Search

The principles governing genotype-phenotype relationships are still emerging(1-3), and detailed translational as well as transcriptomic information is required to understand complex phenotypes, such as the pathogenesis of Alzheimer's disease. For this reason, the proteomics of Alzheimer disease (AD) continues to be studied extensively. Although comparisons between data obtained from humans and mouse models have been reported, approaches that specifically address the between-species statistical comparisons are understudied. Our study investigated the performance of two statistical methods for identification of proteins and biological pathways associated with Alzheimer's disease for cross-species comparisons, taking specific data analysis challenges into account, including collinearity, dimensionality reduction and cross-species protein matching. We used a human dataset from a well-characterized cohort followed for over 22 years with proteomic data available. For the mouse model, we generated proteomic data from whole brains of CVN-AD and matching control mouse models. We used these analyses to determine the reliability of a mouse model to forecast significant proteomic-based pathological changes in the brain that may mimic pathology in human Alzheimer's disease. Compared with LASSO regression, partial least squares discriminant analysis provided better statistical performance for the proteomics analysis. The major biological finding of the study was that extracellular matrix proteins and integrin-related pathways were dysregulated in both the human and mouse data. This approach may help inform the development of mouse models that are more relevant to the study of human late-onset Alzheimer's disease.

Background: The human chromosome 19q13.32 is a gene rich region and has been associated with multiple phenotypes, including late onset Alzheimer's disease (LOAD) and other age-related conditions., Objective: Here we developed the first humanized mouse model that contains the entire TOMM40 and APOE genes with all intronic and intergenic sequences including the upstream and downstream regions. Thus, the mouse model carries the human TOMM40 and APOE genes and their intact regulatory sequences., Methods: We generated the APOE-TOMM40 humanized mouse model in which the entire mouse region was replaced with the human (h)APOE-TOMM40 loci including their upstream and downstream flanking regulatory sequences using recombineering technologies. We then measured the expression of the human TOMM40 and APOE genes in the mice brain, liver, and spleen tissues using TaqMan based mRNA expression assays., Results: We investigated the effects of the '523' polyT genotype (S/S or VL/VL), sex, and age on the human TOMM40- and APOE-mRNAs expression levels using our new humanized mouse model. The analysis revealed tissue specific and shared effects of the '523' polyT genotype, sex, and age on the regulation of the human TOMM40 and APOE genes. Noteworthy, the regulatory effect of the '523' polyT genotype was observed for all studied organs., Conclusion: The model offers new opportunities for basic science, translational, and preclinical drug discovery studies focused on the APOE genomic region in relation to LOAD and other conditions in adulthood.

The Apolipoprotein E ε4 (APOE ε4) haplotype is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). The Translocase of Outer Mitochondrial Membrane-40 (TOMM40) gene maintains cellular bioenergetics, which is disrupted in AD. TOMM40 rs2075650 ('650) G versus A carriage is consistently related to neural and cognitive outcomes, but it is unclear if and how it interacts with APOE. We examined 21 orthogonal neural networks among 8,222 middle-aged to aged participants in the UK Biobank cohort. ANOVA and multiple linear regression tested main effects and interactions with APOE and TOMM40 '650 genotypes, and if age and sex acted as moderators. APOE ε4 was associated with less strength in multiple networks, while '650 G versus A carriage was related to more language comprehension network strength. In APOE ε4 carriers, '650 G-carriage led to less network strength with increasing age, while in non-G-carriers this was only seen in women but not men. TOMM40 may shift what happens to network activity in aging APOE ε4 carriers depending on sex., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Alzheimer's disease is a quintessential 'unmet medical need', accounting for ∼65% of progressive cognitive impairment among the elderly, and 700,000 deaths in the United States in 2020. In 2019, the cost of caring for Alzheimer's sufferers was $244B, not including the emotional and physical toll on caregivers. In spite of this dismal reality, no treatments are available that reduce the risk of developing AD or that offer prolonged mitiagation of its most devestating symptoms. This review summarizes key aspects of the biology and genetics of Alzheimer's disease, and we describe how pioglitazone improves many of the patholophysiological determinants of AD. We also summarize the results of pre-clinical experiments, longitudinal observational studies, and clinical trials. The results of animal testing suggest that pioglitazone can be corrective as well as protective, and that its efficacy is enhanced in a time- and dose-dependent manner, but the dose-effect relations are not monotonic or sigmoid. Longitudinal cohort studies suggests that it delays the onset of dementia in individuals with pre-existing type 2 diabetes mellitus, which small scale, unblinded pilot studies seem to confirm. However, the results of placebo-controlled, blinded clinical trials have not borne this out, and we discuss possible explanations for these discrepancies., Competing Interests: AS is President and CEO of Zinfandel Pharmaceuticals, Inc. DB is Senior Vice President and COO of Zinfandel Pharmaceuticals, Inc. WG has received consulting fees from Zinfandel Pharmaceuticals, Inc., (Copyright © 2021 Saunders, Burns and Gottschalk.)

