintain normal blood glucose levels, counterbalancing the action of insulin at all times.

It is proposed that an important function of leptin is to confine the storage of triglycerides (TG) to the adipocytes, while limiting TG storage in nonadipocytes. Excess TG deposition in nonadipocytes leads to impairment of functions, increased ceramide formation, which triggers nitric oxide-mediated lipotoxicity and lipoapoptosis. The fact that TG content in nonadipocytes normally remains within a very narrow range irrespective of excess caloric intake, while TG content of adipocytes rises, is consistent with a system of fatty acid (FA) homeostasis in nonadipose tissues. When leptin is deficient or leptin receptors are dysfunctional, TG content in nonadipose tissues such as pancreatic islets, heart and skeletal muscle, can increase 10-50-fold, suggesting that leptin controls the putative homeostatic system for intracellular TG. The fact that function and viability of nonadipocytes is compromised when their TG content rises above normal implies that normal homeostasis of their intracellular FA is critical for prevention of complications of obesity. FA overload of skeletal muscle, myocardium and pancreatic islets cause, respectively, insulin resistance, lipotoxic heart disease and adipogenic type 2 diabetes. All can be completely prevented by treatment with antisteatotic agents such as troglitazone. In diet-induced obesity, leptin signaling is normal initially and lipotoxic changes are at first prevented; later, however, post-receptor leptin resistance appears, leading to dysfunction and lipoapoptosis in nonadipose tissues, the familiar complications of obesity.

A central feature of cisternal progression/maturation models for anterograde transport across the Golgi stack is the requirement that the entire population of steady-state residents of this organelle be continuously transported backward to earlier cisternae to avoid loss of these residents as the membrane of the oldest (trans-most) cisterna departs the stack. For this to occur, resident proteins must be packaged into retrograde-directed transport vesicles, and to occur at the rate of anterograde transport, resident proteins must be present in vesicles at a higher concentration than in cisternal membranes. We have tested this prediction by localizing two steady-state residents of medial Golgi cisternae (mannosidase II and N-acetylglucosaminyl transferase I) at the electron microscopic level in intact cells. In both cases, these abundant cisternal constituents were strongly excluded from buds and vesicles. This result suggests that cisternal progression takes place substantially more slowly than most protein transport and therefore is unlikely to be the predominant mechanism of anterograde movement.

Engineered protein aggregates ranging up to 400 nm in diameter were selectively deposited within the cis-most cisternae of the Golgi stack following a 15 degrees C block. These aggregates are much larger than the standard volume of Golgi vesicles, yet they are transported across the stack within 10 min after warming the cells to 20 degrees C. Serial sectioning reveals that during the peak of anterograde transport, about 20% of the aggregates were enclosed in topologically free "megavesicles" which appear to pinch off from the rims of the cisternae. These megavesicles can explain the rapid transport of aggregates without cisternal progression on this time scale.

To determine the mechanism of the cardiac dilatation and reduced contractility of obese Zucker Diabetic Fatty rats, myocardial triacylglycerol (TG) was assayed chemically and morphologically. TG was high because of underexpression of fatty acid oxidative enzymes and their transcription factor, peroxisome proliferator-activated receptor-α. Levels of ceramide, a mediator of apoptosis, were 2–3 times those of controls and inducible nitric oxide synthase levels were 4 times greater than normal. Myocardial DNA laddering, an index of apoptosis, reached 20 times the normal level. Troglitazone therapy lowered myocardial TG and ceramide and completely prevented DNA laddering and loss of cardiac function. In this paper, we conclude that cardiac dysfunction in obesity is caused by lipoapoptosis and is prevented by reducing cardiac lipids.

