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Time-restricted feeding (TRF) has gained attention as a dietary regimen that promotes metabolic health. This study questioned if the health benefits of an intermittent TRF (iTRF) schedule require ketone flux specifically in skeletal and cardiac muscles. Notably, we found that the ketolytic enzyme beta-hydroxybutyrate dehydrogenase 1 (BDH1) is uniquely enriched in isolated mitochondria derived from heart and red/oxidative skeletal muscles, which also have high capacity for fatty acid oxidation (FAO). Using mice with BDH1 deficiency in striated muscles, we discover that this enzyme optimizes FAO efficiency and exercise tolerance during acute fasting. Additionally, iTRF leads to robust molecular remodeling of muscle tissues, and muscle BDH1 flux does indeed play an essential role in conferring the full adaptive benefits of this regimen, including increased lean mass, mitochondrial hormesis, and metabolic rerouting of pyruvate. In sum, ketone flux enhances mitochondrial bioenergetics and supports iTRF-induced remodeling of skeletal muscle and heart., Competing Interests: Declaration of interests D.M.M. is a member of the Cell Metabolism advisory board., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Even-chain acylcarnitine (AC) metabolites, most of which are generated as byproducts of incomplete fatty acid oxidation (FAO), are viewed as biomarkers of mitochondrial lipid stress attributable to one or more metabolic bottlenecks in the β-oxidation pathway. The origins and functional implications of FAO bottlenecks remain poorly understood. Here, we combined a sophisticated mitochondrial phenotyping platform with state-of-the-art molecular profiling tools and multiple two-state mouse models of respiratory function to uncover a mechanism that connects AC accumulation to lipid intolerance, metabolic inflexibility, and respiratory inefficiency in skeletal muscle mitochondria. These studies also identified a short-chain carbon circuit at the C4 node of FAO wherein reverse flux of glucose-derived acetyl CoA through medium-chain ketothiolase enhances lipid tolerance and redox stability in heart mitochondria by regenerating free CoA and NAD + . The findings help to explain why diminished FAO capacity, AC accumulation, and metabolic inflexibility are tightly linked to poor health outcomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Two prominent frontline breast cancer (BC) chemotherapies commonly used in combination, doxorubicin (DOX) and docetaxel (TAX), are associated with long-lasting cardiometabolic and musculoskeletal side effects. Whereas DOX has been linked to mitochondrial dysfunction, mechanisms underlying TAX-induced myotoxicities remain uncertain. Here, the metabolic and functional consequences of TAX ± DOX were investigated using a 3D-bioengineered model of adult human muscle and a drug dosing regimen designed to resemble in vivo pharmacokinetics. DOX potently reduced mitochondrial respiratory capacity, 3D-myobundle size, and contractile force, whereas TAX-induced acetylation and remodeling of the microtubule network led to perturbations in glucose uptake, mitochondrial respiratory sensitivity, and kinetics of fatigue, without compromising tetanic force generation. These findings suggest TAX-induced remodeling of the microtubule network disrupts glucose transport and respiratory control in skeletal muscle and thereby have important clinical implications related to the cardiometabolic health and quality of life of BC patients and survivors., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Nicotinamide riboside supplements (NRS) have been touted as a nutraceutical that promotes cardiometabolic and musculoskeletal health by enhancing nicotinamide adenine dinucleotide (NAD + ) biosynthesis, mitochondrial function, and/or the activities of NAD-dependent sirtuin deacetylase enzymes. This investigation examined the impact of NRS on whole body energy homeostasis, skeletal muscle mitochondrial function, and corresponding shifts in the acetyl-lysine proteome, in the context of diet-induced obesity using C57BL/6NJ mice. The study also included a genetically modified mouse model that imposes greater demand on sirtuin flux and associated NAD + consumption, specifically within muscle tissues. In general, whole body glucose control was marginally improved by NRS when administered at the midpoint of a chronic high-fat diet, but not when given as a preventative therapy upon initiation of the diet. Contrary to anticipated outcomes, the study produced little evidence that NRS increases tissue NAD + levels, augments mitochondrial function, and/or mitigates diet-induced hyperacetylation of the skeletal muscle proteome., Competing Interests: The authors received NR Chloride as a gift from ChromaDex Inc., (© 2021 The Authors.)

