Genetics of T2DM and Its Chronic Complications: Are We Any Closer to the Individual Prediction of Genetic Risk?

Type 2 diabetes mellitus (T2DM) is a complex disease that has risen in global prevalence over recent decades, resulting in concomitant and enormous socio-economic impacts. In addition to the well-documented risk factors of obesity, poor dietary habits and sedentary lifestyles, genetic background plays a key role in the aetiopathogenesis of diabetes and the development of associated micro- and macrovascular complications. Recent advances in genomic research, notably next-generation sequencing and genome- wide association studies, have greatly improved the efficiency with which genetic backgrounds to complex diseases are analysed. To date, several hundred single-nucleotide polymorphisms have been associated with T2DM or its complications. Given the polygenic background to T2DM (and numerous other complex diseases), the degree of genetic predisposition can be treated as a "continuous trait" quantified by a genetic risk score. Focusing mainly on the Central European population, this review summarizes recent state-of-the-art methods that have enabled us to better determine the genetic architecture of T2DM and the utility of genetic risk scores in disease prediction.

The estrogen receptor α-selective agonist propyl pyrazole triol improves glucose tolerance in ob/ob mice: potential molecular mechanisms

The authors and journal apologise for an error in the above paper, which appeared in volume 199 part 2, pages 275­286. The error relates to Fig. 10, given on page 283.

Metabolic and signaling events mediated by cardiotonic steroid ouabain in rat skeletal muscle.

The cardiac glycoside ouabain initiates a cascade of signaling events through Na+,K+-ATPase, leading to an increase in cell growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in skeletal muscle and clarified the mechanisms of ouabain signal transduction. In rat soleus muscle 200 microM ouabain decreased basal glucose uptake without effect on insulin-stimulated glucose uptake. Ouabain increased glycogen synthesis additively to insulin and this effect was abolished in the presence of a MEK1/2 inhibitor (PD98059) or a c-Src inhibitor (PP2). Ouabain exposure reduced glucose oxidation, and this effect was reversed in the presence of PP2. Incubation with ouabain did not affect intramuscular ATP and its metabolites; however acetyl-CoA carboxylase phosphorylation was reduced, with no effect on AMPK phosphorylation. Insulin-stimulated Akt phosphorylation was not affected by ouabain. Ouabain reduced basal and insulin-stimulated phosphorylation of PKC alpha/beta and delta isoforms, whereas phosphorylation of PKCzeta was unchanged. Ouabain exposure increased interaction of 1- and 2-subunits of Na-pump with c-Src, as assessed by co-immunoprecipitation with c-Src. Phosphorylation of ERK1/2, GSK 3 / and p90rsk activity was increased in response to ouabain, and these effects were prevented in the presence of PD98059 and PP2. In conclusion, the cardiac glycoside ouabain stimulates glycogen synthesis additively to insulin in rat skeletal muscle. This effect is mediated by activation of c-Src-, ERK1/2- p90rsk- and GSK3-dependent signaling pathway.

Differences in the ratio of RNA encoding two isoforms of the insulin receptor between control and NIDDM patients. The RNA variant without Exon 11 predominates in both groups.

Two alternative forms of the insulin receptor with different affinities for insulin are expressed as a result of alternative splicing of RNA corresponding to exon 11 of the IR gene. The percentage of IR-RNA molecules without exon 11, encoding the high-affinity isoform, was determined by cDNA-mediated PCR amplification of RNA extracts from the quadriceps femoris muscle of healthy control subjects (n = 9) and NIDDM patients (n = 7). In both patients and control individuals, a majority of the IR-RNA molecules contained exon 11. In addition, the proportion of IR-RNA molecules without exon 11 was decreased in patients (21 +/- 1%) compared with control subjects (31 +/- 3%) (P = 0.018). Careful investigation of the kinetics of the PCR-based assay system, as well as the conditions for separation of the PCR products, allowed us to suggest a possible explanation of the discrepant results concerning the alternative splicing presented in previous reports. The diabetic subjects as a group had higher fasting insulin levels and lower insulin-mediated glucose uptake during a euglycemic-hyperinsulinemic clamp (P = 0.042). However, identification of the regulatory pathways leading to the splicing alteration in NIDDM patients requires further investigation.

Use of a novel impermeable biotinylated photolabeling reagent to assess insulin- and hypoxia-stimulated cell surface GLUT4 content in skeletal muscle from type 2 diabetic patients.

