Heidi K. Ortmeyer

Low Urinary chiro-Inositol Excretion in Non-Insulin-Dependent Diabetes Mellitus

BACKGROUND AND METHODS Inositol is a major component of the intracellular mediators of insulin action. To investigate the possible role of altered inositol metabolism in non-insulin-dependent diabetes mellitus (NIDDM), we used gas chromatography and mass spectrometry to measure the myo-inositol and chiro-inositol content of urine specimens from normal subjects and patients with NIDDM: The study subjects were whites, blacks, and Pima Indians. The type of inositol and its concentration in insulin-mediator preparations from muscle-biopsy specimens from normal subjects and diabetic patients were also determined. RESULTS The urinary excretion of chiro-inositol was much lower in the patients with NIDDM (mean [+/- SE], 1.8 +/- 0.8 mumol per day) than in the normal subjects (mean, 84.9 +/- 26.9 mumol per day; P less than 0.01). In contrast, the mean urinary myo-inositol excretion was higher in the diabetic patients than in the normal subjects (444 +/- 135 vs. 176 +/- 46 mumol per day; P less than 0.05). There was no correlation between chiro-inositol excretion and the age, sex, or weight of the diabetic patients, nor was there any correlation between urinary chiro-inositol and myo-inositol excretion in either group. The results were similar in a primate model of NIDDM, and chiro-inositol excretion was decreased to a lesser extent in animals with prediabetic insulin resistance. chiro-Inositol was undetectable in insulin-mediator preparations from muscle-biopsy samples obtained from patients with NIDDM: Similar preparations from normal subjects contained substantial amounts of chiro-inositol. Furthermore, the chiro-inositol content of such preparations increased after the administration of insulin during euglycemic-hyperinsulinemic-clamp studies in normal subjects but not in patients with NIDDM: CONCLUSIONS NIDDM is associated with decreased chiro-inositol excretion and decreased chiro-inositol content in muscle. These abnormalities seem to reflect the presence of insulin resistance in NIDDM:

Exercise with calorie restriction improves insulin sensitivity and glycogen synthase activity in obese postmenopausal women with impaired glucose tolerance.

Our objective was to compare the effects of in vivo insulin on skeletal muscle glycogen synthase (GS) activity in normal (NGT) vs. impaired glucose-tolerant (IGT) obese postmenopausal women and to determine whether an increase in insulin activation of GS is associated with an improvement in insulin sensitivity (M) following calorie restriction (CR) and/or aerobic exercise plus calorie restriction (AEX + CR) in women with NGT and IGT. We did a longitudinal, clinical intervention study of CR compared with AEX + CR. Overweight and obese women, 49-76 yr old, completed 6 mo of CR (n = 46) or AEX + CR (n = 50) with Vo(2 max), body composition, and glucose tolerance testing. Hyperinsulinemic euglycemic (80 mU·m(-2)·min(-1)) clamps (n = 73) and skeletal muscle biopsies (before and during clamp) (n = 58) were performed before and after the interventions (n = 50). After 120 min of hyperinsulinemia during the clamp, GS fractional activity and insulins effect to increase GS fractional activity (insulin - basal) were significantly lower in IGT vs. NGT (P < 0.01) at baseline. GS total activity increased during the clamp in NGT (P < 0.05), but not IGT, at baseline. CR and AEX + CR resulted in a significant 8% weight loss with reductions in total fat mass, visceral fat, subcutaneous fat, and intramuscular fat. Overall, M increased (P < 0.01), and the change in M (postintervention - preintervention) was associated with the change in insulin-stimulated GS fractional activity (partial r = 0.44, P < 0.005). In IGT, the change (postintervention - preintervention) in insulin-stimulated GS total activity was greater following AEX + CR than CR alone (P < 0.05). In IGT, insulin-stimulated GS-independent (P < 0.005) and fractional activity (P = 0.06) increased following AEX + CR. We conclude that the greatest benefits at the whole body and cellular level (insulin activation of GS) in older women at highest risk for diabetes are derived from a lifestyle intervention that includes exercise and diet.

Regulation of obese (ob) mRNA and Plasma Leptin Levels in Rhesus Monkeys EFFECTS OF INSULIN, BODY WEIGHT, AND NON-INSULIN-DEPENDENT DIABETES MELLITUS

We have cloned the rhesus monkey obese cDNA and have analyzed its expression in monkeys with a wide range of body weights (lean to very obese) and with or without non-insulin-dependent diabetes mellitus to examine the relationship of ob gene expression to obesity and non-insulin-dependent diabetes mellitus. The sequence of monkey ob protein, excluding the signal peptide, showed 91% identity with the human protein. We observed a significant correlation between the level of ob mRNA and body weight. We also found a significant relationship between ob mRNA and fasting plasma insulin concentration; however, insulin stimulation during a 100-140-min euglycemic/hyperinsulinemic clamp did not result in any changes in ob mRNA levels. Circulating levels of the ob gene product leptin were also significantly correlated with body weight. These results show that ob gene expression is related to body weight and is not acutely regulated by insulin.

