Vitamin D deficiency

Vitamin D deficiency is common and may contribute to osteopenia, osteoporosis and falls risk in the elderly. Screening for vitamin D deficiency is important in high-risk patients, especially for patients who suffered minimal trauma fractures. Vitamin D deficiency should be treated according to the severity of the deficiency. In high-risk adults, follow-up serum 25-hydroxyvitamin D concentration should be measured 3–4 months after initiating maintenance therapy to confirm that the target level has been achieved. All patients should maintain a calcium intake of at least 1,000 mg for women aged ≤ 50 years and men ≤ 70 years, and 1,300 mg for women > 50 years and men > 70 years.     

In vivo antiviral activity and pharmacokinetics of (-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine in woodchuck hepatitis virus-infected woodchucks.

The (-) enantiomer of cis-5-fluoro-1l-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine [(-)-FTC)], a substituted oxathiolane compound with anti-hepatitis B virus activity in vitro, was assessed for its efficacy in woodchucks with naturally acquired woodchuck hepatitis virus (WHV) infection. Pharmacokinetics and in vitro anabolism were also determined. (-)-FTC was anabolized to the 5'-triphosphate in a dose-related fashion, reaching a maximum concentration at about 24 h in cultured woodchuck hepatocytes. Following administration of a dose of 10 mg/kg of body weight intraperitoneally (i.p.), the clearance of (-)-FTC from plasma was monoexponential, the terminal half-life was 3.76 +/- 1.4 h, and the systemic clearance was 0.12 +/- 0.06 liters/h/kg. The antiviral efficacy of (-)-FTC in the woodchuck model was assessed by quantitation of serum WHV DNA levels and by WHV particle-associated DNA polymerase activity at two dosages, 30 and 20 mg/kg given i.p. twice daily (b.i.d.), respectively. The level of WHV DNA in serum was reduced 20- to 150-fold (average, 56-fold) in the 30-mg/kg-b.i.d. treatment group and 6- to 49-fold (average, 27-fold) in the 20-mg/kg-b.i.d. treatment group. Viral DNA polymerase levels diminished accordingly. One week after treatment was discontinued, WHV levels returned to pretreatment levels in both studies. These animals were biopsied before and following treatment with 30 mg of (-)-FTC per kg. Their livers were characterized by a mild increase in cytoplasmic lipid levels, but this change was not associated with altered liver enzyme levels. Serum chemistry and hematology results were within the normal ranges for all treated animals. We conclude that (-)-FTC is a potent antihepadnaviral agent and that it has no detectable toxic effects in woodchucks when given for up to 25 days. Further development of (-)-FTC as an anti-hepatitis B virus therapy for patients is warranted.     

Mitochondrial Networking Protects ��-Cells From Nutrient-Induced Apoptosis

OBJECTIVE

Previous studies have reported that β-cell mitochondria exist as discrete organelles that exhibit heterogeneous bioenergetic capacity. To date, networking activity, and its role in mediating β-cell mitochondrial morphology and function, remains unclear. In this article, we investigate β-cell mitochondrial fusion and fission in detail and report alterations in response to various combinations of nutrients.

RESEARCH DESIGN AND METHODS

Using matrix-targeted photoactivatable green fluorescent protein, mitochondria were tagged and tracked in β-cells within intact islets, as isolated cells and as cell lines, revealing frequent fusion and fission events. Manipulations of key mitochondrial dynamics proteins OPA1, DRP1, and Fis1 were tested for their role in β-cell mitochondrial morphology. The combined effects of free fatty acid and glucose on β-cell survival, function, and mitochondrial morphology were explored with relation to alterations in fusion and fission capacity.

RESULTS

β-Cell mitochondria are constantly involved in fusion and fission activity that underlies the overall morphology of the organelle. We find that networking activity among mitochondria is capable of distributing a localized green fluorescent protein signal throughout an isolated β-cell, a β-cell within an islet, and an INS1 cell. Under noxious conditions, we find that β-cell mitochondria become fragmented and lose their ability to undergo fusion. Interestingly, manipulations that shift the dynamic balance to favor fusion are able to prevent mitochondrial fragmentation, maintain mitochondrial dynamics, and prevent apoptosis.

