HI-14K Sigma-AldrichHuman Insulin-Specific RIA

It has previously been shown that misfolded mutant Akita proinsulin in the endoplasmic reticulum engages directly in protein complexes either with nonmutant proinsulin or with hProCpepGFP (human proinsulin bearing emerald-GFP within the C-peptide), impairing the trafficking of these bystander proinsulin molecules (Liu, M., Hodish, I., Rhodes, C. J., and Arvan, P. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 15841-15846). Herein, we generated transgenic mice, which, in addition to expressing endogenous proinsulin, exhibit beta-cell-specific expression of hProCpepGFP via the Ins1 promoter. In these mice, hProCpepGFP protein levels are physiologically regulated, and hProCpepGFP is packaged and processed to CpepGFP that is co-stored in beta-secretory granules. Visualization of CpepGFP fluorescence provides a quantifiable measure of pancreatic islet insulin content that can be followed in live animals in states of health and disease. We examined loss of pancreatic insulin in hProCpepGFP transgenic mice mated to Akita mice that develop neonatal diabetes because of the expression of misfolded proinsulin. Loss of bystander insulin in Akita animals is detected initially as a block in CpepGFP/insulin production with intracellular accumulation of the precursor, followed ultimately by loss of pancreatic beta-cells. The data support that misfolded proinsulin perturbs bystander proinsulin in the endoplasmic reticulum, leading to beta-cell failure.

The purpose of this study is to determine whether moderate-intensity resistance exercise (MOD) lowers postprandial lipemia (PPL) as much as high-intensity resistance exercise (HI) of equal work. Ten healthy men performed three trials, each conducted over 2 days. On day 1 of each treatment, they either did not exercise (CON), performed 3 sets of 16 repetitions of 10 exercises at 50% of 8 repetitions maximum (MOD), or performed 3 sets of 8 repetitions of 10 exercises at 100% of 8 repetitions maximum (HI). On the morning of day 2 at 15.5 h postexercise, participants ate a high-fat meal. Venous blood samples were collected, and metabolic rate was measured at rest and 3 h postprandial. HI reduced fasting triglyceride (TG) and TG area under the curve (AUC) (36%, P = 0.011 and 35%, P = 0.014) compared with CON. MOD tended to reduce fasting TG and TG AUC (21%, P = 0.054 and 26%, P = 0.052) compared with CON, but MOD and HI did not differ in fasting TG or TG AUC. Incremental TG AUC did not differ among treatments. MOD and HI did not change resting metabolic rate. HI increased fat oxidation at rest (21%, P = 0.021) and at 3 h postprandial (39%, P = 0.009) relative to CON. MOD tended to increase fat oxidation at rest (18%, P = 0.060) relative to CON. Fat oxidation and metabolic rate did not differ in MOD and HI. MOD and HI increased the fasting quantitative insulin-sensitivity check index (4%, P = 0.001 and P = 0.004) relative to CON. As MOD and HI resulted in similar reductions in PPL and increases in fat oxidation, resistance exercise intensity does not influence PPL.

The aim of the study was to compare the regulation of ghrelin, leptin, and adiponectin by insulin and glucose during the second and third trimesters of pregnancy in women with diabetes. We studied 9 pregnant women with diabetes. All women were treated with insulin and omitted the morning dose on the day of the test. After collection of baseline fasting samples, we performed 3 successive glucose clamps: 2 euglycemic clamps (glucose, 5 mmol/L; insulin infusion at 20 and 40 mU m(-2) min(-1)) and 1 hyperglycemic clamp (glucose, 10 mmol/L; insulin infusion at 40 mU m(-2) min(-1)). We determined concentrations of acyl and desacyl ghrelin (using a double-antibody sandwich assay that recognizes the full-length molecule), leptin, and adiponectin. Fasting desacyl ghrelin concentrations decreased, whereas insulin and leptin concentrations increased, between the second and third trimesters of pregnancy (P </= .011). During the clamp studies, desacyl ghrelin concentrations decreased by 33% (second trimester, P = .004) and 27% (third trimester, P = .09) with increasing glucose and insulin concentrations, whereas acyl ghrelin, leptin, and adiponectin concentrations were unaffected. Glucose and insulin regulate desacyl ghrelin concentrations in pregnant women with diabetes. Impaired desacyl ghrelin regulation may affect energy metabolism in pregnant women with poorly controlled diabetes.

