Severe recurrent hypoglycaemia in a patient with aggressive melanoma.

We present a case of hypoglycemia in a young patient without diabetes mellitus who presented initially with enlarging neck mass and weight loss, and was found to have aggressive melanoma with metastasis to multiple organs and diffuse lymphadenopathy. He had presented to the emergency room two times with neuroglycopenic symptoms that required admission and intravenous dextrose continuously. Evaluation of hypoglycemia included C-peptide, insulin levels, insulin-like growth factor (IGF) -I and -II, and ß- hydroxybutyrate. Insulin levels were suppressed appropriately during hypoglycemia, however, IGF-II:IGF-I ratio was high, suggesting non-islet tumour induced hypoglycemia. The presence of IGF-II produced by large tumors results in a low hepatic glucose output and increased uptake by skeletal muscle, resulting in hypoglycemia especially in a patient with extremely low appetite such as our patient. Treating the culprit malignancy leads to resolution of hypoglycemia, but corticosteroids have been used to suppress IGF-II levels and alleviate symptoms.

Inhibition of endoplasmic reticulum stress and oxidative stress by vitamin D in endothelial cells.

Endoplasmic reticulum (ER) stress and oxidative stress promote endothelial dysfunction and atherosclerosis. Since vitamin D has been shown in several studies to lower the risk of cardiovascular disease, we examined the effects of vitamin D on ER stress and oxidative stress in endothelial cells. ER stress was measured using the placental secreted alkaline phosphatase assay and oxidative stress was measured by hydroethidine fluorescence. Expression of ER stress markers, including glucose-regulated protein 78, c-jun N-terminal kinase 1 phosphorylation, and eukaryotic initiation factor 2α phosphorylation, as well as X-box binding protein-1 splicing were measured in tunicamycin (TM)-treated human umbilical endothelial cells (HUVEC) treated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and other vitamin D analogs. When TM and 1,25-(OH)2D3 were added simultaneously, 1,25-(OH)2D3 prevented ER stress. However, the effect was much stronger when cells were pre-treated with 1,25-(OH)2D3 for 24-h. However, ER stress was not inhibited by 25-OH vitamin D3 (25-OHD3) or the vitamin D analog EB1089. Both ZK191784 and the vitamin D metabolite 24,25-dihydroxyvitamin D3 were as effective as 1,25-(OH)2D3 in preventing ER stress. Similar effects were observed dextrose-induced stress. All of the compounds tested, except for 25-OHD3, inhibited dextrose-induced (27.5mM) oxidative stress and ER stress. Although TM with and without 1,25-(OH)2D3 had no effect on VDR expression, inhibition of VDR expression via siRNA prevented 1,25-(OH)2D3, ZK191784, EB1089, and 24,25-dihydroxyvitamin D3 from inhibiting dextrose-mediated SO generation. Furthermore, each vitamin D analog, with the exception of 25-OHD3, prevented dextrose-induced toxicity. These results suggest that vitamin D has a protective effect on vascular endothelial cells.

Pro-inflammatory signaling by 24,25-dihydroxyvitamin D3 in HepG2 cells.

The vitamin D metabolite 24,25-dihydroxyvitamin D3 (24, 25[OH]2D3) was shown to induce nongenomic signaling pathways in resting zone chondrocytes and other cells involved in bone remodeling. Recently, our laboratory demonstrated that 24,25-[OH]2D3 but not 25-hydroxyvitamin D3, suppresses apolipoprotein A-I (apo A-I) gene expression and high-density lipoprotein (HDL) secretion in hepatocytes. Since 24,25-[OH]2D3 has low affinity for the vitamin D receptor (VDR) and little is known with regard to how 24,25-[OH]2D3 modulates nongenomic signaling in hepatocytes, we investigated the capacity of 24,25-[OH]2D3 to activate various signaling pathways relevant to apo A-I synthesis in HepG2 cells. Treatment with 24,25-[OH]2D3 resulted in decreased peroxisome proliferator-activated receptor alpha (PPARα) expression and retinoid-X-receptor alpha (RXRα) expression. Similarly, treatment of hepatocytes with 50 nM 24,25-[OH]2D3 for 1-3 h induced PKCα activation as well as c-jun-N-terminal kinase 1 (JNK1) activity and extracellular-regulated kinase 1/2 (ERK1/2) activity. These changes in kinase activity correlated with changes in c-jun phosphorylation, an increase in AP-1-dependent transcriptional activity, as well as repression of apo A-I promoter activity. Furthermore, treatment with 24,25-[OH]2D3 increased IL-1ß, IL-6, and IL-8 expression by HepG2 cells. These observations suggest that 24,25-[OH]2D3 elicits several novel rapid nongenomic-mediated pro-inflammatory protein kinases targeting AP1 activity, increasing pro-inflammatory cytokine expression, potentially impacting lipid metabolism and hepatic function.