Cell surface GLUT4 levels in skeletal muscle from nine type 2 diabetic subjects and nine healthy control subjects have been assessed by a new technique that involves the use of a biotinylated photo-affinity label. A profound impairment in GLUT4 translocation to the skeletal muscle cell surface in response to insulin was observed in type 2 diabetic patients. Levels of insulin-stimulated cell surface GLUT4 above basal in type 2 diabetic patients were only approximately 10% of those observed in healthy subjects. The magnitude of the defect in GLUT4 translocation in type 2 diabetic patients was greater than that observed for glucose transport activity, which was approximately 50% of that in healthy subjects. Reduced GLUT4 translocation is therefore a major contributor to the impaired glucose transport activity in skeletal muscle from type 2 diabetic subjects. When a marked impairment in GLUT4 translocation occurs, the contribution of other transporters to transport activity becomes apparent. In response to hypoxia, marked reductions in skeletal muscle cell surface GLUT4 levels were also observed in type 2 diabetic patients. Therefore, a defect in a common late stage in signal transduction and/or a direct impairment in the GLUT4 translocation process accounts for reduced glucose transport in type 2 diabetic patients.

Characterization of signal transduction and glucose transport in skeletal muscle from type 2 diabetic patients.

We characterized metabolic and mitogenic signaling pathways in isolated skeletal muscle from well-matched type 2 diabetic and control subjects. Time course studies of the insulin receptor, insulin receptor substrate (IRS)-1/2, and phosphatidylinositol (PI) 3-kinase revealed that signal transduction through this pathway was engaged between 4 and 40 min. Insulin-stimulated (0.6-60 nmol/l) tyrosine phosphorylation of the insulin receptor beta-subunit, mitogen-activated protein (MAP) kinase phosphorylation, and glycogen synthase activity were not altered in type 2 diabetic subjects. In contrast, insulin-stimulated tyrosine phosphorylation of IRS-1 and anti-phosphotyrosine-associated PI 3-kinase activity were reduced 40-55% in type 2 diabetic subjects at high insulin concentrations (2.4 and 60 nmol/l, respectively). Impaired glucose transport activity was noted at all insulin concentrations (0.6-60 nmol/l). Aberrant protein expression cannot account for these insulin-signaling defects because expression of insulin receptor, IRS-1, IRS-2, MAP kinase, or glycogen synthase was similar between type 2 diabetic and control subjects. In skeletal muscle from type 2 diabetic subjects, IRS-1 phosphorylation, PI 3-kinase activity, and glucose transport activity were impaired, whereas insulin receptor tyrosine phosphorylation, MAP kinase phosphorylation, and glycogen synthase activity were normal. Impaired insulin signal transduction in skeletal muscle from type 2 diabetic patients may partly account for reduced insulin-stimulated glucose transport; however, additional defects are likely to play a role.

Isomer-specific antidiabetic properties of conjugated linoleic acid. Improved glucose tolerance, skeletal muscle insulin action, and UCP-2 gene expression.

Conjugated linoleic acid (CLA) isomers have a number of beneficial health effects, as shown in biomedical studies with animal models. Previously, we reported that a mixture of CLA isomers improved glucose tolerance in ZDF rats and activated peroxisome proliferator-activated receptor (PPAR)-gamma response elements in vitro. Here, our aim was to elucidate the effect(s) of specific CLA isomers on whole-body glucose tolerance, insulin action in skeletal muscle, and expression of genes important in glucose and lipid metabolism. ZDF rats were fed either a control diet (CON), one of two CLA supplemented diets (1.5% CLA) containing differing isoforms of CLA (47% c9,t11; 47.9% c10,t12, 50:50; or 91% c9,t11, c9,t11 isomers), or were pair-fed CON diet to match the intake of 50:50. The 50:50 diet reduced adiposity and improved glucose tolerance compared with all other ZDF treatments. Insulin-stimulated glucose transport and glycogen synthase activity in skeletal muscle were improved with 50:50 compared with all other treatments. Neither phosphatidlyinositol 3-kinase activity nor Akt activity in muscle was affected by treatment. Uncoupling protein 2 in muscle and adipose tissue was upregulated by c9,t11 and 50:50 compared with ZDF controls. PPAR-gamma mRNA was downregulated in liver of c9,t11 and pair-fed ZDF rats. Thus, the improved glucose tolerance in 50:50 rats is attributable to, at least in part, improved insulin action in muscle, and CLA effects cannot be explained simply by reduced food intake.

