Investigation of liver-targeted peripheral focused ultrasound stimulation (pFUS) and its effect on glucose homeostasis and insulin resistance in type 2 diabetes mellitus: a proof of concept, phase 1 trial.

BACKGROUND: Mechanical waves produced by ultrasound pulses have been shown to activate mechanosensitive ion channels and modulate peripheral nerves. However, while peripheral ultrasound neuromodulation has been demonstrated in vitro and in pre-clinical models, there have been few reports of clinical tests. AIM: We modified a diagnostic imaging system for ultrasound neuromodulation in human subjects. We report the first safety and feasibility outcomes in subjects with type 2 diabetes (T2D) mellitus and discuss these outcomes in relation to previous pre-clinical results. DESIGN: The study was performed as an open label feasibility study to assess the effects of hepatic ultrasound (targeted to the porta hepatis) on glucometabolic parameters in subjects with T2D. Stimulation (peripheral focused ultrasound stimulation treatment) was performed for 3 days (i.e. 15 min per day), preceded by a baseline examination and followed by a 2-week observation period. METHODS: Multiple metabolic assays were employed including measures of fasting glucose and insulin, insulin resistance and glucose metabolism. The safety and tolerability were also assessed by monitoring adverse events, changes in vital signs, electrocardiogram parameters and clinical laboratory measures. RESULTS AND CONCLUSION: We report post-pFUS trends in several outcomes that were consistent with previous pre-clinical findings. Fasting insulin was lowered, resulting in a reduction of HOMA-IR scores (P-value 0.01; corrected Wilcoxon signed-rank test). Additional safety and exploratory markers demonstrated no device-related adverse impact of pFUS. Our findings demonstrate that pFUS represents a promising new treatment modality that could be used as a non-pharmaceutical adjunct or even alternative to current drug treatments in diabetes.

ZAP-70 protein promotes tyrosine phosphorylation of T cell receptor signaling motifs (ITAMs) in immature CD4(+)8(+) thymocytes with limiting p56(lck).

As a result of interaction with epithelial cells in the thymic cortex, immature CD4(+)8(+) (double positive, DP) thymocytes express relatively few T cell receptors (TCRs) and contain diminished numbers of coreceptor-associated p56(lck) (lck) PTK molecules. As a result, TCR signal transduction in DP thymocytes is significantly impaired, despite its importance for repertoire selection. We report here that, in DP thymocytes, tyrosine phosphorylation of TCR signaling motifs (ITAMs) by lck, an early event in TCR signal transduction, is dependent upon ZAP-70 protein independent of ZAP-70's kinase activity. Furthermore, the dependence on ZAP-70 protein for ITAM phosphorylation diminishes as available lck increases. Importantly, ZAP-70's role in ITAM phosphorylation in DP thymocytes is not limited to protecting phosphorylated ITAMs from dephosphorylation. Rather, this study indicates that ZAP-70 protein augments ITAM phosphorylation in DP thymocytes and so compensates in part for the relative deficiency of coreceptor-associated lck.

Association of the adaptor molecule LAT with CD4 and CD8 coreceptors identifies a new coreceptor function in T cell receptor signal transduction.

Linker for activation of T cells (LAT) is an adaptor protein whose tyrosine phosphorylation is critical for transduction of the T cell receptor (TCR) signal. LAT phosphorylation is accomplished by the protein tyrosine kinase ZAP-70, but it is not at all clear how LAT (which is not associated with the TCR) encounters ZAP-70 (which is bound to the TCR). Here we show that LAT associates with surface CD4 and CD8 coreceptors and that its association is promoted by the same coreceptor cysteine motif that mediates Lck binding. In fact, LAT competes with Lck for binding to individual coreceptor molecules but differs from Lck in its preferential association with CD8 rather than CD4 in CD4(+)CD8(+) thymocytes. Importantly, as a consequence of LAT association with surface coreceptors, coengagement of the TCR with surface coreceptors induces LAT phosphorylation and the specific recruitment of downstream signaling mediators to coreceptor-associated LAT molecules. These results point to a new function for CD4 and CD8 coreceptors in TCR signal transduction, namely to promote LAT phosphorylation by ZAP-70 by recruiting LAT to major histocompatibility complex-engaged TCR complexes.

Inferior olive response to passive tactile and visual stimulation with variable interstimulus intervals.