Introduction: Thyroid-associated ophthalmopathy (TAO) is a disfiguring, potentially blinding, and sub-optimally managed autoimmune condition. Current therapy of active TAO consists most frequently of glucocorticoid steroids, orbital radiation, or B-cell depletion; all of which are associated with substantial side effects. Teprotumumab (Tepezza) is a human monoclonal antibody against the insulin-like growth factor type I receptor (IGF-IR), recently evaluated in two clinical trials for active moderate-to-severe TAO that was recently approved by the United States Food and Drug Administration (FDA) for use in TAO., Areas Covered: This article reviews phase II and III placebo-controlled, double-masked, prospective, multicenter studies assessing the efficacy and safety of teprotumumab for the treatment of active, moderate-to-severe TAO., Expert Opinion: Teprotumumab has demonstrated substantial and rapid improvement in Clinical Activity Score and proptosis reduction in TAO compared to placebo. Subjective diplopia and quality of life were also improved in both clinical trials. Teprotumumab exhibited a favorable safety profile, with transient hyperglycemia, muscle cramps, and auditory side effects being associated with the drug; these were usually transient. The trial findings indicate that teprotumumab is a promising, potential first-line therapy for treating TAO.

Purpose: To describe outcomes using umbilical amnion for conjunctival fornix, socket, and eyelid margin reconstruction., Methods: A medical record review was performed to identify patients who had received umbilical amnion over a 2-year period in their department. Patient demographics, disease etiology, and data regarding surgical outcomes were collected. The primary outcome was the success rate of the surgical intervention., Results: Twenty-one patients received umbilical amnion for anophthalmic socket contracture (n = 16), orbital implant exposure (n = 3), ocular surface burn (n = 1), and cicatricial entropion repair (n = 1). The primary outcome was met in 76% of patients overall. In anophthalmic socket contracture, the primary outcome was met in 86% and 0% of patients with acquired and congenital anophthalmia, respectively. The primary outcome was met in all cases of orbital implant exposure and cicatricial entropion. The primary outcome was not met in a Roper-Hall grade IV ocular surface burn., Conclusions: Umbilical amnion is an ideal substrate graft for reconstruction of the conjunctival fornix, socket, and eyelid margin. Umbilical amnion appears to be efficacious for the management of socket contracture in acquired anophthalmia, orbital implant exposure, and cicatricial entropion. Further experience is needed to determine the efficacy of umbilical amnion in ocular surface burns.

A 12-year-old boy and 63-year-old woman were incidentally found to have a solitary, well-defined, raised, ovoid lesion involving the inferomedial palpebral conjunctiva. Both lesions were separate from the lacrimal caruncle with normal conjunctiva surrounding the lesions. Excisional biopsies were consistent with caruncular tissue. In the English literature, supernumerary lacrimal caruncle has only been previously described in adults despite the congenital nature of the lesion.