Inhibition of ABCA1 Protein Expression and Cholesterol Efflux by TNF α in MLO-Y4 Osteocytes.

Hip fracture and myocardial infarction cause significant morbidity and mortality. In vivo studies raising serum cholesterol levels as well as pro-inflammatory cytokines such as TNF α manifest bone loss and atherosclerotic vascular disease, suggesting that abnormalities of cholesterol transport may contribute to osteoporosis. We used the mouse osteocyte cell line (MLO-Y4) to investigate the effects of TNF α on the expression of cholesterol acceptor proteins such as apolipoprotein A-I (apo A-I) and apolipoprotein E (apo E), as well as on the cholesterol transporters ATP-binding cassette-1 (ABCA1), scavenger receptor class B type 1 (SRB1), and cluster of differentiation 36 (CD36). MLO-Y4 cells do not express apo A-I or apo E; however, they do express all three cholesterol transporters (ABCA1, SRB1, and CD36). Treatment of MLO-Y4 cells with TNF α had no effect on SRB1, CD36, and osteocalcin levels; however, TNF α reduced ABCA1 protein levels in a dose-dependent manner and cholesterol efflux to apo A-I. Interestingly, TNF α treatment increased ABCA1 promoter activity and ABCA1 mRNA levels, and increased liver X receptor α protein expression, but had no effect on retinoid X receptor α and retinoic acid receptor α levels. Pharmacological inhibition of p38 mitogen-activated protein (MAP) kinase, but not c-jun-N-terminal kinase 1 or mitogen-activated protein kinase (MEK), restored ABCA1 protein levels in TNF α-treated cells. These results suggest that pro-inflammatory cytokines regulate cholesterol metabolism in osteocytes in part by suppressing ABCA1 levels post-translationally in a p38 MAP kinase-dependent manner.

Myocardial infarction risk among patients with fractures receiving bisphosphonates.

OBJECTIVE: To determine if bisphosphonates are associated with reduced risk of acute myocardial infarction (AMI). PATIENTS AND METHODS: A cohort of 14,256 veterans 65 years or older with femoral or vertebral fractures was selected from national administrative databases operated by the US Department of Veterans Affairs and was derived from encounters at Veterans Affairs facilities between October 1, 1998, and September 30, 2006. The time to first AMI was assessed in relationship to bisphosphonate exposure as determined by records from the Pharmacy Benefits Management Database. Time to event analysis was performed using multivariate Cox proportional hazards regression. An adjusted survival analysis curve and a Kaplan-Meier survival curve were analyzed. RESULTS: After controlling for atherosclerotic cardiovascular disease risk factors and medications, bisphosphonate use was associated with an increased risk of incident AMI (hazard ratio, 1.38; 95% CI, 1.08-1.77; P=.01). The timing of AMI correlated closely with the timing of bisphosphonate therapy initiation. CONCLUSION: Our observations in this study conflict with our hypothesis that bisphosphonates have antiatherogenic effects. These findings may alter the risk-benefit ratio of bisphosphonate use for treatment of osteoporosis, especially in elderly men. However, further analysis and confirmation of these findings by prospective clinical trials is required.

Primary hyperparathyroidism and vitamin D in African Americans.