The extracellular function of chromogranin A (CgA), a glycoprotein widely distributed in secretory vesicles of neurons and neuroendocrine cells, has not been clearly established. To examine whether CgA might modulate the biological properties of epithelial cells, we used an in vitro model of ductal morphogenesis in which mammary epithelial (TAC-2) cells are grown in three-dimensional collagen gels. Whereas under control conditions TAC-2 cells formed thin, branched cords with pointed ends, in the presence of CgA they formed thicker cords with bulbous extremities, reminiscent of growing mammary ducts in vivo. Immunofluorescence analysis demonstrated that CgA increases the deposition of three major basement membrane components, i.e., collagen type IV, laminin, and perlecan, around the surface of the duct-like structures. Similar effects were observed with CgA partially digested with endoproteinase Lys-C, suggesting that one or more fragments of CgA are endowed with the same activity. These findings reveal a hitherto unsuspected activity for CgA, i.e., the ability to alter ductal morphogenesis and to promote basement membrane deposition in mammary epithelial cells.

The prohormone convertases PC2 (SPC2) and PC3/PC1 (SPC3) are the major precursor processing endoproteases in a wide variety of neural and endocrine tissues. Both enzymes are normally expressed in the islet beta cells and participate in proinsulin processing. Recently we generated mice lacking active PC2 due to a disruption of the PC2 gene (Furuta, M., Yano, H., Zhou, A., Rouille, Y., Holst, J. J., Carroll, R. J., Ravazzola, M., Orci, L., Furuta, H., and Steiner, D. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6646-6651). Here we report that these PC2 mutant mice have elevated circulating proinsulin, comprising 60% of immunoreactive insulin-like components. Acid ethanol extractable proinsulin from pancreas is also significantly elevated, representing about 35% of total immunoreactive insulin-like components. These increased amounts of proinsulin are mainly stored in secretory granules, giving rise to an altered appearance on electron microscopy. In pulse-chase experiments, the mutant islets incorporate lesser amounts of isotopic amino acids into insulin-related components than normal islets. In both wild-type and mutant islets, proinsulin I was processed more rapidly to insulin, reflecting the preference of both PC2 and PC3 for substrates having a basic amino acid positioned four residues upstream of the cleavage site. The overall half-time for the conversion of proinsulin to insulin is increased approximately 3-fold in the mutant islets and is associated with a 4-5-fold greater elevation of des-31,32 proinsulin, an intermediate that is formed by the preferential cleavage of proinsulin at the B chain-C-peptide junction by PC3 and is C-terminally processed to remove Arg31 and Arg32 by carboxypeptidase E. The constitutive release of newly synthesized proinsulin from both mutant and wild-type islets during the first 1-2 h of chase was normal (

Epithelial-mesenchymal interactions are responsible for the unique pattern of ductal branching morphogenesis characteristic of the mammary gland. To investigate the factors which control the elongation and branching of lactiferous ducts, we developed an in vitro model of ductal morphogenesis in which clonal mouse mammary epithelial cells (TAC-2 cells) are grown in collagen gels. In this experimental system, fibroblast conditioned medium (CM)3 stimulates the formation of extensively arborized tubules. The molecule responsible for this tubulogenic effect was identified as hepatocyte growth factor/scatter factor (HGF/SF). To determine whether HGF/SF plays a role in mammary gland morphogenesis in vivo, the expression of HGF/SF and its receptor, c-Met, were analyzed in the rat mammary gland during pregnancy, lactation, and involution. Levels of HGF/SF and c-Met transcripts were progressively reduced during pregnancy, were virtually undetectable during lactation, and increased again during involution. Collectively, these in vitro and in vivo findings suggest that HGF/SF is a paracrine mediator of mammary gland ductal morphogenesis. We subsequently investigated the effect of another multifunctional cytokine, namely TGF-beta1, on branching morphogenesis of TAC-2 cells. TGF-beta1 had a striking biphasic effect: whereas relatively high concentrations of this cytokine inhibited colony formation, lower concentrations stimulated extensive elongation and branching of epithelial cords. Taken together, these studies indicate that HGF/SF is a stromal-derived paracrine mediator of mammary ductal morphogenesis, and that when present at low concentrations, TGF-beta1 can contribute to this process.

Electron microscope immunocytochemistry reveals that both anterograde-directed (proinsulin and VSV G protein) and retrograde-directed (the KDEL receptor) cargo are present in COPI-coated vesicles budding from every level of the Golgi stack in whole cells; however, they comprise two distinct populations that together can account for at least 80% of the vesicles budding from Golgi cisternae. Segregation of anterograde- from retrograde-directed cargo into distinct sets of COPI-coated vesicles is faithfully reproduced in the cell-free Golgi transport system, in which VSV G protein and KDEL receptor are packaged into separable vesicles, even when budding is driven by highly purified coatomer and a recombinant ARF protein.