Competitive foods and beverages available for purchase in secondary schools--selected sites, United States, 2004.

School Health Profiles is conducted biennially to assess characteristics of school health programs. State and local departments of education and health select either all public secondary schools within their jurisdictions or a systematic, equal-probability sample of public secondary schools to participate in School Health Profiles. At each school, the principal and lead health education teacher were sent questionnaires to be self-administered and returned to the state or local agency conducting the survey. In 2004, a total of 27 states and 11 large urban school districts obtained weighted data from their survey of principals. The findings in this report indicate that the majority of secondary schools in 27 states and 11 large urban school districts allow students to purchase snack foods or beverages from vending machines or at the school store, canteen, or snack bar. The types of competitive foods and beverages available for purchase varied across states and large urban school districts. Overall, fruits or vegetables were less likely to be available for purchase than the other types of foods or beverages. Bottled water and soft drinks, sports drinks, or fruit drinks that are not 100% juice were most likely to be available for purchase.

High serum retinyl esters are not associated with reduced bone mineral density in the Third National Health And Nutrition Examination Survey, 1988-1994.

Hypervitaminosis A is sometimes associated with abnormalities of calcium metabolism and bone mineral status. A recent study found a negative association between reported dietary vitamin A intake and bone mineral density (BMD). Some segments of the U.S. population have high fasting serum retinyl ester concentrations, a physiological marker that may reflect high and possibly excessive vitamin A intake. We examined the association between fasting serum retinyl esters and BMD in the Third National Health and Nutrition Examination Survey, 1988-1994 (NHANES III), a large, nationally representative sample of the U.S. population. BMD was measured for the femoral neck, trochanter, intertrochanter, and total hip on all nonpregnant participants aged > or = 20 years; 5,790 participants also had complete data on fasting serum retinyl esters and covariates including age, body mass index (BMI), smoking, alcohol consumption, dietary supplement use, diabetes, physical activity, and, among women, parity, menopausal status, and the use of oral contraceptives or estrogen-replacement therapy. The sample included non-Hispanic white, non-Hispanic black, and Mexican American men and women. We examined the association between fasting serum retinyl esters and BMD at each site, controlling for covariates with multiple linear regression. We examined the association with osteopenia and osteoporosis with multiple logistic regression. Although the prevalences of high fasting serum retinyl esters concentration and low BMD were both substantial in this sample, there were no significant associations between fasting serum retinyl esters and any measure of bone mineral status.

Insulin-like growth factor II stimulates glucose transport in human skeletal muscle.

We investigated the effect of insulin-like growth factor II (IGF-II) and insulin-like growth factor binding protein-1 (IGFBP-1) on 3-O-methylglucose transport in incubated human skeletal muscle strips. Increasing physiological concentrations of IGF-II stimulated glucose transport in a dose-dependent manner. Glucose transport was maximally stimulated in the presence of 100 ng/ml (13.4 nM) of IGF-II, which corresponded to the effect obtained by 100 microU/ml (0.6 nM) of insulin. Exposure of muscle strips to IGFBP-1 (500 ng/ml) inhibited the maximal effect of IGF-II on glucose transport by 40%. Thus, it is conceivable that IGF-II and IGFBP-1 are physiological regulators of the glucose transport process in human skeletal muscle.

Cigarette smoking and diabetes mellitus: evidence of a positive association from a large prospective cohort study.

OBJECTIVE: Only a few prospective studies have examined the relationship between the frequency of cigarette smoking and the incidence of diabetes mellitus. The purpose of this study was to determine whether greater frequency of cigarette smoking accelerated the development of diabetes mellitus, and whether quitting reversed the effect. METHODS: Data were collected in the Cancer Prevention Study I, a prospective cohort study conducted from 1959 through 1972 by the American Cancer Society where volunteers recruited more than one million acquaintances in 25 US states. From these over one million original participants, 275,190 men and 434,637 women aged > or = 30 years were selected for the primary analysis using predetermined criteria. RESULTS: As smoking increased, the rate of diabetes increased for both men and women. Among those who smoked > or = 2 packs per day at baseline, men had a 45% higher diabetes rate than men who had never smoked; the comparable increase for women was 74%. Quitting smoking reduced the rate of diabetes to that of non-smokers after 5 years in women and after 10 years in men. CONCLUSIONS: A dose-response relationship seems likely between smoking and incidence of diabetes. Smokers who quit may derive substantial benefit from doing so. Confirmation of these observations is needed through additional epidemiological and biological research.

