Rapamycin and several analogs, such as CCI-779 and RAD001, are currently undergoing clinical evaluation as anticancer agents. In this study, we show that inhibition of mammalian target of rapamycin (mTOR) signaling by rapamycin leads to an increase of Akt phosphorylation in Rh30 and RD human rhabdomyosarcoma cell lines and xenografts, and insulin-like growth factor (IGF)-II-treated C2C12 mouse myoblasts and IGF-II-overexpressing Chinese hamster ovary cells. RNA interference-mediated knockdown of S6K1 also results in an increase of Akt phosphorylation. These data suggest that mTOR/S6K1 inhibition either by rapamycin or small interfering RNA (siRNA) triggers a negative feedback loop, resulting in the activation of Akt signaling. We next sought to investigate the mechanism of this negative feedback regulation from mTOR to Akt. Suppression of insulin receptor substrate (IRS)-1 and tuberous sclerosis complex-1 by siRNAs failed to abrogate rapamycin-induced upregulation of Akt phosphorylation in both Rh30 and RD cells. However, pretreatment with h7C10 antibody directed against insulin-like growth factor-1 receptor (IGF-1R) led to a blockade of rapamycin-induced Akt activation. Combined mTOR and IGF-1R inhibition with rapamycin and h7C10 antibody, respectively, resulted in additive inhibition of cell growth and survival. These data suggest that rapamycin mediates Akt activation through an IGF-1R-dependent mechanism. Thus, combining an mTOR inhibitor and an IGF-1R antibody/inhibitor may be an appropriate strategy to enhance mTOR-targeted anticancer therapy.

New insulin-secreting cell lines (INS-1 and INS-2) were established from cells isolated from an x-ray-induced rat transplantable insulinoma. The continuous growth of these cells was found to be dependent on the reducing agent 2-mercaptoethanol. Removal of this thiol compound caused a 15-fold drop in total cellular glutathione levels. These cells proliferated slowly (population doubling time about 100 h) and, in general, showed morphological characteristics typical of native beta-cells. Most cells stained positive for insulin and did not react with antibodies against the other islet hormones. The content of immunoreactive insulin was about 8 micrograms/10(6) cells, corresponding to 20% of the native beta-cell content. These cells synthesized both proinsulin I and II and displayed conversion rates of the two precursor hormones similar to those observed in rat islets. However, glucose failed to stimulate the rate of proinsulin biosynthesis. In static incubations, glucose stimulated insulin secretion from floating cell clusters or from attached cells. Under perifusion conditions, 10 mM but not 1 mM glucose enhanced secretion 2.2-fold. In the presence of forskolin and 3-isobutyl-1-methylxanthine, increase of glucose concentration from 2.8-20 mM caused a 4-fold enhancement of the rate of secretion. Glucose also depolarized INS-1 cells and raised the concentration of cytosolic Ca2+. This suggests that glucose is still capable of eliciting part of the ionic events at the plasma membrane, which leads to insulin secretion. The structural and functional characteristics of INS-1 cells remained unchanged over a period of 2 yr (about 80 passages). Although INS-2 cells have not been fully characterized, their insulin content was similar to that of INS-1 cells and they also remain partially sensitive to glucose as a secretagogue. INS-1 cells retain beta-cell surface antigens, as revealed by reactivity with the antigangloside monoclonal antibodies R2D6 and A2B5. These findings indicate that INS-1 cells have remained stable and retain a high degree of differentiation which should make them a suitable model for studying various aspects of beta-cell function.