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, both of which may be important for the function of islets of Langerhans. In this study, we have examined the expression of VEGF and its tyrosine kinase receptors (fit and flk-1) in isolated rat islets of Langerhans in vitro. When analyzed by in situ hybridization, islet tissue showed a significant 4.6-fold increase in VEGF mRNA expression over time in culture from 0 to 7 days. Islet tissue exposed to hypoxic/anoxic conditions for a period of 8 hr showed a 3.7-fold increase in VEGF mRNA when analyzed by Northern blot hybridization. Reverse transcriptase-polymerase chain reaction revealed the presence of both fit and flk.l in freshly isolated islets, and two VEGF isoforms, namely VEGF 120 and VEGF 164 . Three rodent β-cell lines derived from insulinomas (RINm5F-2A, INS-1, and MIN6) were also found to express VEGF by Northern blot hybridization. However, neither hypoxia/anoxia nor low (0.3 g/L)- or high (3.0 g/L)-glucose culture conditions modulated their expression of VEGF. VEGF derived from RINm5F-2A cells was bioactive in a three-dimensional in vitro model of angiogenesis, which assays for endothelial cell invasion and capillary morphogenesis. These findings demonstrate, first, that devascularization increases VEGF expression in isolated islet tissue, and they point to VEGF as a potentially important endogenous angiogenic stimulus for subsequent revascularization in vivo. Second, our observations raise the possibility that survival of transplanted islets may be improved by increasing VEGF expression before transplantation.

Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.

The trafficking of proteins within eukaryotic cells is achieved by the capture of cargo and targeting molecules into vesicles that bud from a donor membrane and deliver their contents to a receiving compartment. This process is bidirectional and may involve multiple organelles within a cell. Distinct coat proteins mediate each budding event, serving both to shape the transport vesicle and to select by direct or indirect interaction the desired set of cargo molecules. Secretion, which has been viewed as a default pathway, may require sorting and packaging signals on transported molecules to ensure their rapid delivery to the cell surface.

Extracellular proteolysis is required for matrix degradation and the regulation of cytokine activity during angiogenesis, and this is dependent on a cohort of proteases and protease inhibitors produced by endothelial and nonendothelial cells. The plasminogen activator (PA)/plasmin system has been extensively investigated in these processes, and descriptive studies have demonstrated that urokinase-type PA (uPA), uPA receptor (uPAR) and PA inhibitor-1 (PAI-1) are expressed by endothelial cells during angiogenesis in vivo. In vitro studies have led to the notion that normal capillary morphogenesis is dependent on a protease-antiprotease equilibrium. These findings are discussed in the context of recent observations on uPA-, uPAR-, PAI-1 and plaminogen-deficient mice, in which developmental and physiological angiogenesis appear to occur normally. This has led to a reevaluation of the role of the PA/plasmin system during angiogenesis. In particular, these observations raise the possibility that the role of this system may be limited to situations in which endothelial cells encounter and must degrade fibrin in order to form new capillary sprouts.

The cytosolic yeast proteins Sec13p-Sec31p, Sec23p-Sec24p, and the small GTP-binding protein Sart p generate protein transport vesicles by forming the membrane coat termed COPII We demonstrate by thin section and immunoelectron microscopy that purified COPII components form transport vesicles directly from the outer membrane of isolated yeast nuclei. Another set of yeast cytosolic proteins, coatomer and Arf1p (COPI), also form coated buds and vesicles from the nuclear envelope. Formation of COPI-coated, but not COPII-coated, buds and vesicles on the nuclear envelope is inhibited by the fungal metabolite brefeldin A. The two vesicle populations are distinct. However, both vesicle types are devoid of endoplasmic reticulum (ER) resident proteins, and each contains targeting proteins necessary for docking at the Golgi complex. Our data suggest that COPI and COPII mediate separate vesicular transport pathways from the ER.

