Nazar Mashtalir

The leptin-deficient (ob/ob) mouse: a new animal model of peripheral neuropathy of type 2 diabetes and obesity.

Whereas functional, metabolic, neurotrophic, and morphological abnormalities of peripheral diabetic neuropathy (PDN) have been extensively explored in streptozotocin-induced diabetic rats and mice (models of type 1 diabetes), insufficient information is available on manifestations and pathogenetic mechanisms of PDN in type 2 diabetic models. The latter could constitute a problem for clinical trial design because the vast majority of subjects with diabetes have type 2 (non–insulin dependent) diabetes. This study was aimed at characterization of PDN in leptin-deficient (ob/ob) mice, a model of type 2 diabetes with relatively mild hyperglycemia and obesity. ob/ob mice (∼11 weeks old) clearly developed manifest sciatic motor nerve conduction velocity (MNCV) and hind-limb digital sensory nerve conduction velocity (SNCV) deficits, thermal hypoalgesia, tactile allodynia, and a remarkable (∼78%) loss of intraepidermal nerve fibers. They also had increased sorbitol pathway activity in the sciatic nerve and increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve, spinal cord, and dorsal root ganglion (DRG). Aldose reductase inhibition with fidarestat (16 mg · kg−1 · d−1), administered to ob/ob mice for 6 weeks starting from 5 weeks of age, was associated with preservation of normal MNCV and SNCV and alleviation of thermal hypoalgesia and intraepidermal nerve fiber loss but not tactile allodynia. Sciatic nerve nitrotyrosine immunofluorescence and the number of poly(ADP-ribose)–positive nuclei in sciatic nerve, spinal cord, and DRGs of fidarestat-treated ob/ob mice did not differ from those in nondiabetic controls. In conclusion, the leptin-deficient ob/ob mouse is a new animal model that develops both large motor and sensory fiber and small sensory fiber PDN and responds to pathogenetic treatment. The results support the role for increased aldose reductase activity in functional and structural changes of PDN in type 2 diabetes.

High-Fat Diet–Induced Neuropathy of Pre-Diabetes and Obesity: Effects of “Healthy” Diet and Aldose Reductase Inhibition

OBJECTIVE—Subjects with dietary obesity and pre-diabetes have an increased risk for developing both nerve conduction slowing and small sensory fiber neuropathy. Animal models of this type of neuropathy have not been described. This study evaluated neuropathic changes and their amenability to dietary and pharmacological interventions in mice fed a high-fat diet (HFD), a model of pre-diabetes and alimentary obesity. RESEARCH DESIGN AND METHODS—Female C57BL6/J mice were fed normal diets or HFDs for 16 weeks. RESULTS—HFD-fed mice developed obesity, increased plasma FFA and insulin concentrations, and impaired glucose tolerance. They also had motor and sensory nerve conduction deficits, tactile allodynia, and thermal hypoalgesia in the absence of intraepidermal nerve fiber loss or axonal atrophy. Despite the absence of overt hyperglycemia, the mice displayed augmented sorbitol pathway activity in the peripheral nerve, as well as 4-hydroxynonenal adduct nitrotyrosine and poly(ADP-ribose) accumulation and 12/15-lipoxygenase overexpression in peripheral nerve and dorsal root ganglion neurons. A 6-week feeding with normal chow after 16 weeks on HFD alleviated tactile allodynia and essentially corrected thermal hypoalgesia and sensory nerve conduction deficit without affecting motor nerve conduction slowing. Normal chow containing the aldose reductase inhibitor fidarestat (16 mg · kg−1· day −1) corrected all functional changes of HFD-induced neuropathy. CONCLUSIONS—Similar to human subjects with pre-diabetes and obesity, HFD-fed mice develop peripheral nerve functional, but not structural, abnormalities and, therefore, are a suitable model for evaluating dietary and pharmacological approaches to halt progression and reverse diabetic neuropathy at the earliest stage of the disease.

