Scott Turner

A gene expression network model of type 2 diabetes links cell cycle regulation in islets with diabetes susceptibility

Insulin resistance is necessary but not sufficient for the development of type 2 diabetes. Diabetes results when pancreatic beta-cells fail to compensate for insulin resistance by increasing insulin production through an expansion of beta-cell mass or increased insulin secretion. Communication between insulin target tissues and beta-cells may initiate this compensatory response. Correlated changes in gene expression between tissues can provide evidence for such intercellular communication. We profiled gene expression in six tissues of mice from an obesity-induced diabetes-resistant and a diabetes-susceptible strain before and after the onset of diabetes. We studied the correlation structure of mRNA abundance and identified 105 co-expression gene modules. We provide an interactive gene network model showing the correlation structure between the expression modules within and among the six tissues. This resource also provides a searchable database of gene expression profiles for all genes in six tissues in lean and obese diabetes-resistant and diabetes-susceptible mice, at 4 and 10 wk of age. A cell cycle regulatory module in islets predicts diabetes susceptibility. The module predicts islet replication; we found a strong correlation between (2)H(2)O incorporation into islet DNA in vivo and the expression pattern of the cell cycle module. This pattern is highly correlated with that of several individual genes in insulin target tissues, including Igf2, which has been shown to promote beta-cell proliferation, suggesting that these genes may provide a link between insulin resistance and beta-cell proliferation.

Hepatic gluconeogenic fluxes and glycogen turnover during fasting in humans. A stable isotope study.

Fluxes through intrahepatic glucose-producing metabolic pathways were measured in normal humans during overnight or prolonged (60 h) fasting. The glucuronate probe was used to measure the turnover and sources of hepatic UDP-glucose; mass isotopomer distribution analysis from [2-13C1]glycerol for gluconeogenesis and UDP-gluconeogenesis; [U-13C6]glucose for glucose production (GP) and the direct UDP-glucose pathway; and [1-2H1]galactose for UDP-glucose flux and retention in hepatic glycogen. After overnight fasting, GP (fluxes in milligram per kilogram per minute) was 2.19+/-0.09, of which 0.79 (36%) was from gluconeogenesis, 1.40 was from glycogenolysis, 0.30 was retained in glycogen via UDP-gluconeogenesis, and 0.17 entered hepatic UDP-glucose by the direct pathway. Thus, total flux through the gluconeogenic pathway (1.09) represented 54% of extrahepatic glucose disposal (2.02) and the net hepatic glycogen depletion rate was 0.93 (46%). Prolonging [2-13C1]glycerol infusion slowly increased measured fractional gluconeogenesis. In response to prolonged fasting, GP was lower (1. 43+/-0.06) and fractional and absolute gluconeogenesis were higher (78+/-2% and 1.11+/-0.07, respectively). The small but nonzero glycogen input to plasma glucose (0.32+/-0.03) was completely balanced by retained UDP-gluconeogenesis (0.31+/-0.02). Total gluconeogenic pathway flux therefore accounted for 99+/-2% of GP, but with a glycogen cycle interposed. Prolonging isotope infusion to 10 h increased measured fractional gluconeogenesis and UDP-gluconeogenesis to 84-96%, implying replacement of glycogen by gluconeogenic-labeled glucose. Moreover, after glucagon administration, GP (1.65), recovery of [1-2H1]galactose label in plasma glucose (25%) and fractional gluconeogenesis (91%) increased, such that 78% (0.45/0.59) of glycogen released was labeled (i.e., of recent gluconeogenic origin). In conclusion, hepatic gluconeogenic flux into glycogen and glycogen turnover persist during fasting in humans, reconciling inconsistencies in the literature and interposing another locus of control in the normal pathway of GP.