This study sought to examine the functional significance of mitochondrial protein acetylation using a double knockout (DKO) mouse model harboring muscle-specific deficits in acetyl-CoA buffering and lysine deacetylation, due to genetic ablation of carnitine acetyltransferase and Sirtuin 3, respectively. DKO mice are highly susceptible to extreme hyperacetylation of the mitochondrial proteome and develop a more severe form of diet-induced insulin resistance than either single KO mouse line. However, the functional phenotype of hyperacetylated DKO mitochondria is largely normal. Of the >120 measures of respiratory function assayed, the most consistently observed traits of a markedly heightened acetyl-lysine landscape are enhanced oxygen flux in the context of fatty acid fuel and elevated rates of electron leak. In sum, the findings challenge the notion that lysine acetylation causes broad-ranging damage to mitochondrial quality and performance and raise the possibility that acetyl-lysine turnover, rather than acetyl-lysine stoichiometry, modulates redox balance and carbon flux., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Importance: An increased awareness of the interactions between the medical industry and health care professionals may lead to lower health care costs and more effective health care practices., Objective: To assess the characteristics of industry payments made to ophthalmologists between 2013 and 2017., Design, Setting, and Participants: This analysis included data reported in the June 29, 2018, update of the Centers for Medicare & Medicaid Services Open Payments Database (OPD). The OPD contains public records of industry payments made to physicians and teaching hospitals from August 1, 2013, to December 31, 2017, as reported by the medical industry. All general or research payments distributed to US ophthalmologists and contained in the OPD were included in this study. Data are summarized by practitioner, manufacturer, payment category, and geographic location., Main Outcomes and Measures: Main outcomes were the distribution, quantity, and value of payments made to ophthalmologists practicing in the United States or US territories. The financial characteristics of payment category, manufacturer, product, and location were also assessed., Results: This analysis revealed that the OPD showed industry reporting a total of 20 943 ophthalmologists receiving 736 517 payments worth $543 679 603.53 (1.67% of all industry-reported funds in the OPD). The median payment value was $22.44. Most payments were for food and beverages (581 588 [78.96%]), whereas most funds were allocated toward research ($310 142 151.88 [57.05%]) and consulting fees ($73 565 327.71 [13.51%]). The median payout to each ophthalmologist was $637.75 (interquartile range, $167.33-$2065.54). California was the highest-grossing state, receiving $101 135 980.34 (18.60%) of all payments. Fifteen companies were responsible for 87.68% of all funds distributed ($476 719 470.11) and were mostly involved in the production of pharmaceutical agents (anti-vascular endothelial growth factor agents, glaucoma eyedrops, and ocular lubricants) and surgical devices (cataract and glaucoma)., Conclusions and Relevance: Although there is no way to know the veracity of these reports, the findings suggest the financial ophthalmologist-industry relationship is substantial. These relationships may be adding to health care costs and affecting the quality of care, although those associations were not evaluated in this study.

Porous alloplastic materials such as titanium mesh and porous polyethylene allow for the ingrowth of fibrovascular tissues into the implant; stabilizing the implant and reducing the risk of extrusion and extrusion-related complications (such as hematoma formation). The literature contains 2 specific reports of delayed atraumatic orbital hematomas occurring 8 months and 3 years after orbital floor reconstruction with porous polyethylene implants. In this study, an atraumatic orbital hematoma occurring 16 years after orbital floor reconstruction with a porous polyethylene implant was presented. The hematoma was evacuated and the implant was removed without replacement of a secondary implant. The remaining fibrovascular tissue on the underside of the implant provided enough support of the orbital soft tissues to achieve excellent anatomic and physiologic outcomes.

Purpose: To describe the use of cryopreserved ultra-thick human amniotic membrane for management of anophthalmic socket contracture., Methods: A chart review was performed to identify patients undergoing fornix reconstruction with cryopreserved ultra-thick human amniotic membrane for management of anophthalmic socket contracture. Patient demographics, disease etiology, and data regarding postoperative prosthesis fit and complications were collected., Results: The technique is described in 3 female patients with anophthalmic socket contracture who underwent fornix reconstruction using cryopreserved ultra-thick human amniotic membrane. All patients had excellent prosthesis fit at final follow up (range, 10-14 months). There were no clinically significant complications and no reoperations were performed. One pyogenic granuloma developed and was excised without affecting ocular prosthesis fit., Conclusions: Cryopreserved ultra-thick human amniotic membrane is easy to use, well tolerated, and produces good outcomes for management of anophthalmic socket contracture.