CONCLUSIONS

These data suggest that alterations in mitochondrial fusion and fission play a critical role in nutrient-induced β-cell apoptosis and may be involved in the pathophysiology of type 2 diabetes.Mitochondria mediate β-cell responses to extracellular glucose by generating ATP and initiating a cascade of events culminating in the release of insulin. It is not surprising that β-cell mitochondria have become an important target for investigations into the etiology of type 2 diabetes. Mitochondria are highly dynamic organelles whose morphology is regulated by cycles of fusion and fission, collectively termed mitochondrial dynamics (1,2). Networks are formed when mitochondria undergo fusion events that cause the compartments of participating mitochondria to become continuous. As a result, the constituents of each network share solutes, metabolites, and proteins (3–5) as well as a transmembrane electrochemical gradient (1,6). The disruption of such networks has been shown to have a profound effect on the progression of cells to apoptosis, particularly in cases where reactive oxygen species (ROS) are involved (7). As such, mitochondrial networking is thought to be a potential defense mechanism allowing for the buffering of mitochondrial ROS and calcium overload (8,9).Chronically elevated levels of glucose and fatty acid are thought to contribute to the progression of type 2 diabetes by adversely affecting β-cells and thereby causing a deterioration in insulin secretion (10). In vivo, a reduction in insulin gene expression due to reduced Pdx-1 binding has been observed in rats perifused with glucose and intralipids (11,12). In addition, exposure to high levels of glucose and/or free fatty acid has been shown to affect β-cell viability by inducing mitochondrial apoptosis and has been linked to ROS-induced mitochondrial calcium overload and damage (13). Recent studies indicate that nutrient-induced ROS increases subcellular mitochondrial membrane potential (ΔΨmt) heterogeneity and fragmentation of the mitochondrial architecture (14,15). These findings suggest that mitochondrial fragmentation-defragmentation might play a role in the effects of noxious stimuli. Although the functional significance of these changes has not been studied in β-cells, studies of mitochondrial morphology in other cells have demonstrated that the ability of mitochondria to form networks influences both ROS and calcium handling (7–9).Previous studies have reported that β-cell mitochondria form less elaborate network structures, compared with COS cells for example, and raise doubts on the existence of mitochondrial networking in these cells. Until now, technologies for examining and quantifying the ability of mitochondria to undergo fusion and fission were unavailable.In this work, we show that the densely packed appearance of mitochondria in the β-cell represents the existence of multiple juxtaposed units that do not share continuous matrix lumen but do go through frequent fusion and fission events. We further demonstrate that mitochondrial dynamics are disrupted by exposure to the combination of high fat and glucose, gradually leading to the arrest of fusion activity and complete fragmentation of the mitochondrial architecture. Inhibiting mitochondrial fission preserved mitochondrial morphology and dynamics and prevented β-cell apoptosis.     

Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation

Somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) gene occur in a significant proportion of patients with myeloid malignancies. Although there are extensive genetic data implicating TET2 mutations in myeloid transformation, the consequences of Tet2 loss in hematopoietic development have not been delineated. We report here an animal model of conditional Tet2 loss in the hematopoietic compartment that leads to increased stem cell self-renewal in vivo as assessed by competitive transplant assays. Tet2 loss leads to a progressive enlargement of the hematopoietic stem cell compartment and eventual myeloproliferation in vivo, including splenomegaly, monocytosis, and extramedullary hematopoiesis. In addition, Tet2(+/-) mice also displayed increased stem cell self-renewal and extramedullary hematopoiesis, suggesting that Tet2 haploinsufficiency contributes to hematopoietic transformation in vivo.     

Family study of fibromyalgia

OBJECTIVE: To assess for familial aggregation of fibromyalgia (FM) and measures of tenderness and pain, and for familial coaggregation of FM and major mood disorder (major depressive disorder or bipolar disorder). METHODS: Probands meeting the American College of Rheumatology criteria for FM and control probands with rheumatoid arthritis (RA) and no lifetime diagnosis of FM were recruited from consecutive referrals to 2 community-based rheumatology practices. Probands were ages 40-55 years and had at least 1 first-degree relative age 18 years or older who was available for interview and examination. All probands and interviewed relatives underwent a dolorimeter tender point examination and a structured clinical interview. Interviewed relatives were asked about first-degree relatives who were not available for interview, using a structured family interview. Logistic and linear regression models, adjusting for the correlation of observation within families, were applied to study the aggregation and coaggregation effects. RESULTS: Information was collected for 533 relatives of 78 probands with FM and 272 relatives of 40 probands with RA. FM aggregated strongly in families: the odds ratio (OR) measuring the odds of FM in a relative of a proband with FM versus the odds of FM in a relative of a proband with RA was 8.5 (95% confidence interval [95% CI] 2.8-26, P = 0.0002). The number of tender points was significantly higher, and the total myalgic score was significantly lower in the relatives of probands with FM compared with the relatives of probands with RA. FM coaggregated significantly with major mood disorder: the OR measuring the odds of major mood disorder in a relative of a proband with FM versus the odds of major mood disorder in a relative of a proband with RA was 1.8 (95% CI 1.1-2.9, P = 0.013). CONCLUSION: FM and reduced pressure pain thresholds aggregate in families, and FM coaggregates with major mood disorder in families. These findings have important clinical and theoretical implications, including the possibility that genetic factors are involved in the etiology of FM and in pain sensitivity. In addition, mood disorders and FM may share some of these inherited factors.     

In vivo stabilization of mutant human transthyretin in transgenic mice

Transthyretin (TTR) is a 55 kD homotetrameric serum protein transporter of retinol binding protein charged with retinol and thyroxine (T4). The highly amyloidogenic human TTR variant in which leucine at position 55 is replaced by proline (L55P TTR) is responsible for aggressive fatal amyloidosis with peripheral and autonomic neuropathy, cardiomyopathy and nephropathy. Mice bearing one or two copies of a 19.2 kB human genomic fragment containing the entire coding sequence and the known control regions of the L55P TTR transgene, failed to develop TTR amyloidosis even though their sera contained mutant human TTR. The frequency of TTR tissue deposition was increased when the L55P TTR transgene was bred onto a murine TTR-null background. Denaturation of sera from the transgenic animals and murine TTR-knockouts expressing the human L55P TTR transgene revealed that the TTR tetramer was much more stable in the presence of the murine protein because the TTR circulates as hybrid human/murine heterotetramers. Intraperitoneal administration of diflunisal, a non-steroidal anti-inflammatory drug that binds to TTR in its T4-binding site and inhibits fibril formation in vitro, to human L55P TTR transgenic animals in which the murine TTR gene had been silenced, also stabilizes the circulating mutant protein to in vitro urea denaturation.