Protein ingestion stimulates muscle protein synthesis and improves net muscle protein balance. Insulin resistance has been suggested to result in a reduced muscle protein synthetic response to food intake. As such, we hypothesized that type 2 diabetes patients have a impaired muscle protein synthetic response to food ingestion. To test this hypothesis, 10 male type 2 diabetes patients using their normal oral glucose-lowering medication (68 +/- 2 y) and 10 matched, normoglycemic men (65 +/- 2 y) were randomly assigned to 2 crossover treatments in which whole body and muscle protein synthesis were measured following the consumption of either carbohydrate (CHO) or carbohydrate with a protein hydrolysate (CHO+PRO). Primed, continuous infusions with L-[ring-13C6]phenylalanine and L-[ring-2H2]tyrosine were applied and blood and muscle samples were collected to assess whole-body protein balance and mixed muscle protein fractional synthetic rate over a 6-h period. Whole-body phenylalanine and tyrosine flux were higher after the CHO+PRO treatment compared with the CHO treatment in the diabetes and control group (P 0.01). Protein balance was negative following CHO but positive following CHO+PRO treatment in both groups. Muscle protein synthesis rates were higher in both groups following the CHO+PRO (0.086 +/- 0.014%/h) treatment than in the CHO treatment (0.040 +/- 0.003%/h; P 0.01) with no difference between the diabetes patients and normoglycemic controls. We conclude that the muscle protein synthetic response to CHO or CHO+PRO ingestion is not substantially impaired in longstanding, type 2 diabetes patients treated with oral blood glucose-lowering medication.

Our objective was to investigate the relationship between the E23K genetic variant in the KCNJ11 gene, which encodes for the Kir6.2 subunit of the inward rectifier K+ channel family, and glucose and insulin metabolism and cardiovascular (CV) function in the sedentary state and their responses to exercise training. Two hundred and fourteen healthy sedentary men and women aged 50-75 years old and free of CV disease and type 2 diabetes underwent baseline testing (maximal oxygen consumption (Vo2max), body composition and glucose tolerance). One hundred and sixty-three of them repeated these tests after 24 weeks of exercise training while on a low-fat diet. At baseline, age, height, body fat, resting systolic blood pressure and all glucose and insulin metabolism markers did not differ among E23K genotype groups. In women at baseline, E23K genotype was associated with body weight, body mass index, Vo2max (ml kg(-1) min(-1), l min(-1)) and maximal minute ventilation. In men at baseline, E23K genotype was significantly associated with maximal heart rate, maximal respiratory exchange ratio and diastolic blood pressure at rest. Numerous glucose and insulin metabolism and CV function phenotypes changed significantly with exercise training in the total population. The E23K genotype did not significantly influence any of these training-induced changes. Thus, the common E23K genetic variant at the KCNJ11 gene locus was significantly associated with CV function in the untrained state, although the associations appear to differ between men and women. However, this variant has no significant effect on training-induced CV and glucose and insulin metabolism adaptations.

Previous studies have shown that ventilation patterns affect absorption of inhaled compounds. Thus, the aim of this study was to investigate the effect of large tidal volume ventilation (LTVV) on the absorption of inhaled insulin in rabbits. Mechanically ventilated rabbits were given human insulin via a nebuliser system, and plasma insulin was measured for the following 120min. Ventilation was adjusted to (1) normal tidal volume ventilation (NTVV) for the entire period after dosing (NTVV group), to (2) LTVV for the entire period after dosing (LTVV group), to (3) NTVV except for 15min LTVV immediately after dosing (Early LTVV group), or to (4) NTVV except for 15min LTVV starting at 60min after dosing (late LTVV group). Insulin absorption (AUC(ins(0-120min))) was increased by 149% for the LTVV group compared to NTVV group (p<0.01) with increased maximal insulin concentration (106%, p=0.03). The Early LTVV group showed a changed absorption profile. For the late LTVV group an increase in insulin levels was observed after the LTVV period (not significant compared to the NTVV group). These data could potentially have implications for patients using inhaled insulin in situations where a change in breathing pattern is seen, such as exercise.

As a tool to explore proinsulin (PI) trafficking, a human PI cDNA has been constructed with GFP fused within the C peptide. In regulated secretory cells containing appropriate prohormone convertases, the hProCpepGFP construct undergoes endoproteolytic processing to CpepGFP and native human insulin, which are specifically detected and cosecreted in parallel with endogenous insulin. Expression of C(A7)Y mutant PI results in autosomal dominant diabetes in Akita mice. We directly identify the misfolded PI in Akita islets and also show that C(A7)Y mutant PI, either in the context of the hProCpepGFP chimera or not, engages directly in protein complexes with nonmutant PI, impairing the trafficking and recovery of nonmutant PI. This trapping mechanism decreases insulin production in beta cells. Thereafter we observe a loss of beta cell viability. The data imply that PI misfolding leading to impaired endoplasmic reticulum exit of nonmutant PI may be a key early step in a chain reaction of beta cell dysfunction and demise leading to onset and progression of diabetes.

