A Randomized,Double-Blind,Placebo-Controlled Trial of Adjunctive Metformin Therapy in Overweight/Obese Youth with Type 1 Diabetes

Context

Insulin resistance has been proposed as one of the causes of poor glycemic control in overweight/obese youth with type 1 diabetes (T1D). However, the role of adjunctive metformin, an insulin sensitizer, on glycemic control in these patients is unclear.

Objective

To compare the effect of metformin vs. placebo on hemoglobin A1c (HbA1c), total daily dose (TDD) of insulin, and other parameters in overweight/obese youth with T1D.

Hypothesis

Adjunctive metformin therapy will improve glycemic control in overweight/obese youth with T1D.

Design, Setting, and Participants

A 9-mo randomized, double-blind, placebo controlled trial of metformin and placebo in 28 subjects (13m/15f) of ages 10-20years (y), with HbA1c >8% (64 mmol/mol), BMI >85%, and T1D > 12 months was conducted at a university outpatient facility. The metformin group consisted of 15 subjects (8 m/ 7f), of age 15.0 ± 2.5 y; while the control group was made up of 13 subjects (5m/ 8f), of age 14.5 ± 3.1y. All participants employed a self-directed treat-to-target insulin regimen based on a titration algorithm of (-2)-0-(+2) units to adjust their long-acting insulin dose every 3rd day from -3 mo through +9 mo to maintain fasting plasma glucose (FPG) between 90–120 mg/dL (5.0–6.7 mmol/L). Pubertal maturation was determined by Tanner stage.

Results

Over the course of the 9 months of observation, the between-treatment differences in HbA1c of 0.4% (9.85% [8.82 to 10.88] for placebo versus 9.46% [8.47 to 10.46] for metformin) was not significant (p = 0.903). There were non-significant reduction in fasting plasma glucose (189.4 mg/dL [133.2 to 245.6] for placebo versus 170.5 mg/dL [114.3 to 226.7] for metformin), (p = 0.927); total daily dose (TDD) of short-acting insulin per kg body weight/day(p = 0.936); and the TDD of long-acting insulin per kg body weight per day (1.15 units/kg/day [0.89 to 1.41] for placebo versus 0.90 units/kg/day [0.64 to 1.16] for metformin) (p = 0.221). There was no difference in the occurrence of hypoglycemia between the groups.

Conclusions

This 9-month RCT of adjunctive metformin therapy in overweight and obese youth with T1D resulted in a 0.4% lower HbA1c value in the metformin group compared to the placebo group.

Trial Registration

ClinicalTrial.gov NCT01334125     

Emerging Principles Governing Signal Transduction by Pattern-Recognition Receptors

The problem of recognizing and disposing of non-self-organisms, whether for nutrients or defense, predates the evolution of multicellularity. Accordingly, the function of the innate immune system is often intimately associated with fundamental aspects of cell biology. Here, we review our current understanding of the links between cell biology and pattern-recognition receptors of the innate immune system. We highlight the importance of receptor localization for the detection of microbes and for the initiation of antimicrobial signaling pathways. We discuss examples that illustrate how pattern-recognition receptors influence, and are influenced by, the general membrane trafficking machinery of mammalian cells. In the future, cell biological analysis likely will rival pure genetic analysis as a tool to uncover fundamental principles that govern host–microbe interactions.The innate immune system uses families of pattern-recognition receptors (PRRs) to recognize diverse microbial ligands (Janeway 1989; Janeway and Medzhitov 2002). During infection, these receptors provide signals that up-regulate general antimicrobial features of the innate immune system as well as instruct and initiate adaptive immunity (Iwasaki and Medzhitov 2010). A significant challenge faced by innate immune recognition is the reliable detection of highly diverse, rapidly evolving microbial organisms, many of which possess virulence mechanisms that enable survival within distinct host niches. Moreover, recognition must be linked to induction of contextual signals appropriate for the type of infection. The specificity, signal transduction, and cell biology of PRRs have evolved under these selective pressures to enable broad recognition of microbes within each host niche.Although the collection of PRRs is decidedly less diverse than antigen receptors of the adaptive immune system, the list of players has grown considerably over the past decade (Kawai and Akira 2010). If one classifies these receptors based on common structure and functional domains, then six families emerge: Toll-like receptors (TLRs), C-type lectin receptors (CLRs), RIG-I-like receptors (RLRs), AIM-like receptors (ALRs), Nod-like receptors (NLRs), and OAS-like receptors (OLRs) (Geijtenbeek and Gringhuis 2009; Kawai and Akira 2010; Rathinam and Fitzgerald 2011; Lamkanfi and Dixit 2012; Kranzusch et al. 2013). Collectively, these receptors bind a diverse array of targets, including lipoproteins, polysaccharides, nucleic acids, carbohydrate structures, and a few highly conserved microbial proteins. These ligands are typically shared across large microbial classes, which facilitate broad recognition with such a limited number of PRRs. Moreover, alteration or masking of these ligands to avoid PRR activation often results in reduced microbial fitness.The molecular recognition challenge faced by PRRs is all the more complex when one considers the need to detect microbes within distinct subcellular niches. Microbes can be extracellular or intracellular within membrane-bound organelles, within the cytosol, or in the nucleus. In addition, both the innate and adaptive immune mechanisms appropriate for eliminating microbes within these distinct environments are quite distinct, so it is vital that PRR signaling communicate the location of a microbe as well its nature. We now understand that members of the PRR families highlighted above localize to distinct subcellular compartments, and, in some cases, localization can change in a dynamic fashion that regulates or influences recognition and signaling. Moreover, in some cases, signal transduction and resulting gene induction can be dramatically influenced by the organelle from which signaling initiates. Thus, the innate immune system has harnessed the organization inherent to cells as a means of achieving regulation and signaling specificity. Activation of PRRs can also feed back on basic cell biological processes, such as phagocytosis and autophagy, to enhance or accelerate the response to microbial infection.In the following sections, we discuss these links between cell biology and PRRs of mammalian innate immunity. Our discussions of PRR function and signal transduction will be limited to this theme, as a result, in part, of space constraints but also because in-depth reviews of each PRR family have appeared elsewhere. For discussion purposes, we have grouped the transmembrane PRRs together and the cytosolic PRRs together.     