Immune-based metabolic reprogramming of arginine utilization in the brain contributes to the neuronal pathology associated with Alzheimer's disease (AD). To enable our long-term goals of differentiation of AD mouse model genotypes, ages, and sexes based on activity of this pathway, we describe here the novel dosing (using uniformly labeled ( 13 C 6 15 N 4 ) arginine) and analysis methods using capillary electrophoresis high-resolution accurate-mass mass spectrometry for isotope tracing of metabolic products of arginine. We developed a pseudoprimed infusion-dosing regimen, using repeated injections, to achieve a steady state of uniformly labeled arginine in 135-195 min post bolus dose. Incorporation of stable isotope labeled carbon and nitrogen from uniformly labeled arginine into a host of downstream metabolites was measured in vivo in mice using serially sampled dried blood spots from the tail. In addition to the dried blood spot time course samples, total isotope incorporation into arginine-related metabolites was measured in the whole brain and plasma after 285 min. Preliminary demonstration of the technique identified differences isotope incorporation in arginine metabolites between male and female mice in a mouse-model of sporadic Alzheimer's disease (APOE4/huNOS2). The technique described herein will permit arginine pathway activity differentiation between mouse genotypes, ages, sexes, or drug treatments in order to elucidate the contribution of this pathway to Alzheimer's disease.

Introduction: The study of Alzheimer's disease (AD) has revealed biological pathways with implications for disease neuropathology and pathophysiology. These pathway-level effects may also be mediated by individual characteristics or covariates such as age or sex. Evaluation of AD biological pathways in the context of interactions with these covariates is critical to the understanding of AD as well as the development of model systems used to study the disease., Methods: Gene set enrichment methods are powerful tools used to interpret gene-level statistics at the level of biological pathways. We introduce a method for quantifying gene set enrichment using likelihood ratio-derived test statistics (gsLRT), which accounts for sample covariates like age and sex. We then use our method to test for age and sex interactions with protein expression levels in AD and to compare the pathway results between human and mouse species., Results: Our method, based on nested logistic regressions is competitive with the existing standard for gene set testing in the context of linear models and complex experimental design. The gene sets we identify as having a significant association with AD-both with and without additional covariate interactions-are validated by previous studies. Differences between gsLRT results on mouse and human datasets are observed., Discussion: Characterizing biological pathways involved in AD builds on the important work involving single gene drivers. Our gene set enrichment method finds pathways that are significantly related to AD while accounting for covariates that may be relevant to disease development. The method highlights commonalities and differences between human AD and mouse models, which may inform the development of higher fidelity models for the study of AD., (© 2021 the Alzheimer's Association.)

