Alaina K. Natoli

High-Density Lipoprotein Modulates Glucose Metabolism in Patients With Type 2 Diabetes Mellitus

Background— Low plasma high-density lipoprotein (HDL) is associated with elevated cardiovascular risk and aspects of the metabolic syndrome. We hypothesized that HDL modulates glucose metabolism via elevation of plasma insulin and through activation of the key metabolic regulatory enzyme, AMP-activated protein kinase, in skeletal muscle. Methods and Results— Thirteen patients with type 2 diabetes mellitus received both intravenous reconstituted HDL (rHDL: 80 mg/kg over 4 hours) and placebo on separate days in a double-blind, placebo-controlled crossover study. A greater fall in plasma glucose from baseline occurred during rHDL than during placebo (at 4 hours rHDL=−2.6±0.4; placebo=−2.1±0.3mmol/L; P=0.018). rHDL increased plasma insulin (at 4 hours rHDL=3.4±10.0; placebo= −19.2±7.4 pmol/L; P=0.034) and also the homeostasis model assessment &bgr;-cell function index (at 4 hours rHDL=18.9±5.9; placebo=8.6±4.4%; P=0.025). Acetyl-CoA carboxylase &bgr; phosphorylation in skeletal muscle biopsies was increased by 1.7±0.3-fold after rHDL, indicating activation of the AMP-activated protein kinase pathway. Both HDL and apolipoprotein AI increased glucose uptake (by 177±12% and 144±18%, respectively; P<0.05 for both) in primary human skeletal muscle cell cultures established from patients with type 2 diabetes mellitus (n=5). The mechanism is demonstrated to include stimulation of the ATP-binding cassette transporter A1 with subsequent activation of the calcium/calmodulin-dependent protein kinase kinase and the AMP-activated protein kinase pathway. Conclusions— rHDL reduced plasma glucose in patients with type 2 diabetes mellitus by increasing plasma insulin and activating AMP-activated protein kinase in skeletal muscle. These findings suggest a role for HDL-raising therapies beyond atherosclerosis to address type 2 diabetes mellitus.

Ramipril Reduces Large-Artery Stiffness in Peripheral Arterial Disease and Promotes Elastogenic Remodeling in Cell Culture

Ramipril improves cardiovascular outcome in patients with peripheral arterial disease; however, the precise mechanisms of benefit remain to be elucidated. The effect of ramipril on large-artery stiffness in patients with peripheral arterial disease was examined. In addition, we determined the effect of ramiprilat on extracellular matrix from human aortic smooth muscle cell culture. Forty patients with peripheral arterial disease were randomized to receive ramipril, 10 mg once daily or placebo for 24 weeks. Arterial stiffness was assessed globally via systemic arterial compliance and augmentation index (carotid tonometry and Doppler velocimetry), and regionally via carotid–femoral pulse wave velocity. Angiotensin-converting enzyme inhibition increased arterial compliance by 0.10±0.02 mL/mm Hg, (P<0.001, all probability values relative to placebo) and reduced pulse wave velocity by 1.7±0.2 m/s (P<0.001), augmentation index by 4.1±0.3% (P<0.001), and systolic blood pressure by 5±1 mm Hg (P<0.001). Ramipril did not reduce mean arterial pressure significantly compared with placebo (P=0.59). In cell culture, ramiprilat decreased collagen deposition by >50% and increased elastin and fibrillin-1 deposition by >3- and 4-fold respectively (histochemistry and immunohistochemistry). Fibrillin-1 gene expression was increased 5-fold (real-time reverse-transcriptase polymerase chain reaction). Ramiprilat also reduced gene and protein (Western) expression of both matrix metalloproteinase (MMP)-2 and MMP-3. In conclusion, ramipril promoted an elastogenic matrix profile that may contribute to the observed clinical reduction in large-artery stiffness and carotid pressure augmentation, which occurred independently of mean arterial blood pressure reduction in patients with peripheral arterial disease.

Reduced UCP-1 content in in vitro differentiated beige/brite adipocytes derived from preadipocytes of human subcutaneous white adipose tissues in obesity.