The unique anatomical and electrophysiological features of the inferior olive and its importance to cerebellar function have been recognized for decades. However, understanding the exact function of the inferior olive has been limited by the general lack of correlation between its neural activity and specific behavioral states. Electrophysiological studies in animals showed that the inferior olive response to sensory stimuli is generally invariant to stimulus properties but is enhanced by unexpected stimuli. Using functional magnetic resonance imaging in humans, we have shown that the inferior olive is activated when subjects performed a task requiring perception of visual stimuli with unpredictable timing (Xu et al. J Neurosci 26(22):5990-5995, 2006, Liu et al. J Neurophysiol 100(3):1557-1561, 2008). In the current study, subjects were scanned while passively perceiving visual and tactile stimuli that were rendered unpredictable by continuously varying interstimulus intervals (ISIs). Sequences of visual stimuli and tactile stimuli to the right hand were presented separately within the same scanning session. In addition to the activation of multiple areas in the cerebellar cortex consistent with previous imaging studies, the results show that both tactile and visual stimulation with variable ISIs were effective in activating the inferior olive. Together with our previous findings, the current results are consistent with the electrophysiological studies in animals and further support the view that the inferior olive and the climbing fiber system primarily convey the temporal information of sensory input regardless of the modality.

The motor cortex and the coding of force.

The relation of cellular activity in the motor cortex to the direction of two-dimensional isometric force was investigated under dynamic conditions in monkeys. A task was designed so that three force variables were dissociated: the force exerted by the subject, the net force, and the change in force. Recordings of neuronal activity in the motor cortex revealed that the activity of single cells was directionally tuned and that this tuning was invariant across different directions of a bias force. Cell activity was not related to the direction of force exerted by the subject, which changed drastically as the bias force changed. In contrast, the direction of net force, the direction of force change, and the visually instructed direction all remained quite invariant and congruent and could be the directional variables, alone or in combination, to which cell activity might relate.

Functional magnetic resonance imaging of motor cortex: hemispheric asymmetry and handedness.

A hemispheric asymmetry in the functional activation of the human motor cortex during contralateral (C) and ipsilateral (I) finger movements, especially in right-handed subjects, was documented with nuclear magnetic resonance imaging at high field strength (4 tesla). Whereas the right motor cortex was activated mostly during contralateral finger movements in both right-handed (C/I mean area of activation = 36.8) and left-handed (C/I = 29.9) subjects, the left motor cortex was activated substantially during ipsilateral movements in left-handed subjects (C/I = 5.4) and even more so in right-handed subjects (C/I = 1.3).

Selective impairments in implicit learning in Parkinson's disease.

The basal ganglia are thought to participate in implicit sequence learning. However, the exact nature of this role has been difficult to determine in light of the conflicting evidence on implicit learning in subjects with Parkinson's disease (PD). We examined the performance of PD subjects using a modified form of the serial reaction time task, which ensured that learning remained implicit. Subjects with predominantly right-sided symptoms were trained on a 12-element sequence using the right hand. Although there was no evidence of sequence learning on the basis of response time savings, the subjects showed knowledge of the sequence when performance was assessed in terms of the number of errors made. This effect transferred to the left (untrained) hand as well. Thus, these data demonstrate that PD patients are not impaired at implicitly learning sequential order, but rather at the translation of sequence knowledge into rapid motor performance. Furthermore, the results suggest that the basal ganglia are not essential for implicit sequence learning in PD.

Functional imaging of the motor system.

Recent studies of the motor system using functional imaging have served to emphasize the complexity of the control of even relatively simple movements. The results of these studies suggest that the behavioral context of the movement is an important determinant of functional activation within cortical motor areas.

Cortical control of motor behavior at the cellular level.

The studies reviewed in this paper describe the relations of single-cell activity in central motor structures to complex visuomotor tasks and document the fact that various cortical areas process visuomotor information in parallel. Moreover, the studies provide clear evidence that the map in the motor cortex is modifiable and dynamically maintained.

The retention of calcium, iron, phosphorus, and magnesium during pregnancy: the adequacy of prenatal diets with and without supplementation.

Vitamin and mineral supplementation is often prescribed by physicians to meet the additional nutrient requirements of pregnancy. In order to partially ascertain the effectiveness of these prenatal supplements, the retention of calcium and iron was determined in pregnant women consuming supplemented or unsupplemented self-selected diets. The retention of phosphorus and magnesium, minerals not included in the prenatal supplements, was also determined. Seven-day metabolic balance experiments spaced periodically throughout the pregnancy were conducted on 10 healthy pregnant white women. The retention of calcium by the supplemented group was comparable to that of the unsupplemented group, while the retention of iron was more dependent on the magnitude of the iron intake than on its source. Although no supplement contained phosphate, the intake of phosphorus met the recommended allowances for this mineral. Significantly related to the intake of dietary calcium, the adequate phosphorus intakes reflected diets providing adequate calcium. The mean magnesium intake was only 60% of the recently established recommended dietary allowance. Although the calcium and iron salts provided by the prenatal supplements were well utilized, the intakes of phosphorus and magnesium indicate that the reliance on the effectiveness of mineral supplements should not lessen the emphasis by the physician on the importance of a good prenatal diet.

Acquired pendular nystagmus in toluene addiction.

We studied the ocular motor abnormalities in 4 patients chronically addicted to sniffing glue containing toluene. They showed acquired pendular nystagmus with horizontal and vertical components. One patient also showed saccadic oscillations. The pendular nystagmus may be a manifestation of a disturbance in brainstem-cerebellar connections secondary to the toxic effect of toluene on white matter.