The TOMM40 poly-T is a polymorphism in intron 6 of the TOMM40 gene, which is adjacent to and in linkage disequilibrium with APOE. Roses et al. identified the association between the length of TOMM40 poly-T with the risk and age of onset of late-onset Alzheimer's disease (LOAD). Following the original discovery, additional studies found associations between the TOMM40 poly-T and LOAD-related phenotypes independent of APOE genotypes, while others did not replicate these associations. Furthermore, the identity of the TOMM40 poly-T risk allele has been controversial between different LOAD-related phenotypes. Here, we propose a framework to address the conflicting findings with respect to the TOMM40 poly-T allele associations with LOAD phenotypes and their functional effects. The framework is used to interpret previous studies as means to gain insights regarding the nature of the risk allele, very long versus short. We suggest that the identity of the TOMM40 poly-T risk allele depends on the phenotype being evaluated, the ages of the study subjects at the time of assessment, and the context of the APOE genotypes. In concluding remarks, we outline future studies that will inform the mechanistic interpretation of the genetic data., (Copyright © 2018 the Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

A variable-length poly-T variant in intron 6 of the TOMM40 gene, rs10524523, is associated with risk and age-of-onset of sporadic (late-onset) Alzheimer's disease. In Caucasians, the three predominant alleles at this locus are Short (S), Long (L) or Very long (VL). On an APOE ε3/3 background, the S/VL and VL/VL genotypes are more protective than S/S. The '523 poly-T has regulatory properties, in that the VL poly-T results in higher expression than the S poly-T in luciferase expression systems. The aim of the current work was to identify effects on cellular bioenergetics of increased TOM40 protein expression. MitoTracker Green fluorescence and autophagic vesicle staining was the same in control and over-expressing cells, but TOM40 over-expression was associated with increased expression of TOM20, a preprotein receptor of the TOM complex, the mitochondrial chaperone HSPA9, and PDHE1a, and increased activities of the oxidative phosphorylation complexes I and IV and of the TCA member α-ketoglutaric acid dehydrogenase. Consistent with the complex I findings, respiration was more sensitive to inhibition by rotenone in control cells than in the TOM40 over-expressing cells. In the absence of inhibitors, total cellular ATP, the mitochondrial membrane potential, and respiration were elevated in the over-expressing cells. Spare respiratory capacity was greater in the TOM40 over-expressing cells than in the controls. TOM40 over-expression blocked Ab-elicited decreases in the mitochondrial membrane potential, cellular ATP levels, and cellular viability in the control cells. These data suggest elevated expression of TOM40 may be protective of mitochondrial function., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Chromosome 19q13.32 is a gene rich region, and has been implicated in multiple human phenotypes in adulthood including lipids traits, Alzheimer's disease, and longevity. Peroxisome Proliferator Activated Receptor Gamma (PPARγ) is a ligand-activated nuclear transcription factor that plays a role in human complex traits that are also genetically associated with the chromosome 19q13.32 region. Here, we study the effects of PPARγ on the regional expression regulation of the genes clustered within chromosome 19q13.32, specifically TOMM40, APOE, and APOC1, applying two complementary approaches. Using the short hairpin RNA (shRNA) method in the HepG2 cell-line we knocked down PPARγ expression and measured the effect on mRNA expression. We discovered PPARγ knock down increased the levels of TOMM40-, APOE-, and APOC1-mRNAs, with the highest increase in expression observed for APOE-mRNA. To complement the PPARγ knockdown findings we also examined the effects of low doses of PPARγ agonists (nM range) on mRNA expression of these genes. Low (nM) concentrations of pioglitazone (Pio) decreased transcription of TOMM40, APOE, and APOC1 genes, with the lowest mRNA levels for each gene observed at 1.5nM. Similar to the effect of PPARγ knockdown, the strongest response to pioglitazone was also observed for APOE-mRNA, and rosiglitazone (Rosi), another PPARγ agonist, produced results that were consistent with these. In conclusion, our results further established a role for PPARγ in regional transcriptional regulation of chr19q13.32, underpinning the association between PPARγ, the chr19q13.32 genes cluster, and human complex traits and disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Introduction: In this article we discuss several human neurological diseases and their relationship to specific highly polymorphic small structural variants (SVs). Unlike genome-wide association analysis (GWAS), this methodology is not a genome screen to define new possibly associated genes, requiring statistical corrections for a million association tests. SVs provide local mapping information at a specific locus. Used with phylogenetic analysis, the specific association of length variants can be mapped and recognized., Areas Covered: This experimental strategy provides identification of DNA variants, particularly variable length Simple Sequence Repeats (SSRs or STRs or microsatellites) that provide specific local association data at the SV locus. Phylogenetic analysis that includes the specific appearance of different length SV variations can differentiate specific phenotypic risks in a population such as age of onset related to variable length polymorphisms and risk of phenotypic variations associated with several adjacent structural variations (SVs). We focus on data for three recent examples associated with Alzheimer's disease, Levy Bodies, and Parkinson's disease., Expert Opinion: SVs are understudied, but have led directly to mechanism of pathogenesis studies involving the regulation of gene expression. The identification of specific length polymorphisms associated with clinical disease has led to translational advances and new drug discovery.

