Douglas N. Ishii

Implication of insulin-like growth factors in the pathogenesis of diabetic neuropathy

Neuropathy can be a highly debilitating complication for about 10-15% of diabetic individuals. Unfortunately, the complex syndrome has proven difficult to explain and a consensus as to its cause has not emerged. It has recently come to light that insulin and insulin-like growth factors (IGFs) have neurotrophic actions on sensory, sympathetic and motor neurons. These are the main types of neurons afflicted in this disorder. Moreover, IGF activity is reduced in both clinical and experimental diabetes. The premise that insulin, IGF-I and IGF-II provide redundant neurotrophic support underlies the following new theory for pathogenesis of diabetic neural disturbances: a loss of insulin activity leads to a secondary partial decline in IGF-I activity. Although most of the redundant neurotrophic support is thereby eliminated, IGF-II activity continues to support the nervous system. The final enemy is time and the relentless age- and duration-dependent run-down of IGF activity is suggested to contribute to the age- and duration-dependent neuropathy. Weight loss or anorexia nervosa are independent risk factors that can cause a rapid, painful neuropathy to develop as a result of a rapid loss of IGF activity. A distinguishing feature of this new theory is that hyperglycemia is not considered to be the main culprit. The following critical predictions from the theory were tested in diabetic rats: (i) IGF activity is reduced in diabetic neural tissues; (ii) conduction velocity is impaired in the diabetic spinal cord; (iii) replacement therapy with IGF can prevent neuropathy in diabetic nerves; and (iv) IGFs can prevent diabetic neuropathy, despite hyperglycemia. All of these predictions have been validated. It is hoped that a fresh perspective will stimulate renewed study into the causation of this most unfortunate disorder.

Role of insulin-like growth factors in peripheral nerve regeneration

Prolonged denervation results in atrophy of target organs and increased risk of permanent paralysis. A better understanding of the mechanism responsible for nerve regeneration may one day lead to improved rates of nerve regeneration and diminished risk of loss of function. Neurobiologists have known for decades that soluble neurotrophic activity is present in nerves and nerve targets. Until recently, the soluble molecules that regulate the rate of nerve regeneration have eluded identification. Insulin-like growth factor (IGF) gene expression is correlated with synapse formation during development and regeneration. IGFs are now identified as the first soluble nerve- and muscle-derived neurotrophic factors found to regulate the rate of peripheral nerve regeneration. The roles of IGFs and other neurotrophic factors in peripheral nerve regeneration, motor nerve terminal sprouting and synapse formation are reviewed.

Systemic insulin-like growth factor-I administration prevents cognitive impairment in diabetic rats, and brain IGF regulates learning/memory in normal adult rats

Diabetic patients have impaired learning/memory, brain atrophy, and two‐fold increased risk of dementia. The cause of cognitive disturbances that progress to dementia is unknown. Because neurotrophic insulin‐like growth factor (IGF) levels are reduced in diabetic patients and rodents, and IGF can cross the blood‐central nervous system barrier (B‐CNS‐B), the hypothesis was tested that IGF administered systemically can prevent cognitive disturbances, independently of hyperglycemia and a generalized catabolic state. Latency to escape to a hidden platform in the Morris Water Maze is used widely to test spatial memory, a hippocampus‐dependent task. Adult rats were rendered diabetic with streptozotocin and implanted 4 weeks later with subcutaneous pumps that released either vehicle (D + Veh) or 20 μg/day IGF‐I (D + IGF). Latency to escape to the hidden platform was prolonged in (D + Veh) versus non‐diabetic rats (P < 0.003) 10.5 weeks after the onset of diabetes. Such prolongation was prevented in (D + IGF) versus (D + Veh) rats (P < 0.03). The data show that IGF‐I can act across the B‐CNS‐B to prevent loss of cognition‐related performance in the water maze independently of ongoing hyperglycemia and reduction in brain (P < 0.001) and whole body weight (P < 0.001) in diabetic rats. The hypothesis that brain IGF contributes to learning/memory was tested. An anti‐IGF antibody, or preimmune serum, was infused into the lateral ventricles in non‐diabetic rats. Learning in a passive avoidance task was impaired significantly in the IGF antibody versus preimmune serum‐treated groups on test Days 1, 2, and 3 (P = 0.04, 0.02 and 0.004, respectively). The data together are consistent with a model in which brain IGF is essential for learning/memory, and a loss of IGF activity due to diabetes may contribute to cognitive disturbances.

Insulin-like growth factors reverse or arrest diabetic neuropathy: effects on hyperalgesia and impaired nerve regeneration in rats.