Physical activity, food choice, and weight management goals and practices among US college students.

INTRODUCTION: Physical activity and a healthy diet have been recommended to help reverse the increasing prevalence of overweight among adolescents and adults in the United States. METHODS: Data is from the 1995 National College Health Risk Behavior Survey. A representative sample of US undergraduate college students (n = 4609) were analyzed to examine associations of physical activity and food choice with weight management goals and practices. RESULTS: Based on self-reported height and weight, 35% of students were overweight or obese (body mass index > or = 25.0). Nearly half (46%) of all students reported they were trying to lose weight. Female students were less likely than male students to be overweight, but more likely to be trying to lose weight. Among female and male students, using logistic regression to control for demographics, trying to lose weight was associated with participation in vigorous physical activity and strengthening exercises, and consumption of < or = 2 servings/ day of high-fat foods. Female and male students who reported using exercise to lose weight or to keep from gaining weight were more likely than those who did not to participate in vigorous, strengthening, and moderate physical activity, and were more likely to eat > or = 5 servings/day of fruits and vegetables and < or = 2 servings/day of high-fat foods. Among students who were trying to lose weight, only 54% of females and 41% of males used both exercise and diet for weight control. CONCLUSION: Colleges should implement programs to increase student awareness of healthy weight management methods and the importance of physical activity combined with a healthy diet.

Effects of non-esterified fatty acids on insulin-stimulated glucose transport in isolated skeletal muscle from patients with type 2 (non-insulin-dependent) diabetes mellitus.

The influence of elevated levels of oleate on insulin-stimulated 3-0-methylglucose transport was assessed in vitro, in isolated skeletal muscle obtained from patients with type 2 (non-insulin-dependent) diabetes mellitus (n = 7) and control subjects (n = 8). An increase in oleate levels from 0.3 to 1.0 mmol/l induced a 3.7-fold increase in the rate of oleate oxidation (P < 0.01) in skeletal muscle from control subjects. However, the rate of insulin-stimulated 3-0-methylglucose transport was not altered in isolated skeletal muscle from the control subjects or the type 2 diabetic patients following exposure to 1.0 mmol/l oleate. This observation indicates that elevation of non-esterified fatty acids to a high physiological level has no inhibitory effect on glucose transport.

The epidemiology of recent involuntary weight loss in the United States population.

BACKGROUND: Although recent involuntary weight loss (RIWL) has been associated with mortality, no national studies described the prevalence among the general population, characteristics and long-term outcomes of people with RIWL. METHODS: The authors analyzed data from the NHANES II Mortality Study of 5838 individuals 50-74.9 years old who between 1976-1980 underwent a physical examination that included height and weight measurements, biochemical tests and responded to questions about involuntary weight loss within the past six months. Vital status was determined through 1992. Logistic regression was used to examine characteristics associated with RIWL and Cox proportional hazard modeling was used to measure associations between RIWL and mortality. RESULTS: 13.3% of the population reported RIWL with 6.9% reporting > or = 5% RIWL. Obese individuals were at significantly higher risk of RIWL of > or = 5% compared to those with BMI 19-24.9 (OR=1.57. 95% CI: 1.13, 2.18). Other significant risk factors for RIWL included; poor self-reported health, cancer, high white blood cell count, low albumin and low hemoglobin levels, age and current smoking status. RIWL of > or = 5% was significantly associated with mortality (RR=1.24, 95% CI: 1.01, 1.53). CONCLUSION: In summary, RIWL is fairly common among community-dwelling older adults, occurs disproportionately among obese individuals, is associated with characteristics of poor health and independently associated with mortality. These results indicate that RIWL needs to be considered an adverse health indicator even among obese individuals and despite the absence of several clinical indicators of disease.

Insulin signaling and glucose transport in insulin resistant skeletal muscle. Special reference to GLUT4 transgenic and GLUT4 knockout mice.