Pioglitazone (PIO) is a peroxisome proliferator-activated receptor-γ (PPARγ) agonist in clinical use for treatment of type 2 diabetes (T2DM). Accumulating evidence suggests PPARγ agonists may be useful for treating or delaying the onset of Alzheimer's disease (AD), possibly via actions on mitochondria, and that dose strengths lower than those clinically used for T2DM may be efficacious. Our major objective was to determine if low doses of pioglitazone, administered orally, impacted brain activity. We measured blood-oxygenation-level dependent (BOLD) low-frequency fluctuations in conscious rats to map changes in brain resting-state functional connectivity due to daily, oral dosing with low-dose PIO. The connectivity in two neural circuits exhibited significant changes compared with vehicle after two days of treatment with PIO at 0.08 mg/kg/day. After 7 days of treatment with a range of PIO dose-strengths, connections between 17 pairs of brain regions were significantly affected. Functional connectivity with the CA1 region of the hippocampus, a region that is involved in memory and is affected early in the progression of AD, was specifically investigated in a seed-based analysis. This approach revealed that the spatial pattern of CA1 connectivity was consistent among all dose groups at baseline, prior to treatment with PIO, and in the control group imaged on day 7. Compared to baseline and controls, increased connectivity to CA1 was observed regionally in the hypothalamus and ventral thalamus in all PIO-treated groups, but was least pronounced in the group treated with the highest dose of PIO. These data support our hypothesis that PIO modulates neuronal and/or cerebrovascular function at dose strengths significantly lower than those used to treat T2DM and therefore may be a useful therapy for neurodegenerative diseases including AD.

Background: Several studies have demonstrated a lower apolipoprotein E4 (APOE ε4) allele frequency in African-Americans, but yet an increased age-related prevalence of AD. An algorithm for prevention clinical trials incorporating TOMM40'523 (Translocase of Outer Mitochondria Membrane) and APOE depends on accurate TOMM40'523-APOE haplotypes., Methods: We have compared the APOE and TOMM40'523 phased haplotype frequencies of a 9.5 kb TOMM40/APOE genomic region in West African, Caucasian, and African-American cohorts., Results: African-American haplotype frequency scans of poly-T lengths connected in phase with either APOE ε4 or APOE ε3 differ from both West Africans and Caucasians and represent admixture of several distinct West African and Caucasian haplotypes. A new West African TOMM40'523 haplotype, with APOE ε4 connected to a short TOMM40'523 allele, is observed in African-Americans but not Caucasians., Conclusion: These data have therapeutic implications for the age of onset risk algorithm estimates and the design of a prevention trial for African-Americans or other mixed ethnic populations., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondrial dysfunction is an important factor in the pathogenesis of age-related diseases, including neurodegenerative diseases like Alzheimer's and Parkinson's spectrum disorders. A polymorphism in Translocase of the Outer Mitochondrial Membrane - 40 kD ( TOMM40 ) is associated with risk and age-of onset of late-onset AD, and is the only nuclear- encoded gene identified in genetic studies to date that presumably contributes to LOAD-related mitochondria dysfunction. In this review, we describe the TOM40-mediated mitochondrial protein import mechanism, and discuss the evidence linking TOM40 with Alzheimer's (AD) and Parkinson's (PD) diseases. All but 36 of the >~1,500 mitochondrial proteins are encoded by the nucleus and are synthesized on cytoplasmic ribosomes, and most of these are imported into mitochondria through the TOM complex, of which TOM40 is the central pore, mediating communication between the cytoplasm and the mitochondrial interior. APP enters and obstructs the TOM40 pore, inhibiting import of OXPHOS-related proteins and disrupting the mitochondrial redox balance. Other pathogenic proteins, such as Aβ and alpha-synuclein, readily pass through the pore and cause toxic effects by directly inhibiting mitochondrial enzymes. Healthy mitochondria normally import and degrade the PD-related protein Pink1, but Pink1 exits mitochondria if the membrane potential collapses and initiates Parkin-mediated mitophagy. Under normal circumstances, this process helps clear dysfunctional mitochondria and contributes to cellular health, but PINK1 mutations associated with PD exit mitochondria with intact membrane potentials, disrupting mitochondrial dynamics, leading to pathology. Thus, TOM40 plays a central role in the mitochondrial dysfunction that underlies age-related neurodegenerative diseases. Learning about the factors that control TOM40 levels and activity, and how TOM40, specifically, and the TOM complex, generally, interacts with potentially pathogenic proteins, will provide deeper insights to AD and PD pathogenesis, and possibly new targets for preventative and/or therapeutic treatments.