Introduction Brown adipose tissue (BAT) is a potential therapeutic target to reverse obesity. The purpose of this study was to determine whether primary precursor cells isolated from human adult subcutaneous white adipose tissue (WAT) can be induced to differentiate in-vitro into adipocytes that express key markers of brown or beige adipose, and whether the expression level of such markers differs between lean and obese young adult males. Methods Adipogenic precursor cells were isolated from lean and obese individuals from subcutaneous abdominal WAT biopsies. Cells were grown to confluence, differentiated for 2.5 weeks then harvested for measurement of gene expression and UCP1 protein. Results There was no difference between groups with respect to differentiation into adipocytes, as indicated by oil red-O staining, rates of lipolysis, and expression of adipogenic genes (FABP4, PPARG). WAT genes (HOXC9, RB1) were expressed equally in the two groups. Post differentiation, the beige adipose specific genes CITED1 and CD137 were significantly increased in both groups, but classic BAT markers ZIC1 and LHX8 decreased significantly. Cell lines from both groups also equally increased post-differentiation expression of the thermogenic-responsive gene PPARGC1A (PGC-1α). UCP1 gene expression was undetectable prior to differentiation, however after differentiation both gene expression and protein content were increased in both groups and were significantly greater in cultures from lean compared with obese individuals (p<0.05). Conclusion Human subcutaneous WAT cells can be induced to attain BAT characteristics, but this capacity is reduced in WAT cells from obese individuals.

Effects of High-Density Lipoprotein Elevation With Cholesteryl Ester Transfer Protein Inhibition on Insulin Secretion

Rationale: High-density lipoprotein cholesterol elevation via cholesteryl ester transfer protein (CETP) inhibition represents a novel therapy for atherosclerosis, which also may have relevance for type 2 diabetes mellitus. Objective: The current study assessed the effects of a CETP inhibitor on postprandial insulin, ex vivo insulin secretion, and cholesterol efflux from pancreatic &bgr;-cells. Methods and Results: Healthy participants received a daily dose of CETP inhibitor (n=10) or placebo (n=15) for 14 days in a randomized double-blind study. Insulin secretion and cholesterol efflux from MIN6N8 &bgr;-cells were determined after incubation with treated plasma. CETP inhibition increased plasma high-density lipoprotein cholesterol, apolipoprotein AI, and postprandial insulin. MIN6N8 &bgr;-cells incubated with plasma from CETP inhibitor–treated individuals (compared with placebo) exhibited an increase in both glucose-stimulated insulin secretion and cholesterol efflux over the 14-day treatment period. Conclusions: CETP inhibition increased postprandial insulin and promoted ex vivo &bgr;-cell glucose-stimulated insulin secretion, potentially via enhanced &bgr;-cell cholesterol efflux.Rationale: High-density lipoprotein (HDL) cholesterol elevation via cholesteryl ester transfer protein (CETP) inhibition represents a novel therapy for atherosclerosis, which may also have relevance for type 2 diabetes. Objective: The current study assessed the effects of a CETP inhibitor (CETPi) on postprandial insulin, ex vivo insulin secretion and cholesterol efflux from pancreatic β-cells. Methods and Results: Healthy participants received a daily dose of CETPi (n=10) or placebo (n=15) for 14 days in a randomized, double-blind study. Insulin secretion and cholesterol efflux from MIN6N8 β-cells was determined following incubation with treated plasma. CETP inhibition increased plasma HDL cholesterol, apoAI and postprandial insulin. MIN6N8 β-cells incubated with plasma from CETPi-treated individuals (vs placebo) exhibited an increase in both glucose-stimulated insulin secretion (GSIS) and cholesterol efflux over the 14 day treatment period. Conclusions: CETP inhibition increased postprandial insulin and promoted ex vivo β-cell GSIS, potentially via enhanced β-cell cholesterol efflux.

Effects of HDL Elevation with CETP Inhibition on Insulin Secretion

Rationale: High-density lipoprotein cholesterol elevation via cholesteryl ester transfer protein (CETP) inhibition represents a novel therapy for atherosclerosis, which also may have relevance for type 2 diabetes mellitus. Objective: The current study assessed the effects of a CETP inhibitor on postprandial insulin, ex vivo insulin secretion, and cholesterol efflux from pancreatic &bgr;-cells. Methods and Results: Healthy participants received a daily dose of CETP inhibitor (n=10) or placebo (n=15) for 14 days in a randomized double-blind study. Insulin secretion and cholesterol efflux from MIN6N8 &bgr;-cells were determined after incubation with treated plasma. CETP inhibition increased plasma high-density lipoprotein cholesterol, apolipoprotein AI, and postprandial insulin. MIN6N8 &bgr;-cells incubated with plasma from CETP inhibitor–treated individuals (compared with placebo) exhibited an increase in both glucose-stimulated insulin secretion and cholesterol efflux over the 14-day treatment period. Conclusions: CETP inhibition increased postprandial insulin and promoted ex vivo &bgr;-cell glucose-stimulated insulin secretion, potentially via enhanced &bgr;-cell cholesterol efflux.Rationale: High-density lipoprotein (HDL) cholesterol elevation via cholesteryl ester transfer protein (CETP) inhibition represents a novel therapy for atherosclerosis, which may also have relevance for type 2 diabetes. Objective: The current study assessed the effects of a CETP inhibitor (CETPi) on postprandial insulin, ex vivo insulin secretion and cholesterol efflux from pancreatic β-cells. Methods and Results: Healthy participants received a daily dose of CETPi (n=10) or placebo (n=15) for 14 days in a randomized, double-blind study. Insulin secretion and cholesterol efflux from MIN6N8 β-cells was determined following incubation with treated plasma. CETP inhibition increased plasma HDL cholesterol, apoAI and postprandial insulin. MIN6N8 β-cells incubated with plasma from CETPi-treated individuals (vs placebo) exhibited an increase in both glucose-stimulated insulin secretion (GSIS) and cholesterol efflux over the 14 day treatment period. Conclusions: CETP inhibition increased postprandial insulin and promoted ex vivo β-cell GSIS, potentially via enhanced β-cell cholesterol efflux.