Epithelial tubulogenesis is responsible for the exquisitely intricate organization of functional units of parenchymal organs. We have previously demonstrated that hepatocyte growth factor (HGF--also known as scatter factor) is a stroma-derived epithelial morphogen, which induces tubulogenesis by kidney-derived epithelial cells in vitro. The mammary gland provides a particularly attractive model for the study of epithelial morphogenesis, since its development in postnatal life involves elongation and branching of epithelial tubules. The aim of the present studies was to assess the expression and modulation of HGF and its receptor c-Met in the rat mammary gland during pregnancy, lactation, and involution. By ribonuclease protection assay, we demonstrate that levels of both HGF and c-met transcripts are progressively reduced during pregnancy, are virtually undetectable during lactation, and increase during the phase of involution to prepregnancy levels. The reduction in HGF and c-met expression corresponds to periods in which functions other than tubulogenesis predominate in the mammary gland, namely alveologenesis (mid to late pregnancy) and milk protein synthesis (lactation). Using a murine mammary gland-derived epithelial cell line, we demonstrate that levels of c-met mRNA are significantly reduced by exogenously added prolactin, providing a possible explanation for the reduction in c-met in the rat mammary gland during lactation. The potential significance of down-regulation of HGF/c-met during lactation is discussed.

Ontogenic relationships between the different types of endocrine cells in the islets of Langerhans were explored by generating transgenic mouse embryos in which cells transcribing the glucagon, insulin, or pancreatic polypeptide genes were destroyed through the promoter-targeted expression of the diphtheria toxin A chain. Embryos lacking glucagon- or insulin-containing cells did not exhibit alterations in the development of the nontargeted islet cell types, whereas embryos lacking pancreatic polypeptide gene-expressing cells also lacked pancreatic insulin- and somatostatin-containing cells. These results show that neither glucagon nor insulin gene-expressing cells are essential for the differentiation of the other islet endocrine-cell types. These results also suggest that pancreatic polypeptide gene-expressing cells are indispensable for the differentiation of islet beta and delta cells because the former produce a necessary paracrine or endocrine factor and/or operate through a cell-lineage relationship.

A specialized region of the endoplasmic reticulum--the BFA body--is defined by the site of accumulation of coatomer when nonclathrin coat protein (COP)-coated vesicle assembly is prevented by the drug brefeldin A (BFA). BFA bodies are formed by part smooth, part rough domains of endoplasmic reticulum that are cis to the classical transitional endoplasmic reticulum and to BFA-induced Golgi remnants.

We have investigated the expression of three gap junction proteins and their corresponding mRNAs by secretory cells of a variety of endocrine and exocrine rat glands. By immunostaining cryostat sections (indirect immunofluorescence) with antibodies against connexins (Cx) 26, 32, and 43 and by hybridizing total glandular RNA (Northern blot) with cRNAs for these proteins, we have found that several endocrine glands (pituitary, parathyroid, pancreatic islets, and adrenal) express Cx43, variable levels of Cx26, and no Cx32, whereas several exocrine glands (lacrimal gland, salivary glands, pancreas, prostate, and seminal vesicle) express high levels of Cx32 and variable levels of Cx26, but no Cx43. Thus, different sets of proteins comprise the gap junctions of endocrine and exocrine glands. Together with the findings that an endocrine gland (thyroid) that discharges secretory products extracellularly before releasing them in the vascular compartment expresses both Cx43 and Cx32 and that an exocrine gland (preputial gland) that has a pheromonal role expresses Cx43, these observations suggest that the differential expression of gap junction connexins may be required to specify the endocrine or exocrine differentiation of a secretory cell.

The internalization of signaling receptors such as the insulin receptor is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin receptor and to establish which receptor domains are implicated in each of these by the use of receptors possessing in vitro mutations. We find that kinase activation and autophosphorylation of all three regulatory tyrosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the COOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the receptor from microvilli where initial binding occurs to the nonvillous domain of the cell. Early intracellular steps in insulin signal transduction involving the activation of phosphatidylinositol 3'-kinase are not required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin- coated pits. In contrast to the first ligand specific step, this step is common to many receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but requires a juxta-membrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent mechanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit.