A number of recent studies have not replicated the association of the translocase of the outer mitochondrial membrane pore subunit (TOMM40) rs10524523 polymorphism, which is in linkage disequilibrium with apolipoprotein E (APOE), with age of onset of Alzheimer's disease (AD). This perspective describes the differences between these later studies and the original experiments. We highlight the necessity for using standardized and informative assessment tools and processes when determining the age of development of AD or AD symptoms, and also stress that this clinical phenotype is best measured reliably in prospective studies during which subjects are monitored over time. This is true when assessing potential biomarkers for age of onset and when assessing the therapeutic potential of medicines that may delay the onset or progression of this disease., (Copyright © 2013 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.)

Curing Alzheimer's disease (AD) remains an elusive goal; indeed, it may even prove to be impossible, given the nature of the disease. Although modulating disease progression is an attractive target and will alleviate the burden of the most severe stages, this strategy will not reduce the prevalence of the disease itself. Preventing or (as described in this article) delaying the onset of cognitive impairment and AD will provide the greatest benefit to individuals and society by pushing the onset of disease into the later years of life. Because of the high variability in the age of onset of the disease, AD prevention studies that do not stratify participants by age-dependent disease risk will be operationally challenging, being large in size and of long duration. We present a composite genetic biomarker to stratify disease risk so as to facilitate clinical studies in high-risk populations. In addition, we discuss the rationale for the use of pioglitazone to delay the onset of AD in individuals at high risk.

Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain-CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined alpha-helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket.

Type 2 diabetes is associated with insulin resistance in peripheral tissues, such as muscle and fat, impaired glucose-stimulated insulin secretion from pancreatic beta-cells and elevated hepatic gluconeogenesis. Current pharmacotherapy does not adequately address the metabolic defects underlying this disease. Thus, novel targets are being explored that enhance insulin action at target tissues, stimulate carbohydrate and fat catabolism, decrease endogenous glucose production and increase pancreatic beta-cell neogenesis and glucose-dependent insulin secretion. This article reviews recent developments in research on several of these targets, namely acetyl-CoA carboxylase 2 (ACC2), I kappa kinase (IKK) beta, dipeptidyl peptidase IV (DPP-IV) and glucagon-like peptide-1 receptor (GLP-1R).