Integrin-linked kinase (ILK) is an ankyrin-repeat containing serine-threonine protein kinase capable of interacting with the cytoplasmic domains of integrin beta1, beta2, and beta3 subunits. Overexpression of ILK in epithelial cells disrupts cell-extracellular matrix as well as cell-cell interactions, suppresses suspension-induced apoptosis (also called Anoikis), and stimulates anchorage-independent cell cycle progression. In addition, ILK induces nuclear translocation of beta-catenin, where the latter associates with a T cell factor/lymphocyte enhancer-binding factor 1 (TCF/LEF-1) to form an activated transcription factor. We now demonstrate that ILK activity is rapidly, but transiently, stimulated upon attachment of cells to fibronectin, as well as by insulin, in a phosphoinositide-3-OH kinase [Pi(3)K]-dependent manner. Furthermore, phosphatidylinositol(3,4,5)trisphosphate specifically stimulates the activity of ILK in vitro, and in addition, membrane targetted constitutively active Pi(3)K activates ILK in vivo. We also demonstrate here that ILK is an upstream effector of the Pi(3)K-dependent regulation of both protein kinase B (PKB/AKT) and glycogen synthase kinase 3 (GSK-3). Specifically, ILK can directly phosphorylate GSK-3 in vitro and when stably, or transiently, overexpressed in cells can inhibit GSK-3 activity, whereas the overexpression of kinase-deficient ILK enhances GSK-3 activity. In addition, kinase-active ILK can phosphorylate PKB/AKT on serine-473, whereas kinase-deficient ILK severely inhibits endogenous phosphorylation of PKB/AKT on serine-473, demonstrating that ILK is involved in agonist stimulated, Pi(3)K-dependent, PKB/AKT activation. ILK is thus a receptor-proximal effector for the Pi(3)K-dependent, extracellular matrix and growth factor mediated, activation of PKB/AKT, and inhibition of GSK-3.

BACKGROUND

Childhood obesity, epidemic in the United States, has been accompanied by an increase in the prevalence of type 2 diabetes among children and adolescents. We determined the prevalence of impaired glucose tolerance in a multiethnic cohort of 167 obese children and adolescents.

METHODS

All subjects underwent a two-hour oral glucose-tolerance test (1.75 g [DOSAGE ERROR CORRECTED] of glucose per kilogram of body weight), and glucose, insulin, and C-peptide levels were measured. Fasting levels of proinsulin were obtained, and the ratio of proinsulin to insulin was calculated. Insulin resistance was estimated by homeostatic model assessment, and beta-cell function was estimated by calculating the ratio between the changes in the insulin level and the glucose level during the first 30 minutes after the ingestion of glucose.

RESULTS

Impaired glucose tolerance was detected in 25 percent of the 55 obese children (4 to 10 years of age) and 21 percent of the 112 obese adolescents (11 to 18 years of age); silent type 2 diabetes was identified in 4 percent of the obese adolescents. Insulin and C-peptide levels were markedly elevated after the glucose-tolerance test in subjects with impaired glucose tolerance but not in adolescents with diabetes, who had a reduced ratio of the 30-minute change in the insulin level to the 30-minute change in the glucose level. After the body-mass index had been controlled for, insulin resistance was greater in the affected cohort and was the best predictor of impaired glucose tolerance.

CONCLUSIONS

Impaired glucose tolerance is highly prevalent among children and adolescents with severe obesity, irrespective of ethnic group. Impaired oral glucose tolerance was associated with insulin resistance while beta-cell function was still relatively preserved. Overt type 2 diabetes was linked to beta-cell failure.

MicroRNAs have essential functional roles in brain development and neuronal specification but their roles in neurodegenerative diseases such as Alzheimer's disease (AD) is unknown. Using a sensitive qRT-PCR platform we identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain microRNAs are biomarkers for known and novel pathways in AD pathogenesis related to amyloid processing, neurogenesis, insulin resistance, and innate immunity. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease.

BACKGROUND

The scarcity of data addressing the health effects of popular diets is an important public health concern, especially since patients and physicians are interested in using popular diets as individualized eating strategies for disease prevention.

OBJECTIVE

To assess adherence rates and the effectiveness of 4 popular diets (Atkins, Zone, Weight Watchers, and Ornish) for weight loss and cardiac risk factor reduction.

METHODS

A single-center randomized trial at an academic medical center in Boston, Mass, of overweight or obese (body mass index: mean, 35; range, 27-42) adults aged 22 to 72 years with known hypertension, dyslipidemia, or fasting hyperglycemia. Participants were enrolled starting July 18, 2000, and randomized to 4 popular diet groups until January 24, 2002.

METHODS

A total of 160 participants were randomly assigned to either Atkins (carbohydrate restriction, n=40), Zone (macronutrient balance, n=40), Weight Watchers (calorie restriction, n=40), or Ornish (fat restriction, n=40) diet groups. After 2 months of maximum effort, participants selected their own levels of dietary adherence.

