Assessment of DNA quality for whole genome library preparation.

In recent years, more sophisticated DNA technologies for genotyping have enabled considerable progress in various fields such as clinical genetics, archaeogenetics and forensic genetics. DNA samples previously rejected as too challenging to analyze due to low amounts of degraded DNA can now provide useful information. To increase the chances of success with the new methodologies, it is crucial to know the fragment size of the template DNA molecules, and whether the DNA in a sample is mostly single or double stranded. With this knowledge, an appropriate library preparation method can be chosen, and the DNA shearing parameters of the protocol can be adjusted to the DNA fragment size in the sample. In this study, we first developed and evaluated a user-friendly fluorometry-based protocol for estimation of DNA strandedness. We also evaluated different capillary electrophoresis methods for estimation of DNA fragmentation levels. Next, we applied the developed methodologies to a broad variety of DNA samples processed with different DNA extraction protocols. Our findings show that both the applied DNA extraction method and the sample type affect the DNA strandedness and fragmentation. The established protocols and the gained knowledge will be applicable for future sequencing-based high-density SNP genotyping in various fields.

A single mechanism can explain network-wide insulin resistance in adipocytes from obese patients with type 2 diabetes.

The response to insulin is impaired in type 2 diabetes. Much information is available about insulin signaling, but understanding of the cellular mechanisms causing impaired signaling and insulin resistance is hampered by fragmented data, mainly obtained from different cell lines and animals. We have collected quantitative and systems-wide dynamic data on insulin signaling in primary adipocytes and compared cells isolated from healthy and diabetic individuals. Mathematical modeling and experimental verification identified mechanisms of insulin control of the MAPKs ERK1/2. We found that in human adipocytes, insulin stimulates phosphorylation of the ribosomal protein S6 and hence protein synthesis about equally via ERK1/2 and mTORC1. Using mathematical modeling, we examined the signaling network as a whole and show that a single mechanism can explain the insulin resistance of type 2 diabetes throughout the network, involving signaling both through IRS1, PKB, and mTOR and via ERK1/2 to the nuclear transcription factor Elk1. The most important part of the insulin resistance mechanism is an attenuated feedback from the protein kinase mTORC1 to IRS1, which spreads signal attenuation to all parts of the insulin signaling network. Experimental inhibition of mTORC1 using rapamycin in adipocytes from non-diabetic individuals induced and thus confirmed the predicted network-wide insulin resistance.

Insulin signaling in type 2 diabetes: experimental and modeling analyses reveal mechanisms of insulin resistance in human adipocytes.

Type 2 diabetes originates in an expanding adipose tissue that for unknown reasons becomes insulin resistant. Insulin resistance reflects impairments in insulin signaling, but mechanisms involved are unclear because current research is fragmented. We report a systems level mechanistic understanding of insulin resistance, using systems wide and internally consistent data from human adipocytes. Based on quantitative steady-state and dynamic time course data on signaling intermediaries, normally and in diabetes, we developed a dynamic mathematical model of insulin signaling. The model structure and parameters are identical in the normal and diabetic states of the model, except for three parameters that change in diabetes: (i) reduced concentration of insulin receptor, (ii) reduced concentration of insulin-regulated glucose transporter GLUT4, and (iii) changed feedback from mammalian target of rapamycin in complex with raptor (mTORC1). Modeling reveals that at the core of insulin resistance in human adipocytes is attenuation of a positive feedback from mTORC1 to the insulin receptor substrate-1, which explains reduced sensitivity and signal strength throughout the signaling network. Model simulations with inhibition of mTORC1 are comparable with experimental data on inhibition of mTORC1 using rapamycin in human adipocytes. We demonstrate the potential of the model for identification of drug targets, e.g. increasing the feedback restores insulin signaling, both at the cellular level and, using a multilevel model, at the whole body level. Our findings suggest that insulin resistance in an expanded adipose tissue results from cell growth restriction to prevent cell necrosis.

Getting the conclusive lead with investigative genetic genealogy - A successful case study of a 16 year old double murder in Sweden.