Effect of Iron Chelation on Myocardial Infarct Size and Oxidative Stress in ST-Elevation–Myocardial Infarction

Background— Experimental studies suggest that deferoxamine (DFO) limits the generation of reactive oxygen species by chelating redox-active iron and thereby may reduce ischemia-reperfusion injury and myocardial infarct (MI) size. We investigated whether DFO administered before reperfusion by primary percutaneous coronary intervention (PPCI) would ameliorate oxidative stress and MI size. Methods and Results— We randomly assigned 60 patients with ST-elevation–MI to receive an intravenous bolus of DFO (500 mg) immediately before PPCI followed by a 12-hour infusion (50 mg/kg of body weight) (n=28) or normal saline bolus and infusion (placebo group, n=32). MI size was measured by contrast-enhanced cardiac MRI (CMRI; day 3±1), creatine kinase and troponin I area-under-the-curve, and severity of wall motion abnormality on echocardiography. Clinical follow-up including repeat CMRI and echocardiography were performed at 3 months (100±17 days). Oxidative stress was assessed by plasma F2-isoprostane levels. DFO and placebo groups were well balanced with respect to baseline characteristics, symptom- and door-to-balloon times, pre-PPCI coronary patency, and infarct-related artery location. Serum iron levels were decreased with DFO treatment after PPCI compared with placebo (3.0±2.5 versus 12.6±5.5 &mgr;mol/L, P<0.0001), which persisted until the end of the infusion. In DFO-treated patients, there was a significant reduction in plasma F2-isoprostane levels immediately after PPCI (2878±1461 versus 2213±579 pmol/L, P=0.04). However, there was no difference in CMRI-determined infarct size (DFO, 17.4±10.8%, versus placebo, 18.6±10.2%; P=0.73), myocardial salvage index at 3 days or at 3 months, or the area-under-the-curve for creatine kinase or troponin I. Conclusions— Adjunctive DFO treatment after the onset of ischemia and continued periprocedurally ameliorates oxidative stress without limiting infarct size. Clinical Trial Registration— URL: http://www.anzctr.org.au/. Unique identifier: ACTRN12608000308392.

Reconstituted high-density lipoprotein infusion modulates fatty acid metabolism in patients with type 2 diabetes mellitus

We recently demonstrated that reconstituted high-density lipoprotein (rHDL) modulates glucose metabolism in humans via both AMP-activated protein kinase (AMPK) in muscle and by increasing plasma insulin. Given the key roles of both AMPK and insulin in fatty acid metabolism, the current study investigated the effect of rHDL infusion on fatty acid oxidation and lipolysis. Thirteen patients with type 2 diabetes received separate infusions of rHDL and placebo in a randomized, cross-over study. Fatty acid metabolism was assessed using steady-state tracer methodology, and plasma lipids were measured by mass spectrometry (lipidomics). In vitro studies were undertaken in 3T3-L1 adipocytes. rHDL infusion inhibited fasting-induced lipolysis (P = 0.03), fatty acid oxidation (P < 0.01), and circulating glycerol (P = 0.04). In vitro, HDL inhibited adipocyte lipolysis in part via activation of AMPK, providing a possible mechanistic link for the apparent reductions in lipolysis observed in vivo. In contrast, circulating NEFA increased after rHDL infusion (P < 0.01). Lipidomic analyses implicated phospholipase hydrolysis of rHDL-associated phosphatidylcholine as the cause, rather than lipolysis of endogenous fat stores. rHDL infusion inhibits fasting-induced lipolysis and oxidation in patients with type 2 diabetes, potentially through both AMPK activation in adipose tissue and elevation of plasma insulin. The phospholipid component of rHDL also has the potentially undesirable effect of increasing circulating NEFA.

MicroRNA-194 Modulates Glucose Metabolism and Its Skeletal Muscle Expression Is Reduced in Diabetes.