The Ubiquitin Carboxyl Hydrolase BAP1 Forms a Ternary Complex with YY1 and HCF-1 and Is a Critical Regulator of Gene Expression

ABSTRACT The candidate tumor suppressor BAP1 is a deubiquitinating enzyme (DUB) involved in the regulation of cell proliferation, although the molecular mechanisms governing its function remain poorly defined. BAP1 was recently shown to interact with and deubiquitinate the transcriptional regulator host cell factor 1 (HCF-1). Here we show that BAP1 assembles multiprotein complexes containing numerous transcription factors and cofactors, including HCF-1 and the transcription factor Yin Yang 1 (YY1). Through its coiled-coil motif, BAP1 directly interacts with the zinc fingers of YY1. Moreover, HCF-1 interacts with the middle region of YY1 encompassing the glycine-lysine-rich domain and is essential for the formation of a ternary complex with YY1 and BAP1 in vivo. BAP1 activates transcription in an enzymatic-activity-dependent manner and regulates the expression of a variety of genes involved in numerous cellular processes. We further show that BAP1 and HCF-1 are recruited by YY1 to the promoter of the cox7c gene, which encodes a mitochondrial protein used here as a model of BAP1-activated gene expression. Our findings (i) establish a direct link between BAP1 and the transcriptional control of genes regulating cell growth and proliferation and (ii) shed light on a novel mechanism of transcription regulation involving ubiquitin signaling.

Poly(ADP-ribose) polymerase inhibition alleviates experimental diabetic sensory neuropathy

Poly(ADP-ribose) polymerase (PARP) activation is emerging as a fundamental mechanism in the pathogenesis of diabetes complications including diabetic neuropathy. This study evaluated the role of PARP in diabetic sensory neuropathy. The experiments were performed in control and streptozotocin-induced diabetic rats treated with or without the PARP inhibitor 1,5-isoquinolinediol (ISO; 3 mg · kg−1 · day−1 i.p.) for 2 weeks after 2 weeks without treatment. Diabetic rats developed thermal hyperalgesia (assessed by paw-withdrawal and tail-flick tests), mechanical hyperalgesia (von Frey anesthesiometer/rigid filaments and Randall-Sellito tests), tactile allodynia (flexible von Frey filaments), and increased flinching behavior in phases 1 and 2 of the 2% formalin pain test. They also had clearly manifest increase in nitrotyrosine and poly(ADP-ribose) immunoreactivities in the sciatic nerve and increased superoxide formation (hydroxyethidine method) and nitrotyrosine immunoreactivity in vasa nervorum. ISO treatment alleviated abnormal sensory responses, including thermal and mechanical hyperalgesia and tactile allodynia as well as exaggerated formalin flinching behavior in diabetic rats, without affecting the aforementioned variables in the control group. Poly(ADP-ribose) and, to a lesser extent, nitrotyrosine abundance in sciatic nerve, as well as superoxide and nitrotyrosine formation in vasa nervorum, were markedly reduced by ISO therapy. Apoptosis in dorsal root ganglion neurons (transferase-mediated dUTP nick-end labeling assay) was not detected in any of the groups. In conclusion, PARP activation contributes to early diabetic sensory neuropathy by mechanisms that may include oxidative stress but not neuronal apoptosis.

Adipogenic Human Adenovirus Ad-36 Induces Commitment, Differentiation, and Lipid Accumulation in Human Adipose-Derived Stem Cells

Human adenovirus Ad‐36 is causatively and correlatively linked with animal and human obesity, respectively. Ad‐36 enhances differentiation of rodent preadipocytes, but its effect on adipogenesis in humans is unknown. To indirectly assess the role of Ad‐36‐induced adipogenesis in human obesity, the effect of the virus on commitment, differentiation, and lipid accumulation was investigated in vitro in primary human adipose‐derived stem/stromal cells (hASC). Ad‐36 infected hASC in a time‐ and dose‐dependent manner. Even in the presence of osteogenic media, Ad‐36‐infected hASC showed significantly greater lipid accumulation, suggestive of their commitment to the adipocyte lineage. Even in the absence of adipogenic inducers, Ad‐36 significantly increased hASC differentiation, as indicated by a time‐dependent expression of genes within the adipogenic cascade—CCAAT/Enhancer binding protein‐β, peroxisome proliferator‐activated receptor‐γ, and fatty acid‐binding protein—and consequentially increased lipid accumulation in a time‐ and viral dose‐dependent manner. Induction of hASC to the adipocyte state by Ad‐36 was further supported by increased expression of lipoprotein lipase and the accumulation of its extracellular fraction. hASC from subjects harboring Ad‐36 DNA in their adipose tissue due to natural infection had significantly greater ability to differentiate compared with Ad‐36 DNA‐negative counterparts, which offers a proof of concept. Thus, Ad‐36 has the potential to induce adipogenesis in hASC, which may contribute to adiposity induced by the virus.

