Deletion distal to the PAX6 coding region reveals a novel basis for familial cosegregation of aniridia and diabetes mellitus.

AIMS: Analyze cosegregation of aniridia and diabetes to identify genetic criteria for detection and early treatment of diabetes-susceptible aniridia patients. METHODS: We assessed a two-generation family: three individuals with aniridia, two previously diagnosed as type 2 diabetes. One individual with aniridia, with unknown diabetes status, was evaluated by oral glucose tolerance test. Genetic analysis of aniridia-associated genes was performed on all available family members. Candidate genes were functionally tested by gene silencing in MIN6 pancreatic ß-cells. RESULTS: A 25 year old male with aniridia had a diabetic oral glucose tolerance test despite a normal fasting blood glucose. A 484-630 kb deletion ∼120 kb distal to PAIRED BOX 6 (PAX6) showed dominant cosegregation with aniridia and diabetes in all affected family members. The deleted region contains regulatory elements for PAX6 expression and four additional coding regions. Knockdown of two of the deleted genes (Dnajc24 or Immp1l) with Pax6 impaired glucose-stimulated insulin secretion. CONCLUSIONS: We demonstrate dominant cosegregation of diabetes and aniridia with a deletion distal to PAX6, which is clinically distinct from the mild glucose intolerance previously reported with PAX6 coding mutations. Asymptomatic aniridia individuals appear at risk of diabetes (and its complications) and could benefit from earlier diagnosis and treatment.

Effects of dietary fat subtypes on glucose homeostasis during pregnancy in rats.

BACKGROUND: Dietary n-3 and n-6 polyunsaturated fatty acids (PUFAs) have an impact on insulin secretion and sensitivity but whether and how these may be related to maternal glucose homeostasis during pregnancy is unclear. METHODS: Female Wistar rats (240-250 g) were assigned to laboratory CHOW or high fat diets rich in either n-6 (safflower oil; n-6 group) or n-6 + n-3 (safflower oil + fish oil; n-3 group) PUFAs. After 10 days half of the animals in each diet group were inseminated and confirmed pregnant. An overnight fasted intravenous glucose tolerance test (500 mg glucose/kg body weight) was performed on chronically cannulated non-pregnant and 20-day pregnant rats. Indices of insulin secretion (ß) and insulin sensitivity (S) were calculated from the plasma glucose and insulin responses. The fatty acid composition of phospholipids was determined in samples of liver and two skeletal muscles (soleus and red quadriceps). RESULTS: Pregnancy in the CHOW group significantly increased ß (P < 0.001) and decreased S (P < 0.01). In contrast, both n-6 and n-3 diets abolished both the pregnancy-induced decrease in S and pregnancy-induced increase in ß with the n-3 diet having a more potent effect on both S and ß. S was positively correlated with the sum of n-3 fatty acids, with docosahexaenoic acid (22:6 n-3) the major contributor, in liver (r = 0.485; P < 0.001), red quadriceps (r = 0.421; P = 0.004) and soleus (r = 0.476; P < 0.001). In contrast S was inversely related to arachidonic acid (20:4n-6) levels in liver and red quadriceps across all groups and these relationships were particularly powerful in pregnancy (liver: r = -0.785; red quadriceps: r = -0.754, both P < 0.0001). CONCLUSIONS: The results demonstrate potent effects of dietary fat amount and profile on glucoregulation during pregnancy and emphasize the importance of the balance between dietary n-3 and n-6 PUFAs.

Effect of acute and chronic glucocorticoid therapy on insulin sensitivity and postprandial vascular function.