One of the phenotypic hallmarks of migrating endothelial cells, both in vivo and in vitro, is expression of the urokinase-type plasminogen activator (u-PA), a key mediator of extracellular proteolysis. In the study reported here, we have used an in vitro model of endothelial cell migration to explore the mechanism of this phenomenon. We have found that wounding of an endothelial cell monolayer triggers a marked, rapid and sustained increase in expression of a specific high-affinity receptor for u-PA (u-PAr) on the surface of migrating cells. Migrating cells displayed an increase in the levels of u-PA and u-PAr mRNAs, and this increase was mediated by endogenous basic fibroblast growth factor (bFGF). We also show that the increase in u-PA activity on migrating cells can be accounted for by an increase in receptor-bound u-PA, and that the increase in activity is also dependent on endogenous bFGF. These results demonstrate that the expression of plasmin-mediated proteolytic activity by migrating endothelial cells is a consequence of increased production of both u-PA and its receptor, and that this in turn is mediated by endogenous bFGF. This suggests that u-PA, produced at increased levels by migrating cells, binds to u-PAr whose expression is upregulated on the same cells. These observations are in accord with the postulated role of u-PAr in mediating efficient and spatially restricted extracellular proteolysis, particularly in the context of cell migration.

GLUT2 underexpression has been reported in the +-cells of Zucker diabetic fatty rats and db/db mice, models of spontaneously occurring NIDDM with antecedent obesity. To determine whether the +-cells of a nonobese rodent model of NIDDM exhibit the same abnormalities in GLUT2, we studied Goto-Kakizaki rats. In these mildly diabetic animals glucose-stimulated insulin secretion was reduced at all ages examined from 8 to 48 wk. In normal control Wistar rats, immunostainable GLUT2 was present on all insulin-positive cells in the pancreatic islets. Only 85% of +-cells were GLUT2-positive in GK rats at 12 wk of age, and only 34% were positive at 48 wk of age. GLUT2 mRNA was 50% of normal in 12-wk-old GK rats. In the latter age-group, glucose-stimulated insulin secretion was only 28% of normal at a time when 85% of +-cells were GLUT2-positive and initial 3-O-methyl-D-glucose transport rate was 77% of the control value. We conclude that although GLUT2 is underexpressed, neither the magnitude of the underexpression of GLUT2 nor of the reduction in GLUT2 transport function in islets of GK rats is sufficient by itself to explain the profound reduction in glucose-stimulated insulin secretion.

Do the coats on vesicles budded from the Golgi apparatus actually cause the budding, or do they simply coat buds (Fig. 1)? One view (the membrane-mediated budding hypothesis) is that budding is an intrinsic property of Golgi membranes not requiring extrinsic coat proteins. Assembly of coats from dispersed subunits is super-imposed upon the intrinsic budding process and is proposed to convert the tips of tubules into vesicles. The alternative view (the coat-mediated budding hypothesis) is that coat formation provides the essential driving force for budding. The membrane-mediated budding hypothesis was inspired by the microtubule-dependent extension of apparently uncoated, 90-nm-diameter membrane tubules from the Golgi apparatus and other organelles in vivo after treatment with brefeldin A, a drug that inhibits the assembly of coat proteins onto Golgi membranes. This hypothesis predicts that tubules will be extended when coat proteins are unavailable to convert tubule-derived membrane into vesicles. Here we use a cell-free system in which coated vesicles are formed from Golgi cisternae to show that, on the contrary, when budding diminishes as a result of immunodepletion of coat protein pools, tubules are not formed at the expense of vesicles. We conclude that coat proteins are required for budding from Golgi membranes.