On the morning of October 19, 2004, an eight-year-old boy and a 56-year-old woman were stabbed to death on an open street in the city of Linköping, Sweden. The perpetrator left his DNA at the crime scene, and after 15 years of various investigation efforts, including more than 9000 interrogations and mass DNA screening of more than 6000 men, there were still no clues about the identity of the unknown murderer. The successful application of investigative genetic genealogy (IGG) in the US raised the interest for this tool within the Swedish Police Authority. After legal consultations it was decided that IGG could be applied in this double murder case as a pilot case study. From extensive DNA analysis, including whole-genome sequencing and genotype imputation, DNA data sets were established and searched within both GEDmatch and FamilyTree DNA genealogy databases. A number of fairly distant relatives were found from which family trees were created. The genealogy work resulted in two candidates, two brothers, one of whom matched the crime scene samples by routine STR profiling. The suspect confessed the murders at the initial police hearing and was later convicted of the murders. In this paper we describe the successful application of an emerging technology. We disclose details of the DNA analyses which, due to the poor quality and low quantity of the DNA, required reiterative sequencing and genotype imputation efforts. The successful application of IGG in this double murder case exemplifies its applicability not only in the US but also in Europe. The pressure is now high on the involved authorities to establish IGG as a tool for cold case criminal investigations and for missing person identifications. There is, however, a continuous need to accommodate legal, social and ethical aspects as well.

Short-term overeating induces insulin resistance in fat cells in lean human subjects.

Insulin resistance and type 2 diabetes (T2D) are closely linked to obesity. Numerous prospective studies have reported on weight gain, insulin resistance, and insulin signaling in experimental animals, but not in humans. We examined insulin signaling in adipocytes from lean volunteers, before and at the end of a 4-wk period of consuming a fast-food, high-calorie diet that led to weight gain. We also examined adipocytes from patients with T2D. During the high-calorie diet, subjects gained 10% body weight and 19% total body fat, but stayed lean (body mass index = 24.3 kg/m(2)) and developed moderate systemic insulin resistance. Similarly to the situation in T2D subjects, in subjects on the high-calorie diet, the amount of insulin receptors was reduced and phosphorylation of IRS1 at tyrosine and at serine-307 (human sequence, corresponding to murine serine-302) were impaired. The amount of insulin receptor substrate protein-1 (IRS1) and the phosphorylation of IRS1 at serine-312 (human sequence, corresponding to murine serine-307) were unaffected by the diet. Unlike the T2D subjects, in subjects on the high-calorie diet, likely owing to the ongoing weight-gain, phosphorylation of MAP-kinases ERK1/2 became hyperresponsive to insulin. To our knowledge this study is the first to investigate insulin signaling during overeating in humans, and it demonstrates that T2D effects on intracellular insulin signaling already occur after 4 wks of a high-calorie diet and that the effects in humans differ from those in laboratory animals.

Phosphorylation of IRS1 at serine 307 in response to insulin in human adipocytes is not likely to be catalyzed by p70 ribosomal S6 kinase.

The insulin receptor substrate-1 (IRS1) is phosphorylated on serine 307 (human sequence, corresponding to murine serine 302) in response to insulin as part of a feedback loop that controls IRS1 phosphorylation on tyrosine residues by the insulin receptor. This in turn directly affects downstream signaling and is in human adipocytes implicated in the pathogenesis of insulin resistance and type 2 diabetes. The phosphorylation is inhibited by rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR) in complex with raptor (mTORC1). The mTORC1-downstream p70 ribosomal protein S6 kinase (S6K1), which is activated by insulin, can phosphorylate IRS1 at serine 307 in vitro and is considered the physiological protein kinase. Because the IRS1 serine 307-kinase catalyzes a critical step in the control of insulin signaling and constitutes a potential target for treatment of insulin resistance, it is important to know whether S6K1 is the physiological serine 307-kinase or not. We report that, by several criteria, S6K1 does not phosphorylate IRS1 at serine 307 in response to insulin in intact human primary adipocytes: (i) The time-courses for phosphorylation of S6K1 and its phosphorylation of S6 are not compatible with the phosphorylation of IRS1 at serine 307; (ii) A dominant-negative construct of S6K1 inhibits the phosphorylation of S6, without effect on the phosphorylation of IRS1 at serine 307; (iii) The specific inhibitor of S6K1 PF-4708671 inhibits the phosphorylation of S6, without effect on phosphorylation of IRS1 at serine 307. mTOR-immunoprecipitates from insulin-stimulated adipocytes contains an unidentified protein kinase specific for phosphorylation of IRS1 at serine 307, but it is not mTOR or S6K1.