Background The regulation of microRNAs (miRNAs) at different stages of the progression of type 2 diabetes mellitus (T2DM) and their role in glucose homeostasis was investigated. Methods Microarrays were used to assess miRNA expression in skeletal muscle biopsies taken from healthy individuals and patients with pre-diabetes or T2DM, and insulin resistant offspring of rat dams fed a high fat diet during pregnancy. Results Twenty-three miRNAs were differentially expressed in patients with T2DM, and 7 in the insulin resistant rat offspring compared to their controls. Among these, only one miRNA was similarly regulated: miR-194 expression was significantly reduced by 25 to 50% in both the rat model and in human with pre-diabetes and established diabetes. Knockdown of miR-194 in L6 skeletal muscle cells induced an increase in basal and insulin-stimulated glucose uptake and glycogen synthesis. This occurred in conjunction with an increased glycolysis, indicated by elevated lactate production. Moreover, oxidative capacity was also increased as we found an enhanced glucose oxidation in presence of the mitochondrial uncoupler FCCP. When miR-194 was down-regulated in vitro, western blot analysis showed an increased phosphorylation of AKT and GSK3β in response to insulin, and an increase in expression of proteins controlling mitochondrial oxidative phosphorylation. Conclusions Type 2 diabetes mellitus is associated with regulation of several miRNAs in skeletal muscle. Interestingly, miR-194 was a unique miRNA that appeared regulated across different stages of the disease progression, from the early stages of insulin resistance to the development of T2DM. We have shown miR-194 is involved in multiple aspects of skeletal muscle glucose metabolism from uptake, through to glycolysis, glycogenesis and glucose oxidation, potentially via mechanisms involving AKT, GSK3 and oxidative phosphorylation. MiR-194 could be down-regulated in patients with early features of diabetes as an adaptive response to facilitate tissue glucose uptake and metabolism in the face of insulin resistance.

Frequent interruptions of sedentary time modulates contraction- and insulin-stimulated glucose uptake pathways in muscle: Ancillary analysis from randomized clinical trials

Epidemiological studies have observed associations between frequent interruptions of sitting time with physical activity bouts and beneficial metabolic outcomes, even in individuals who regularly exercise. Frequent interruptions to prolonged sitting reduce postprandial plasma glucose. Here we studied potential skeletal muscle mechanisms accounting for this improved control of glycemia in overweight adults under conditions of one day uninterrupted sitting and sitting interrupted with light-intensity or moderate-intensity walking every 20-min (n = 8); and, after three days of either uninterrupted sitting or light-intensity walking interruptions (n = 5). Contraction- and insulin-mediated glucose uptake signaling pathways as well as changes in oxidative phosphorylation proteins were examined. We showed that 1) both interventions reduce postprandial glucose concentration, 2) acute interruptions to sitting over one day stimulate the contraction-mediated glucose uptake pathway, 3) both acute interruptions to sitting with moderate-intensity activity over one day and light-intensity activity over three days induce a transition to modulation of the insulin-signaling pathway, in association with increased capacity for glucose transport. Only the moderate-intensity interruptions resulted in greater capacity for glycogen synthesis and likely for ATP production. These observations contribute to a mechanistic explanation of improved postprandial glucose metabolism with regular interruptions to sitting time, a promising preventive strategy for metabolic diseases.

The effect of the nitric oxide donor sodium nitroprusside on glucose uptake in human primary skeletal muscle cells.

Nitric oxide (NO) has been implicated as an important signaling molecule in the insulin-independent, contraction-mediated glucose uptake pathway and may represent a novel strategy for blood glucose control in patients with type 2 diabetes (T2DM). The current study sought to determine whether the NO donor, sodium nitroprusside (SNP) increases glucose uptake in primary human skeletal muscle cells (HSkMC) derived from both healthy individuals and patients with T2DM. Vastus lateralis muscle cell cultures were derived from seven males with T2DM (aged 54 +/-2 years, BMI 31.7 +/-1.2 kg/m(2), fasting plasma glucose 9.52+/-0.80 mmol/L) and eight healthy individuals (aged 46 +/-2 years, BMI 27.1 +/- 1.5 kg/m(2), fasting plasma glucose 4.69+/-0.12 mmol/L). Cultures were treated with both therapeutic (0.2 and 2 microM) and supratherapeutic (3, 10 and 30 mM) concentrations of SNP. An additional NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) was also examined at a concentration of 50 microM. Glucose uptake was significantly increased following both 30 and 60 min incubations with the supratherapeutic SNP treatments (P=0.03) but not the therapeutic SNP doses (P=0.60) or SNAP (P=0.54). There was no difference in the response between the healthy and T2DM cell lines with any treatment or dose. The current study demonstrates that glucose uptake is elevated by supratherapeutic, but not therapeutic doses of SNP in human primary skeletal muscle cells derived from both healthy volunteers and patients with T2D. These data confirm that nitric oxide donors have potential therapeutic utility to increase glucose uptake in humans, but that SNP only achieves this in supratherapeutic doses. Further study to delineate mechanisms and the therapeutic window is warranted.