Hyperinsulinemia is associated with altered insulin receptor mRNA splicing in muscle of the spontaneously obese diabetic rhesus monkey.

The human insulin receptor has two isoforms derived from alternative splicing of exon 11 of the insulin receptor gene. The type B (containing exon 11, or exon 11+) isoform binds insulin with twofold lower affinity than the type A (lacking exon 11, or exon 11-) isoform. In efforts to resolve the controversy over whether altered splicing is involved in the development of insulin resistance and non-insulin-dependent diabetes mellitus (NIDDM), the spontaneously obese diabetic rhesus monkey, a unique model that is extraordinarily similar to human NIDDM, was used. Cross-sectional studies of insulin receptor mRNA splicing variants in vastus lateralis muscle were performed on 19 rhesus monkeys. When monkeys were divided into four groups based upon the known stages of progression to NIDDM: normal (normoglycemic/normoinsulinemic), prediabetic (normoglycemic/hyperinsulinemic), early NIDDM (hyperglycemic/hyperinsulinemic), and late NIDDM (hyperglycemic/hypoinsulinemic), both hyperinsulinemic groups had significantly higher percentages of the exon 11- mRNA splicing variant compared to the normal (74.8 +/- 1.7 vs 59.0 +/- 2.3%; P < 0.005) and late NIDDM groups (74.8 +/- 1.7 vs 64.2 +/- 3.9%; P < 0.05). Our findings provide the first direct evidence linking hyperinsulinemia to alterations in insulin receptor mRNA splicing, and suggest that alterations of insulin receptor mRNA splicing in muscle is an early molecular marker that may play an important role in NIDDM.

Relationship of skeletal muscle glucose 6-phosphate to glucose disposal rate and glycogen synthase activity in insulin-resistant and non-insulin-dependent diabetic rhesus monkeys

SummaryReduced insulin action on skeletal muscle glycogen synthase activity and reduced whole-body insulin-mediated glucose disposal rates in insulin-resistant subjects may be associated with an alteration in muscle glucose transport (or phosphorylation) or with a defect distal to glucose 6-phosphate. To examine this issue we determined the glucose 6-phosphate concentration and glycogen synthase activity in muscle samples obtained under basal and euglycaemic hyperinsulinaemic clamp conditions in 27 rhesus monkeys (Macaca mulatta). They ranged from metabolically normal (n = 11) to insulin-resistant (n = 8) to overtly diabetic (non-insulin-dependent) (n = 8). The glucose 6-phosphate measured under insulin-stimulated conditions was inversely correlated to insulin-stimulated glycogen synthase independent activity (r = −0.54, p < 0.005), the change in glycogen synthase independent activity (insulin-stimulated minus basal) (r = −0.58, p < 0.002) and to whole-body insulin-mediated glucose disposal rate (r = −0.60, p < 0.002). The insulin-resistant and diabetic monkeys had significantly higher insulinstimulated glucose 6-phosphate concentrations (0.57 ± 0.11 and 0.62 ± 0.11 nmol/mg dry weight, respectively) compared to the normal monkeys (0.29 ± 0.05 nmol/mg dry weight) (p’s < 0.05). We conclude that under euglycaemic/hyperinsulinaemic conditions, a defect distal to glucose 6-phosphate is a major contributor to reduced whole-body insulin-mediated glucose disposal rates and to reduced insulin action on glycogen synthase in insulin-resistant and diabetic monkeys.

Relationships of PPARγ and PPARγ2 mRNA levels to obesity, diabetes and hyperinsulinaemia in rhesus monkeys

OBJECTIVE: To examine the expression of peroxisome proliferator-activated receptor γ (PPARγ) together with CCAAT/enhancer binding protein α (C/EBPα), lipoprotein lipase (LPL) and glucose transporter (GLUT4) mRNA in adipose tissue of rhesus monkeys in relation to obesity.DESIGN: Cloning of the PPARγ1 and γ2 cDNAs and analysis of PPARγ, C/EBPα, LPL and GLUT4 mRNA levels in the adipose tissue of lean and obese monkeys.SUBJECTS: 28 rhesus monkeys (Macaca mulatta) with a wide range of body weights (9.2–22.6 kg) and with or without type 2 diabetes.MEASUREMENTS: Sequence of PPARγ1 and γ2. Tissue distribution of PPARγ1 and γ2. The mRNA levels of PPARγ, C/EBPα, LPL and GLUT4 in adipose tissue. The ratio of PPARγ2 mRNA to total PPARγ mRNA.RESULTS: The monkey PPARγ2 protein showed 99% identity with the human protein. PPARγ1 mRNA was shown to be expressed in various tissues and most abundantly in adipose tissue. PPARγ2 existed mainly in adipose tissue. A significant correlation between the ratio of PPARγ2 mRNA to total PPARγ mRNA and obesity was observed, whereas total PPARγ mRNA levels showed no significant relationships to obesity. There was also a significant relationship between the ratio of PPARγ2 mRNA to total PPARγ mRNA and fasting plasma insulin concentration. The mRNA levels of C/EBPα, LPL and GLUT4 were highly correlated to that of total PPARγ mRNA. They were also significantly correlated to the mRNA levels of PPARγ1 and PPARγ2.CONCLUSIONS: The ratio of PPARγ2 mRNA to total PPARγ mRNA is related to obesity in the rhesus monkey and mRNA expression of PPARγ1, PPARγ2, C/EBPα, LPL and GLUT4 appear to be coordinated in vivo.