The identification of a series of novel, soluble non-peptidic neuropeptide Y Y2 receptor antagonists

The identification and subsequent optimisation of a selective non-peptidic NPY Y2 antagonist series is described. This led to the development of amine 2, a selective, soluble NPY Y2 receptor antagonist with enhanced CNS exposure.     

The Characterization of Twenty Sequenced Human Genomes

We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten “case” genomes from individuals with severe hemophilia A and ten “control” genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs) discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.     

Inhibition of phospholipase C disrupts cytoskeletal organization and gravitropic growth in Arabidopsis roots

The phospholipase protein superfamily plays an important role in hormonal signalling and cellular responses to environmental stimuli. There is also growing evidence for interactions between phospholipases and the cytoskeleton. In this report we used a pharmacological approach to investigate whether inhibiting a member of the phospholipase superfamily, phospholipase C (PLC), affects microtubules and actin microfilaments as well as root growth and morphology of Arabidopsis thaliana seedlings. Inhibiting PLC activity using the aminosteroid U73122 significantly inhibited root elongation and disrupted root morphology in a concentration-dependent manner, with the response being saturated at 5 μM, whereas the inactive analogue U73343 was ineffective. The primary root appeared to lose growth directionality accompanied by root waving and formation of curls. Immunolabelling of roots exposed to increasingly higher U73122 concentrations revealed that the normal transverse arrays of cortical microtubules in the elongation zone became progressively more disorganized or depolymerized, with the disorganization appearing within 1 h of incubation. Likewise, actin microfilament arrays also were disrupted. Inhibiting PLC using an alternative inhibitor, neomycin, caused similar disruptions to both cytoskeletal organization and root morphology. In seedlings gravistimulated by rotating the culture plates by 90°, both U73122 and neomycin disrupted the normal gravitropic growth of roots and etiolated hypocotyls. The effects of PLC inhibitors are therefore consistent with the notion that, as with phospholipases A and D, PLC likewise interacts with the cytoskeleton, alters growth morphology, and is involved in gravitropism.     

7,12-Dimethylbenz[A]Anthracene Induces Sertoli�CLeydig-Cell Tumors in the Follicle-Depleted Ovaries of Mice Treated with 4-Vinylcyclohexene Diepoxide