OBJECTIVES: Vitamin D deficiency is more common in African Americans than in the general population or other ethnicities. Vitamin D deficiency also occurs more frequently in patients with primary hyperparathyroidism (PHPT) than in the general population. Currently, the limited data on vitamin D deficiency in African Americans with primary hyperparathyroidism (PHPT) is inconsistent as to whether the vitamin D deficiency observed in PHPT is yet even more pronounced in Africans with PHPT relative to non-African Americans with PHPT. METHODS: On the basis of biochemical, radiological, and surgical (adenoma weight) parameters, African Americans have been reported to have a more severe form of PHPT than non-African Americans. However, comparative clinical manifestations of PHPT in African Americans have not been well described. RESULTS: Current guidelines recommend vitamin D repletion in mild, asymptomatic PHPT when levels of 25-hydroxyvitamin D are less than 20 ng/mL. Studies that reported vitamin D repletion with ergocalciferol or cholecalciferol in PHPT have not stratified data according to ethnicity. Discrepancies therefore exist between repleting vitamin D in African Americans who may potentially have a more severe PHPT profile, but simultaneously a more pronounced vitamin D deficiency. CONCLUSION: Effectively designed clinical trials are necessary to evaluate the indications, efficacy, and safety of vitamin D in African Americans with PHPT.

The emerging evidence for vitamin D-mediated regulation of apolipoprotein A-I synthesis.

Ischemic heart disease and cerebrovascular ischemia are leading causes of mortality in industrialized countries. The pathogenesis of these diseases involves the formation of atherosclerotic plaques with eventual rupture and superimposed thrombosis. This process is inhibited by high-density lipoprotein (HDL), the main protein component of which is apolipoprotein A-I (apo A-I). Vitamin D3 is a hormone produced by sun-exposed skin but is acquired also in the diet. The Framingham Offspring Study and the Third National Health and Nutritional Examination Survey showed a link between vitamin D3 intake and cardiovascular risk factors. The link between 25-hydroxyvitamin D3 and HDL cholesterol (HDLc) and apo A-I is not as clear. Studies in vitamin D receptor knockout mice demonstrated higher HDLc and hepatic apo A-I messenger RNA expression relative to wild type. Experiments in cultured hepatocytes supported these observations. Human studies evaluating the relationship between vitamin D3 and apo A-I and HDLc have yielded conflicting results, but most suggest a positive link between increasing vitamin D3 levels and plasma apo A-I and HDLc. The purpose of this review is to examine the evidence linking vitamin D status and cardiovascular disease, to determine if there is a relationship between vitamin D levels and development of an atherogenic lipid profile. Our objectives are to determine if plasma vitamin D levels correlate with plasma HDLc and apo A-I and, if so, offer speculation as to how apo A-I in the context of high vitamin D levels provides enhanced atheroprotection.

24, 25-dihydroxycholecalciferol but not 25-hydroxycholecalciferol suppresses apolipoprotein A-I gene expression.

AIMS: Ligands for the vitamin D receptor (VDR) regulate apolipoprotein A-I (apo A-I) gene expression in a tissue-specific manner. The vitamin D metabolite 24, 25-dihydroxycholecalciferol (24, 25-(OH)(2)D(3)) has been shown to possess unique biological effects. To determine if 24, 25-(OH)(2)D(3) modulates apo A-I gene expression, HepG2 hepatocytes and Caco-2 intestinal cells were treated with 24, 25-(OH)(2)D(3) or its precursor 25-OHD(3). MAIN METHODS: Apo A-I protein levels and mRNA levels were measured by Western and Northern blotting, respectively. Changes in apo A-I promoter activity were measured using the chlorampenicol acetytransferase assay. KEY FINDINGS: Treatment with 24, 25-(OH)(2)D(3), but not 25-OHD(3), inhibited apo A-I secretion in HepG2 and Caco-2 cells and apo A-I mRNA levels and apo A-I promoter activity in HepG2 cells. To determine if 24, 25-(OH)(2)D(3) represses apo A-I gene expression through site A, the nuclear receptor binding element that is essential for VDRs effects on apo A-I gene expression, HepG2 cells were transfected with plasmids containing or lacking site A. While the site A-containing plasmid was suppressed by 24, 25-(OH)(2)D(3), the plasmid lacking site A was not. Likewise, treatment with 24, 25-(OH)(2)D(3) suppressed reporter gene expression in cells transfected with a plasmid containing site A in front of a heterologous promoter. Finally, antisense-mediated VDR depletion failed to reverse the silencing effects of 24, 25-(OH)(2)D(3) on apo A-I expression. SIGNIFICANCE: These results suggest that the vitamin D metabolite 24, 25-(OH)(2)D(3) is an endogenous regulator of apo A-I synthesis through a VDR-independent signaling mechanism.