Peroxisome proliferator-activated receptor gamma (PPAR gamma) agonists, including the glitazone class of drugs, are insulin sensitizers that reduce glucose and lipid levels in patients with type 2 diabetes mellitus. To more fully understand the molecular mechanisms underlying their therapeutic actions, we have characterized the effects of the potent, tyrosine-based PPAR gamma ligand GW1929 on serum glucose and lipid parameters and gene expression in Zucker diabetic fatty rats. In time-course studies, GW1929 treatment decreased circulating FFA levels before reducing glucose and triglyceride levels. We used a comprehensive and unbiased messenger RNA profiling technique to identify genes regulated either directly or indirectly by PPAR gamma in epididymal white adipose tissue, interscapular brown adipose tissue, liver, and soleus skeletal muscle. PPAR gamma activation stimulated the expression of a large number of genes involved in lipogenesis and fatty acid metabolism in both white adipose tissue and brown adipose tissue. In muscle, PPAR gamma agonist treatment decreased the expression of pyruvate dehydrogenase kinase 4, which represses oxidative glucose metabolism, and also decreased the expression of genes involved in fatty acid transport and oxidation. These changes suggest a molecular basis for PPAR gamma-mediated increases in glucose utilization in muscle. In liver, PPAR gamma activation coordinately decreased the expression of genes involved in gluconeogenesis. We conclude from these studies that the antidiabetic actions of PPAR gamma agonists are probably the consequence of 1) their effects on FFA levels, and 2), their coordinate effects on gene expression in multiple insulin-sensitive tissues.

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of serine/threonine residues of nuclear and cytoplasmic proteins. We determined whether insulin or coinfusion of glucosamine (GlcN) with insulin alters O-GlcNAc of skeletal muscle proteins. Three groups of conscious fasted rats received 6-hour infusions of either saline (BAS), insulin 18 mU/kg.min and saline (INS), or insulin and GlcN 30 micromol/kg.min (GLCN) during maintenance of normoglycemia. At 6 hours, the concentrations of muscle UDP-GlcNAc, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), glycogen, and N and O-linked GlcNAc (galactosyltransferase labeling followed by beta elimination) were measured in freeze-clamped abdominis muscle. Insulin increased whole-body glucose uptake from 49 +/- 5 to 239 +/- 8 micromol/kg.min (P < .001) and glycogen in abdominis muscle from 138 +/- 11 to 370 +/- 26 mmol/kg dry weight (P < .001). Insulin increased the amount of cytosolic N - and O-linked GlcNAc by 56% from 362 +/- 30 to 564 +/- 45 dpm/microg protein . 100 min (P < .02), and O-GlcNAc from 221 +/- 16 to 339 +/- 27 dpm/microg . 100 min (P < .02). Glycogen content was positively correlated with the amount of total (r = .90, P < .005) and O-linked GlcNAc in insulin-infused animals. Coinfusion of GlcN with insulin increased muscle UDP-GlcNAc about fourfold (100 +/- 6 nmol/g) compared with insulin (27 +/- 1, P < .001) or saline (25 +/- 1, P < .001) infusion. GlcN also decreased glucose uptake over 6 hours by 30% to 168 +/- 8 micromol/kg . min (P < .001 for GLCN v INS) and muscle glycogen to 292 +/- 24 mmol/kg dry weight (P < .05 for GLCN v INS). Both total (635 +/- 60 dpm/microg . 100 min, P < .002) and O-linked GlcNAc (375 +/- 36 dpm/microg . 100 min, P < .002) in the cytosol were significantly higher in GLCN rats (635 +/- 60 dpm/microg) versus BAS rats (P < .002). As in INS rats, muscle glycogen and O-GlcNAc were positively correlated in GLCN rats (r = .54, P < .05). Variation in total and O-linked GlcNAc in GLCN rats was due both to GlcN (P < .02) and to variation in the glycogen content (P < .005).