Glucose homeostasis is impaired in patients with non-insulin dependent diabetes mellitus (NIDDM) and this defect in due in part, to defects in glucose transport in skeletal muscle. Intense interest is now focused on whether reduced insulin-mediated glucose transport in muscle from NIDDM patients results from alterations in the insulin signal transduction pathway or from alterations in traffic and/or translocation of GLUT4 to the plasma membrane. Recently, potential targets for impaired traffic/translocation of GLUT4 have been reported to include defective phosphorylation of IRS-1 and reduced PI-3 kinase activity. In addition to insulin signaling defects, impaired glucose transport may result from a defect(s) in the activation or functional capacity of GLUT4. Because GLUT4 is dysregulated in skeletal muscle from NIDDM patients, it is an attractive target for gene therapy. Overexpression of GLUT4 in muscle results in increased glucose uptake and metabolism, and protects against the development of insulin resistance in transgenic mice. Genetic ablation of GLUT4 results in impaired insulin tolerance and defects in glucose metabolism in skeletal muscle. Because impaired muscle glucose transport leads to reduced whole body glucose uptake and hyperglycemia, understanding the molecular regulation of glucose transport in skeletal muscle is necessary to develop effective strategies to prevent or reduce the incidence of NIDDM.

Achieving a healthy lifestyle among United States adults: a long way to go.

OBJECTIVE: The United States population has been advised to engage in various healthy lifestyle factors known to be associated with reduced morbidity and mortality from various chronic conditions. These include not smoking, adequate fruit and vegetable intake, adequate physical activity, and normal body weight. Little is known about the prevalence of United States adults who engage in all four of these behaviors, however. DESIGN: Cross-sectional analysis. SETTING: The third National Health and Nutrition Examination Survey (1988-1994). PARTICIPANTS: 16,176 participants aged > or = 21 years. MAIN OUTCOME MEASURES: Percentage of participants engaging in four healthy lifestyle factors. RESULTS: Overall, 6.8% of the US population engaged in all four healthy lifestyle factors. Women were more likely than men (P = .001), and Whites and participants of "other race or ethnicity" were more likely than African Americans and Mexican Americans to engage in all four healthy lifestyle factors. A significant positive educational gradient was also evident (P for linear trend <.001). The highest percentages of participants engaging in all four lifestyle factors occurred among men (15.8%) and women (18.4%) of "other" race who had at least 13 years of education. The lowest percentages were observed for White men (1.1%) and African-American women (0.9%) with little education. CONCLUSIONS: The small proportion of US adults engaging in four healthy lifestyle factors demonstrates the enormity of the task that awaits the public health community in persuading Americans to adopt a multidimensional healthy lifestyle.

The estrogen receptor {alpha}-selective agonist propyl pyrazole triol improves glucose tolerance in ob/ob mice; potential molecular mechanisms.

The aim of this study was to validate the role of estrogen receptor alpha (ERalpha) signaling in the regulation of glucose metabolism, and to compare the molecular events upon treatment with the ERalpha-selective agonist propyl pyrazole triol (PPT) or 17beta-estradiol (E(2)) in ob/ob mice. Female ob/ob mice were treated with PPT, E(2) or vehicle for 7 or 30 days. Intraperitoneal glucose and insulin tolerance tests were performed, and insulin secretion was determined from isolated islets. Glucose uptake was assayed in isolated skeletal muscle and adipocytes. Gene expression profiling in the liver was performed using Affymetrix microarrays, and the expression of selected genes was studied by real-time PCR analysis. PPT and E(2) treatment improved glucose tolerance and insulin sensitivity. Fasting blood glucose levels decreased after 30 days of PPT and E(2) treatment. However, PPT and E(2) had no effect on insulin secretion from isolated islets. Basal and insulin-stimulated glucose uptake in skeletal muscle and adipose tissue were similar in PPT and vehicle-treated ob/ob mice. Hepatic lipid content was decreased after E(2) treatment. In the liver, treatment with E(2) and PPT increased and decreased the respective expression levels of the transcription factor signal transducer and activator of transcription 3, and of glucose-6-phosphatase. In summary, our data demonstrate that PPT exerts anti-diabetic effects, and these effects are mediated via ERalpha.

Secular trends in desired weight of adults.

CONTEXT: The prevalence of overweight and obese adults in the United States is at record levels. OBJECTIVE: The primary purpose is to describe secular trends in desired weight among adults from 1994 to 2003, and secondarily, to examine the hypothetical impact of achieving desired weight on obesity prevalence. DESIGN: Data were from the Behavioral Risk Factor Surveillance System (1994, 1996, 1998, 2000, 2003), a random-digit-dialed telephone survey. SETTING: Sample included respondents from 47 states and the District of Columbia. PARTICIPANTS: Non-institutionalized adults aged 18 years or older were included (N=703 286). MAIN OUTCOME MEASURES: Primary outcome measures included reported weight and desired weight. RESULTS: Means for desired weight increased 2.3 kg between 1994 and 2003, and reported weights increased 3.9 kg. The increased trend was observed across several subgroups for age, race/ethnicity and education. Within subgroups of weight status, the trend has remained relatively stable, particularly when examined in relation to the difference between reported and desired weight as a percentage of reported body weight. Generally, overweight men desired weights approximately 4.5% less than their reported weight, and obese men desired weights approximately 15% less than their reported weight for each corresponding year. For women, approximate values of desired weight were 12% less than reported weight for overweight women and 24% less for obese women. The prevalence of obesity would decrease to 4.4% if individuals weighed their desired weight. CONCLUSIONS: Americans are shifting their desired weight upward, concomitantly with an increase in their reported body weight.