Genetic manipulation via transgene overexpression, RNAi, or Cas9-based methods is central to biomedical research. Unfortunately, use of these tools is often limited by vector options. We have created a modular platform (pMVP) that allows a gene of interest to be studied in the context of an array of promoters, epitope tags, conditional expression modalities, and fluorescent reporters, packaged in 35 custom destination vectors, including adenovirus, lentivirus, PiggyBac transposon, and Sleeping Beauty transposon, in aggregate >108,000 vector permutations. We also used pMVP to build an epigenetic engineering platform, pMAGIC, that packages multiple gRNAs and either Sa-dCas9 or x-dCas9(3.7) fused to one of five epigenetic modifiers. Importantly, via its compatibility with adenoviral vectors, pMAGIC uniquely enables use of dCas9/LSD1 fusions to interrogate enhancers within primary cells. To demonstrate this, we used pMAGIC to target Sa-dCas9/LSD1 and modify the epigenetic status of a conserved enhancer, resulting in altered expression of the homeobox transcription factor PDX1 and its target genes in pancreatic islets and insulinoma cells. In sum, the pMVP and pMAGIC systems empower researchers to rapidly generate purpose-built, customized vectors for manipulation of gene expression, including via targeted epigenetic modification of regulatory elements in a broad range of disease-relevant cell types., (© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Acyl CoA metabolites derived from the catabolism of carbon fuels can react with lysine residues of mitochondrial proteins, giving rise to a large family of post-translational modifications (PTMs). Mass spectrometry-based detection of thousands of acyl-PTMs scattered throughout the proteome has established a strong link between mitochondrial hyperacylation and cardiometabolic diseases; however, the functional consequences of these modifications remain uncertain. Here, we use a comprehensive respiratory diagnostics platform to evaluate three disparate models of mitochondrial hyperacylation in the mouse heart caused by genetic deletion of malonyl CoA decarboxylase (MCD), SIRT5 demalonylase and desuccinylase, or SIRT3 deacetylase. In each case, elevated acylation is accompanied by marginal respiratory phenotypes. Of the >60 mitochondrial energy fluxes evaluated, the only outcome consistently observed across models is a ∼15% decrease in ATP synthase activity. In sum, the findings suggest that the vast majority of mitochondrial acyl PTMs occur as stochastic events that minimally affect mitochondrial bioenergetics., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

An elderly female with progressive proptosis was found to have an aggressive retrobulbar solid orbital mass. The mass was distinct from the optic nerve sheath and intracranial meninges, and produced concave erosion of the sphenoid wing. Operative findings demonstrated an orbital mass adherent to the dura of the superior orbital fissure. The mass did not demonstrate meningeal violation, infiltrate the superior orbital fissure, or display intracranial spread. The dura remained intact after gross total resection. Histopathology revealed a malignant meningioma with papillary and focal rhabdoid morphology and bony invasion (WHO grade III). The patient received 2500cGy of stereotactic radiotherapy in addition to gross total resection. Postoperatively, the signs and symptoms of orbital mass effect resolved (proptosis, relative afferent papillary defect, and periorbital edema) and the vision improved. There was no orbital recurrence or intracranial extension. The follow-up time was limited to eight months secondary to the patient succumbing to metastatic lung adenocarcinoma, which was demonstrated to be a separate process from the orbital meningioma. We propose the etiology of this tumor to be most consistent with an orbital malignant primary extradural meningioma - the first case reported in the literature.

Levels of certain circulating short-chain dicarboxylacylcarnitine (SCDA), long-chain dicarboxylacylcarnitine (LCDA) and medium chain acylcarnitine (MCA) metabolites are heritable and predict cardiovascular disease (CVD) events. Little is known about the biological pathways that influence levels of most of these metabolites. Here, we analyzed genetics, epigenetics, and transcriptomics with metabolomics in samples from a large CVD cohort to identify novel genetic markers for CVD and to better understand the role of metabolites in CVD pathogenesis. Using genomewide association in the CATHGEN cohort (N = 1490), we observed associations of several metabolites with genetic loci. Our strongest findings were for SCDA metabolite levels with variants in genes that regulate components of endoplasmic reticulum (ER) stress (USP3, HERC1, STIM1, SEL1L, FBXO25, SUGT1) These findings were validated in a second cohort of CATHGEN subjects (N = 2022, combined p = 8.4x10-6-2.3x10-10). Importantly, variants in these genes independently predicted CVD events. Association of genomewide methylation profiles with SCDA metabolites identified two ER stress genes as differentially methylated (BRSK2 and HOOK2). Expression quantitative trait loci (eQTL) pathway analyses driven by gene variants and SCDA metabolites corroborated perturbations in ER stress and highlighted the ubiquitin proteasome system (UPS) arm. Moreover, culture of human kidney cells in the presence of levels of fatty acids found in individuals with cardiometabolic disease, induced accumulation of SCDA metabolites in parallel with increases in the ER stress marker BiP. Thus, our integrative strategy implicates the UPS arm of the ER stress pathway in CVD pathogenesis, and identifies novel genetic loci associated with CVD event risk.

