Citations

In vivo studies have reported conflicting effects of insulin on mixed tissue protein synthesis rates. To test the hypothesis that insulin has differential effects on synthesis rates of various protein fractions in different organs, we infused miniature swine (n = 8 per group) with saline, insulin alone (at 0.7 mU/kg−1 · min−1), or insulin plus an amino acid mixture for 8 h. Fractional synthesis rate (FSR) of mitochondrial and cytoplasmic proteins in liver, heart, and skeletal muscle, as well as myosin heavy chain (MHC) in muscle, were measured using l-[1-13C]leucine as a tracer. The FSR of mitochondrial and cytoplasmic proteins were highest in liver, followed by heart and then muscle. Mitochondrial FSR in muscle was higher during insulin and insulin plus amino acid infusions than during saline. Insulin had no significant effect on FSR of MHC in muscle. In contrast, FSR of both mitochondrial and cytoplasmic proteins were not stimulated by insulin in liver. Insulin also did not increase FSR of mitochondrial in heart, whereas insulin and amino acid stimulated FSR of cytoplasmic protein. In conclusion, insulin stimulates the synthesis of muscle mitochondrial proteins, with no significant stimulatory effect on synthesis of sarcoplasmic and MHC. These results demonstrate that insulin has different effects on synthesis rates of specific protein fractions in the liver, heart, and skeletal muscle.

The metabolic response to fasting involves a series of hormonal and metabolic adaptations leading to protein conservation. An increase in the serum level of growth hormone (GH) during fasting has been well substantiated. The present study was designed to test the hypothesis that GH may be a principal mediator of protein conservation during fasting and to assess the underlying mechanisms. Eight normal subjects were examined on four occasions: 1) in the basal postabsorptive state (basal), 2) after 40 h of fasting (fast), 3) after 40 h of fasting with somatostatin suppression of GH (fast-GH), and 4) after 40 h of fasting with suppression of GH and exogenous GH replacement (fast+GH). The two somatostatin experiments were identical in terms of hormone replacement (except for GH), meaning that somatostatin, insulin, glucagon and GH were administered for 28 h; during the last 4 h, substrate metabolism was investigated. Compared with the GH administration protocol, IGF-I and free IGF-I decreased 35 and 70%, respectively, during fasting without GH. Urinary urea excretion and serum urea increased when participants fasted without GH (urea excretion: basal 392 ± 44, fast 440 ± 32, fast-GH 609 ± 76, and fast+GH 408 ± 36 mmol/24 h, P < 0.05; serum urea: basal 4.6 ± 0.1, fast 6.2 ± 0.1, fast-GH 7.0 ± 0.2, and fast+GH 4.3 ± 0.2 mmol/l, P < 0.01). There was a net release of phenylalanine across the forearm, and the negative phenylalanine balance was higher during fasting with GH suppression (balance: basal 9 ± 3, fast 15 ± 6, fast-GH 17 ± 4, and fast+GH 11 ± 5 nmol/min, P < 0.05). Muscle-protein breakdown was increased among participants who fasted without GH (phenylalanine rate of appearance: basal 17 ± 4, fast 26 ± 9, fast-GH 33 ± 7, fast+GH 25 ± 6 nmol/min, P < 0.05). Levels of free fatty acids and oxidation of lipid decreased during fasting without GH (P < 0.01). In summary, we find that suppression of GH during fasting leads to a 50% increase in urea-nitrogen excretion, together with an increased net release and appearance rate of phenylalanine across the forearm. These results demonstrate that GH—possibly by maintenance of circulating concentrations of free IGF-I—is a decisive component of protein conservation during fasting and provide evidence that the underlying mechanism involves a decrease in muscle protein breakdown.