Ovarian cancer is associated with high mortality due to its late onset of symptoms and lack of reliable screening methods for early detection. Furthermore, the incidence of ovarian cancer is higher in postmenopausal women. Mice rendered follicle-depleted through treatment with 4-vinylcyclohexene diepoxide (VCD) are a model of ovary-intact menopause. The present study was designed to induce ovarian neoplasia in this model by treating mice with 7,12-dimethylbenz[a]anthracene (DMBA). Female B6C3F1 mice (age, 28 d) received intraperitoneal sesame oil (vehicle; VCD– groups) as a control or VCD (160 mg/kg; VCD+ groups) daily for 20 d to cause ovarian failure. Four months after the onset of dosing, mice from each group received a single injection of DMBA (VCD–DMBA+ and VCD+DMBA+ groups, n = 15 per group) or vehicle control (VCD–DMBA–, n = 15; VCD+ DMBA–, n = 14) under the bursa of the right ovary. Ovaries were collected 3 or 5 mo after injection and processed for histologic evaluation. Immunohistochemistry was used to confirm classification of neoplasms. None of the animals in the VCD–DMBA– and VCD–DMBA+ groups (that is, mice still undergoing estrus) had tumors at either time point. At the 3-mo time point, 12.5% of the VCD+DMBA+ mice had ovarian tumors; at 5 mo, 57.1% of the VCD+DMBA+ and 14.3% of VCD+DMBA– ovaries had neoplasms. Neoplasms stained positively for inhibin α (granulosa cells) and negatively for keratin 7 (surface epithelium), thus confirming classification of the lesions as Sertoli–Leydig cell tumors. These findings provide evidence for an increased incidence of DMBA-induced ovarian neoplasms in the ovaries of follicle-depleted mice compared with that in age-matched cycling controls.Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; OSE, ovarian surface epithelium; VCD, 4-vinylcyclohexene diepoxideApproximately 20,000 women are diagnosed with ovarian cancer annually, of whom 15,000 are anticipated to die of the disease. Ovarian cancer ranks fifth in deaths by all cancers and first in cancers of the reproductive system.¹² The survival rate of ovarian cancer patients improves greatly when the disease is detected early,² but fewer than 20% of ovarian cancers are found at an early stage due to the lack of reliable screening methods for early detection. Because approximately two-thirds of ovarian cancer cases are diagnosed in women older than 55 y, the incidence of ovarian cancer is increased in peri- and postmenopausal women.¹² For this reason, research using relevant animal models of menopause is needed to advance the understanding of the biology of neoplasms in the postmenopausal ovary.Ovarian cancer can be due to transformation of surface epithelial cells, germ cells, or sex cord and stromal cells. Almost 90% of all ovarian cancers are thought to be derived from the flat-to-cuboidal epithelial cells (that is, the ovarian surface epithelium [OSE]) that cover the ovary.^6,49 Alternatively, fewer than 5% of ovarian cancers are classified as sex cord–stromal tumors, which include granulosa cell tumors, and Sertoli–Leydig cell tumors.¹⁸ The incidence of sex cord–stromal ovarian cancers is highest in women older than 50 y, but has also been diagnosed in premenopausal women.¹⁸ The etiology of ovarian cancer is not completely understood, but factors associated with development of the disease include ovulation, altered levels of gonadotropins (luteinizing and follicle-stimulating hormones) and steroid hormones (estrogens and androgens), germ-cell or follicle depletion, altered expression of oncogenes and tumor suppressor genes, altered levels of growth factors and cytokines, and exposure to environmental agents.⁴¹Recently, an ovary-intact mouse model of menopause was developed by using the occupational chemical 4-vinylcyclohexene diepoxide (VCD).^24,25,27 Repeated daily dosing of mice and rats with VCD selectively destroys ovarian primordial and primary follicles by accelerating the natural process of follicular atresia.^14,15,42,44 Because VCD does not target larger follicles, the animal continues to ovulate normally until no more follicles can be recruited. Thus, ovarian follicular depletion in the VCD-treated mouse is gradual. As with women undergoing perimenopause, VCD-treated mice show increased levels of follicle-stimulating hormone,²⁷ irregular estrous cycles, and declining levels of estrogen²⁴ as they become follicle-depleted. In addition, residual ovarian tissue is retained after ovarian failure. Therefore, preservation of residual ovarian tissue in the VCD-treated follicle-depleted mouse makes this model ideal for studying the physiology of the postmenopausal ovary. The VCD-treated mouse model of peri- and postmenopause has been used to study several menopause-related disorders including atherosclerotic lesion development,²⁸ diabetic kidney disease,²⁰ osteoporosis,⁵¹ and metabolic syndrome.³⁹ Because the VCD-treated mouse has been shown to be relevant for studies related to both perimenopausal and postmenopausal stages,⁵⁰ it is a useful candidate for studies of ovarian cancer.Even though spontaneous ovarian tumors in rodents have been reported,³⁶ the paucity of these cases precludes their use in modeling ovarian cancer. Therefore, much effort has been put into developing relevant animal models for ovarian cancers. One such model involves the use of the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA),^8,21,23,43 a polycyclic aromatic hydrocarbon that induces carcinogenic mutations by forming DNA adducts.⁹ Recently, the DMBA model of carcinogenesis has been combined with the VCD model of menopause to cause ovarian cancer in F344 rats.^13,19 However, no studies have characterized the combined use of both chemicals in mice. Developing this combined model in mice is important because of the existence of various genetically engineered mice that have potential relevance to enhancing our understanding of the biology of ovarian cancer.The present study was designed to determine whether ovarian failure affects susceptibility to the development of ovarian neoplasms in mice and to model DMBA-induced ovarian neoplasia in VCD-treated follicle-depleted mice. VCD-treated follicle-depleted mice and cycling controls received ovarian injections with DMBA to induce neoplasia. The incidence of neoplasms was determined by histologic evaluation, and the lesions were classified through immunostaining for keratin 7 and inhibin α.     