Diabetic neuropathy is a debilitating disorder whose causation is poorly understood. A new theory proposes that neuropathy may arise as a consequence of loss of neurotrophic insulin-like growth factor (IGF) activity due to diabetes, superimposed on a slow continual loss due to aging. The prediction that IGF-I and IGF-II gene expression are reduced in diabetic nerves was recently tested and validated. Here we tested the prediction that IGF administration can prevent or reverse diabetic sensory neuropathy. Subcutaneous infusion of IGF-I or IGF-II, but not vehicle, halted (P < 0.01) the progression of hyperalgesia in streptozotocin-diabetic rats. Moreover, impaired sensory nerve regeneration was partially reversed within 2 weeks after treatment of diabetic rats with IGFs (P < 0.01). Impaired regeneration could also be prevented by daily subcutaneous IGF injections. The low replacement doses of IGFs were effective despite unabated hyperglycemia and weight loss. These results show that IGF replacement therapy can reverse or prevent diabetic sensory neuropathy independently of hyperglycemia or weight loss.

Insulin-like growth factor II increases the rate of sciatic nerve regeneration in rats

A slow rate of nerve regeneration conspires together with atrophy and degeneration of denervated organs to increase the risk of permanent disability following injury to the mammalian peripheral nervous system. Therefore, it is of both practical and theoretical interest to identify those endogenous factors that determine the spontaneous velocity of nerve regeneration, and to discover exogenous factors which hold promise for augmenting the rate. We report that locally infused insulin-like growth factor II significantly increases the speed of sensory axon regeneration in rat sciatic nerves. It appeared that 1 microgram/ml insulin-like growth factor II acted through insulin-like growth factor receptors, because a comparable concentration of insulin had little effect. Furthermore, there was a sustained reduction in regeneration rate when an anti-insulin-like growth factor II antiserum was continuously infused near a window in the epineurium located just below a site of nerve crush, indicating that the spontaneous regeneration rate was continuously dependent on endogenous insulin-like growth factor activity. These results show that exogenously administered insulin-like growth factor II can increase the rate of peripheral nerve regeneration, and that the endogenous insulin-like growth factors in nerves are required to maintain the normal rate of regeneration. These in vivo data complement previous observations showing that insulin-like growth factors can increase neurite outgrowth in cultured neurons, and that insulin-like growth factor II gene expression is correlated with synapse development. They further support the hypothesis that insulin-like growth factors play a role in nerve regeneration.

Stabilization of tubulin mRNAs by insulin and insulin-like growth factor I during neurite formation.

Neurotrophic factors may increase axon and dendrite growth in part by regulating the content of cytoskeletal elements such as microtubules, which are comprised of tubulin subunits. The mechanism by which insulin, insulin-like growth factors (IGFs), and nerve growth factor (NGF) can increase the relative abundance of tubulin mRNAs as a prelude to neurite formation was studied. Insulin significantly increased the abundance of tubulin mRNAs relative to total RNA in cultured human neuroblastoma SH-SY5Y cells. This increase was not the result of a generalized elevation of all transcripts, because tubulin mRNAs were elevated relative to poly(A)+ RNA as well. Moreover, whereas polymerases I and III were elevated in activity, polymerase II was not. Tubulin mRNAs were stabilized against degradation in the presence of actinomycin D by both insulin and IGF-I. In contrast, actin and histone 3.3 mRNAs were neither increased nor stabilized. Insulin did not alter alpha- or beta-tubulin gene transcription rates in nuclear run-off experiments, and did increase the relative synthesis of tubulin proteins. These results suggest that tubulin mRNA levels are increased mainly through selective stabilization by insulin and IGFs. Because NGF is known to stabilize tubulin mRNA levels also, stabilization of tubulin mRNAs is suggested to be a common event in the pathway leading to neurite elongation directed by neuritogenic polypeptides.

Effects of insulin and insulin-like growth factors on neurofilament mRNA and tubulin mRNA content in human neuroblastoma SH-SY5Y cells

Insulin-like growth factors (IGFs) are implicated in the development of the vertebrate neural circuitry, and increase neurite growth in vitro and in vivo. The construction of the cytoskeleton is necessary for growth of axons and dendrites, and the neurofilament (NF) 68 kDa and 170 kDa proteins assemble to help form major fibrillar elements of the neurite cytoskeleton. We report that physiological concentrations of insulin, IGF-I or IGF-II increased the contents of 68 kDa NF, 170 kDa NF, alpha-tubulin, and beta-tubulin mRNAs, relative to total RNA, in cultured human neuroblastoma SH-SY5Y cells. In contrast, the relative contents of histone 3.3 mRNA, and poly(A)+ RNA were not increased. Ligand concentrations which increased NF mRNAs were very similar to those which increased neurite outgrowth. Although each gene was evidently independently regulated, the 68 kDa NF, 170 kDa NF, alpha-tubulin, and beta-tubulin mRNAs were nevertheless all transiently elevated over approximately the same time interval in response to insulin. These data, when considered together with studies by others with nerve growth factor, show that the 68 kDa and 170 kDa NF mRNAs are elevated in a biochemical pathway activated in common during neurite outgrowth directed by insulin, IGF-I, IGF-II, and nerve growth factor.