METHODS

One-year changes in baseline weight and cardiac risk factors, and self-selected dietary adherence rates per self-report.

RESULTS

Assuming no change from baseline for participants who discontinued the study, mean (SD) weight loss at 1 year was 2.1 (4.8) kg for Atkins (21 [53%] of 40 participants completed, P = .009), 3.2 (6.0) kg for Zone (26 [65%] of 40 completed, P = .002), 3.0 (4.9) kg for Weight Watchers (26 [65%] of 40 completed, P < .001), and 3.3 (7.3) kg for Ornish (20 [50%] of 40 completed, P = .007). Greater effects were observed in study completers. Each diet significantly reduced the low-density lipoprotein/high-density lipoprotein (HDL) cholesterol ratio by approximately 10% (all P<.05), with no significant effects on blood pressure or glucose at 1 year. Amount of weight loss was associated with self-reported dietary adherence level (r = 0.60; P<.001) but not with diet type (r = 0.07; P = .40). For each diet, decreasing levels of total/HDL cholesterol, C-reactive protein, and insulin were significantly associated with weight loss (mean r = 0.36, 0.37, and 0.39, respectively) with no significant difference between diets (P = .48, P = .57, P = .31, respectively).

CONCLUSIONS

Each popular diet modestly reduced body weight and several cardiac risk factors at 1 year. Overall dietary adherence rates were low, although increased adherence was associated with greater weight loss and cardiac risk factor reductions for each diet group.

In both type 1 and type 2 diabetes, diabetic complications in target organs arise from chronic elevations of glucose. The pathogenic effect of high glucose, possibly in concert with fatty acids, is mediated to a significant extent via increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and subsequent oxidative stress. ROS and RNS directly oxidize and damage DNA, proteins, and lipids. In addition to their ability to directly inflict damage on macromolecules, ROS and RNS indirectly induce damage to tissues by activating a number of cellular stress-sensitive pathways. These pathways include nuclear factor-kappaB, p38 mitogen-activated protein kinase, NH(2)-terminal Jun kinases/stress-activated protein kinases, hexosamines, and others. In addition, there is evidence that in type 2 diabetes, the activation of these same pathways by elevations in glucose and free fatty acid (FFA) levels leads to both insulin resistance and impaired insulin secretion. Therefore, we propose here that the hyperglycemia-induced, and possibly FFA-induced, activation of stress pathways plays a key role in the development of not only the late complications in type 1 and type 2 diabetes, but also the insulin resistance and impaired insulin secretion seen in type 2 diabetes.

OBJECTIVE

Numerous studies have identified an increased risk of cancer in type 2 diabetes. We explored the association between antidiabetic therapies and cancer-related mortality in patients with type 2 diabetes, postulating that agents that increase insulin levels might promote cancer.

METHODS

This was a population-based cohort study using administrative databases from Saskatchewan Health. Cancer-related mortality was compared among inception cohorts of metformin users and sulfonylurea monotherapy users. Multivariate Cox regression was used to estimate the hazard ratio (HR) of cancer-related mortality, after adjusting for age, sex, insulin use, and chronic disease score. All statistical tests were two-sided.

RESULTS

We identified 10,309 new users of metformin or sulfonylureas with an average follow-up of 5.4 +/- 1.9 years (means +/- SD). The mean age for the cohort was 63.4 +/- 13.3 years, and 55% were men. Cancer mortality over follow-up was 4.9% (162 of 3,340) for sulfonylurea monotherapy users, 3.5% (245 of 6,969) for metformin users, and 5.8% (84 of 1,443) for subjects who used insulin. After multivariate adjustment, the sulfonylurea cohort had greater cancer-related mortality compared with the metformin cohort (adjusted HR 1.3 [95% CI 1.1-1.6]; P = 0.012). Insulin use was associated with an adjusted HR of cancer-related mortality of 1.9 (95% CI 1.5-2.4; P < 0.0001).

CONCLUSIONS

Patients with type 2 diabetes exposed to sulfonylureas and exogenous insulin had a significantly increased risk of cancer-related mortality compared with patients exposed to metformin. It is uncertain whether this increased risk is related to a deleterious effect of sulfonylurea and insulin or a protective effect of metformin or due to some unmeasured effect related to both choice of therapy and cancer risk.

