Issue published October 1, 2000

P revious linkage studies in Mexican-Americans localized a major susceptibility locus for type 2 diabetes, NIDDM1, to chromosome 2q. This evidence for linkage to type 2 diabetes was recently found to be associated with a common G→A polymorphism (UCSNP-43) within the CAPN10 gene. The at-risk genotype was homozygous for the UCSNP-43 G allele. In the present study among Pima Indians, the UCSNP-43 G/G genotype was not associated with an increased prevalence of type 2 diabetes. However, Pima Indians with normal glucose tolerance, who have a G/G genotype at UCSNP-43, were found to have decreased rates of postabsorptive and insulin-stimulated glucose turnover that appear to result from decreased rates of glucose oxidation. In addition, G/G homozygotes were found to have reduced CAPN10 mRNA expression in their skeletal muscle. A decreased rate of insulin-mediated glucose turnover, or insulin resistance, is one mechanism by which the polymorphism in CAPN10 may increase susceptibility to type 2 diabetes mellitus in older persons.

V EGF is a secreted mitogen associated with angiogenesis and is also a potent vascular permeability factor. The biological role of VEGF in the ischemic brain remains unknown. This study was undertaken to investigate whether VEGF enhances cerebral microvascular perfusion and increases blood-brain barrier (BBB) leakage in the ischemic brain. Using magnetic resonance imaging (MRI), three-dimensional laser-scanning confocal microscope, and functional neurological tests, we measured the effects of administrating recombinant human VEGF₁₆₅ (rhVEGF₁₆₅) on angiogenesis, functional neurological outcome, and BBB leakage in a rat model of focal cerebral embolic ischemia. Late (48 hours) administration of rhVEGF₁₆₅ to the ischemic rats enhanced angiogenesis in the ischemic penumbra and significantly improved neurological recovery. However, early postischemic (1 hour) administration of rhVEGF₁₆₅ to ischemic rats significantly increased BBB leakage, hemorrhagic transformation, and ischemic lesions. Administration of rhVEGF₁₆₅ to ischemic rats did not change BBB leakage and cerebral plasma perfusion in the contralateral hemisphere. Our results indicate that VEGF can markedly enhance angiogenesis in the ischemic brain and reduce neurological deficits during stroke recovery and that inhibition of VEGF at the acute stage of stroke may reduce the BBB permeability and the risk of hemorrhagic transformation after focal cerebral ischemia.

D espite prolonged treatment with highly active antiretroviral therapy (HAART), infectious HIV-1 continues to replicate and to reside latently in resting memory CD4⁺ T lymphocytes, creating a major obstacle to HIV-1 eradication. It is therefore not surprising to observe a prompt viral rebound after discontinuation of HAART. The nature of the rebounding virus, however, remains undefined. We now report on the genetic characterization of rebounding viruses in eight patients in whom plasma viremia was undetectable throughout about 3 years of HAART. Taking advantage of the extensive length polymorphism in HIV-1 env, we found that in five patients who did not show HIV-1 replication during treatment, the rebound virus was identical to those isolated from the latent reservoir. In three other patients, two of whom had been free of plasma viremia but had showed some residual viral replication, the rebound virus was genetically different from the latent reservoir virus, corresponding instead to minor viral variants detected during the course of treatment in lymphoid tissues. We conclude that in cases with apparent complete HIV-1 suppression by HAART, viral rebound after cessation of therapy could have originated from the activation of virus from the latent reservoir. In patients with incomplete suppression by chemotherapy, however, the viral rebound is likely triggered by ongoing, low-level replication of HIV-1, perhaps occurring in lymphoid tissues.

C ardiac mitochondrial function is altered in a variety of inherited and acquired cardiovascular diseases. Recent studies have identified the transcriptional coactivator peroxisome proliferator–activated receptor γ coactivator-1 (PGC-1) as a regulator of mitochondrial function in tissues specialized for thermogenesis, such as brown adipose. We sought to determine whether PGC-1 controlled mitochondrial biogenesis and energy-producing capacity in the heart, a tissue specialized for high-capacity ATP production. We found that PGC-1 gene expression is induced in the mouse heart after birth and in response to short-term fasting, conditions known to increase cardiac mitochondrial energy production. Forced expression of PGC-1 in cardiac myocytes in culture induced the expression of nuclear and mitochondrial genes involved in multiple mitochondrial energy-transduction/energy-production pathways, increased cellular mitochondrial number, and stimulated coupled respiration. Cardiac-specific overexpression of PGC-1 in transgenic mice resulted in uncontrolled mitochondrial proliferation in cardiac myocytes leading to loss of sarcomeric structure and a dilated cardiomyopathy. These results identify PGC-1 as a critical regulatory molecule in the control of cardiac mitochondrial number and function in response to energy demands.