5A Apolipoprotein Mimetic Peptide Promotes Cholesterol Efflux and Reduces Atherosclerosis in Mice

Intravenous administration of apolipoprotein (apo) A-I complexed with phospholipid has been shown to rapidly reduce plaque size in both animal models and humans. Short synthetic amphipathic peptides can mimic the antiatherogenic properties of apoA-I and have been proposed as alternative therapeutic agents. In this study, we investigated the atheroprotective effect of the 5A peptide, a bihelical amphipathic peptide that specifically effluxes cholesterol from cells by ATP-binding cassette transporter 1 (ABCA1). 5A stimulated a 3.5-fold increase in ABCA1-mediated efflux from cells and an additional 2.5-fold increase after complexing it with phospholipid (1:7 mol/mol). 5A-palmitoyl oleoyl phosphatidyl choline (POPC), but not free 5A, was also found to promote cholesterol efflux by ABCG1. When incubated with human serum, 5A-POPC bound primarily to high-density lipoprotein (HDL) but also to low-density lipoprotein (LDL) and promoted the transfer of cholesterol from LDL to HDL. Twenty-four hours after intravenous injection of 5A-POPC (30 mg/kg) into apoE-knockout (KO) mice, both the cholesterol (181%) and phospholipid (219%) content of HDL significantly increased. By an in vivo cholesterol isotope dilution study and monitoring of the flux of cholesterol from radiolabeled macrophages to stool, 5A-POPC treatment was observed to increase reverse cholesterol transport. In three separate studies, 5A when complexed with various phospholipids reduced aortic plaque surface area by 29 to 53% (n = 8 per group; p < 0.02) in apoE-KO mice. No signs of toxicity from the treatment were observed during these studies. In summary, 5A promotes cholesterol efflux both in vitro and in vivo and reduces atherosclerosis in apoE-KO mice, indicating that it may be a useful alternative to apoA-I for HDL therapy.

Hepatic glucose sensing is required to preserve β cell glucose competence

Liver glucose metabolism plays a central role in glucose homeostasis and may also regulate feeding and energy expenditure. Here we assessed the impact of glucose transporter 2 (Glut2) gene inactivation in adult mouse liver (LG2KO mice). Loss of Glut2 suppressed hepatic glucose uptake but not glucose output. In the fasted state, expression of carbohydrate-responsive element-binding protein (ChREBP) and its glycolytic and lipogenic target genes was abnormally elevated. Feeding, energy expenditure, and insulin sensitivity were identical in LG2KO and control mice. Glucose tolerance was initially normal after Glut2 inactivation, but LG2KO mice exhibited progressive impairment of glucose-stimulated insulin secretion even though β cell mass and insulin content remained normal. Liver transcript profiling revealed a coordinated downregulation of cholesterol biosynthesis genes in LG2KO mice that was associated with reduced hepatic cholesterol in fasted mice and reduced bile acids (BAs) in feces, with a similar trend in plasma. We showed that chronic BAs or farnesoid X receptor (FXR) agonist treatment of primary islets increases glucose-stimulated insulin secretion, an effect not seen in islets from Fxr(-/-) mice. Collectively, our data show that glucose sensing by the liver controls β cell glucose competence and suggest BAs as a potential mechanistic link.

Measurement of Reverse Cholesterol Transport Pathways in Humans: In Vivo Rates of Free Cholesterol Efflux, Esterification, and Excretion