Mechanistic explanations for counter-intuitive phosphorylation dynamics of the insulin receptor and insulin receptor substrate-1 in response to insulin in murine adipocytes.

Insulin signaling through insulin receptor (IR) and insulin receptor substrate-1 (IRS1) is important for insulin control of target cells. We have previously demonstrated a rapid and simultaneous overshoot behavior in the phosphorylation dynamics of IR and IRS1 in human adipocytes. Herein, we demonstrate that in murine adipocytes a similar overshoot behavior is not simultaneous for IR and IRS1. The peak of IRS1 phosphorylation, which is a direct consequence of the phosphorylation and the activation of IR, occurs earlier than the peak of IR phosphorylation. We used a conclusive modeling framework to unravel the mechanisms behind this counter-intuitive order of phosphorylation. Through a number of rejections, we demonstrate that two fundamentally different mechanisms may create the reversed order of peaks: (i) two pools of phosphorylated IR, where a large pool of internalized IR peaks late, but phosphorylation of IRS1 is governed by a small plasma membrane-localized pool of IR with an early peak, or (ii) inhibition of the IR-catalyzed phosphorylation of IRS1 by negative feedback. Although (i) may explain the reversed order, this two-pool hypothesis alone requires extensive internalization of IR, which is not supported by experimental data. However, with the additional assumption of limiting concentrations of IRS1, (i) can explain all data. Also, (ii) can explain all available data. Our findings illustrate how modeling can potentiate reasoning, to help draw nontrivial conclusions regarding competing mechanisms in signaling networks. Our work also reveals new differences between human and murine insulin signaling.

Rapid insulin-dependent endocytosis of the insulin receptor by caveolae in primary adipocytes.

BACKGROUND: The insulin receptor is localized in caveolae and is dependent on caveolae or cholesterol for signaling in adipocytes. When stimulated with insulin, the receptor is internalized. METHODOLOGY/PRINCIPAL FINDINGS: We examined primary rat adipocytes by subcellular fractionation to examine if the insulin receptor was internalized in a caveolae-mediated process. Insulin induced a rapid, t(1/2)<3 min, endocytosis of the insulin receptor in parallel with receptor tyrosine autophosphorylation. Concomitantly, caveolin-1 was phosphorylated at tyrosine(14) and endocytosed. Vanadate increased the phosphorylation of caveolin-1 without affecting insulin receptor phosphorylation or endocytosis. Immunocapture of endosomal vesicles with antibodies against the insulin receptor co-captured caveolin-1 and immunocapture with antibodies against tyrosine(14)-phosphorylated caveolin-1 co-captured the insulin receptor, demonstrating that the insulin receptor was endocytosed together with tyrosine(14)-phosphorylated caveolin-1. By immunogold electron microscopy the insulin receptor and caveolin-1 were colocalized in endosome vesicles that resembled caveosomes. Clathrin was not endocytosed with the insulin receptor and the inhibitor of clathrin-coated pit-mediated endocytosis, chlorpromazine, did not inhibit internalization of the insulin receptor, while transferrin receptor internalization was inhibited. CONCLUSION: It is concluded that in response to insulin stimulation the autophosphorylated insulin receptor in primary adipocytes is rapidly endocytosed in a caveolae-mediated process, involving tyrosine phosphorylation of caveolin-1.