Neuron activity and insulin release were measured simultaneously from 33 preparations of intrapancreatic canine ganglia and pancreatic parenchyma adjacent to the ganglia. The electrical activity of single neurons of the ganglia was recorded with intracellular microelectrodes, and insulin release from the attached islets was determined with an enzyme-linked immunosorbent assay. Insulin release was 62 ± 18 fmol preparation/min in the presence of 10 mmol/l glucose and pulsatile (3.7 ± 0.4 min/pulse). Corresponding measurements of neuronal electrical activity showed a stable membrane potential of -53.5 ± 0.6 mV. Short, high-frequency (20 Hz) preganglionic nerve stimulation evoked action potentials and, in 46% of the preparations, a threefold rise in the insulin secretory rate associated with increased amplitude of the insulin pulses. The effects were blocked by 10 μmol/l tetrodotoxin (TTX). In other preparations, continuous low-frequency (0.05-0.5 Hz) preganglionic nerve stimulation evoked action potentials and, in 50% of the preparations, a gradual increase of insulin release associated with augmentation of insulin pulse amplitude without alteration of the duration. The effects were blocked by 50 μmol/l hexamethonium (HEX). In the remaining preparations, no change in insulin release was observed during nerve stimulation. In the absence of stimulation, neither TTX nor HEX affected the membrane potential or insulin secretion. These first simultaneous measurements of intrapancreatic ganglion activity and insulin secretion are consistent with amplitude modulation of pulsatile insulin secretion induced by changes in electrical activity in a population of intrapancreatic ganglion neurons.

Although it is well established that glucagon-like peptide 1(7-36) amide (GLP-1) is a potent stimulator of insulin secretion, its effects on insulin action and glucose effectiveness are less clear. To determine whether GLP-1 increases insulin action and glucose effectiveness, subjects with type 2 diabetes were studied on two occasions. Insulin was infused during the night on both occasions to ensure that baseline glucose concentrations were comparable. On the morning of study, either GLP-1 (1.2 pmol x kg(-1) x min(-1)) or saline were infused along with somatostatin and replacement amounts of glucagon. Glucose also was infused in a pattern mimicking that typically observed after a carbohydrate meal. Insulin concentrations were either kept constant at basal levels (n = 6) or varied so as to create a prandial insulin profile (n = 6). The increase in glucose concentration was virtually identical on the GLP-1 and saline study days during both the basal (1.21 +/- 0.15 vs. 1.32 +/- 0.19 mol/l per 6 h) and prandial (0.56 +/- 0.14 vs. 0.56 +/- 0.10 mol/l per 6 h) insulin infusions. During both the basal and prandial insulin infusions, glucose disappearance promptly increased after initiation of the glucose infusion to rates that did not differ on the GLP-1 and saline study days. Suppression of endogenous glucose production also was comparable on the GLP-1 and saline study days during both the basal (-2.7 +/- 0.3 vs. -3.1 +/- 0.2 micromol/kg) and prandial (-3.1 +/- 0.4 vs. -3.0 +/- 0.6 pmol/kg) insulin infusions. We conclude that when insulin and glucagon concentrations are matched, GLP-1 has negligible effects on either insulin action or glucose effectiveness in people with type 2 diabetes. These data strongly support the concept that GLP-1 improves glycemic control in people with type 2 diabetes by increasing insulin secretion, by inhibiting glucagon secretion, and by delaying gastric emptying rather than by altering extrapancreatic glucose metabolism.

Specific HLA DQ and DR alleles have been associated with susceptibility to type 1 diabetes. HLA DQ8 and DQ2 have been shown to strongly predispose to disease and to be in linkage disequilibrium with at-risk DR4 and DR3 alleles, respectively. Inheritance of a mixed DR3/DR4 haplotype confers the greatest risk. A double transgenic mouse expressing both DR3 and DQ8 was generated to investigate potential major histocompatibility complex class II interactions. The DR3/DQ8 transgenic mice developed a spontaneous loss of tolerance to GAD65, in which the T-cell response to GAD65 was restricted by HLA DR. Although the mice also showed spontaneous insulitis, they did not progress to overt diabetes. Mice expressing either transgene (DQ8 or DR3) alone showed mild infiltration of their islets, which disappeared when DQ8 or DR3 was co-expressed with a resistant DR2 allele or the neutral DQ6 allele. Therefore, in a fashion analogous to human diabetes, the murine model demonstrated a requirement for a combination of at-risk DR and DQ allotypes for the initiation of spontaneous autoimmunity.