An observational study of musculoskeletal pain among patients receiving bisphosphonate therapy.

OBJECTIVE: To seek evidence for the association of bisphosphonate use with diffuse musculoskeletal pain (MSKP) in a large national cohort, controlling for conditions associated with MSKP. PATIENTS AND METHODS: This retrospective cohort study enrolled all US veterans aged 65 years or older with a vertebral or hip fracture who were treated for at least 1 year between October 1, 1998, and September 30, 2006 (N=26,545). All International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnostic codes, demographics, and pharmaceutical data were obtained from national databases. A composite end point, based on ICD-9-CM codes compatible with diffuse MSKP, was constructed. The primary outcome was time until MSKP. We performed regression analysis using the Cox proportional hazards model, controlling for age, sex, race, alcoholism, depression, anxiety, smoking, recent 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (statin) use, rheumatic disease, and comorbidity score. RESULTS: The univariate regression identified an association of bisphosphonate exposure and MSKP (hazard ratio, 1.22; 95% confidence interval, 1.04-1.44). In the multivariate regression, however, patients prescribed a bisphosphonate were not more likely to be assigned an ICD-9-CM code compatible with diffuse MSKP (hazard ratio, 1.10; 95% confidence interval, 0.93-1.30). Consistent with prior studies, we found that female sex, depression, anxiety, comorbidity score, and the presence of a rheumatic disease were all associated with a greater risk of a diagnosis of diffuse MSKP. There was no demonstrable association with statin exposure. CONCLUSION: Bisphosphonate use was not associated with a statistically higher rate of MSKP in this cohort. Individual patients may rarely report MSKP while taking bisphosphonates; however, for our studied cohort, incident MSKP does not appear to explain bisphosphonate discontinuation rates.

Obesity-related changes in high-density lipoprotein metabolism.

Obesity is associated with a 3-or-more-fold increase in the risk of fatal and nonfatal myocardial infarction (1,2,3,4,5,6). The American Heart Association has reclassified obesity as a major, modifiable risk factor for coronary heart disease (7). The increased prevalence of premature coronary heart disease in obesity is attributed to multiple factors (8,9,10). A principal contributor to this serious morbidity is the alterations in plasma lipid and lipoprotein levels. The dyslipidemia of obesity is commonly manifested as high plasma triglyceride levels, low high-density lipoprotein cholesterol (HDLc), and normal low-density lipoprotein cholesterol (LDLc) with preponderance of small dense LDL particles (7,8,9,10). However, there is a considerable heterogeneity of plasma lipid profile in overweight and obese people. The precise cause of this heterogeneity is not entirely clear but has been partly attributed to the degree of visceral adiposity and insulin resistance. The emergence of glucose intolerance or a genetic predisposition to familial combined hyperlipidemia will further modify the plasma lipid phenotype in obese people (11,12,13,14,15).

Differential regulation of apolipoprotein A-I gene expression by vitamin D receptor modulators.