Uptake of circulating insulin-like growth factor-I into the cerebrospinal fluid of normal and diabetic rats and normalization of IGF-II mRNA content in diabetic rat brain.

Brain injury has been prevented recently by systemic administration of human insulin‐like growth factor‐I (hIGF‐I). It is widely believed that protein neurotrophic factors do not enter the brain from blood, and the mechanism by which circulating hIGF‐I may be neuroprotective is uncertain. This investigation tested the hypothesis that hIGF‐I is taken up into cerebrospinal fluid (CSF) from the circulation. 125I‐hIGF‐I was injected subcutaneously into rats. The 125I‐IGF‐I recovered from CSF and plasma were indistinguishable in size from authentic 125I‐hIGF‐I on SDS‐PAGE. An ELISA was used that detected immunoreactive hIGF‐I, but not rat IGF‐I, rat IGF‐II, human IGF‐II, or insulin. Osmotic minipumps were implanted for constant subcutaneous infusion of various hIGF‐I doses. Uptake into CSF reached a plateau at plasma concentrations above approximately 150 ng/ml hIGF‐I; the plateau was consistent with carrier‐mediated uptake. The plasma, but not CSF, hIGF‐I level was significantly reduced in streptozotocin diabetic vs. nondiabetic rats, and uptake of hIGF‐I into CSF was nonlinear with respect to plasma hIGF‐I concentrations. Nonlinear uptake excluded leakage or transmembrane diffusion of IGF‐I from blood into CSF as a dominant route for entry, but the site and mechanism of uptake remain to be established. The IGF‐II mRNA content per milligram brain (P < 0.02) as well as per poly(A)+ RNA (P < 0.05) was significantly increased towards normal in diabetic rats treated by subcutaneous administration of hIGF‐I vs. vehicle. This effect of circulating hIGF‐I may have been due to regulation of IGF‐II gene expression in the choroid plexus and leptomeninges, structures at least in part outside of the blood–central nervous system barrier. These data support the hypothesis that circulating IGF‐I supports the brain indirectly through regulation of IGF‐II gene expression as well as by uptake into the CSF. J. Neurosci. Res. 59:649–660, 2000

Elevated insulin-like growth factor (IGF) gene expression in sciatic nerves during IGF-supported nerve regeneration

Nerve regeneration is augmented by neurotrophic activity, which has long been known to be increased in lesioned nerves. Of identified soluble nerve-derived neurotrophic factors, to date only insulin-like growth factors (IGFs) have been observed to increase the rate of axon regeneration in peripheral nerves. We report that IGF-I and IGF-II mRNA contents were significantly increased (P < 0.0005) distal to the site of crush in rat sciatic nerves, and decreased following axon regeneration. In transected nerves in which axon regeneration was prevented, IGF mRNAs remained elevated. IGF-I mRNAs per mg tissue were increased more in lesioned nerves than denervated muscles, whereas IGF-II mRNAs were increased more in denervated muscles than lesioned nerves. This suggested that IGF-I and IGF-II each play distinct regulatory roles during regeneration. These data bolster the hypothesis that increased IGF mRNA content in nerves supports the rate of nerve regeneration in mammals.

Uptake of Circulating Insulin-Like Growth Factors (IGFs) into Cerebrospinal Fluid Appears to Be Independent of the IGF Receptors as Well as IGF-Binding Proteins1

Peripheral administration of human insulin-like growth factor (hIGF) results in both uptake of hIGF into the cerebrospinal fluid (CSF) and amelioration of brain injury. We tested the hypotheses that IGF uptake into CSF is independent of IGF receptors and IGF-binding proteins (IGFBP). Adult rats were injected sc with various concentrations of hIGF-I or structural analogs, and serum and CSF were withdrawn for assay 90 min later. An enzyme-linked immunoassay was used that detected immunoreactive hIGF-I and its analogs, but not rat IGF-I, IGF-II, or insulin. Plasma hIGF-I levels increased linearly (r = 0.97) with hIGF-I dose between 25-300 microgram/rat. By contrast, uptake into CSF reached saturation above 100 microgram, suggesting carrier-mediated uptake. hIGF-II reduced the uptake of hIGF-I into CSF (P < 0.02). Des(1-3)hIGF-I is a hIGF-I analog missing the N-terminal tripeptide, resulting in greatly reduced affinity for IGFBP-1, -3, -4, and -5. Nevertheless, des(1-3)hIGF-I was taken up into CSF. [Leu(24)]hIGF-I and [Leu(60)]hIGF-I have 20- to 85-fold reduced affinity for the type I IGF receptor, yet both were taken up into CSF in amounts similar to hIGF-I. In addition, hIGF-I and des(1-3)hIGF-I were taken up into CSF, although binding to the type II receptor is extremely weak. These data suggest that uptake of circulating IGF-I into CSF is independent of the type I or II IGF receptors as well as IGF sequestration to IGFBP-1, -3, -4, or -5.