This study used mice with muscle-specific overexpression of PGC-1α, a transcriptional coactivator that promotes mitochondrial biogenesis, to determine whether increased oxidative potential facilitates metabolic improvements in response to lifestyle modification. MCK-PGC1α mice and nontransgenic (NT) littermates were fed a high-fat diet (HFD) for 10 weeks, followed by stepwise exposures to voluntary wheel running (HFD+Ex) and then 25% caloric restriction with exercise (Ex/CR), each for an additional 10 weeks with continued HFD. Running and CR improved weight and glucose control similarly in MCK-PGC1α and NT mice. Sedentary MCK-PGC1α mice were more susceptible to diet-induced glucose intolerance, and insulin action measured in isolated skeletal muscles remained lower in the transgenic compared with the NT group, even after Ex/CR. Comprehensive profiling of >200 metabolites and lipid intermediates revealed dramatic group-specific responses to the intervention but did not produce a lead candidate that tracked with changes in glucose tolerance irrespective of genotype. Instead, principal components analysis identified a chemically diverse metabolite cluster that correlated with multiple measures of insulin responsiveness. These findings challenge the notion that increased oxidative capacity defends whole-body energy homeostasis and suggest that the interplay between mitochondrial performance, lipotoxicity, and insulin action is more complex than previously proposed., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes.

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family member involved in redox sensing and metabolic control. Growing evidence links TXNIP to mitochondrial function, but the molecular nature of this relationship has remained poorly defined. Herein, we employed targeted metabolomics and comprehensive bioenergetic analyses to evaluate oxidative metabolism and respiratory kinetics in mouse models of total body (TKO) and skeletal muscle-specific (TXNIP(SKM-/-)) Txnip deficiency. Compared with littermate controls, both TKO and TXNIP(SKM-/-) mice had reduced exercise tolerance in association with muscle-specific impairments in substrate oxidation. Oxidative insufficiencies in TXNIP null muscles were not due to perturbations in mitochondrial mass, the electron transport chain, or emission of reactive oxygen species. Instead, metabolic profiling analyses led to the discovery that TXNIP deficiency causes marked deficits in enzymes required for catabolism of branched chain amino acids, ketones, and lactate, along with more modest reductions in enzymes of β-oxidation and the tricarboxylic acid cycle. The decrements in enzyme activity were accompanied by comparable deficits in protein abundance without changes in mRNA expression, implying dysregulation of protein synthesis or stability. Considering that TXNIP expression increases in response to starvation, diabetes, and exercise, these findings point to a novel role for TXNIP in coordinating mitochondrial fuel switching in response to nutrient availability.

Vasopressin regulates water excretion, in part, by controlling the abundances of the water channel aquaporin-2 (AQP2) protein and regulatory proteins in the renal collecting duct. To determine whether vasopressin-induced alterations in protein abundance result from modulation of protein production, protein degradation, or both, we used protein mass spectrometry with dynamic stable isotope labeling in cell culture to achieve a proteome-wide determination of protein half-lives and relative translation rates in mpkCCD cells. Measurements were made at steady state in the absence or presence of the vasopressin analog, desmopressin (dDAVP). Desmopressin altered the translation rate rather than the stability of most responding proteins, but it significantly increased both the translation rate and the half-life of AQP2. In addition, proteins associated with vasopressin action, including Mal2, Akap12, gelsolin, myosin light chain kinase, annexin-2, and Hsp70, manifested altered translation rates. Interestingly, desmopressin increased the translation of seven glutathione S-transferase proteins and enhanced protein S-glutathionylation, uncovering a previously unexplored vasopressin-induced post-translational modification. Additional bioinformatic analysis of the mpkCCD proteome indicated a correlation between protein function and protein half-life. In particular, processes that are rapidly regulated, such as transcription, endocytosis, cell cycle regulation, and ubiquitylation are associated with proteins with especially short half-lives. These data extend our understanding of the mechanisms underlying vasopressin signaling and provide a broad resource for additional investigation of collecting duct function (http://helixweb.nih.gov/ESBL/Database/ProteinHalfLives/index.html).

