Ping H. Wang

Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases.

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+CCR7−CD45RA− effector memory T cells (TEM cells) with elevated Kv1.3 potassium channel expression. In contrast, T cells with other antigen specificities from these patients, or autoreactive T cells from healthy individuals and disease controls, express low levels of Kv1.3 and are predominantly naïve or central-memory (TCM) cells. In TEM cells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvβ2, SAP97, ZIP, p56lck, and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation, they suppress Ca2+-signaling, cytokine production, and proliferation of autoantigen-specific TEM cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted.

Relationship between obesity and diabetes in a US adult population: findings from the National Health and Nutrition Examination Survey, 1999-2006.

BackgroundObesity is one of the most important modifiable risk factors for the prevention of type 2 diabetes. The aim of this study was to examine the prevalence of diabetes with increasing severity of obesity and the distribution of HbA1c levels in diabetics participating in the latest National Health and Nutrition Examination Survey (NHANES).MethodsData from a representative sample of adults with diabetes participating in the NHANES between 1999 and 2006 were reviewed. The prevalence of diabetes and levels of fasting glucose, insulin, c-peptide, and HbA1c were examined across different weight classes with normal weight, overweight, and obesity classes 1, 2, and 3 were defined as body mass index (BMI) of <25.0, 25.0–29.9, 30.0–34.9, 35.0–39.9, and equal to 40.0, respectively. The distribution of HbA1c levels among adults with diabetes was also examined.ResultsThere were 2,894 adults with diabetes (13.6%) among the 21,205 surveyed participants. Among the adults with diabetes, the mean age was 59xa0years, the mean fasting glucose was 155u2009±u20092xa0mg/dl, and the mean HbA1c was 7.2%; 80.3% of diabetics were considered overweight (BMIu2009≥u200925) and 49.1% of diabetics were considered obese (BMIu2009≥u200930). The prevalence of adults with diabetes increased with increasing weight classes, from 8% for normal weight individuals to 43% for individuals with obesity class 3; the distribution of HbA1c levels were considered as good (<7.0%) in 60%, fair (7.0–8.0%) in 17%, and poor (>8.0%) in 23%. The mean fasting glucose and HbA1c levels were highest for diabetics with BMI <25.0, suggesting a state of higher severity of disease. Mean insulin and c-peptide levels were highest for diabetics with BMIu2009=u200935.0, suggesting a state of insulin resistance.ConclusionsIn a nationally representative sample of US adults, the prevalence of diabetes increases with increasing weight classes. Nearly one fourth of adults with diabetes have poor glycemic control and nearly half of adult diabetics are considered obese suggesting that weight loss is an important intervention in an effort to reduce the impact of diabetes on the health care system.

Hsp10 and Hsp60 modulate Bcl-2 family and mitochondria apoptosis signaling induced by doxorubicin in cardiac muscle cells.

The development of doxorubicin cardiomyopathy involves apoptosis of cardiac muscle cells. This study was carried out to define the roles of two heat-shock proteins, Hsp10 and Hsp60, on doxorubicin-induced apoptosis in primary cardiomyocytes. Doxorubicin induces apoptosis of cardiomyocytes by activating mitochondria apoptosis signaling. Transducing cardiomyocytes with Hsp10 or Hsp60 with adenoviral vector suppressed the occurrence of apoptosis in the doxorubicin-treated cardiomyocytes. Overexpression of Hsp10 and Hsp60 increased the abundance of the anti-apoptotic Bcl-xl and Bcl-2, and reduced the protein content of the pro-apoptotic Bax. Hsp60 overexpression also significantly reduced doxorubicin induction of Bad, whereas overexpression of Hsp10 did not alter the expression of Bad in the doxorubicin-treated cells. Overexpression of Hsp10 and Hsp60, respectively, stabilized mitochondrial cross-membrane potential, inhibited Caspase 3, and suppressed PARP. These findings indicate that overexpression of Hsp10 and Hsp60 differentially modulated Bcl-2 family and in turn attenuate doxorubicin-induced cardiac muscle death. The effects of Hsp10 and Hsp60 on Bcl-2 family could not be explained by the abundance of Bcl-2 family mRNA levels. Hsp60 interacted with Bcl-xl and Bax in the cardiomyocytes in vivo. The effect of Hsp10 and Hsp60 on the abundance of Bcl-xl could not be blocked by cycloheximide. Moreover, Hsp10 and Hsp60 inhibited ubiquitination of Bcl-xl. These findings suggest that Hsp10 and Hsp60 modulated post-translational modification of Bcl-xl. Antisense Hsp60 reduced the abundance of endogenous Hsp60 in cardiomyocytes and amplified the cytotoxicity of doxorubicin. These data provide a novel link between Hsp10/Hsp60 and cardiac protection in doxorubicin cardiomyopathy.