M MP activity with disruption of structural collagen has been implicated in the pathophysiology of dilated cardiomyopathy. To examine the role of this enzyme in cardiac function, a transgenic mouse was created that constitutively expressed human collagenase (MMP-1) in the heart. At 6 months of age, these animals demonstrated compensatory myocyte hypertrophy with an increase in the cardiac collagen concentration due to elevated transcription of type III collagen. Chronic myocardial expression of MMP-1 produced loss of cardiac interstitial collagen coincident with a marked deterioration of systolic and diastolic function at 12 months of age. This is the first animal model demonstrating that direct disruption of the extracellular matrix in the heart reproduces the changes observed in the progression of human heart failure.

I n North America, liver disease due to alcohol consumption is an important cause of death in adults, although its pathogenesis remains obscure. Despite the fact that resident hepatic macrophages are known to contribute to early alcohol-induced liver injury via oxidative stress, the exact source of free radicals has remained a mystery. To test the hypothesis that NADPH oxidase is the major source of oxidants due to ethanol, we used p47^phox knockout mice, which lack a critical subunit of this major source of reactive oxygen species in activated phagocytes. Mice were treated with ethanol chronically, using a Tsukamoto-French protocol, for 4 weeks. In wild-type mice, ethanol caused severe liver injury via a mechanism involving gut-derived endotoxin, CD14 receptor, production of electron spin resonance–detectable free radicals, activation of the transcription factor NF-κB, and release of cytotoxic TNF-α from activated Kupffer cells. In NADPH oxidase–deficient mice, neither an increase in free radical production, activation of NF-κB, an increase in TNF-α mRNA, nor liver pathology was observed. These data strongly support the hypothesis that free radicals from NADPH oxidase in hepatic Kupffer cells play a predominant role in the pathogenesis of early alcohol-induced hepatitis by activating NF-κB, which activates production of cytotoxic TNF-α.

A ntithrombin is a plasma protease inhibitor that inhibits thrombin and contributes to the maintenance of blood fluidity. Using targeted gene disruption, we investigated the role of antithrombin in embryogenesis. Mating mice heterozygous for antithrombin gene (ATIII) disruption, ATIII^+/–, yielded the expected Mendelian distribution of genotypes until 14.5 gestational days (gd). However, approximately 70% of the ATIII^–/– embryos at 15.5 gd and 100% at 16.5 gd had died and showed extensive subcutaneous hemorrhage. Histological examination of those embryos revealed extensive fibrin(ogen) deposition in the myocardium and liver, but not in the brain or lung. Furthermore, no apparent fibrin(ogen) deposition was detected in the extensive hemorrhagic region, suggesting that fibrinogen might be decreased due to consumptive coagulopathy and/or liver dysfunction. These findings suggest that antithrombin is essential for embryonic survival and that it plays an important role in regulation of blood coagulation in the myocardium and liver.

W e previously reported that active sensitization of rats resulted in the appearance of a unique system for rapid and specific antigen uptake across intestinal epithelial cells. The current studies used rats sensitized to horseradish peroxidase (HRP) to define the essential components of this antigen transport system. Sensitization of rats to HRP stimulated increased HRP uptake into enterocytes (significantly larger area of HRP-containing endosomes) and more rapid transcellular transport compared with rats sensitized to an irrelevant protein or naive control rats. Whole serum but not IgE-depleted serum from sensitized rats was able to transfer the enhanced antigen transport phenomenon. Immunohistochemistry demonstrated that sensitization induced expression of CD23, the low-affinity IgE receptor (FcεRII), on epithelial cells. The number of immunogold-labeled CD23 receptors on the enterocyte microvillous membrane was significantly increased in sensitized rats and was subsequently reduced after antigen challenge when CD23 and HRP were localized within the same endosomes. Finally, pretreatment of tissues with luminally added anti-CD23 antibody significantly inhibited both antigen transport and the hypersensitivity reaction. Our results provide evidence that IgE antibodies bound to low-affinity receptors on epithelial cells are responsible for the specific and rapid nature of this novel antigen transport system.