Glutamine:fructose 6-phosphate amidotransferase (GFA) is rate-limiting for hexosamine biosynthesis, while a UDP-GlcNAc beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) catalyses final O-linked attachment of GlcNAc to serine and threonine residues on intracellular proteins. Increased activity of the hexosamine pathway is a putative mediator of glucose-induced insulin resistance but the mechanisms are unclear. We determined whether O-GlcNAc transferase is found in insulin-sensitive tissues and compared its activity to that of GFA in rat tissues. We also determined whether non-insulin-dependent diabetes mellitus (NIDDM) or acute hyperinsulinaemia alters O-GlcNAc transferase activity in human skeletal muscle. O-GlcNAc transferase was measured using 3H-UDP-GlcNAc and a synthetic cationic peptide substrate containing serine and threonine residues, and GFA was determined by measuring a fluorescent derivative of GlcN6P by HPLC. O-GlcNAc transferase activities were 2-4 fold higher in skeletal muscles and the heart than in the liver, which had the lowest activity, while GFA activity was 14-36-fold higher in submandibular gland and 5-18 fold higher in the liver than in skeletal muscles or the heart. In patients with NIDDM (n = 11), basal O-GlcNAc transferase in skeletal muscle averaged 3.8 +/- 0.3 nmol/mg.min, which was not different from that in normal subjects (3.3 +/- 0.4 nmol/mg.min). A 180-min intravenous insulin infusion (40 mU/m2.min) did not change muscle O-GlcNAc transferase activity in either group. We conclude that O-GlcNAc transferase is widely distributed in insulin-sensitive tissues in the rat and is also found in human skeletal muscle. These findings suggest the possibility that O-linked glycosylation of intracellular proteins is involved in mediating glucose toxicity. O-GlcNAc transferase does not, however, appear to be regulated by either NIDDM or acute hyperinsulinaemia, suggesting that mass action effects determine the extent of O-linked glycosylation under hyperglycaemic conditions.

Insulin-like growth factor 1 (IGF-1) had no detectable effect on pyruvate dehydrogenase (PDH) in NIH 3T3 cells that stably overexpressed normal human IGF-1 receptors. Insulin stimulated PDH activity 3-4--fold in cells that overexpressed normal human insulin receptors, but not in cells expressing TMI receptors, composed of the ligand binding domain of the insulin receptor coupled to the transmembrane and intracellular components of the IGF-1 receptor, or CEX receptors, in which the carboxy terminal 110 amino acid portion of the insulin receptor was exchanged for the corresponding portion of the IGF-1 receptor. In contrast, insulin stimulated glucose uptake in the control cell line and in each of the chimeric receptor-expressing lines with similar dose-response characteristics. These findings suggest the carboxy terminal portion of the IR may play a role in mediating the stimulation of PDH activity.

We studied the effects of insulin on the turnover of glucosamine-labeled lipids in embryonic RAT fibroblasts that overexpressed either normal human insulin receptors or insulin receptors with defective tyrosine kinase domains. Fractionation of organic extracts by thin layer chromatography in chloroform/acetone/methanol/acetic acid/water (50/20/10/10/5, v/v) revealed two insulin-sensitive glucosaminyl lipid fractions, the TLC origin (the Rf 0.0 fraction) and a fraction that migrated with Rf 0.18-0.2 (the Rf 0.2 fraction). The insulin-sensitive molecules in both fractions could also be labeled with D-[6-3H]galactose, but not with myo-[2-3H]inositol. Methanolysis and exposure to methylamine, phospholipase A2, or phosphatidylinositol-specific PLC destroyed the insulin-sensitive lipids in the Rf 0.0 fraction, but had no effect on the Rf 0.2 fraction lipid. The Rf 0.2 fraction lipid was destroyed by endoglycoceraminidase. Insulin caused a rapid loss of label from the Rf 0.0 fraction and an equally rapid increased labeling of the Rf 0.2 fraction, with similar time courses and dependencies on insulin concentration. The turnover of both lipids exhibited the same the insulin dose-response characteristics in cultures which overexpressed insulin receptors with defective tyrosine kinase domains as in cultures that overexpressed normal human insulin receptors. This result supports the conclusions that a number of signaling pathways diverge from the insulin receptor and that not all of those pathways are regulated by the insulin receptor tyrosine kinase.