Previous studies in yeast have supported the view that post-transcriptional regulation of protein abundances may be more important than previously believed. Here we ask the question: "In a physiological regulatory process (the response of mammalian kidney cells to the hormone vasopressin), what fraction of the expressed proteome undergoes a change in abundance and what fraction of the regulated proteins have corresponding changes in mRNA levels?" In humans and other mammals, vasopressin fulfills a vital homeostatic role (viz. regulation of renal water excretion) by regulating the water channel aquaporin-2 in collecting duct cells. To address the question posed, we utilized large-scale quantitative protein mass spectrometry (LC-MS/MS) employing stable isotopic labeling in cultured mpkCCD cells ('SILAC') coupled with transcriptomic profiling using oligonucleotide expression arrays (Affymetrix). Preliminary studies analyzing two nominally identical control samples by SILAC LC-MS/MS yielded a relative S.D. of 13% (for ratios), establishing the precision of the SILAC approach in our hands. We quantified nearly 3000 proteins with nontargeted SILAC LC-MS/MS, comparing vasopressin- versus vehicle-treated samples. Of these proteins 786 of them were quantified in each of 3 experiments, allowing statistical analysis and 188 of these showed significant vasopressin-induced changes in abundance, including aquaporin-2 (20-fold increase). Among the proteins with statistically significant abundance changes, a large fraction (at least one-third) was found to lack changes in the corresponding mRNA species (despite sufficient statistical power), indicating that post-transcriptional regulation of protein abundance plays an important role in the vasopressin response. Bioinformatic analysis of the regulated proteins (versus all transcripts) shows enrichment of glutathione S-transferase isoforms as well as proteins involved in organization of the actin cytoskeleton. The latter suggests that long-term regulatory processes may contribute to actomyosin-dependent trafficking of the water channel aquaporin-2. The results provide impetus for increased focus on translational regulation and regulation of protein degradation in physiological control in mammalian epithelial cells.

Because previous studies of three-dimensional skeletal muscle cultures have shown limited differentiation, the goal of this study was to establish conditions that would produce mature sarcomeres in a mammalian-derived skeletal muscle construct. We evaluated the differentiation of bioartificial muscles generated from C(2)C(12) myoblasts in a collagen gel cultured under steady, passive tension for up to 36 days. Staining for alpha-actinin, myosin, and F-actin indicated the presence of striated fibers as early as 6 days post-differentiation. Electron microscopy at 16 days post-differentiation revealed multinucleated myotubes with ordered, striated myofibers. At 33 days, the cultures contained collagen fibers and showed localization of paxillin at the fiber termini, suggesting that myotendinous junctions were forming. The present study demonstrates mature muscle synthesis in a three-dimensional system using a pure mammalian myoblast cell line. Our results suggest that this culture model can be used to evaluate the effects of various mechanical and biochemical cues on muscle development under normal and pathological conditions.