The peripheral nerve of experimental diabetic neuropathy (EDN) is reported to be ischemic and hypoxic, with an increased dependence on anaerobic metabolism, requiring increased energy substrate stores. When glucose stores become reduced, fiber degeneration has been reported. We evaluated glucose uptake, nerve energy metabolism, the polyol pathway, and protein kinase C (PKC) activity in EDN induced by streptozotocin. Control and diabetic rats received lipoic acid (0, 10, 25, 50, 100 mg/kg). Duration of diabetes was 1 month, and alpha-lipoic acid was administered intraperitoneally 5 times per week for the final week of the experiment. Nerve glucose uptake was reduced to 60, s 37, and 30% of control values in the sciatic nerve, L5 dorsal root ganglion, and superior cervical ganglion (SCG), respectively, in rats with EDN. Alpha-lipoic acid supplementation had no effect on glucose uptake in normal nerves at any dose, but reversed the deficit in EDN, with a threshold between 10 and 25 mg/kg. Endoneurial glucose, fructose, sorbitol, and myo-inositol were measured in sciatic nerve. Alpha-lipoic acid had no significant effect on either energy metabolism or polyol pathway of normal nerves. In EDN, endoneurial glucose, fructose, and sorbitol were significantly increased, while myo-inositol was significantly reduced. Alpha-lipoic acid had a biphasic effect: it dose-dependently increased fructose, glucose, and sorbitol, peaking at 25 mg/kg, and then fell beyond that dose, and it dose-dependently increased myo-inositol. Sciatic nerve cytosolic PKC was increased in EDN. ATP, creatine phosphate, and lactate were measured in sciatic nerve and SCG. Alpha-lipoic acid prevented the reduction in SCG creatine phosphate. We conclude that glucose uptake is reduced in EDN and that this deficit is dose-dependently reversed by alpha-lipoic acid, a change associated with an improvement in peripheral nerve function.

Studies were conducted to determine whether regional free fatty acid (FFA) release is differentially regulated by insulin. Systemic, leg, and splanchnic palmitate rate of appearance ([9,10-(3)H]palmitate) was measured in 26 healthy adults using the euglycemic-hyperinsulinemic clamp technique to achieve a physiological range of plasma insulin concentrations. We found that insulin inhibited systemic, leg, and splanchnic palmitate release in a dose-dependent fashion over the range of insulin infused (0-1.0 mU x kg(-1) x min(-1)). Progressive hyperinsulinemia changed the leg from a net producer to a net FFA consumer, whereas the splanchnic bed converted from a net FFA consumer to a net producer. At the 0.5 mU x kg(-1) x min(-1) insulin infusion rate, leg FFA release was almost completely suppressed, whereas even with the 1.0 mU x kg(-1) x min(-1) insulin infusion rate, splanchnic FFA release decreased by only approximately 65% (P < 0.05 leg vs. splanchnic). These results demonstrate the regional heterogeneity of insulin-regulated FFA release in vivo, and indicate that visceral adipose tissue lipolysis is more resistant to insulin suppression than is leg lipolysis in humans.