Apolipoprotein E is associated with age-related risk for Alzheimer's disease and plays critical roles in Abeta homeostasis. We report that ApoE plays a role in facilitating the proteolytic clearance of soluble Abeta from the brain. The endolytic degradation of Abeta peptides within microglia by neprilysin and related enzymes is dramatically enhanced by ApoE. Similarly, Abeta degradation extracellularly by insulin-degrading enzyme is facilitated by ApoE. The capacity of ApoE to promote Abeta degradation is dependent upon the ApoE isoform and its lipidation status. The enhanced expression of lipidated ApoE, through the activation of liver X receptors, stimulates Abeta degradation. Indeed, aged Tg2576 mice treated with the LXR agonist GW3965 exhibited a dramatic reduction in brain Abeta load. GW3965 treatment also reversed contextual memory deficits. These data demonstrate a mechanism through which ApoE facilitates the clearance of Abeta from the brain and suggest that LXR agonists may represent a novel therapy for AD.

Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease with a strong inflammatory component. The latest studies indicate that innate immunity and inflammatory mediators have a much broader role in T1DM than initially assumed. Inflammation might contribute to early induction and amplification of the immune assault against pancreatic beta cells and, at later stages, to the stabilization and maintenance of insulitis. Inflammatory mediators probably contribute to the suppression of beta-cell function and subsequent apoptosis; they may also inhibit or stimulate beta-cell regeneration and might cause peripheral insulin resistance. The different effects of inflammation take place in different phases of the course of T1DM, and should be considered in the context of a 'dialog' between invading immune cells and the target beta cells. This dialog is mediated both by cytokines and chemokines that are released by beta cells and immune cells, and by putative, immunogenic signals that are delivered by dying beta cells. In this Review, we divided the role of inflammation in T1DM into three arbitrary stages: induction, amplification and maintenance or resolution of insulitis. These stages, and their progression or resolution, might depend on a patient's genetic background, which contributes to disease heterogeneity.

Obesity is associated with insulin resistance and a state of abnormal inflammatory response. The Toll-like receptor (TLR)4 has an important role in inflammation and immunity, and its expression has been reported in most tissues of the body, including the insulin-sensitive ones. Because it is activated by lipopolysaccharide and saturated fatty acids, which are inducers of insulin resistance, TLR4 may be a candidate for participation in the cross-talk between inflammatory and metabolic signals. Here, we show that C3H/HeJ mice, which have a loss-of-function mutation in TLR4, are protected against the development of diet-induced obesity. In addition, these mice demonstrate decreased adiposity, increased oxygen consumption, a decreased respiratory exchange ratio, improved insulin sensitivity, and enhanced insulin-signaling capacity in adipose tissue, muscle, and liver compared with control mice during high-fat feeding. Moreover, in these tissues, control mice fed a high-fat diet show an increase in IkappaB kinase complex and c-Jun NH(2)-terminal kinase activity, which is prevented in C3H/HeJ mice. In isolated muscles from C3H/HeJ mice, protection from saturated fatty acid-induced insulin resistance is observed. Thus, TLR4 appears to be an important mediator of obesity and insulin resistance and a potential target for the therapy of these highly prevalent medical conditions.

BACKGROUND

Trials comparing the effectiveness and safety of weight-loss diets are frequently limited by short follow-up times and high dropout rates.

METHODS

In this 2-year trial, we randomly assigned 322 moderately obese subjects (mean age, 52 years; mean body-mass index [the weight in kilograms divided by the square of the height in meters], 31; male sex, 86%) to one of three diets: low-fat, restricted-calorie; Mediterranean, restricted-calorie; or low-carbohydrate, non-restricted-calorie.