OBJECTIVE: Postprandial hyperglycaemia is associated with increased arterial stiffness and cardiovascular events. Low-dose prednisolone causes insulin resistance that typically manifests as postprandial hyperglycaemia. We investigated whether prednisolone causes postprandial vascular dysfunction in a cohort of patients with rheumatoid arthritis. DESIGN: An open interventional and cross-sectional study was undertaken. PATIENTS AND MEASUREMENTS: Eighteen subjects with rheumatoid arthritis who had not taken oral glucocorticoids for ≥6 months were studied before and after prednisolone 6 mg/day for 7 days to determine the acute effects of prednisolone. Pre-prednisolone data were compared to 18 subjects with rheumatoid arthritis taking long-term (>6 months) prednisolone (6·5 ± 1·8 mg/day) to assess the chronic effects of prednisolone. Augmentation index (by applanation tonometry) and reactive hyperaemia index (by peripheral artery tonometry) were measured before and after a mixed-meal (10 kcal/kg, 45% carbohydrate, 15% protein, 40% fat). Insulin sensitivity was estimated by the Matsuda index and sympathetic nervous system activity from urinary noradrenaline excretion. RESULTS: Matsuda index was lower after acute (2·0 ± 1·0 vs 3·6 ± 1·1, P = 0·01) and chronic (1·9 ± 1·0 vs 3·6 ± 1·1, P = 0·04) prednisolone. Postprandial augmentation index was lower after acute prednisolone (2551 ± 197 vs 2690 ± 272%*min, P ≤ 0·001), but not chronic prednisolone. There were no significant differences in reactive hyperaemia index with acute or chronic prednisolone. Noradrenaline excretion was lower after acute (54 ± 8 vs 93 ± 23 nmol/6 h, P = 0·02), but not chronic, prednisolone. CONCLUSIONS: Prednisolone-induced insulin resistance is not associated with postprandial vascular dysfunction in patients with rheumatoid arthritis. Reduced sympathetic activity may contribute to the reduction in postprandial arterial stiffness with acute prednisolone.

Segregation of a latent high adiposity phenotype in families with a history of type 2 diabetes mellitus implicates rare obesity-susceptibility genetic variants with large effects in diabetes-related obesity.

BACKGROUND: We recently reported significantly greater weight gain in non-diabetic healthy subjects with a 1(st) degree family history (FH+) of type 2 diabetes mellitus (T2DM) than in a matched control group without such history (FH-) during voluntary overfeeding, implying co-inheritance of susceptibilities to T2DM and obesity. We have estimated the extent and mode of inheritance of susceptibility to increased adiposity in FH+. METHODS: Normoglycaemic participants were categorised either FH+ (≥1 1(st) degree relative with T2DM, 50 F/30 M, age 45 ± 14 (SD) yr) or FH- (71F/51M, age 43 ± 14 yr). Log-transformed anthropometric measurements (height, hip and waist circumferences) and lean, bone and fat mass (Dual Energy X-ray Absorptiometry) data were analysed by rotated Factor Analysis. The age- and gender-adjusted distributions of indices of adiposity in FH+ were assessed by fits to a bimodal model and by relative risk ratios (RR, FH+/FH-) and interpreted in a purely genetic model of FH effects. RESULTS: The two orthogonal factors extracted, interpretable as Frame and Adiposity accounted for 80% of the variance in the input data. FH+ was associated with significantly higher Adiposity scores (p<0.01) without affecting Frame scores. Adiposity scores in FH+ conformed to a bimodal normal distribution, consistent with dominant expression of major susceptibility genes with 59% (95% CI 40%, 74%) of individuals under the higher mode. Calculated risk allele frequencies were 0.09 (0.02, 0.23) in FH-, 0.36 (0.22, 0.48) in FH+ and 0.62 (0.36, 0.88) in unobserved T2DM-affected family members. CONCLUSIONS: The segregation of Adiposity in T2DM-affected families is consistent with dominant expression of rare risk variants with major effects, which are expressed in over half of FH+ and which can account for most T2DM-associated obesity in our population. The calculated risk allele frequency in FH- suggests that rare genetic variants could also account for a substantial fraction of the prevalent obesity in this society.

Effects of low-dose prednisolone on hepatic and peripheral insulin sensitivity, insulin secretion, and abdominal adiposity in patients with inflammatory rheumatologic disease.