Cell culture models that mimic long-term exposure to microgravity provide important insights into the cellular biological adaptations of human skeletal muscle to long-term residence in space. We developed insert scaffolding for the NASA-designed rotating cell culture system (RCCS) in order to study the effects of time-averaged microgravity on the proliferation and differentiation of anchorage-dependent skeletal muscle myocytes. We hypothesized that prolonged microgravity exposure would result in the retardation of myocyte differentiation. Microgravity exposure in the RCCS resulted in increased cellular proliferation. Despite shifting to media conditions promoting cellular differentiation, 5 d later, there was an increase in cell number of approximately 62%, increases in total cellular protein (52%), and cellular proliferating cell nuclear antigen (PCNA) content (2.7 times control), and only a modest (insignificant) decrease (10%) in sarcomeric myosin protein expression. We grew cells in an inverted orientation on membrane inserts. Changes in cell number and PCNA content were the converse to those observed for cells in the RCCS. We also grew cells on inserts at unit gravity with constant mixing. Mixing accounted for part, but not all, of the effects of microgravity exposure on skeletal muscle cell cultures (53% of the RCCS effect on PCNA at 4-6 d). In summary, the mechanical effects of simulated microgravity exposure in the RCCS resulted in the maintenance of cellular proliferation, manifested as increases in cell number and expression of PCNA relative to control conditions, with only a modest reciprocal inhibition of cellular differentiation. Therefore, this model provides conditions wherein cellular differentiation and proliferation appear to be uncoupled.

Insulin-like growth factor binding protein-2 (IGFBP-2) production as a function of cell number by intestinal epithelial cells (IEC-6) was regulated such that the IGFBP-2 concentration in 24-h conditioned medium reached a maximum, which was maintained despite increasing cell number. Northern blot analysis revealed that this effect could largely be attributed to decreasing IGFBP-2 mRNA. In contrast to IGFBP-2, secretion of IGF-II and accumulation of IGF-II mRNA by IEC-6 cells correlated positively with cell number. The highest level of IGF-II protein detected by immunoblotting of conditioned medium occurred in post-confluent cells. IGF-I stimulated the cells to grow to a high cellular density and inhibited IGFBP-2 secretion in a concentration-dependent fashion. We conclude that expression of IGF-II and IGFBP-2 are regulated in IEC-6 cells by cellular density, and IGF-II may act as a survival factor at high cell density., (Copyright 1999 Harcourt Publishers Ltd.)

Insulin-like growth factor II (IGF-II) and phosphomannosylated glycoproteins bind to distinct sites on the same receptor, the IGF-II/mannose 6-phosphate receptor (IGF2R). Analysis of truncated receptors (minireceptors) has been used to map the IGF-II binding site within the receptor's extracytoplasmic domain, which consists of 15 homologous repeats. A minireceptor consisting of repeat 11 contained the minimal elements for binding IGF-II, but with 5- to 10-fold lower relative binding affinity than the full-length receptor. We hypothesized that the complete, high-affinity IGF-II binding site is formed by interaction between the primary site in repeat 11 and a putative affinity-enhancing domain. To determine the minimum portion of the IGF2R's extracytoplasmic domain needed for expression of high-affinity IGF-II binding, a nested set of FLAG epitope-tagged minireceptors encompassing repeats 11 through 15 was prepared and transiently expressed in 293T cells. Minireceptors containing repeats 11-13 or 11-15 exhibited high affinity, comparable to the full-length receptor (IC50 = 1-2 nM), whereas constructs containing repeat 11 only or repeats 11-12 did not (IC50 = 10-20 nM). These data suggested that the affinity-enhancing domain is located within repeat 13, which contains a unique 43-residue insert that has approximately 50% sequence identity to the type II repeat of fibronectin. Although a repeat 13 minireceptor did not bind IGF-II on its own, an 11-13 minireceptor containing a deletion of the 43-residue insert exhibited low IGF-II binding affinity (IC50 = 10-20 nM). Expression of mutant receptors from a full-length IGF2R construct bearing a deletion of the 43-residue insert was very low relative to wild type. Depletion assays using IGF-II-Sepharose showed that the mutant receptor had lower affinity for IGF-II than the wild-type receptor. This study reveals that two independent receptor domains are involved in the formation of a high-affinity binding site for IGF-II, and that a complete repeat 13 is required for high-affinity IGF-II binding.