The effect of insulin on pyruvate dehydrogenase activity was examined in two different cell types that over expressed either normal or defective human insulin receptors, RAT 1 embryonic fibroblasts and Chinese hamster ovary (CHO) cells. Insulin stimulated pyruvate dehydrogenase activity in cells that expressed normal insulin receptors (RAT 1 HIRc, and CHO-WT and CHO-T cells), or receptors in which lysine 1018 in the ATP-binding site of the tyrosine kinase domain was exchanged for alanine (RAT 1 A/K1018 and CHO-mut cells). For both rat and hamster cell lines, the insulin dose-response curves from cells that expressed the mutant receptors were identical to those from the appropriate controls that over expressed the normal insulin receptors. Insulin failed to stimulate pyruvate dehydrogenase activity in CHO-delta cells, which expressed a mutant human insulin receptor that was truncated by 112 amino acids at the carboxyl terminal of the beta chain. Control studies verified that all the cells used in this study exhibited the expected phenotypes with respect to the number of insulin receptors which they expressed, insulin-stimulated tyrosine kinase activity, and the biological consequences of inactivating the insulin receptor tyrosine kinase. These findings show that the insulin receptor tyrosine kinase does not play an obligatory role in the insulin signaling pathway that stimulates pyruvate dehydrogenase activity.

Blastocladiella emersonii zoospore maintenance factor (ZMF), released into the medium during zoospore production, mediates a reversible developmental block to zoospore encystment (Gottschalk, W. K., and Sonneborn, D. R. (1981) Exp. Mycol. 5, 1-14 and (1982) Dev. Biol. 93, 165-180). Crude ZMF and purified ZMF display indistinguishable sensitivities/insensitivities to inactivations by several different chemical or enzymatic treatments. Such data have provided additional support for the conclusion (Gottschalk, W. K., and Sonneborn, D. R. (1985) J. Biol. Chem. 260, 6588-6591) that ZMF biological activity resides in a single molecular species. The inactivation analyses have provided substantial evidence that ZMF is a newly discovered SH-containing cyclic ribotide. At least one SH-containing side group and at least one free amino group linked to an imidazole, as well as a ribosyl moiety containing a cyclic 3',5'-phosphate, a 2'-free hydroxyl, and a 1'-linkage to the imidazole, appear to be essential structural requirements for ZMF-mediated encystment blockage. The proposed structure of biologically functional ZMF is similar to that of a key intermediate in the de novo pathway of purine nucleotide biosynthesis (5'-phosphoribosyl-5-aminoimidazole-4-N-succino-carboxamide), except that ZMF, and not 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide, contains a cyclic phosphate and at least one reduced SH group.

The water mold Blastocladiella emersonii releases zoospore maintenance factor into the medium during zoosporogenesis. Extracellular factor mediates a reversible developmental block that maintains the motile, cell wall-less zoospore phenotype. A method for purifying the factor is reported that results in 75-120% recovery of biological activity. Analyses of purified factor by thin layer chromatography support the conclusion that factor activity resides in a single organic, low molecular weight molecular species. Other data (Gottschalk, W.K. & Sonneborn, D. R. (1985) J. Biol. Chem. 260, 6592-6599) independently support this conclusion and, in addition, support the conclusion that biological activity resides in an SH-containing cyclic ribotide.

The polar head group that was released by treating an insulin-sensitive glycophospholipid with a phosphatidylinositol-specific phospholipase C (PI-PLC) stimulated pyruvate dehydrogenase (PDH) in both subcellular and whole cell assays. Stimulation of PDH activity in the subcellular assay was detected after gel filtration chromatography of the polar head group. This stimulation was not due to the presence of contaminating calcium and magnesium. The PDH-stimulating activity was proportional to the amount of polar head group added to the assay. The effect of the polar head group on PDH in the subcellular assay was blocked by sodium fluoride, suggesting that the polar head group activated the PDH phosphatase. In the whole cell assay, the polar head group stimulated PDH activity to an equal or greater extent as a physiological concentration of insulin. The effect of the polar head group was detected at 5 min, peaked at 10 min, and declined thereafter. In contrast, insulin stimulated PDH activity more slowly, but consistently. The PDH-stimulating activity eluted after bacitracin but ahead of ATP during gel filtration chromatography, and it was destroyed by exposure to NH4OH or alkaline phosphatase and by boiling in water. These data support the proposal that an early step in insulin action is the release of insulinomimetic polar head group from the insulin-sensitive glycophospholipid.