OBJECTIVE: The metabolic effects of low-dose prednisolone and optimal management of glucocorticoid-induced diabetes are poorly characterized. The aims were to investigate the acute effects of low-dose prednisolone on carbohydrate metabolism and whether long-term low-dose prednisolone administration increases visceral adiposity, amplifying metabolic perturbations. RESEARCH DESIGN AND METHODS: Subjects with inflammatory rheumatologic disease without diabetes mellitus were recruited. Nine subjects (age, 59 ± 11 years) not using oral glucocorticoids were studied before and after a 7- to 10-day course of oral prednisolone 6 mg daily. Baseline data were compared with 12 subjects (age, 61 ± 8 years) using continuous long-term prednisolone (6.3 ± 2.2 mg/day). Basal endogenous glucose production (EGP) was estimated by 6,6-(2)H2 glucose infusion, insulin sensitivity was estimated by two-step hyperinsulinemic-euglycemic clamp, insulin secretion was estimated by intravenous glucose tolerance test, and adipose tissue areas were estimated by computed tomography. RESULTS: Prednisolone acutely increased basal EGP (2.44 ± 0.46 to 2.65 ± 0.35 mg/min/kg; P = 0.05) and reduced insulin suppression of EGP (79 ± 7 to 67 ± 14%; P = 0.03), peripheral glucose disposal (8.2 ± 2.4 to 7.0 ± 1.6 mg/kg/min; P = 0.01), and first-phase (5.9 ± 2.0 to 3.9 ± 1.6 mU/mmol; P = 0.01) and second-phase (4.6 ± 1.7 to 3.6 ± 1.4 mU/mmol; P = 0.02) insulin secretion. Long-term prednisolone users had attenuated insulin suppression of EGP (66 ± 14 vs. 79 ± 7%; P = 0.03) and nonoxidative glucose disposal (44 ± 24 vs. 62 ± 8%; P = 0.02) compared with nonglucocorticoid users, whereas basal EGP, insulin secretion, and adipose tissue areas were not significantly different. CONCLUSIONS: Low-dose prednisolone acutely perturbs all aspects of carbohydrate metabolism. Long-term low-dose prednisolone induces hepatic insulin resistance and reduces peripheral nonoxidative glucose disposal. We conclude that hepatic and peripheral insulin sensitivity should be targeted by glucose-lowering therapy for glucocorticoid-induced diabetes.

Chronic hepatitis C is associated with peripheral rather than hepatic insulin resistance.

BACKGROUND & AIMS: Chronic hepatitis C (CHC) is associated with insulin resistance (IR), liver steatosis (genotype 3), and increased diabetes risk. The site and mechanisms of IR are unclear. METHODS: We compared cross-sectionally 29 nonobese, normoglycemic males with CHC (genotypes 1 and 3) to 15 adiposity and age-matched controls using a 2-step hyperinsulinemic-euglycemic clamp with [6,6-(2)H(2)] glucose to assess insulin sensitivity in liver and peripheral tissues and (1)H-magnetic resonance spectroscopy to evaluate liver and intramyocellular lipid. Insulin secretion was assessed after intravenous glucose. RESULTS: Insulin secretion was not impaired in CHC. Peripheral insulin sensitivity was 35% higher in controls vs CHC (P < .001) during high-dose (264.3 +/- 25 [standard error] mU/L) insulin (P < .001); this was negatively associated with viral load (R(2) = .12; P = .05) and subcutaneous fat (R(2) = .41; P < .001). IR was similar in both genotypes despite 3-fold increased hepatic fat in genotype 3 (P < .001). Hepatic glucose production (P = .25) and nonesterified free fatty acid (P = .84) suppression with insulin were not different between CHC and controls inferring no adipocyte IR, and suggesting IR is mainly in muscle. In CHC, intramyocellular lipid was nonsignificantly increased but levels of glucagon (73.8 +/- 3.6 vs 52.8 +/- 3.1 ng/mL; P < .001), soluble tumor necrosis factor receptor 2 (3.1 +/- 0.1 vs 2.3 +/- 0.1 ng/mL; P < .001), and Lipocalin-2 (36.4 +/- 2.9 vs 19.6 +/- 1.6 ng/mL; P < .001) were elevated. CONCLUSIONS: CHC represents a unique infective/inflammatory model of IR, which is predominantly in muscle, correlates with subcutaneous, not visceral, adiposity, and is independent of liver fat.

Obesity is an important determinant of baseline serum C-reactive protein concentration in monozygotic twins, independent of genetic influences.