Evidence that oestrogen receptor-alpha plays an important role in the regulation of glucose homeostasis in mice: insulin sensitivity in the liver.

AIMS/HYPOTHESIS: We used oestrogen receptor-alpha (ERalpha) knockout (ERKO) and receptor-beta (ERbeta) knockout (BERKO) mice to investigate the mechanism(s) behind the effects of oestrogens on glucose homeostasis. METHODS: Endogenous glucose production (EGP) was measured in ERKO mice using a euglycaemic-hyperinsulinaemic clamp. Insulin secretion was determined from isolated islets. In isolated muscles, glucose uptake was assayed by using radiolabelled isotopes. Genome-wide expression profiles were analysed by high-density oligonucleotide microarray assay, and the expression of the genes encoding steroyl-CoA desaturase and the Leptin receptor (Scd1 and Lepr, respectively) was confirmed by RT-PCR. RESULTS: ERKO mice had higher fasting blood glucose, plasma insulin levels and IGT. The plasma leptin level was increased, while the adiponectin concentration was decreased in ERKO mice. Levels of both glucose- and arginine-induced insulin secretion from isolated islets were similar in ERKO and wild-type mice. The euglycaemic-hyperinsulinaemic clamp revealed that suppression of EGP by increased insulin levels was blunted in ERKO mice, which suggests a pronounced hepatic insulin resistance. Microarray analysis revealed that in ERKO mice, the genes involved in hepatic lipid biosynthesis were upregulated, while genes involved in lipid transport were downregulated. Notably, hepatic Lepr expression was decreased in ERKO mice. In vitro studies showed a modest decrease in insulin-mediated glucose uptake in soleus and extensor digitorum longus (EDL) muscles of ERKO mice. BERKO mice demonstrated normal glucose tolerance and insulin release. CONCLUSIONS/INTERPRETATION: We conclude that oestrogens, acting via ERalpha, regulate glucose homeostasis mainly by modulating hepatic insulin sensitivity, which can be due to the upregulation of lipogenic genes via the suppression of Lepr expression.

Is caffeine associated with bone mineral density in young adult women?

BACKGROUND: By increasing the urinary excretion of calcium, caffeine consumption may reduce bone mineral density (BMD) and subsequently increase the risk for osteoporotic fracture. Although negative associations between caffeine consumption and BMD have been reported for postmenopausal women, in particular for those who consume low amounts of dietary calcium, the relation between caffeine and BMD in younger women is unclear. Therefore, we evaluated the association between caffeine consumption and BMD in a cross-sectional study of 177 healthy white women, age 19-26 years, who attended a Midwestern university. METHODS: Average caffeine intake (milligrams per day) was calculated from self-reports of the consumption of coffee, decaffeinated coffee, tea, colas, chocolate products, and select medications during the previous 12 months (mean caffeine intake = 99. 9 mg/day). BMD (grams per square centimeter) at the femoral neck and the lumbar spine was measured by dual-energy X-ray absorptiometry. RESULTS: After adjusting in linear regression models for potential confounders, including height, body mass index, age at menarche, calcium intake, protein consumption, alcohol consumption, and tobacco use, caffeine consumption was not a significant predictor of BMD. For every 100 mg of caffeine consumed, femoral neck BMD decreased 0.0069 g/cm(2) (95% confidence in terval [CI] = -0.0215, 0. 0076) and lumbar spine BMD decreased 0.0119 g/cm(2) (95% CI = -0. 0271, 0.0033). No single source of caffeine was significantly associated with a decrease in BMD. Furthermore, the association between caffeine consumption and BMD at either site did not differ significantly between those who consumed low levels of calcium (< or =836 mg/day) and those who consumed high levels of calcium (>836 mg/day). CONCLUSIONS: Caffeine intake in the range consumed by young adult women is not an important risk factor for low BMD.