This study examined postprandial plasma insulin and glucose responses after co-ingestion of an insulinotropic protein (Pro) hydrolysate with and without additional free leucine with a single bolus of carbohydrate (Cho). Male patients with long-standing Type 2 diabetes (n = 10) and healthy controls (n = 10) participated in 3 trials in which plasma glucose, insulin, and amino acid responses were determined after the ingestion of beverages of different composition (Cho: 0.7 g/kg carbohydrate, Cho+Pro: 0.7 g/kg carbohydrate with 0.3 g/kg protein hydrolysate, or Cho+Pro+Leu: 0.7 g/kg carbohydrate, 0.3 g/kg protein hydrolysate and 0.1 g/kg free leucine). Plasma insulin responses [expressed as area under the curve (AUC)] were 141 and 204% greater in patients with Type 2 diabetes and 66 and 221% greater in the controls in the Cho+Pro and Cho+Pro+Leu trials, respectively, compared with those in the Cho trial (P 0.05). The concomitant plasma glucose responses were 15 and 12% lower in the patients with Type 2 diabetes and 92 and 97% lower in the control group in the Cho+Pro and Cho+Pro+Leu trials, respectively, compared with those in the Cho trial (P 0.05). Plasma leucine concentrations correlated with the insulin response in all subjects (r = 0.43, P 0.001). We conclude that co-ingestion of a protein hydrolysate with or without additional free leucine strongly augments the insulin response after ingestion of a single bolus of carbohydrate, thereby significantly reducing postprandial blood glucose excursions in patients with long-standing Type 2 diabetes.

OBJECTIVES: Given that adverse effects of chronic sympathetic activation are mediated by all three adrenergic receptor subtypes (beta1, beta2, alpha1), we examined the effects of standard doses of carvedilol and metoprolol succinate (metoprolol controlled release/extended release [CR/XL]) on hemodynamics, myocardial metabolism, and regional organ perfusion. BACKGROUND: Both beta1 selective and combined adrenergic blockade reduce morbidity and mortality in heart failure. Whether there are advantages of one class over the other remains controversial, even in the wake of the Carvedilol Or Metoprolol European Trial (COMET). Similarly, the mechanistic basis for the relative differences is incompletely understood. METHODS: Thirty-three conscious, chronically instrumented dogs with pacing-induced (240 min(-1) for 4 weeks) dilated cardiomyopathy (DCM) were randomized to carvedilol (25 mg twice daily, Coreg, Glaxo Smith Kline, Research Triangle, North Carolina) or metoprolol succinate (100 mg qd, Toprol XL, Astra Zeneca, Wilmington, Delaware). Left ventricular and systemic hemodynamics, myocardial substrate uptake, and norepinephrine spillover were measured before and after three days of treatment. Regional (renal, hepatic, skeletal muscle) blood flows were measured using neutron-activated microspheres. RESULTS: Both agents had comparable heart rate effects. However, carvedilol-treated dogs showed significantly greater increases in stroke volume and cardiac output and decreases in left ventricular end-diastolic pressure and systemic vascular resistance. Carvedilol increased renal, hepatic, and skeletal muscle blood flow. Carvedilol increased myocardial glucose uptake and suppressed norepinephrine and glucagon. Carvedilol antagonized the response to exogenous norepinephrine to a greater extent than metoprolol CR/XL. CONCLUSIONS: At doses inducing comparable heart rate reductions, short-term treatment with carvedilol had superior hemodynamic and metabolic effects compared with metoprolol CR/XL. These data suggest important advantages of blocking all three adrenergic receptor subtypes in DCM.

BACKGROUND: Epidemiologic evidence suggests that serum carotenoids are potent antioxidants and may play a protective role in the development of chronic diseases including cancers, cardiovascular disease, and inflammatory diseases. The role of these antioxidants in the pathogenesis of diabetes mellitus remains unclear. OBJECTIVE: This study examined data from a cross-sectional survey to investigate the association between serum carotenoids and type 2 diabetes. DESIGN: Study participants were adults aged > or = 25 y (n = 1597) from 6 randomly selected cities and towns in Queensland, Australia. Study examinations conducted between October and December 2000 included fasting plasma glucose, an oral-glucose-tolerance test, and measurement of the serum concentrations of 5 carotenoid compounds. RESULTS: Mean 2-h postload plasma glucose and fasting insulin concentrations decreased significantly with increasing quintiles of the 5 serum carotenoids--alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein/zeaxanthin, and lycopene. Geometric mean concentrations for all serum carotenoids decreased (all decreases were significant except that of lycopene) with declining glucose tolerance status. Beta-carotene had the greatest decrease, to geometric means of 0.59, 0.50, and 0.42 micromol/L in persons with normal glucose tolerance, impaired glucose metabolism, and type 2 diabetes, respectively (P 0.01 for linear trend), after control for potential confounders. CONCLUSIONS: Serum carotenoids are inversely associated with type 2 diabetes and impaired glucose metabolism. Randomized trials of diets high in carotenoid-rich vegetables and fruit are needed to confirm these results and those from other observational studies. Such evidence would have very important implications for the prevention of diabetes.