BACKGROUND: C-reactive protein (CRP) values predict atherothrombotic cardiovascular disease and type 2 diabetes mellitus. Associations between CRP and obesity, predominantly assessed anthropometrically, may partly explain these observations. Previous studies have been unable to control for genetic influences on CRP and obesity. The aim of this study was to examine the relationship between CRP and accurately measured body fat, lipids, apolipoproteins, blood pressure, and environmental and behavioral factors, independent of genetic influences. METHODS AND RESULTS: One hundred ninety-four healthy female twins (age 57.2+/-7 years) were studied after excluding pairs with CRP values >10 mg/L. Total body fat and central abdominal fat (CAF) were measured by dual-energy x-ray absorptiometry. CRP concentration was strongly related to surrogate and direct measures of body fat (r=0.31 to 0.54, P<0.001), diastolic blood pressure (r=0.20, P=0.003), and lipid and apolipoprotein levels (r=0.21 to 0.51, P<0.008). Light-to-moderate alcohol consumers and nonusers of hormone replacement therapy (HRT) had lower CRP levels than abstainers and HRT users, respectively. In stepwise multiple regression analysis, CAF, triglycerides, apolipoprotein B, and HRT use explained 46% of the variance in circulating CRP. In analyses controlling for genetic influences in monozygotic twins, within-pair differences in CRP were associated with within-pair differences in total body fat (r=0.39, P<0.001), CAF (r=0.34, P=0.002), diastolic blood pressure (r=0.24, P=0.03), apolipoprotein AI (r=-0.33, P=0.01), HDL cholesterol (r=-0.42, P=0.001), and triglycerides (r=0.35, P=0.007). CONCLUSIONS: CRP was strongly related to total and central abdominal obesity, blood pressure, and lipid levels, independent of genetic influences. These relationships are likely to contribute significantly to prospective associations between CRP and type 2 diabetes and coronary events.

Interactions between temperature and human leptin physiology in vivo and in vitro.

To investigate the possibility that environmental temperature may exert physiologically significant direct, local effects on subcutaneous adipose tissue temperatures, and its secretion of leptin, we exposed healthy males ( n=12) to repeated cold-water immersion (study 1), and also incubated surgically removed human subcutaneous adipose tissue samples ( n=7) at 27 degrees, 32 degrees and 37 degrees C (study 2). In vivo immersions were conducted over 15 days (60-90 min at 18 degrees C). Regional body temperatures and plasma leptin concentrations were measured before and during immersion. Acute cold exposure suppressed plasma leptin concentration (25 min: -14%, 60 min: -22%, P=0.0001), whilst repeated cold-water immersion was associated with an increase of plasma leptin concentration relative to test day 1 (+19% day 8, +13% day 15, overall P=0.03). Leptin secretion in vitro decreased 3.7-fold as the incubation temperature decreased from 37 degrees to 27 degrees C ( P=0.001). In a compartmental model of leptin turnover in vivo, the measured (local) temperature effect on leptin secretion in vitro was more than able to account for the observed cold-induced decrease in leptin concentration in vivo. We therefore conclude that acute and repeated cold-water immersions have separate and opposing effects on circulating leptin concentrations in humans. Under our experimental conditions, the local effects of reduced subcutaneous adipose tissue temperature may be a more important contributor to the acute effects observed in vivo, than the sympathetically mediated suppression of leptin secretion.

The impact of usual dietary patterns, selection of significant foods and cuisine choices on changing dietary fat under 'free living' conditions.

Dietary guidelines for the general population and for the management of obesity, diabetes and heart disease suggest a reduction in dietary fat, and in particular dietary saturated fatty acids (SFA). In order to achieve the recommended levels, changes in food choice patterns are required. Foods are consumed in combination with other foods, and these combinations are often recognizable as cuisine patterns. In this study we examined the food choice patterns of a group of 63 adults with existing type 2 diabetes mellitus who completed a 12 month dietary intervention trial aimed at changing dietary fat under 'free living' conditions. In both lower fat (LF, 27%) and modified fat (MF, 37%E) groups, a reduction in dietary SFA and an increase in polyunsaturated fat were required, with an additional requirement to increase dietary monounsaturated fat in the MF group. The usual diets of the study sample were on average low in total fat (27%E), but high in saturated fat (12%E). Those already consuming total fat at the level concordant with their allocation (LF or MF) achieved targets faster than those with a discordant allocation, but there was no significant effect of usual diet on time of target achievement at 12 months. At 6 months, those achieving dietary fat targets had changed to low fat dairy products and leaner meats, were having more spreads, oils, and nuts and were consuming takeaway meals less than twice a week. Contributions to dietary fat shifted from takeaway foods, meat, dairy products and cakes to spreads, oils and nuts. The modified fat and low fat groups chose more Mediterranean and South East Asian cuisines respectively. In this study sample, usual dietary patterns had an initial impact on change in the diet, but identifiable changes in food choice patterns and the adoption of certain cuisines that combined foods indicative of the dietary guidelines resulted in successful achievement of dietary fat targets.