Background Reverse cholesterol transport from peripheral tissues is considered the principal atheroprotective mechanism of high-density lipoprotein, but quantifying reverse cholesterol transport in humans in vivo remains a challenge. We describe here a method for measuring flux of cholesterol though 3 primary components of the reverse cholesterol transport pathway in vivo in humans: tissue free cholesterol (FC) efflux, esterification of FC in plasma, and fecal sterol excretion of plasma-derived FC. Methods and Results A constant infusion of [2,3-13C2]-cholesterol was administered to healthy volunteers. Three-compartment SAAM II (Simulation, Analysis, and Modeling software; SAAM Institute, University of Washington, WA) fits were applied to plasma FC, red blood cell FC, and plasma cholesterol ester 13C–enrichment profiles. Fecal sterol excretion of plasma-derived FC was quantified from fractional recovery of intravenous [2,3-13C2]-cholesterol in feces over 7 days. We examined the key assumptions of the method and evaluated the optimal clinical protocol and approach to data analysis and modeling. A total of 17 subjects from 2 study sites (n=12 from first site, age 21 to 75 years, 2 women; n=5 from second site, age 18 to 70 years, 2 women) were studied. Tissue FC efflux was 3.79±0.88 mg/kg per hour (mean ± standard deviation), or ≍8 g/d. Red blood cell–derived flux into plasma FC was 3.38±1.10 mg/kg per hour. Esterification of plasma FC was ≍28% of tissue FC efflux (1.10±0.38 mg/kg per hour). Recoveries were 7% and 12% of administered [2,3-13C2]-cholesterol in fecal bile acids and neutral sterols, respectively. Conclusions Three components of systemic reverse cholesterol transport can be quantified, allowing dissection of this important function of high-density lipoprotein in vivo. Effects of lipoproteins, genetic mutations, lifestyle changes, and drugs on these components can be assessed in humans. (J Am Heart Assoc. 2012;1:e001826 doi: 10.1161/JAHA.112.001826.)

Reverse cholesterol transport fluxes.

Purpose of review Reverse cholesterol transport (RCT) is considered a significant component of the atheroprotective effects of HDL. Methods for quantifying flux through the RCT pathway have not been available until recently. There is a need to improve our understanding of HDL function, including the role of RCT in general and individual steps of RCT in particular, on atherosclerosis. This review highlights new information about cholesterol flux through the RCT pathway. Recent findings Recent clinical studies have demonstrated several important quantitative features of cholesterol fluxes in vivo, providing insight into variability and control of specific components of the RCT pathway. The findings illustrate the independent nature of individual steps in the RCT pathway and their apparently weak relationship to plasma HDL cholesterol levels. Nonclinical studies provide some mechanistic data re-enforcing the importance of apoB particles in RCT and role roles for serum albumin and erythrocytes in free cholesterol flux. These findings suggest that the HDL-centric view of RCT may need revision. Summary The constellation of known lipoproteins and other players involved in this pathway continues to increase. Further research, particularly in humans, is needed in order to understand which parts of the RCT pathway are most relevant to the pathophysiology and treatment of atherosclerosis.

Helix stabilization of amphipathic peptides by hydrocarbon stapling increases cholesterol efflux by the ABCA1 transporter

Apolipoprotein mimetic peptides are short amphipathic peptides that efflux cholesterol from cells by the ABCA1 transporter and are being investigated as therapeutic agents for cardiovascular disease. We examined the role of helix stabilization of these peptides in cholesterol efflux. A 23-amino acid long peptide (Ac-VLEDSFKVSFLSALEEYTKKLNTQ-NH2) based on the last helix of apoA-I (A10) was synthesized, as well as two variants, S1A10 and S2A10, in which the third and fourth and third and fifth turn of each peptide, respectively, were covalently joined by hydrocarbon staples. By CD spectroscopy, the stapled variants at 24 °C were more helical in aqueous buffer than A10 (A10 17%, S1A10 62%, S2A10 97%). S1A10 and S2A10 unlike A10 were resistant to proteolysis by pepsin and chymotrypsin. S1A10 and S2A10 showed more than a 10-fold increase in cholesterol efflux by the ABCA1 transporter compared to A10. In summary, hydrocarbon stapling of amphipathic peptides increases their helicity, makes them resistant to proteolysis and enhances their ability to promote cholesterol efflux by the ABCA1 transporter, indicating that this peptide modification may be useful in the development of apolipoprotein mimetic peptides.

The effect of phospholipid composition of reconstituted HDL on its cholesterol efflux and anti-inflammatory properties.