RESULTS

The rate of adherence to a study diet was 95.4% at 1 year and 84.6% at 2 years. The Mediterranean-diet group consumed the largest amounts of dietary fiber and had the highest ratio of monounsaturated to saturated fat (P<0.05 for all comparisons among treatment groups). The low-carbohydrate group consumed the smallest amount of carbohydrates and the largest amounts of fat, protein, and cholesterol and had the highest percentage of participants with detectable urinary ketones (P<0.05 for all comparisons among treatment groups). The mean weight loss was 2.9 kg for the low-fat group, 4.4 kg for the Mediterranean-diet group, and 4.7 kg for the low-carbohydrate group (P<0.001 for the interaction between diet group and time); among the 272 participants who completed the intervention, the mean weight losses were 3.3 kg, 4.6 kg, and 5.5 kg, respectively. The relative reduction in the ratio of total cholesterol to high-density lipoprotein cholesterol was 20% in the low-carbohydrate group and 12% in the low-fat group (P=0.01). Among the 36 subjects with diabetes, changes in fasting plasma glucose and insulin levels were more favorable among those assigned to the Mediterranean diet than among those assigned to the low-fat diet (P<0.001 for the interaction among diabetes and Mediterranean diet and time with respect to fasting glucose levels).

CONCLUSIONS

Mediterranean and low-carbohydrate diets may be effective alternatives to low-fat diets. The more favorable effects on lipids (with the low-carbohydrate diet) and on glycemic control (with the Mediterranean diet) suggest that personal preferences and metabolic considerations might inform individualized tailoring of dietary interventions. (ClinicalTrials.gov number, NCT00160108.)

OBJECTIVE

Nonalcoholic fatty liver disease is frequently associated with type 2 diabetes mellitus, obesity, and dyslipidemia, but some patients have normal glucose tolerance or normal weight. We tested the hypothesis that there is an association between nonalcoholic fatty liver disease and insulin resistance that is independent of diabetes and obesity.

METHODS

We measured anthropometric and metabolic variables in 46 patients with chronically elevated serum aminotransferase levels, "bright liver" on ultrasound scan, and normal glucose tolerance. Indexes of insulin resistance and secretion were determined using the homeostasis model assessment method. They were compared with 92 normal subjects who were matched for age and sex.

RESULTS

Patients with nonalcoholic fatty liver disease were characterized by fasting and glucose-induced hyperinsulinemia, insulin resistance, postload hypoglycemia, and hypertriglyceridemia. Insulin resistance [odds ratio (OR) = 15 per percent increase, 95% confidence interval (CI): 3.0 to 70], fasting triglyceride level (OR = 3.1 per mmol/liter increase, 95% CI: 1.1 to 8.9), 180-minute blood glucose level (OR = 4.3 per mmol/ liter decrease, 95% CI: 1.6 to 12), and average insulin concentration in response to oral glucose (OR = 3.0 per 100 pmol/liter increase, 95% CI: 1.5 to 6.2) were independently associated with nonalcoholic fatty liver disease. The exclusion of overweight and obese subjects did not change the results.

CONCLUSIONS

Nonalcoholic fatty liver disease is associated with insulin resistance and hyperinsulinemia even in lean subjects with normal glucose tolerance. Genetic factors that reduce insulin sensitivity and increase serum triglyceride levels may be responsible for its development.

BACKGROUND

Low birth weight is a risk factor for coronary heart disease. It is uncertain how postnatal growth affects disease risk.

METHODS

We studied 8760 people born in Helsinki from 1934 through 1944. Childhood growth had been recorded. A total of 357 men and 87 women had been admitted to the hospital with coronary heart disease or had died from the disease. Coronary risk factors were measured in a subset of 2003 people.

RESULTS

The mean body size of children who had coronary events as adults was below average at birth. At two years of age the children were thin; subsequently, their body-mass index (BMI) increased relative to that of other children and had reached average values by 11 years of age. In simultaneous regressions, the hazard ratios associated with a 1 SD increase in BMI were 0.76 (95 percent confidence interval, 0.66 to 0.87; P<0.001) at 2 years and 1.14 (95 percent confidence interval, 1.00 to 1.31; P=0.05) at 11 years among the boys. The corresponding figures for the girls were 0.62 (95 percent confidence interval, 0.46 to 0.82; P=0.001) and 1.35 (95 percent confidence interval, 1.02 to 1.78; P=0.04). Low BMI at 2 years of age and increased BMI from 2 to 11 years of age were also associated with raised fasting insulin concentrations (P<0.001 for both).