Human adenovirus Ad-36 induces adipogenesis via its E4 orf-1 gene.

Objective:Understanding the regulation of adipocyte differentiation by cellular and extracellular factors is crucial for better management of chronic conditions such as obesity, insulin resistance and lipodystrophy. Experimental infection of rats with a human adenovirus type 36 (Ad-36) improves insulin sensitivity and promotes adipogenesis, reminiscent of the effect of thiozolinediones. Therefore, we investigated the role of Ad-36 as a novel regulator of the adipogenic process.Design and Results:Even in the absence of adipogenic inducers, infection of 3T3-L1 preadipocytes and human adipose-derived stem cells (hASC) by Ad-36, but not Ad-2 that is another human adenovirus, modulated regulatory points that spanned the entire adipogenic cascade ranging from the upregulation of cAMP, phosphatidylinositol 3-kinase and p38 signaling pathways, downregulation of Wnt10b expression, and increased expression of CCAAT/enhancer binding protein-β and peroxisome proliferator-activated receptor γ2 and consequential lipid accumulation. Next, we identified that E4 open reading frame (orf)-1 gene of the virus is necessary and sufficient for Ad-36-induced adipogenesis. Selective knockdown of E4 orf-1 by RNAi abrogated Ad-36-induced adipogenic signaling cascade in 3T3-L1 cells and hASC. Compared to the null vector, selective expression of Ad-36 E4 orf-1 in 3T3-L1 induced adipogenesis, which was abrogated when the PDZ-binding domain of the protein was deleted.Conclusion:Thus, Ad-36 E4 orf-1 is a novel inducer of rodent and human adipocyte differentiation process.

Human Adenovirus Type 36 Enhances Glucose Uptake in Diabetic and Nondiabetic Human Skeletal Muscle Cells Independent of Insulin Signaling

OBJECTIVE—Human adenovirus type 36 (Ad-36) increases adiposity but improves insulin sensitivity in experimentally infected animals. We determined the ability of Ad-36 to increase glucose uptake by human primary skeletal muscle (HSKM) cells. RESEARCH DESIGN AND METHODS—The effect of Ad-36 on glucose uptake and cell signaling was determined in HSKM cells obtained from type 2 diabetic and healthy lean subjects. Ad-2, another human adenovirus, was used as a negative control. Gene expression and proteins of GLUT1 and GLUT4 were measured by real-time PCR and Western blotting. Role of insulin and Ras signaling pathways was determined in Ad-36–infected HSKM cells. RESULTS—Ad-36 and Ad-2 infections were confirmed by the presence of respective viral mRNA and protein expressions. In a dose-dependent manner, Ad-36 significantly increased glucose uptake in diabetic and nondiabetic HSKM cells. Ad-36 increased gene expression and protein abundance of GLUT1 and GLUT4, GLUT4 translocation to plasma membrane, and phosphatidylinositol 3-kinase (PI 3-kinase) activity in an insulin-independent manner. In fact, Ad-36 decreased insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation and IRS-1–and IRS-2–associated PI 3-kinase activities. On the other hand, Ad-36 increased Ras gene expression and protein abundance, and Ras siRNA abrogated Ad-36–induced PI 3-kinase activation, GLUT4 protein abundance, and glucose uptake. These effects were not observed with Ad-2 infection. CONCLUSIONS—Ad-36 infection increases glucose uptake in HSKM cells via Ras-activated PI 3-kinase pathway in an insulin-independent manner. These findings may provide impetus to exploit the role of Ad-36 proteins as novel therapeutic targets for improving glucose handling.