The goal of this study was to understand how the reconstituted HDL (rHDL) phospholipid (PL) composition affects its cholesterol efflux and anti-inflammatory properties. An ApoA-I mimetic peptide, 5A, was combined with either SM or POPC. Both lipid formulations exhibited similar in vitro cholesterol efflux by ABCA1, but 5A-SM exhibited higher ABCG1- and SR-BI-mediated efflux relative to 5A-POPC (P < 0.05). Injection of both rHDLs in rats resulted in mobilization of plasma cholesterol, although the relative potency was 3-fold higher for the same doses of 5A-SM than for 5A-POPC. Formation of preβ HDL was observed following incubation of rHDLs with both human and rat plasma in vitro, with 5A-SM inducing a higher extent of preβ formation relative to 5A-POPC. Both rHDLs exhibited anti-inflammatory properties, but 5A-SM showed higher inhibition of TNF-α, IL-6, and IL-1β release than did 5A-POPC (P < 0.05). Both 5A-SM and 5A-POPC showed reduction in total plaque area in ApoE−/− mice, but only 5A-SM showed a statistically significant reduction over placebo control and baseline (P < 0.01). The type of PL used to reconstitute peptide has significant influence on rHDL’s anti-inflammatory and anti-atherosclerosis properties.

Craniometric variation in a population of mantled howler monkeys (Alouatta palliata): Evidence of size selection in females and growth in dentally mature males

A large body of work on monkey cranial metrics (involving conclusions about interspecific variation, sexual dimorphism, and ontogeny) depends on the assumptions that growth effectively ceases with dental maturity and that intraspecific variation is negligible. We test these assumptions by examining variation in 39 measurements of 166 dentally mature Alouatta palliata skulls from animals found dead on Barro Colorado Island (BCI), Panama. We also investigate whether this population is under size-based selection, since our found-dead sample reflects the natural mortality in this population. The sample was divided into three age stages by occlusal wear (A-C, least to most wear). Female stage A means are significantly smaller than female stage B means for three cranial measures. Female stage B means are significantly smaller than female stage C means for five cranial measures. Male stage A means are significantly smaller than male stage B means for 21 cranial measures. Multivariate analyses confirm this trend of expansion between adult age stages. The dental metric and suture closure data suggest that the cranial expansion in females is due to size-based selection, while the cranial expansion in males is due to significant growth after dental maturity. Sexual dimorphism ratios are highly variable across different samples of A. palliata, indicating that dimorphism varies between populations of this species. These results provide insight into the selective forces operating on the BCI howlers and challenge the validity of the many studies which pool subspecies and assume growth ceases with maturity.

Plasma plant sterols serve as poor markers of cholesterol absorption in man

The validation of the use of plasma plant sterols as a marker of cholesterol absorption is frail. Nevertheless, plant sterol concentrations are routinely used to describe treatment-induced changes in cholesterol absorption. Their use has also been advocated as a clinical tool to tailor cholesterol-lowering therapy. Prior to wider implementation, however, the validity of plant sterols as absorption markers needs solid evaluation. Therefore, we compared plasma plant sterol concentrations to gold-standard stable isotope-determined cholesterol absorption. Plasma campesterol/TC concentrations (camp/TC) were measured in a population of 175 mildly hypercholesterolemic individuals (age: 59.7 ± 5.6 years; BMI: 25.5 ± 2.9kg/m2; LDL-C: 4.01 ± 0.56 mmol/l). We compared cholesterol absorption according to the plasma dual-isotope method in subjects with the highest camp/TC concentrations (N = 41, camp/TC: 2.14 ± 0.68 μg/mg) and the lowest camp/TC concentrations (N = 39, camp/TC: 0.97 ± 0.22 μg/mg). Fractional cholesterol absorption did not differ between the groups (24 ± 12% versus 25 ± 16%, P = 0.60), nor was it associated with plasma camp/TC concentrations in the total population of 80 individuals (β = 0.13; P = 0.30, adjusted for BMI and plasma triglycerides). Our findings do not support a relation between plasma plant sterol concentrations and true cholesterol absorption and, therefore, do not favor the use of these sterols as markers of cholesterol absorption. This bears direct consequences for the interpretation of earlier studies, as well as for future studies targeting intestinal regulation of cholesterol metabolism.