Mitochondrial DNA variant associated with Leber hereditary optic neuropathy and high-altitude Tibetans

The distinction between mild pathogenic mtDNA mutations and population polymorphisms can be ambiguous because both are homoplasmic, alter conserved functions, and correlate with disease. One possible explanation for this ambiguity is that the same variant may have different consequences in different contexts. The NADH dehydrogenase subunit 1 (ND1) nucleotide 3394 T > C (Y30H) variant is such a case. This variant has been associated with Leber hereditary optic neuropathy and it reduces complex I activity and cellular respiration between 7% and 28% on the Asian B4c and F1 haplogroup backgrounds. However, complex I activity between B4c and F1 mtDNAs, which harbor the common 3394T allele, can also differ by 30%. In Asia, the 3394C variant is most commonly associated with the M9 haplogroup, which is rare at low elevations but increases in frequency with elevation to an average of 25% of the Tibetan mtDNAs (odds ratio = 23.7). In high-altitude Tibetan and Indian populations, the 3394C variant occurs on five different macrohaplogroup M haplogroup backgrounds and is enriched on the M9 background in Tibet and the C4a4 background on the Indian Deccan Plateau (odds ratio = 21.9). When present on the M9 background, the 3394C variant is associated with a complex I activity that is equal to or higher than that of the 3394T variant on the B4c and F1 backgrounds. Hence, the 3394C variant can either be deleterious or beneficial depending on its haplogroup and environmental context. Thus, this mtDNA variant fulfills the criteria for a common variant that predisposes to a “complex” disease.

Insulin stimulates Akt translocation to mitochondria: Implications on dysregulation of mitochondrial oxidative phosphorylation in diabetic myocardium

Mitochondrial oxidative phosphorylation is the major source of energy in cardiac muscle. In the streptozotocin-induced diabetic (STZ-DM) mice, myocardial oxidative phosphorylation was perturbated and oxidative phosphorylation complex V (ATP synthase) activity was significantly reduced. To determine the independent effects of hyperglycemia and insulin deficiency on the changes of myocardial complex V, we used phlorizin (Ph) to normalize blood glucose in the diabetic mice. Ph treatment did not improve myocardial complex V activity in the STZ-DM mice, whereas insulin treatment normalized myocardial complex V activity in the diabetic mice. Therefore, the reduction of complex V activity was caused by insulin deficiency and not by hyperglycemia in STZ-DM myocardium. Acute insulin stimulation induced phosphorylation of Akt and translocation of Akt to mitochondria in myocardium. Translocation of phospho-Akt to mitochondria was enhanced in the STZ-DM mice and was blunted in the diet-induced diabetic mice. In parallel, insulin activation of complex V was enhanced in the STZ-DM myocardium and suppressed in the diet-induced diabetic myocardium. In vivo inhibition of Akt blocked insulin stimulation of phospho-Akt translocation and blunted activation of complex V. Insulin-activated Akt translocation to mitochondria in cardiac muscle is a novel paradigm that may have important implications on myocardial bioenergetics.

Mechanisms of Mitochondrial Damage in Keratinocytes by Pemphigus Vulgaris Antibodies