CONCLUSIONS

On average, adults who had a coronary event had been small at birth and thin at two years of age and thereafter put on weight rapidly. This pattern of growth during childhood was associated with insulin resistance in later life. The risk of coronary events was more strongly related to the tempo of childhood gain in BMI than to the BMI attained at any particular age.

There is currently substantial confusion between the conceptual definition of the metabolic syndrome and the clinical screening parameters and cut-off values proposed by various organizations (NCEP-ATP III, IDF, WHO, etc) to identify individuals with the metabolic syndrome. Although it is clear that in vivo insulin resistance is a key abnormality associated with an atherogenic, prothrombotic, and inflammatory profile which has been named by some the "metabolic syndrome" or by others "syndrome X" or "insulin resistance syndrome", it is more and more recognized that the most prevalent form of this constellation of metabolic abnormalities linked to insulin resistance is found in patients with abdominal obesity, especially with an excess of intra-abdominal or visceral adipose tissue. We have previously proposed that visceral obesity may represent a clinical intermediate phenotype reflecting the relative inability of subcutaneous adipose tissue to act as a protective metabolic sink for the clearance and storage of the extra energy derived from dietary triglycerides, leading to ectopic fat deposition in visceral adipose depots, skeletal muscle, liver, heart, etc. Thus, visceral obesity may partly be a marker of a dysmetabolic state and partly a cause of the metabolic syndrome. Although waist circumference is a better marker of abdominal fat accumulation than the body mass index, an elevated waistline alone is not sufficient to diagnose visceral obesity and we have proposed that an elevated fasting triglyceride concentration could represent, when waist circumference is increased, a simple clinical marker of excess visceral/ectopic fat. Finally, a clinical diagnosis of visceral obesity, insulin resistance, or of the metabolic syndrome is not sufficient to assess global risk of cardiovascular disease. To achieve this goal, physicians should first pay attention to the classical risk factors while also considering the additional risk resulting from the presence of abdominal obesity and the metabolic syndrome, such global risk being defined as cardiometabolic risk.

The insulin-like growth factors (IGFs) are mitogens that play a pivotal role in regulating cell proliferation, differentiation, and apoptosis. The effects of IGFs are mediated through the IGF-I receptor, which is also involved in cell transformation induced by tumor virus proteins and oncogene products. Six IGF-binding proteins (IGFBPs) can inhibit or enhance the actions of IGFs. These opposing effects are determined by the structures of the binding proteins. The effects of IGFBPs on IGFs are regulated in part by IGFBP proteases. Laboratory studies have shown that IGFs exert strong mitogenic and antiapoptotic actions on various cancer cells. IGFs also act synergistically with other mitogenic growth factors and steroids and antagonize the effect of antiproliferative molecules on cancer growth. The role of IGFs in cancer is supported by epidemiologic studies, which have found that high levels of circulating IGF-I and low levels of IGFBP-3 are associated with increased risk of several common cancers, including those of the prostate, breast, colorectum, and lung. Evidence further suggests that certain lifestyles, such as one involving a high-energy diet, may increase IGF-I levels, a finding that is supported by animal experiments indicating that IGFs may abolish the inhibitory effect of energy restriction on cancer growth. Further investigation of the role of IGFs in linking high energy intake, increased cell proliferation, suppression of apoptosis, and increased cancer risk may provide new insights into the etiology of cancer and lead to new strategies for cancer prevention.

Based on the fact that aging is associated with a reciprocal decrease of osteogenesis and an increase of adipogenesis in bone marrow and that osteoblasts and adipocytes share a common progenitor, this study investigated the role of PPARgamma, a key regulator of adipocyte differentiation, in bone metabolism. Homozygous PPARgamma-deficient ES cells failed to differentiate into adipocytes, but spontaneously differentiated into osteoblasts, and these were restored by reintroduction of the PPARgamma gene. Heterozygous PPARgamma-deficient mice exhibited high bone mass with increased osteoblastogenesis, but normal osteoblast and osteoclast functions, and this effect was not mediated by insulin or leptin. The osteogenic effect of PPARgamma haploinsufficiency became prominent with aging but was not changed upon ovariectomy. The PPARgamma haploinsufficiency was confirmed to enhance osteoblastogenesis in the bone marrow cell culture but did not affect the cultures of differentiated osteoblasts or osteoclast-lineage cells. This study demonstrates a PPARgamma-dependent regulation of bone metabolism in vivo, in that PPARgamma insufficiency increases bone mass by stimulating osteoblastogenesis from bone marrow progenitors.