We have found that 1,25-dihydroxy-cholecalciferol (1,25-(OH)(2)D(3)) represses the expression of the apolipoprotein A-I (apo A-I) gene in hepatocytes. In this manuscript we examined the effects of the vitamin D receptor (VDR) modulators EB1089 (EB) and ZK191784 (ZK) on expression of the apo A-I gene in liver (HepG2) and in intestinal (Caco-2) cells. In HepG2 cells, EB and ZK induced apo A-I secretion and gene promoter activity in a dose-dependent manner. This induction did not require the VDR since antisense-mediated inhibition of VDR had no appreciable effect on apo A-I promoter activity in cells treated with EB or ZK. Although repression of apo A-I gene expression by 1,25-(OH)(2)D(3) in hepatocytes required nuclear receptor binding to site A in the promoter, this cis-element was insufficient for induction of apo-AI by EB and ZK. In Caco-2 cells, treatment with 1,25-(OH)(2)D(3) had no effect on apo A-I protein secretion or promoter activity while EB induced and ZK inhibited apo A-I gene expression. Gel shift assays showed that none of the treatments resulted in a change in site A binding activity. These results indicate that VDR modulators in hepatocytes and intestinal cells differentially regulate expression of the apo A-I gene.

Inhibition of apolipoprotein AI gene expression by 1, 25-dihydroxyvitamin D3.

Members of the steroid receptor superfamily are known to alter the transcription of apolipoprotein AI (apo AI), the major apoprotein of high-density lipoprotein (HDL). To assess the role of vitamin D receptor (VDR) in apo AI gene expression, we investigated the effect of 1alpha, 25-dihydroxycholecalciferol (1, 25-(OH)2 D3) as well as the vitamin D antagonist ZK-191784 (ZK), on apo AI gene expression and promoter activity in the human hepatoma cell line HepG2. Apo AI secretion and mRNA levels were both suppressed in a dose-dependent manner in HepG2 cells treated 1, 25-(OH)2 D3. This was accompanied by a similar decrease in apo AI promoter activity. Mapping of the vitamin D response element showed that suppression required a region of the apo AI gene promoter identified previously to contain site A. However, vitamin D treatment had no effect on nuclear factor binding to site A of the apo AI promoter. Treatment with vitamin D receptor antagonist ZK inhibited the ability of 1, 25-(OH)2 D3 to repress apo AI promoter activity, while higher doses of ZK increased apo AI promoter activity. ZK did not alter estradiol stimulated apo AI promoter activity. The VDR antisense ODN had no effect on apo AI promoter activity in control cells, however, it reversed the repression normally seen in cells treated with 1, 25-(OH)2D3. It is concluded that 1, 25-(OH)2 D3 suppresses apo A1 gene expression at the transcriptional level, possibly by altering coactivators or corepressors. This effect requires the VDR as well as a vitamin D response element in the apo AI promoter.

Nonalcoholic steatohepatitis: histologic features and clinical correlations with 30 blinded biopsy specimens.

Thirty overweight patients with clinically characterized and biopsy proven nonalcoholic steatohepatitis (NASH) were enrolled in a 48-week treatment trial with rosiglitazone, a peroxisome proliferator-activator receptor (PPAR)-gamma agonist that enhances insulin sensitivity. Improvement in laboratory liver tests, insulin resistance and liver fat content were documented; blinded biopsy review demonstrated decreases in necroinflammatory activity or grade and in individual components of grade, and changes in the relationship of lobular and portal inflammation as well as in the nature of perisinusoidal fibrosis. The current study identified correlations of histological features of the protocol entry biopsy specimens with contemporaneous laboratory and imaging tests. Significant correlations with histologically assessed steatosis were liver fat, evaluated by computed tomography (P = 0.001); mean HbA1C, a measure of glycemic control (P = 0.004); and QUICKI, a measure of insulin sensitivity (P = 0.05). Histologically determined grades of steatohepatitis (SH) correlated with HbA1C (P = 0.01), and a trend toward elevated fasting glucose levels was seen. No subject in the study was cirrhotic at entry; fibrosis scores of the 30 subjects did not significantly correlate with age, gender, body mass index, or clinical tests. All subjects underwent 3 biopsies (prior, entry, and posttreatment), and all had undergone a prior biopsy with diagnostic SH. By blinded analysis, 7 study entry biopsy specimens did not fulfill published strict criteria for SH. Laboratory results from these subjects included normal fasting glucose level and, compared with the 23 subjects with criteria for SH, lower mean alanine aminotransferase and aspartate aminotransferase levels (P = 0.02 for both), less insulin resistance (P = 0.03), and lower mean HbA1C (P = 0.001). We conclude that biopsy findings determined by blinded analysis correlated with image-detected steatosis, laboratory markers of hepatic inflammation, insulin resistance, and long-term glycemia; the findings confirm the usefulness of strict histological criteria in the evaluation of NASH.