Background: Previous studies suggested that mitochondrial antibodies contribute to pemphigus vulgaris (PV). Results: PV sera elicited mitochondrial damage, and mitochondria-protecting drugs exhibited protective effect in cell culture and mouse skin. Conclusion: PV antibodies altered O2 respiration, disrupted electron transfer chain, and increased reactive oxygen species. Significance: Results provide the mechanism of therapeutic action and justify the use of mitochondria-protecting drugs in PV. The development of nonhormonal treatment of pemphigus vulgaris (PV) has been hampered by a lack of clear understanding of the mechanisms leading to keratinocyte (KC) detachment and death in pemphigus. In this study, we sought to identify changes in the vital mitochondrial functions in KCs treated with the sera from PV patients and healthy donors. PV sera significantly increased proton leakage from KCs, suggesting that PV IgGs increase production of reactive oxygen species. Indeed, measurement of intracellular reactive oxygen species production showed a drastic increase of cell staining in response to treatment by PV sera, which was confirmed by FACS analysis. Exposure of KCs to PV sera also caused dramatic changes in the mitochondrial membrane potential detected with the JC-1 dye. These changes can trigger the mitochondria-mediated intrinsic apoptosis. Although sera from different PV patients elicited unique patterns of mitochondrial damage, the mitochondria-protecting drugs nicotinamide (also called niacinamide), minocycline, and cyclosporine A exhibited a uniform protective effect. Their therapeutic activity was validated in the passive transfer model of PV in neonatal BALB/c mice. The highest efficacy of mitochondrial protection of the combination of these drugs found in mitochondrial assay was consistent with the ability of the same drug combination to abolish acantholysis in mouse skin. These findings provide a theoretical background for clinical reports of the efficacy of mitochondria-protecting drugs in PV patients. Pharmacological protection of mitochondria and/or compensation of an altered mitochondrial function may therefore become a novel approach to development of personalized nonhormonal therapies of patients with this potentially lethal autoimmune blistering disease.

Mitochondrial Akt-regulated mitochondrial apoptosis signaling in cardiac muscle cells

We recently reported translocation and activation of Akt in cardiac mitochondria. This study was to determine whether activation of Akt in mitochondria could inhibit apoptosis of cardiac muscle cells. Insulin stimulation induced translocation of phosphorylated Akt to the mitochondria in primary cardiomyocytes. A mitochondria-targeted constitutively active Akt was overexpressed via adenoviral vector and inhibited efflux of cytochrome c and apoptosis-inducing factor from mitochondria to cytosol and partially prevented loss of mitochondria cross-membrane electrochemical gradient. Activation of caspase 3 was suppressed in the cardiomyocytes transduced with mitochondria-targeted active Akt, whereas a mitochondria-targeted dominant negative Akt enhanced activation of caspase 3. Terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling assay showed that mitochondrial activation of Akt significantly reduced the number of apoptotic cells. When the endogenous Akt was abolished by LY294002, the antiapoptotic actions of mitochondrial Akt remained effective. These experiments suggested that mitochondrial Akt suppressed apoptosis signaling independent of cytosolic Akt in cardiac muscle cells.

18F-Fallypride PET of Pancreatic Islets: In Vitro and In Vivo Rodent Studies

Islet cell loss in the pancreas results in diabetes. A noninvasive method that measures islet cell loss and also tracks the fate of transplanted islets would facilitate the development of novel therapeutics and improve the management of diabetes. We describe a novel dopamine D2/D3 receptor (D2/D3R)–based PET method to study islet cells in the rat pancreas and in islet cell transplantation. Methods: 18F-fallypride binding to isolated rat islets and pancreas was evaluated in the absence and presence of the D2/D3R inhibitor haloperidol. After intravenous 18F-fallypride (28–37 MBq) administration, normal rats and rats pretreated with haloperidol were imaged in a PET/CT scanner and subsequently studied ex vivo for 18F-fallypride localization in the pancreas. A streptozotocin-treated diabetic rat model was used to study localization of 18F-fallypride in the pancreas, in vitro and ex vivo. Rat islet cells were transplanted into the spleen and visualized using 18F-fallypride PET. Results: 18F-fallypride bound to isolated islet cells and pancreatic sections with an endocrine or exocrine selectivity of approximately 4; selectivity was reduced by haloperidol, suggesting that binding was D2/D3R-specific. Chemical destruction of islets by streptozotocin decreased 18F-fallypride binding in pancreas by greater than 50%, paralleling the decrease in insulin immunostaining. Uptake of 18F-fallypride in the pancreas was confirmed by radiochromatography and was 0.05% injected dose/cm3 as measured by PET/CT. The ratio of 18F-fallypride uptake in the pancreas to reference tissue (erector spinae muscle) was 5.5. Rat islets transplanted into the spleen were visualized in vivo by 18F-fallypride and confirmed by immunostaining. The ratio of spleen-transplanted islets to erector spinae muscle was greater than 5, compared with a ratio of 2.8 in untransplanted rats. Conclusion: These studies demonstrate the potential utility of 18F-fallypride as a PET agent for islet cells.

Critical Role of the Neonatal Fc Receptor (FcRn) in the Pathogenic Action of Antimitochondrial Autoantibodies Synergizing with Anti-desmoglein Autoantibodies in Pemphigus Vulgaris.