Moderate alcohol consumption, dietary fat composition, and abdominal obesity in women: evidence for gene-environment interaction.

We examined relationships among alcohol intake, dietary fat composition, and total body fat (TBF) and central abdominal fat (CAF), independent of genetic confounders, and evaluated the modulating effect of genetic susceptibility. We studied 334 female twins (57.7 +/- 6.7 yr) after excluding dietary underreporters. Diet was assessed by Food-Frequency Questionnaire and body fat by dual-energy x-ray absorptiometry. Moderate alcohol consumers (12-17.9 g/d) had less TBF (20.6 +/- 5.6 vs. 24.8 +/- 8.4 kg, P = 0.03) and CAF (1.2 +/- 0.6 vs. 1.6 +/- 0.7 kg, P = 0.03) than abstainers. In multiple regression, alcohol consumption remained independently associated with body fat distribution. In cotwin case-control (monozygotic twin) analysis, moderate alcohol consumption accounted for 300 g less CAF, independent of genetic and other environmental factors. Gene-environment interaction analysis indicated that this association was limited to subjects at high genetic risk of abdominal obesity. There was no relationship between dietary fat composition and adiposity. However, in women at low genetic risk of abdominal obesity, subjects with polyunsaturated fat intakes in the highest tertile had about 50% less CAF than subjects with intakes in the lowest tertile (0.9 +/- 0.4 vs. 1.6 +/- 0.4 kg, P = 0.0007), an association absent in subjects with high genetic risk. In conclusion, genetic risk modulates relationships between dietary factors and adiposity. Lower abdominal fat may mediate associations between dietary intake and type 2 diabetes risk.

Moderate alcohol consumption, estrogen replacement therapy, and physical activity are associated with increased insulin sensitivity: is abdominal adiposity the mediator?

OBJECTIVE: To investigate 1). associations between environmental factors (alcohol consumption, hormone replacement therapy [HRT], and physical activity) and insulin resistance and secretion, independent of genetic influences; 2). the contribution of abdominal adiposity to these relationships; and 3). whether gene-environment interactions mediate these associations. RESEARCH DESIGN AND METHODS: Reported effects of lifestyle factors on insulin resistance and secretion are inconsistent, possibly due to difficulty in dissecting environmental from genetic influences and to confounding by adiposity. We examined these relationships in 798 nondiabetic female twins. Insulin resistance and secretion were estimated by modified homeostasis model assessment (HOMA-R' and HOMA-beta', respectively). Percent total body fat and percent central abdominal fat (CAF) were measured by dual-energy X-ray absorptiometry. RESULTS: All categories of alcohol consumption were associated with lower insulin levels and HOMA-beta' than abstinence. Only moderate alcohol consumers (11-20 units/week) had lower HOMA-R' than abstainers (-0.16 +/- 0.09 vs. 0.14 +/- 0.13 SD, P = 0.048). This difference was attenuated after controlling for percent CAF (P = 0.57), which was lower in moderate drinkers. Controlling for genetic and smoking effects in cotwin case-control analysis, monozygotic pairs discordant for alcohol consumption had greater within-pair differences in HOMA-R' than concordant pairs (P = 0.02). Postmenopausal women using estrogen-only HRT had lower HOMA-R' than non-HRT users (-0.33 +/- 0.16 vs. 0.17 +/- 0.08 SD, P = 0.003), even after controlling for percent CAF. Lower fasting glucose levels and insulin resistance and secretion indexes in physically active subjects were partly explained by lower abdominal adiposity. CONCLUSIONS: Moderate alcohol consumption, estrogen replacement, and physical activity are associated with increased insulin sensitivity in female twins. The favorable effects of moderate alcohol consumption and physical activity on insulin sensitivity are partly mediated by lower abdominal adiposity.