Although the existence of an increasing number of angiogenesis-regulating cytokines is well documented, the response elicited by combinations of these cytokines is largely unknown. Using an in vitro model in which microvascular endothelial cells can be induced to form capillary-like tubes within three-dimensional collagen or fibrin gels, we have investigated the effect of transforming growth factor-beta 1 (TGF-beta 1) on basic fibroblast growth factor (bFGF)-induced and vascular endothelial growth factor (VEGF)-induced angiogenesis. Endothelial cell invasion and capillary lumen formation were inhibited by TGF-beta 1 at relatively high concentrations (5-10 ng/ml), while lower concentrations (100 pg/ml-1 ng/ml) of TGF-beta 1 potentiated the effect of bFGF- and VEGF-induced invasion. The optimal potentiating effect was observed at 200-500 pg/ml TGF-beta 1. At invasion-potentiating doses of TGF-beta 1, lumen size in fibrin gels was markedly reduced compared to that in cultures treated with bFGF alone. These results show that TGF-beta 1 exerts a biphasic effect on bFGF- and VEGF-induced angiogenesis in vitro. Our studies support the notion that the nature of the angiogenic response elicited by a specific cytokine is contextual, i.e., depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell.

Efficient sorting of proteins is essential to allow transport between intracellular compartments while maintaining their specific composition. During endocytosis, membrane proteins can be concentrated in endocytic vesicles by specific interactions between their cytoplasmic domains and cytosolic coat proteins. It is, however, unclear whether they can be excluded from transport vesicles and what the determinants for this sorting could be. Here, we show that in the absence of cytosolic sorting signals, transmembrane domains control the access of surface proteins to endosomal compartments. They act in particular by determining the degree of exclusion of membrane proteins from endocytic clathrin-coated vesicles. When cytosolic endocytosis signals are present, it is the combination of cytosolic and transmembrane determinants that ultimately controls the efficiency with which a given transmembrane protein is endocytosed.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a recently characterized endothelial-specific mitogen which is angiogenic in vivo. Here we demonstrate that VEGF is angiogenic in vitro: when added to microvascular endothelial cells grown on the surface of three-dimensional collagen gels, VEGF induces the cells to invade the underlying matrix and to form capillary-like tubules, with an optimal effect at approximately 2.2nM (100ng/ml). When compared to basic fibroblast growth factor (bFGF) at equimolar (0.5nM) concentrations, VEGF was about half as potent. The most striking effect was seen in combination with bFGF: when added simultaneously, VEGF and bFGF induced an in vitro angiogenic response which was far greater than additive, and which occurred with greater rapidity than the response to either cytokine alone. These results demonstrate that like bFGF, VEGF induces an angiogenic response via a direct effect on endothelial cells, and that by acting in concert, these two cytokines have a potent synergistic effect on the induction of angiogenesis in vitro. We suggest that the synergism between VEGF and bFGF plays an important role in the control of angiogenesis in vivo.

We have recently demonstrated that fibroblast-conditioned medium induces Madin-Darby canine kidney (MDCK) epithelial cells to form branching tubules when grown in three-dimensional collagen or fibrin gels (Montesano, R., Schaller, G., and Orci, L. (1991) Cell 66, 697-711), and that this morphogenetic effect is mediated by hepatocyte growth factor (HGF), also known as scatter factor (Montesano, R., Matsumoto, K., Nakamura, T., and Orci, L. (1991) Cell 67, 901-908). In fibrin gels, this effect is inhibited by addition of exogenous serine protease inhibitors, which suggests a role for plasminogen activators (PAs) in the matrix remodeling required for tubulogenesis. In the studies reported in this paper, we have investigated the effect of fibroblast-conditioned medium (CM) and HGF on the production of PAs by MDCK cells. We have found that urokinase-type PA (u-PA) activity and mRNA are increased 4.9-fold by CM from human MRC-5 fibroblasts, which has tubulogenic activity, but not by CM from human Detroit-550 fibroblasts, which lacks tubulogenic activity. The u-PA inductive property of MRC-5 CM was completely inhibited by preincubation with antibodies to recombinant human HGF (rhHGF). Exogenously added rhHGF also increased u-PA activity and mRNA 5.9-fold in MDCK cells, with an optimal effect at approximately 10 ng/ml. MRC-5 CM also increased u-PA receptor mRNA 34.9-fold in MDCK cells, an effect which was inhibited by 71% by preincubating the CM with antibodies to rhHGF, and which was mimicked by exogenously added rhHGF (31.3-fold increase). These results demonstrate that HGF, which induces tubulogenesis by MDCK cells in vitro, also increases u-PA and u-PA receptor expression in these cells. Taken together with our previous observations, this suggests that the resulting increase in extracellular proteolysis, appropriately localized to the cell surface, is required for epithelial morphogenesis.