Improved nonalcoholic steatohepatitis after 48 weeks of treatment with the PPAR-gamma ligand rosiglitazone.

Insulin resistance (IR) commonly is associated with nonalcoholic steatohepatitis (NASH). To establish whether IR causes NASH, this study was undertaken to determine if improving IR would improve the histologic features that define NASH. Thirty adults with prior biopsy evidence of NASH were enrolled to receive rosiglitazone, 4 mg twice daily for 48 weeks. All patients were overweight (body mass index [BMI] > 25 kg/m(2)) and 23% were severely obese (BMI > 35 kg/m(2)); 50% had impaired glucose tolerance or diabetes. Liver biopsy specimens were obtained before beginning treatment and at treatment completion. Twenty-six patients had posttreatment biopsies; of these, 22 had initial protocol liver biopsies that met published criteria for NASH on subsequent blinded evaluation. Within this initial NASH group, the mean global necroinflammatory score significantly improved with treatment and biopsies of 10 patients (45%) no longer met published criteria for NASH after treatment. Significant improvement in hepatocellular ballooning and zone 3 perisinusoidal fibrosis also occurred. Five patients withdrew early; the 25 patients completing 48 weeks of treatment had significantly improved insulin sensitivity and mean serum alanine aminotransferase (ALT) levels (104 initially, 42 U/L at the end of treatment). Adverse effects led to withdrawal of 3 patients (10%). Weight gain occurred in 67% of patients and the median weight increase was 7.3%. Within 6 months of completing treatment, liver enzyme levels had increased to near pretreatment levels. In conclusion, improving insulin sensitivity with rosiglitazone resulted in improved histologic markers of NASH, an observation suggesting that insulin resistance contributes to its development and that improving insulin sensitivity may be important in treating this liver disease.

Interim results of a pilot study demonstrating the early effects of the PPAR-gamma ligand rosiglitazone on insulin sensitivity, aminotransferases, hepatic steatosis and body weight in patients with non-alcoholic steatohepatitis.

BACKGROUND/AIMS: Hyperinsulinemia may cause hepatic steatosis and non-alcoholic steatohepatitis (NASH). The aims of this pilot study were to examine the safety of using the insulin-sensitizing peroxisomal proliferator activated receptor (PPAR) gamma ligand rosiglitazone in patients with NASH and determine whether improved insulin sensitivity correlates with improved fatty liver. METHODS: Thirty subjects with NASH and elevated alanine aminotransferase (ALT) levels received rosiglitazone, 4 mg twice daily for 48 weeks; the preliminary results presented here were obtained at 24 weeks. Insulin sensitivity was measured using fasting insulin and glucose levels and liver fat content was estimated by CT imaging. RESULTS: By 24 weeks, rosiglitazone improved insulin sensitivity and reduced liver fat content. The mean ALT decreased from 86 to 37 U/l (P<0.01). Four subjects (13%) withdrew, one because of a rise in ALT from 59 to 277 U/l that coincided with concomitant prednisone use. Subjects experienced a mean weight gain of 3.5% and hemoglobin drop of 1.1 g/dl. CONCLUSIONS: Treatment of NASH with rosiglitazone for 24 weeks improved insulin sensitivity, reduced liver fat content and improved biochemical evidence of hepatocellular injury. These preliminary data provide evidence that hyperinsulinemia may be a cause of NASH. Strategies to improve insulin sensitivity as a treatment of NASH deserve further investigation.