Physical activity reduces genetic susceptibility to increased central systolic pressure augmentation: a study of female twins.

OBJECTIVES: We sought to examine associations between the augmentation index (AI) and metabolic, adiposity, and lifestyle factors, independent of genetic influences, and to determine whether gene-environment interactions modulate these relationships. BACKGROUND: Reported associations between AI, an index of systemic arterial stiffness, and metabolic, adiposity, and lifestyle factors remain contradictory. The modulating effect of genetic risk is unknown. METHODS: We studied 684 female twins (age 18 to 71 years); AI was derived from the pressure waveform measured at the radial artery by applanation tonometry. Percentage of total body fat (TBF) and percentage of central abdominal fat (CAF) were assessed by dual-energy X-ray absorptiometry. RESULTS: In univariate analysis, age-adjusted AI was significantly associated with fasting triglyceride levels (r = 0.1, P = 0.03), apolipoprotein-B/A1 (r = 0.1, P = 0.04), percentage of TBF (r = 0.11, P = 0.006), and percentage of CAF (r = 0.11, P = 0.004). In co-twin case-control (monozygotic twin) analysis, a 3.1% absolute within-pair difference in percentage of CAF accounted for a 6% within-pair difference in AI, independent of genetic effects. Smokers and subjects with alcohol intakes >15 U/week had higher AI than nonsmokers (p = 0.01) and nondrinkers (p = 0.02), respectively. Forty percent of the variance in AI was explained by age, central mean arterial pressure, heart rate, height, percentage of CAF, and smoking. In gene-environment interaction analysis, subjects at high genetic risk of increased AI participating in regular leisure-time physical activity had AI values similar to low genetic risk subjects. CONCLUSIONS: Central abdominal adiposity is a significant determinant of AI in female twins, independent of hemodynamic, lifestyle, and, importantly, genetic effects. Smoking is associated with increased AI, even after controlling for abdominal obesity and other AI determinants. Physical activity reduces genetic predisposition to increased AI.

Target-seeking behavior of plasma glucose with exercise in type 1 diabetes.

OBJECTIVE: To investigate the reproducibility of the plasma glucose (PG) response to exercise in subjects with type 1 diabetes on a nonintensive insulin regimen. RESEARCH DESIGN AND METHODS: Subjects cycled for 45 min at 50% VO(2max) on two occasions (studies 1 and 2) either 1 h after lunch and usual insulin (protocol A) or after overnight fasting without morning insulin (protocol B). Identical diet, activity, and insulin (twice daily neutral and intermediate) were maintained before and during each study day. A total of 13 type 1 diabetic subjects (6 men and 7 women, BMI 24.0 +/- 0.9 kg/m(2) [means +/- SE], age 42.6 +/- 2.7 years, diabetes duration 14.1 +/- 2.8 years) completed protocol A, and 7 (3 men and 4 women, BMI 25.8 +/- 1.3 kg/m(2), age 39.7 +/- 1.3 years, diabetes duration 14 +/- 4.4 years) completed protocol B. RESULTS: In protocol A (fed), the fall in PG during exercise was 4.5 +/- 1.0 and 5.0 +/- 0.8 mmol/l in studies 1 and 2, respectively, whereas in protocol B (fasted), it was 0.6 +/- 0.8 and 3.4 +/- 1.6 mmol/l. Regression analysis of the change in PG in protocol A in study 1 versus study 2 showed poor reproducibility (r(2) = 0.12, P = 0.25) despite uniform conditions. In protocol B, the fall in PG was more reproducible (r(2) = 0.81, P = 0.006). In fed subjects, there was better (P = 0.01) and clinically useful reproducibility of the PG at exercise completion (r(2) = 0.77, P = 0.0001) compared with preexercise. CONCLUSIONS: These results indicate poor reproducibility of the change in PG during exercise after feeding in type 1 diabetes on nonintensive insulin regimens but reasonable reproducibility when fasting. Exercise apparently decreases the glycemic variability after feeding, so that PG concentrations after exercise seek a reproducible "target." Thus, the absolute PG level after a typical bout of exercise in the fed state should be a good guide to carbohydrate or insulin adjustment on subsequent occasions.