Metabolically Favorable Remodeling of Human Adipose Tissue by Human Adenovirus Type 36

OBJECTIVE—Experimental infection of rats with human adenovirus type 36 (Ad-36) promotes adipogenesis and improves insulin sensitivity in a manner reminiscent of the pharmacologic effect of thiozolinediones. To exploit the potential of the viral proteins as a therapeutic target for treating insulin resistance, this study investigated the ability of Ad-36 to induce metabolically favorable changes in human adipose tissue. RESEARCH DESIGN AND METHODS—We determined whether Ad-36 increases glucose uptake in human adipose tissue explants. Cell-signaling pathways targeted by Ad-36 to increase glucose uptake were determined in the explants and human adipose–derived stem cells. Ad-2, a nonadipogenic human adenovirus, was used as a negative control. As a proof of concept, nondiabetic and diabetic subjects were screened for the presence of Ad-36 antibodies to ascertain if natural Ad-36 infection predicted improved glycemic control. RESULTS—Ad-36 increased glucose uptake by adipose tissue explants obtained from nondiabetic and diabetic subjects. Without insulin stimulation, Ad-36 upregulated expressions of several proadipogenic genes, adiponectin, and fatty acid synthase and reduced the expression of inflammatory cytokine macrophage chemoattractant protein-1 in a phosphotidylinositol 3-kinase (PI3K)-dependent manner. In turn, the activation of PI3K by Ad-36 was independent of insulin receptor signaling but dependent on Ras signaling recruited by Ad-36. Ad-2 was nonadipogenic and did not increase glucose uptake. Natural Ad-36 infection in nondiabetic and diabetic subjects was associated with significantly lower fasting glucose levels and A1C, respectively. CONCLUSIONS—Ad-36 proteins may provide novel therapeutic targets that remodel human adipose tissue to a more metabolically favorable profile.

Autodeubiquitination Protects the Tumor Suppressor BAP1 from Cytoplasmic Sequestration Mediated by the Atypical Ubiquitin Ligase UBE2O

The tumor suppressor BAP1 interacts with chromatin-associated proteins and regulates cell proliferation, but its mechanism of action and regulation remain poorly defined. We show that the ubiquitin-conjugating enzyme UBE2O multi-monoubiquitinates the nuclear localization signal of BAP1, thereby inducing its cytoplasmic sequestration. This activity is counteracted by BAP1 autodeubiquitination through intramolecular interactions. Significantly, we identified cancer-derived BAP1 mutations that abrogate autodeubiquitination and promote its cytoplasmic retention, indicating that BAP1 autodeubiquitination ensures tumor suppression. The antagonistic relationship between UBE2O and BAP1 is also observed during adipogenesis, whereby UBE2O promotes differentiation and cytoplasmic localization of BAP1. Finally, we established a putative targeting consensus sequence of UBE2O and identified numerous chromatin remodeling factors as potential targets, several of which tested positive for UBE2O-mediated ubiquitination. Thus, UBE2O defines an atypical ubiquitin-signaling pathway that coordinates the function of BAP1 and establishes a paradigm for regulation of nuclear trafficking of chromatin-associated proteins.

Crosstalk between O-GlcNAcylation and proteolytic cleavage regulates the host cell factor-1 maturation pathway

Host Cell Factor 1 (HCF-1) plays critical roles in regulating gene expression in a plethora of physiological processes. HCF-1 is first synthesized as a precursor, and subsequently specifically proteolytically cleaved within a large middle region termed the proteolytic processing domain (PPD). Although the underlying mechanism remains enigmatic, proteolysis of HCF-1 regulates its transcriptional activity and is important for cell cycle progression. Here we report that HCF-1 proteolysis is a regulated process. We demonstrate that a large proportion of the signaling enzyme O-linked-N-acetylglucosaminyl transferase (OGT) is complexed with HCF-1 and this interaction is essential for HCF-1 cleavage. Moreover, HCF-1 is, in turn, required for stabilizing OGT in the nucleus. We provide evidence indicating that OGT regulates HCF-1 cleavage via interaction with and O-GlcNAcylation of the HCF-1 PPD. In contrast, although OGT also interacts with the basic domain in the HCF-1 amino-terminal subunit, neither the interaction nor the O-GlcNAcylation of this region are required for proteolysis. Moreover, we show that OGT-mediated modulation of HCF-1 impacts the expression of the herpes simplex virus immediate-early genes, targets of HCF-1 during the initiation of viral infection. Together the data indicate that O-GlcNAcylation of HCF-1 is a signal for its proteolytic processing and reveal a unique crosstalk between these posttranslational modifications. Additionally, interactions of OGT with multiple HCF-1 domains may indicate that OGT has several functions in association with HCF-1.