BACKGROUND

Rosiglitazone is an insulin sensitiser used in combination with metformin, a sulfonylurea, or both, for lowering blood glucose in people with type 2 diabetes. We assessed cardiovascular outcomes after addition of rosiglitazone to either metformin or sulfonylurea compared with the combination of the two over 5-7 years of follow-up. We also assessed comparative safety.

METHODS

In a multicentre, open-label trial, 4447 patients with type 2 diabetes on metformin or sulfonylurea monotherapy with mean haemoglobin A(1c) (HbA(1c)) of 7.9% were randomly assigned to addition of rosiglitazone (n=2220) or to a combination of metformin and sulfonylurea (active control group, n=2227). The primary endpoint was cardiovascular hospitalisation or cardiovascular death, with a hazard ratio (HR) non-inferiority margin of 1.20. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00379769.

RESULTS

321 people in the rosiglitazone group and 323 in the active control group experienced the primary outcome during a mean 5.5-year follow-up, meeting the criterion of non-inferiority (HR 0.99, 95% CI 0.85-1.16). HR was 0.84 (0.59-1.18) for cardiovascular death, 1.14 (0.80-1.63) for myocardial infarction, and 0.72 (0.49-1.06) for stroke. Heart failure causing admission to hospital or death occurred in 61 people in the rosiglitazone group and 29 in the active control group (HR 2.10, 1.35-3.27, risk difference per 1000 person-years 2.6, 1.1-4.1). Upper and distal lower limb fracture rates were increased mainly in women randomly assigned to rosiglitazone. Mean HbA(1c) was lower in the rosiglitazone group than in the control group at 5 years.

CONCLUSIONS

Addition of rosiglitazone to glucose-lowering therapy in people with type 2 diabetes is confirmed to increase the risk of heart failure and of some fractures, mainly in women. Although the data are inconclusive about any possible effect on myocardial infarction, rosiglitazone does not increase the risk of overall cardiovascular morbidity or mortality compared with standard glucose-lowering drugs.

BACKGROUND

GlaxoSmithKline plc, UK.

Evidence is reviewed that free fatty acids (FFAs) are one important link between obesity and insulin resistance and NIDDM. First, plasma FFA levels are elevated in most obese subjects. Second, physiological elevations in plasma FFA concentrations inhibit insulin stimulated peripheral glucose uptake in a dose-dependent manner in normal controls and in patients with NIDDM. Two possible mechanisms are identified: 1) a fat-related inhibition of glucose transport or phosphorylation, which appears after 3-4 h of fat infusion, and 2) a decrease in muscle glycogen synthase activity, which appears after 4-6 h of fat infusion. Third, FFAs stimulate insulin secretion in nondiabetic individuals. Some of this insulin is transmitted in the peripheral circulation and is able to compensate for FFA-mediated peripheral insulin resistance. FFA-mediated portal hyperinsulinemia counteracts the stimulation of FFAs on hepatic glucose production (HGP) and thus prevents hepatic glucose overproduction. We speculate that, in obese individuals who are genetically predisposed to develop NIDDM, FFAs will eventually fail to promote insulin secretion. The stimulatory effect of FFAs on HGP would then become unchecked, resulting in hyperglycemia. Hence, continuously elevated levels of plasma FFAs may play a key role in the pathogenesis of NIDDM in predisposed individuals by impairing peripheral glucose utilization and by promoting hepatic glucose overproduction.