To define the mechanism of insulin's anticatabolic action, the effects of three different dosages of insulin (0.25, 0.5, and 1.0 mU x kg(-1) x min(-1)) versus saline on protein dynamics across splanchnic and skeletal muscle (leg) beds were determined using stable isotopes of phenylalanine, tyrosine, and leucine in 24 healthy subjects. After an overnight fast, protein breakdown in muscle exceeded protein synthesis, causing a net release of amino acids from muscle bed, while in the splanchnic bed protein synthesis exceeded protein breakdown, resulting in a net uptake of these amino acids. Insulin decreased (P < 0.003) muscle protein breakdown in a dose-dependent manner with no effect on muscle protein synthesis, thus decreasing the net amino acid release from the muscle bed. In contrast, insulin decreased protein synthesis (P < 0.03) in the splanchnic region with no effect on protein breakdown, thereby decreasing the net uptake of the amino acids. In addition, insulin also decreased (P < 0.001) leucine nitrogen flux substantially more than leucine carbon flux, indicating increased leucine transamination (an important biochemical process for nitrogen transfer between amino acids and across the organs), in a dose-dependent manner, with the magnitude of effect being greater on skeletal muscle than on the splanchnic bed. In conclusion, muscle is in a catabolic state in human subjects after an overnight fast and provides amino acids for synthesis of essential proteins in the splanchnic bed. Insulin achieves amino acid balance across splanchnic and skeletal muscle beds through its differential effects on protein dynamics in these tissue beds.

The catabolic state of poorly controlled type 1 diabetes has largely been attributed to insulin deficiency. However, the role of hyperglucagonemia, which occurs concomitantly with insulin deficiency, has not been fully investigated. We studied the effects of hyperglucagonemia during insulin deprivation on energy expenditure (using indirect calorimetry) and protein metabolism (using L-[1-(13)C,15N]leucine and L-[1-(13)C]leucine as tracers) in 12 type 1 diabetic subjects. Five protocols were used: insulin treatment, insulin deprivation, insulin deprivation with suppression of endogenous glucagon with somatostatin (SRIH) and growth hormone replacement, insulin deprivation with endogenous glucagon suppression with SRIH (no growth hormone replacement), and insulin deprivation with SRIH and a high level of glucagon replacement (no growth hormone replacement). It was observed that leucine oxidation and the resting metabolic rate (RMR) were significantly lower during insulin treatment and insulin deprivation with concomitant SRIH infusion (lowering glucagon) than during insulin deprivation alone. Replacement of glucagon at a high level during SRIH infusion in the insulin-deprived state increased leucine oxidation and the RMR. Hyperglucagonemia was also associated with a trend for decreased protein synthesis. Hyperglucagonemia did not affect leucine transamination. Insulin replacement decreased leucine flux and oxidation. Leucine oxidation (R2 = 0.79) and the RMR (R2 = 0.81) were seen, by multiple regression analysis, to correlate with glucagon levels and not with other hormones. We conclude that while insulin deficiency increases protein breakdown, hyperglucagonemia is primarily responsible for the increased leucine oxidation and RMR seen during insulin deprivation.

Highly informative yet simple protocols to assess insulin secretion and action would considerably enhance the quality of epidemiological and large-scale clinical trials. In an effort to develop such protocols, a 5-h, 11-sample oral glucose tolerance test (OGTT) was performed in 100 individuals and a 7-h, 21-sample meal in another 100. Plasma glucose, insulin, and C-peptide concentrations were measured. We show that virtually the same minimal model assessment of β-cell responsivity (dynamic [Φd] and static [Φs]), insulin sensitivity (Si), and disposition index (DI) can be obtained with a reduced seven-sample 2-h protocol: Φd, reduced versus full: 871.50 vs. 873.32, r = 0.98 in OGTT and 494.88 vs. 477.99 10−9, r = 0.91 in meal; Φs: 42.36 vs. 44.35, r = 0.88 in OGTT and 35.31 vs. 35.37 10−9 min−1, r = 0.90 in meal; Si: 24.33 vs. 22.77 10−5 dl · kg−1 · min−1 per pmol/l, r = 0.89 in OGTT and 19.03 vs. 19.77 10−5 dl · kg−1 · min−1 per pmol/l, r = 0.85 in meal; and DI: 1,282.26 vs. 1,273.23, r = 0.84 in OGTT and 726.92 vs. 776.97 10−14 dl · kg−1 · min−2 per pmol/l, r = 0.84 in meal. This reduced protocol will facilitate the study of insulin secretion and action under physiological conditions in nondiabetic humans.