Microbial community biofabrics in a geothermal mine adit

Speleothems such as stalactites and stalagmites are usually considered to be mineralogical in composition and origin; however, microorganisms have been implicated in the development of some speleothems. We have identified and characterized the biological and mineralogical composition of mat-like biofabrics in two novel kinds of speleothems from a 50 degrees C geothermal mine adit near Glenwood Springs, CO. One type of structure consists of 2- to 3-cm-long, 3- to 4-mm-wide, leather-like, hollow, soda straw stalactites. Light and electron microscopy indicated that the stalactites are composed of a mineralized biofabric with several cell morphotypes in a laminated form, with gypsum and sulfur as the dominant mineral components. A small-subunit rRNA gene phylogenetic community analysis along the stalactite length yielded a diverse gradient of organisms, with a relatively simple suite of main constituents: Thermus spp., crenarchaeotes, Chloroflexi, and Gammaproteobacteria. PCR analysis also detected putative crenarchaeal ammonia monooxygenase subunit A (amoA) genes in this community, the majority related to sequences from other geothermal systems. The second type of speleothem, dumpling-like rafts floating on a 50 degrees C pool on the floor of the adit, showed a mat-like fabric of evidently living organisms on the outside of the dumpling, with a multimineral, amorphous, gypsum-based internal composition. These two novel types of biofabrics are examples of the complex roles that microbes can play in mineralization, weathering, and deposition processes in karst environments.     

The 'human revolution' in lowland tropical Southeast Asia: the antiquity and behavior of anatomically modern humans at Niah Cave (Sarawak, Borneo)

Recent research in Europe, Africa, and Southeast Asia suggests that we can no longer assume a direct and exclusive link between anatomically modern humans and behavioral modernity (the 'human revolution'), and assume that the presence of either one implies the presence of the other: discussions of the emergence of cultural complexity have to proceed with greater scrutiny of the evidence on a site-by-site basis to establish secure associations between the archaeology present there and the hominins who created it. This paper presents one such case study: Niah Cave in Sarawak on the island of Borneo, famous for the discovery in 1958 in the West Mouth of the Great Cave of a modern human skull, the 'Deep Skull,' controversially associated with radiocarbon dates of ca. 40,000 years before the present. A new chronostratigraphy has been developed through a re-investigation of the lithostratigraphy left by the earlier excavations, AMS-dating using three different comparative pre-treatments including ABOX of charcoal, and U-series using the Diffusion-Absorption model applied to fragments of bones from the Deep Skull itself. Stratigraphic reasons for earlier uncertainties about the antiquity of the skull are examined, and it is shown not to be an 'intrusive' artifact. It was probably excavated from fluvial-pond-desiccation deposits that accumulated episodically in a shallow basin immediately behind the cave entrance lip, in a climate that ranged from times of comparative aridity with complete desiccation, to episodes of greater surface wetness, changes attributed to regional climatic fluctuations. Vegetation outside the cave varied significantly over time, including wet lowland forest, montane forest, savannah, and grassland. The new dates and the lithostratigraphy relate the Deep Skull to evidence of episodes of human activity that range in date from ca. 46,000 to ca. 34,000 years ago. Initial investigations of sediment scorching, pollen, palynomorphs, phytoliths, plant macrofossils, and starch grains recovered from existing exposures, and of vertebrates from the current and the earlier excavations, suggest that human foraging during these times was marked by habitat-tailored hunting technologies, the collection and processing of toxic plants for consumption, and, perhaps, the use of fire at some forest-edges. The Niah evidence demonstrates the sophisticated nature of the subsistence behavior developed by modern humans to exploit the tropical environments that they encountered in Southeast Asia, including rainforest.