Background: Patients with pemphigus vulgaris develop antibodies to both cell adhesion molecules and intracellular proteins. Results: On the keratinocyte cell membrane, pemphigus autoantibodies form complexes with FcRn internalizing and trafficking them to mitochondria. Conclusion: The pathogenic role of antimitochondrial autoantibodies is complementary to that of anti-desmoglein autoantibodies, because both are required to disrupt the epidermal barrier. Significance: FcRn represents a common acceptor protein for internalization of autoantibodies to intracellular proteins. Pemphigus vulgaris (PV) is a life-long, potentially fatal IgG autoantibody-mediated blistering disease targeting mucocutaneous keratinocytes (KCs). PV patients develop pathogenic anti-desmoglein (Dsg) 3 ± 1 and antimitochondrial antibodies (AMA), but it remained unknown whether and how AMA enter KCs and why other cell types are not affected in PV. Therefore, we sought to elucidate mechanisms of cell entry, trafficking, and pathogenic action of AMA in PV. We found that PVIgGs associated with neonatal Fc receptor (FcRn) on the cell membrane, and the PVIgG-FcRn complexes entered KCs and reached mitochondria where they dissociated. The liberated AMA altered mitochondrial membrane potential, respiration, and ATP production and induced cytochrome c release, although the lack or inactivation of FcRn abolished the ability of PVIgG to reach and damage mitochondria and to cause detachment of KCs. The assays of mitochondrial functions and keratinocyte adhesion demonstrated that although the pathobiological effects of AMA on KCs are reversible, they become irreversible, leading to epidermal blistering (acantholysis), when AMA synergize with anti-Dsg antibodies. Thus, it appears that AMA enter a keratinocyte in a complex with FcRn, become liberated from the endosome in the cytosol, and are trafficked to the mitochondria, wherein they trigger pro-apoptotic events leading to shrinkage of basal KCs uniquely expressing FcRn in epidermis. During recovery, KCs extend their cytoplasmic aprons toward neighboring cells, but anti-Dsg antibodies prevent assembly of nascent desmosomes due to steric hindrance, thus rendering acantholysis irreversible. In conclusion, FcRn is a common acceptor protein for internalization of AMA and, perhaps, for PV autoantibodies to other intracellular antigens, and PV is a novel disease paradigm for investigating and elucidating the role of FcRn in this autoimmune disease and possibly other autoimmune diseases.

Pemphigus vulgaris antibodies target the mitochondrial nicotinic acetylcholine receptors that protect keratinocytes from apoptolysis

The mechanism of detachment and death of keratinocytes in pemphigus vulgaris (PV) involves pro-apoptotic action of constellations of autoantibodies determining disease severity and response to treatment. The presence of antibodies to nicotinic acetylcholine receptors (nAChRs) and the therapeutic efficacy of cholinomimetics in PV is well-established. Recently, adsorption of anti-mitochondrial antibodies abolished the ability of PVIgGs to cause acantholysis, demonstrating their pathophysiological significance. Since, in addition to cell membrane, nAChRs are also present on the mitochondrial outer membrane, wherein they act to prevent activation of intrinsic (mitochondrial apoptosis), we hypothesized that mitochondrial (mt)-nAChRs might be targeted by PVIgGs. To test this hypothesis, we employed the immunoprecipitation-western blot assay of keratinocyte mitochondrial proteins that visualized the α3, α5, α7, α9, α10, β2 and β4 mt-nAChR subunits precipitated by PV IgGs, suggesting that functions of mt-nAChRs are compromised in PV. To pharmacologically counteract the pro-apoptotic action of anti-mitochondrial antibodies in PV, we exposed naked keratinocyte mitochondria to PVIgGs in the presence of the nicotinic agonist nicotine ± antagonists, and measured cytochrome c (CytC) release. Nicotine abolished PVIgG-dependent CytC release, showing a dose-dependent effect, suggesting that protection of mitochondria can be a novel mechanism of therapeutic action of nicotinic agonists in PV. The obtained results indicated that the mt-nAChRs targeted by anti-mitochondrial antibodies produced by PV patients are coupled to inhibition of CytC release, and that nicotinergic stimulation can abolish PVIgG-dependent activation of intrinsic apoptosis in KCs. Future studies should determine if and how the distinct anti-mt-nAChR antibodies penetrate KCs and correlate with disease severity.