It has been proposed that a finely tuned protease-anti-protease equilibrium must be maintained during processes of cell migration in order to limit extracellular proteolysis to the close proximity of the cell surface, and thereby to prevent excessive extracellular matrix degradation. We have previously shown that urokinase-type plasminogen activator (u-PA) activity is induced in microvascular endothelial cells migrating from the edges of a wounded monolayer in vitro (Pepper et al., J. Cell Biol., 105:2535-2541, 1987). By Northern analysis, we now demonstrate that plasminogen activator inhibitor 1 (PAI-1) mRNA is increased in multiple-wounded monolayers of bovine microvascular (BME) or aortic (BAE) endothelial cells, with a maximal 7- and 9-fold increase 4 h after wounding, respectively. By in situ hybridization, we demonstrate that the increase in PAI-1 mRNA is localized to cells at the edge of a wounded BME or BAE cell monolayer. The increase in PAI-1 mRNA observed in BME cells is independent of cell division and is inhibited by antibodies to basic fibroblast growth factor (bFGF), suggesting that PAI-1 induction in migrating cells is mediated by the autocrine activity of bFGF. Taken together with our previous observations on the induction of u-PA, these results support the hypothesis that the proteolytic balance in the pericellular environment of migrating cells is regulated through the concomitant production of proteases and protease inhibitors.

Endothelial cells of the microvasculature differ both structurally and functionally from endothelial cells of larger vessels. To assess whether these cells also differ in terms of direct cell-to-cell communication, we compared gap junction-mediated intercellular coupling and connexin (Cx) expression in monolayer cultures of bovine microvascular and large vessel (aortic and pulmonary artery) endothelial cells. In confluent monolayers, junctional communication (as assessed by transfer of Lucifer Yellow) was greater between large vessel than between microvascular endothelial cells. Basal levels of connexin 43 (Cx43) and Cx43 mRNA were also greater in large vessel than in microvascular endothelial cells. When monolayers of microvascular endothelial cells were mechanically wounded, junctional communication was increased between migrating cells at the wound edge. In contrast, coupling between large vessel endothelial cells was not increased after wounding. The wound-induced increase in coupling between microvascular endothelial cells was accompanied by an increase in Cx43 and Cx43 mRNA. In contrast, Cx43 expression was unaltered after wounding monolayers of large vessel endothelial cells. These studies revealed differences in basal and wound-induced levels of coupling and Cx43 expression in microvascular and large vessel endothelial cells in vitro, raising the possibility that the role of coupling in endothelial cell function may be different in these different cell types.

In the 20th century industrialized nations have become afflicted with an unprecedented pandemic of increased adiposity. In the United States, the epicenter of the epidemic, over 2/3 of the population, is overweight and 1 of every 6 Americans carries the diagnosis of metabolic syndrome. Although genes determine susceptibility to environmental factors, the epidemic is clearly due to increased consumption of calorie-dense, highly lipogenic foods, coupled with a marked decrease in physical exertion resulting from modern technologies. If this lifestyle continues, morbid consequences are virtually inevitable. They include type II diabetes and a cluster of disorders known as "the metabolic syndrome" usually appearing in middle age. The morbid consequences of the chronic caloric surplus are buffered before middle age by the partitioning of these calories as fat in the adipocyte compartment which is specifically designed to store triglycerides. Leptin has been proposed as the major hormonal regulator of the partitioning of surplus calories. However, multiple factors can determine the storage capacity of the fat tissue and when it is exceeded ectopic lipid deposition begins. The organs affected in metabolic syndrome include skeletal muscle, liver, heart and pancreas, which are now known to contain abnormal levels of triglycerides. While neutral fat is probably harmless, it is an index of ectopic lipid overload. Fatty acid derivatives can interfere with the function of the cell and ultimately lead to its demise through lipoapoptosis, the consequences of which are gradual organ failure.