T o investigate the role of IL-6 in alcohol-mediated osteoporosis, we measured a variety of bone remodeling parameters in wild-type (il6^+/+) or IL-6 gene knockout (il6^–/–) mice that were fed either control or ethanol liquid diets for 4 months. In the il6^+/+ mice, ethanol ingestion decreased bone mineral density, as determined by dual-energy densitometry; decreased cancellous bone volume and trabecular width and increased trabecular spacing and osteoclast surface, as determined by histomorphometry of the femur; increased urinary deoxypyridinolines, as determined by ELISA; and increased CFU-GM formation and osteoclastogenesis as determined ex vivo in bone marrow cell cultures. In contrast, ethanol ingestion did not alter any of these parameters in the il6^–/– mice. Ethanol increased receptor activator of NF-κB ligand (RANKL) mRNA expression in the bone marrow of il6^+/+ but not il6^–/– mice. Additionally, ethanol decreased several osteoblastic parameters including osteoblast perimeter and osteoblast culture calcium retention in both il6^+/+ and il6^–/– mice. These findings demonstrate that ethanol induces bone loss through IL-6. Furthermore, they suggest that IL-6 achieves this effect by inducing RANKL and promoting CFU-GM formation and osteoclastogenesis.

A TP-sensitive potassium channels play a major role in linking metabolic signals to the exocytosis of insulin in the pancreatic β cell. These channels consist of two types of protein subunit: the sulfonylurea receptor SUR1 and the inward rectifying potassium channel Kir6.2. Mutations in the genes encoding these proteins are the most common cause of congenital hyperinsulinism (CHI). Since 1973, we have followed up 38 pediatric CHI patients in Finland. We reported previously that a loss-of-function mutation in SUR1 (V187D) is responsible for CHI of the most severe cases. We have now identified a missense mutation, E1506K, within the second nucleotide binding fold of SUR1, found heterozygous in seven related patients with CHI and in their mothers. All patients have a mild form of CHI that usually can be managed by long-term diazoxide treatment. This clinical finding is in agreement with the results of heterologous coexpression studies of recombinant Kir6.2 and SUR1 carrying the E1506K mutation. Mutant K_ATP channels were insensitive to metabolic inhibition, but a partial response to diazoxide was retained. Five of the six mothers, two of whom suffered from hypoglycemia in infancy, have developed gestational or permanent diabetes. Linkage and haplotype analysis supported a dominant pattern of inheritance in a large pedigree. In conclusion, we describe the first dominantly inherited SUR1 mutation that causes CHI in early life and predisposes to later insulin deficiency.

C hromogranin A (CgA) is the major soluble protein in the core of catecholamine-storage vesicles and is also distributed widely in secretory vesicles throughout the neuroendocrine system. CgA contains the sequences for peptides that modulate catecholamine release, but the proteases responsible for the release of these bioactive peptides from CgA have not been established. We show here that the major fibrinolytic enzyme, plasmin, can cleave CgA to form a series of large fragments as well as small trichloroacetic acid-soluble peptides. Peptides generated by plasmin-mediated cleavage of CgA significantly inhibited nicotinic cholinergic stimulation of catecholamine release from PC12 cells and primary bovine adrenal chromaffin cells. We also show that the zymogen, plasminogen, as well as tissue plasminogen activator bind saturably and with high capacity to catecholaminergic (PC12) cells. Occupancy of cell surface binding sites promoted the cleavage of CgA by plasmin. Positive and negative modulation of the local cellular fibrinolytic system resulted in substantial alterations in catecholamine release. These results suggest that catecholaminergic cells express binding sites that localize fibrinolytic molecules on their surfaces to promote plasminogen activation and proteolytic processing of CgA in the environment into which CgA is secreted to generate peptides which may regulate neuroendocrine secretion. Interactions between CgA and plasmin(ogen) define a previously unrecognized autocrine/paracrine system that may have a dramatic impact upon catecholamine secretion.