Complete understanding of the functional significance of insulin-like growth factor II (IGF-II) binding by the IGF-II/mannose-6-phosphate (Man-6-P) receptor requires mapping and ultimately mutational analysis of the receptor's IGF-II binding domain. Recent advances have localized the IGF-II binding site to extracytoplasmic repeats 10-11. To improve resolution of the binding site map, a nested set of epitope-tagged, truncated forms of the human IGF-II/Man-6-P receptor were transiently expressed in COS-7 cells. The IGF-II binding properties of truncated receptors immunoprecipitated from cell lysates and conditioned media were determined by affinity cross-linking. From the largest truncated receptor, encompassing extracytoplasmic repeats 8-11 (M(r) 68 K), through the smallest, comprised primarily of repeat 11 (M(r) 23 K), all were able to bind and cross-link to IGF-II. As a group, the truncated receptors had similar affinities for IGF-II, but with relative binding affinities 5-to 10-fold lower than those of full-length receptors. A point mutation substituting threonine for isoleucine at residue 1572, located in the NH2-terminal half of repeat 11, completely abolished IGF-II binding. We conclude that repeat 11 of the IGF-II/Man-6-P receptor's extracytoplasmic domain contains the minimal elements required for binding and cross-linking to IGF-II, and that lle1572 and other residues within the NH2-terminal half of repeat 11 are particularly important for IGF-II interaction.

The components of the insulin-like growth factor (IGF) axis and their roles in regulating proliferation and differentiation of the human colon adenocarcinoma cell line, Caco-2, have been investigated. Caco-2 cells proliferated in serum-free medium at 75% the rate observed in medium containing 10% fetal bovine serum. IGF-I (10 nM) increased Caco-2 cell growth in serum-free medium, but not to the rate seen with serum. Multiple IGF-II mRNA species were produced by Caco-2 cells, but IGF-I mRNA was undetectable. Secretion of radioimmunoassayable IGF-II corresponded with steady-state levels of IGF-II mRNA, neither of which was observed to change markedly over the course of 16 days of Caco-2 cell differentiation. Levels of sucrase-isomaltase mRNA, a marker for enterocytic differentiation, increased 12-fold between days 5 and 16 of culture. Northern blotting of total RNA and ligand blot and immunoblot analyses of serum-free conditioned medium revealed that Caco-2 cells produce several IGF binding proteins (IGFBPs), including IGFBP-2, -3, and -4, as well as a 31,000 M(r) species that was not identified. The pattern of IGFBP secretion changed dramatically during Caco-2 cell differentiation: IGFBP-3 and IGFBP-2 increased 8.5-fold and 5-fold, respectively, whereas IGFBP-4 and the 31,000 M(r) species decreased 43% and 90%. Caco-2 cell clones stably transfected with a human IGFBP-4 cDNA construct exhibited a 60% increase in steady-state level of IGFBP-4 mRNA, and secreted twice as much IGFBP-4 protein as controls. Moreover, IGFBP-4-overexpressing cells proliferated at only 25% the rate of control cells in serum-free medium, in conjunction with a 70% increase in expression of sucrase-isomaltase. In summary, these studies indicate that a complex IGF axis is involved in autocrine regulation of Caco-2 cell proliferation and differentiation.

IEC-6 cells, an intestinal epithelial cell line, produce insulin-like growth factor (IGF)-II and IGF-binding protein-2 (IGFBP-2). Exogenous IGFs stimulate and IGFBP-2 attenuates DNA synthesis. To investigate whether endogenously secreted IGFBP-2 inhibits proliferation, IEC-6 cells were transfected with a full-length rat IGFBP-2 cDNA antisense expression construct. The steady-state level of IGFBP-2 mRNA decreased by 54% in the antisense cDNA-transfected cells (denoted revBP2) compared with vector-transfected controls. Moreover, revBP2 cells secreted less IGFBP-2 than controls (maximally a 68% decrease). In serum-containing medium, revBP2 cells exhibited a 35% increase in log-phase proliferation rate and growth-arrested at a maximum density 14% higher than controls. We conclude that endogenous IGFBP-2 inhibits proliferation of IEC-6 cells, probably by sequestering IGFs.