It has been proposed that hematopoietic and endothelial cells are derived from a common cell, the hemangioblast. In this study, we demonstrate that a subset of CD34(+) cells have the capacity to differentiate into endothelial cells in vitro in the presence of basic fibroblast growth factor, insulin-like growth factor-1, and vascular endothelial growth factor. These differentiated endothelial cells are CD34(+), stain for von Willebrand factor (vWF), and incorporate acetylated low-density lipoprotein (LDL). This suggests the possible existence of a bone marrow-derived precursor endothelial cell. To demonstrate this phenomenon in vivo, we used a canine bone marrow transplantation model, in which the marrow cells from the donor and recipient are genetically distinct. Between 6 to 8 months after transplantation, a Dacron graft, made impervious to prevent capillary ingrowth from the surrounding perigraft tissue, was implanted in the descending thoracic aorta. After 12 weeks, the graft was retrieved, and cells with endothelial morphology were identified by silver nitrate staining. Using the di(CA)n and tetranucleotide (GAAA)n repeat polymorphisms to distinguish between the donor and recipient DNA, we observed that only donor alleles were detected in DNA from positively stained cells on the impervious Dacron graft. These results strongly suggest that a subset of CD34+ cells localized in the bone marrow can be mobilized to the peripheral circulation and can colonize endothelial flow surfaces of vascular prostheses.

Insulin stimulates the rapid translocation of intracellular glucose transporters of the GLUT4 isotype to the plasma membrane in fat and muscle cells. The connections between known insulin signaling pathways and the protein machinery of this membrane-trafficking process have not been fully defined. Recently, we identified a 160-kDa protein in adipocytes, designated AS160, that is phosphorylated by the insulin-activated kinase Akt. This protein contains a GTPase-activating domain (GAP) for Rabs, which are small G proteins required for membrane trafficking. In the present study we have identified six sites of in vivo phosphorylation on AS160. These sites lie in the motif characteristic of Akt phosphorylation, and insulin treatment increased phosphorylation at five of the sites. Expression of AS160 with two or more of these sites mutated to alanine markedly inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Moreover, this inhibition did not occur when the GAP function in the phosphorylation site mutant was inactivated by a point mutation. These findings strongly indicate that insulin-stimulated phosphorylation of AS160 is required for GLUT4 translocation and that this phosphorylation signals translocation through inactivation of the Rab GAP function.

Recent muscle biopsy studies have shown a relation between intramuscular lipid content and insulin resistance. The aim of this study was to test this relation in humans by using a novel proton nuclear magnetic resonance (1H NMR) spectroscopy technique, which enables non-invasive and rapid (approximately 45 min) determination of intramyocellular lipid (IMCL) content. Normal weight non-diabetic adults (n = 23, age 29+/-2 years. BMI = 24.1+/-0.5 kg/m2) were studied using cross-sectional analysis. Insulin sensitivity was assessed by a 2-h hyperinsulinaemic (approximately 450 pmol/l)-euglycaemic (approximately 5 mmol/l) clamp test. Intramyocellular lipid concentrations were determined by using localized 1H NMR spectroscopy of soleus muscle. Simple linear regression analysis showed an inverse correlation (r = -0.579, p = 0.0037) [corrected] between intramyocellular lipid content and M-value (100-120 min of clamp) as well as between fasting plasma non-esterified fatty acid concentration and M-value (r = -0.54, p = 0.0267). Intramyocellular lipid content was not related to BMI, age and fasting plasma concentrations of triglycerides, non-esterified fatty acids, glucose or insulin. These results show that intramyocellular lipid concentration, as assessed non invasively by localized 1H NMR spectroscopy, is a good indicator of whole body insulin sensitivity in non-diabetic, non-obese humans.

Stearoyl-CoA desaturase (SCD) is a central lipogenic enzyme catalyzing the synthesis of monounsaturated fatty acids, mainly oleate (C18:1) and palmitoleate (C16:1), which are components of membrane phospholipids, triglycerides, wax esters, and cholesterol esters. Several SCD isoforms (SCD1-3) exist in the mouse. Here we show that mice with a targeted disruption of the SCD1 isoform have reduced body adiposity, increased insulin sensitivity, and are resistant to diet-induced weight gain. The protection from obesity involves increased energy expenditure and increased oxygen consumption. Compared with the wild-type mice the SCD1-/- mice have increased levels of plasma ketone bodies but reduced levels of plasma insulin and leptin. In the SCD1-/- mice, the expression of several genes of lipid oxidation are up-regulated, whereas lipid synthesis genes are down-regulated. These observations suggest that a consequence of SCD1 deficiency is an activation of lipid oxidation in addition to reduced triglyceride synthesis and storage.