Increased Skeletal Muscle Capillarization Independently Enhances Insulin Sensitivity in Older Adults After Exercise Training and Detraining

Intramuscular signaling and glucose transport mechanisms contribute to improvements in insulin sensitivity after aerobic exercise training. This study tested the hypothesis that increases in skeletal muscle capillary density (CD) also contribute to exercise-induced improvements in whole-body insulin sensitivity (insulin-stimulated glucose uptake per unit plasma insulin [M/I]) independent of other mechanisms. The study design included a 6-month aerobic exercise training period followed by a 2-week detraining period to eliminate short-term effects of exercise on intramuscular signaling and glucose transport. Before and after exercise training and detraining, 12 previously sedentary older (65 ± 3 years) men and women underwent research tests, including hyperinsulinemic-euglycemic clamps and vastus lateralis biopsies. Exercise training increased Vo2max (2.2 ± 0.2 vs. 2.5 ± 0.2 L/min), CD (313 ± 13 vs. 349 ± 18 capillaries/mm2), and M/I (0.041 ± 0.005 vs. 0.051 ± 0.007 μmol/kg fat-free mass/min) (P < 0.05 for all). Exercise training also increased the insulin activation of glycogen synthase by 60%, GLUT4 expression by 16%, and 5′ AMPK-α1 expression by 21%, but these reverted to baseline levels after detraining. Conversely, CD and M/I remained 15% and 18% higher after detraining, respectively (P < 0.05), and the changes in M/I (detraining minus baseline) correlated directly with changes in CD in regression analysis (partial r = 0.70; P = 0.02). These results suggest that an increase in CD is one mechanism contributing to sustained improvements in glucose metabolism after aerobic exercise training.

Adipose tissue glycogen synthase activation by in vivo insulin in spontaneously insulin-resistant and Type 2 (non-insulin-dependent) diabetic rhesus monkeys

SummaryIn skeletal muscle, a defect in the covalent activation of glycogen synthase by insulin has been identified in insulin resistance and in Type 2 (non-insulin-dependent) diabetes mellitus, but a similar defect in insulin action at the adipose tissue has not been demonstrated. We sought to determine whether this defect in insulin action in muscle was also present in the same pathway in adipose tissue. We examined the effect of in vivo insulin on adipose tissue glycogen synthase and phosphorylase activity in normal (n=11), hyperinsulinaemic (n=8), and impaired glucose tolerant and Type 2 diabetic (n=8) rhesus monkeys. Adipose tissue samples were obtained before and during a euglycaemic hyperinsulinaemic clamp. Glycogen synthase fractional velocity, independent and total activities were significantly higher in the insulin-stimulated samples compared to the basal samples in the normal group (p<0.05, respectively). In the hyperinsulinaemic group, however, insulin had no effect on glycogen synthase fractional velocity or independent activity, but did increase the total activity of glycogen synthase and phosphorylase (p<0.05, respectively). Furthermore, both the basal and the insulin-stimulated total activities of these two enzymes were significantly greater in the hyperinsulinaemic group as compared to both the normal and the diabetic groups (p<0.05, respectively). In the diabetic group, insulin was without effect on glycogen synthase fractional velocity, independent activity or total activity. We conclude that the covalent activation of adipose tissue glycogen synthase by insulin is absent in both obese hyperinsulinaemic and in spontaneously diabetic monkeys.

In vivo D-chiroinositol activates skeletal muscle glycogen synthase and inactivates glycogen phosphorylase in rhesus monkeys

Chiroinositol, a component of an inositol phosphoglycan, has been found to be reduced in the urine of humans and monkeys with non-insulin-dependent diabetes mellitus. To determine whether in vivo repletion of this inositol could improve the action of insulin on skeletal muscle glycogen synthase and glycogen phosphorylase, D-chiroinositol was administered intravenously to six monkeys during a euglycemic hyperinsulinemic clamp. Enzyme activities were determined in vastus lateralis which was freeze-clamped in situ at three time periods: immediately prior to initiation of the clamp, after steady-state glucose and insulin concentrations had been reached and the glucose disposal rate determined, and 30 min after the administration of D-chiroinositol which was superimposed on the steady-state clamp condition. D-chiroinositol increased the activity (independent and fractional velocity) of glycogen synthase ( P P

Aerobic exercise + weight loss decreases skeletal muscle myostatin expression and improves insulin sensitivity in older adults

To determine whether aerobic exercise training + weight loss (AEX + WL) would affect the expression of myostatin and its relationship with insulin sensitivity in a longitudinal, clinical intervention study.