Antagonist discrimination of two muscarinic responses elicited by applied agonists and orthodromic stimuli in superior cervical ganglion of rabbit.

Pirenzepine and gallamine selectively and differentially antagonized two muscarinic responses, in the superior cervical ganglion of the rabbit, whether elicited by the muscarinic agonist methacholine or by orthodromic stimulation. Methacholine elicited a biphasic ganglionic response, consisting of hyperpolarizing and depolarizing components that were the agonist-induced equivalents of the slow-inhibitory and slow-excitatory postsynaptic potentials elicited by orthodromic stimulation. Superfusion of ganglia with pirenzepine resulted in a concentration-dependent suppression of depolarization induced by methacholine with no suppressant action on ganglionic hyperpolarization. In contrast, superfusion of ganglia with gallamine resulted in a concentration-dependent suppression of ganglionic hyperpolarization and the slow-inhibitory postsynaptic potential. These effects occurred without appreciable suppression of ganglionic depolarization or the slow-excitatory postsynaptic potential. The action of gallamine was specific for muscarinic hyperpolarization. Hyperpolarizations produced by superfusion with dopamine or norepinephrine were unaffected by gallamine, at concentrations that suppressed the muscarinic slow-inhibitory post-synaptic potential. Incubation with anti-cholinesterases produced a parallel shift, to the right, of concentration-response curves for suppression by gallamine of the slow-inhibitory postsynaptic potential. This was presumably the consequence of an increase in the acetylcholine available for interaction with the muscarinic receptor. The evidence suggests that the ability of gallamine and pirenzepine to suppress selectively the slow-inhibitory and slow-excitatory postsynaptic potentials, as previously demonstrated, is through an action at muscarinic receptors. Furthermore, the data suggest that these pharmacological agents produce their effects by interaction at different muscarinic recognition sites.

Differential and selective antagonism of the slow-inhibitory postsynaptic potential and slow-excitatory postsynaptic potential by gallamine and pirenzepine in the superior cervical ganglion of the rabbit.

Two cholinergic antagonists, gallamine and pirenzepine, agents that have been shown to bind selectively to different subpopulations of the muscarinic receptor, were found to antagonize selectively and differentially the amplitudes of the slow-inhibitory and slow-excitatory postsynaptic potentials in the superior cervical ganglion of the rabbit. Incubation of ganglia with gallamine resulted in a concentration-dependent suppression of the slow-inhibitory postsynaptic potential. The pharmacological action of gallamine was highly specific. At concentrations which reduced the amplitude of the slow-inhibitory postsynaptic potential by as much as 70-90%, there was no reduction of the amplitudes of the muscarinic slow-excitatory postsynaptic potential, the nicotinic fast-excitatory postsynaptic potential, noncholinergic slow-slow-excitatory postsynaptic potential, or post-stimulus hyperpolarizing afterpotentials. The amplitude of the slow-excitatory postsynaptic potential was actually facilitated in the presence of gallamine, presumably as a result of suppression of the overlapping slow-inhibitory postsynaptic potential. In contrast to the action of gallamine, pirenzepine produced a selective suppression of the amplitude of the slow-excitatory postsynaptic potential. Pirenzepine had very little influence on the amplitude of the slow-inhibitory postsynaptic potential at concentrations sufficient to reduce the amplitude of the slow-excitatory postsynaptic potential by as much as 50%, and had no effect on the amplitudes of the nicotinic fast-excitatory postsynaptic potential or noncholinergic slow-slow-excitatory postsynaptic potential. The evidence presented suggests that multiple muscarinic recognition sites, previously identified by studies of the affinities of pharmacological agents for the muscarinic receptor, may actually be involved in synaptic transmission and functionally coupled to cellular effector mechanisms.

Metoclopramide mimics a D-1 type of dopamine action in rabbit superior cervical ganglion.

Metoclopramide (MCP) in a sufficiently high concentration (100 microM) induced a large and persisting potentiation of slow-excitatory postsynaptic potentials (s-epsp) and slow-inhibitory postsynaptic potentials (s-epsp) but depressed in fast epsp. This modulatory action of metoclopramide was markedly suppressed by (+)-butaclamol (7 microM) and, to a lesser extent, by spiroperidol (2.5-4 microM). Metoclopramide also possessed weak anti-acetylcholinesterase activity(I50% = 245 microM; measured by Dr N. Inestrosa), but this was shown not to account for the potentiating actions of metoclopramide. Thus, although metoclopramide is a D-2 antagonist, it appears to mimic the D-1 action of dopamine in modulating the slow psps.

Differential effects of the muscarinic M2 antagonists, AF-DX 116 and gallamine, on single neurons of rabbit sympathetic ganglia.

Intracellular recording techniques were used to compare the effects of the M2 muscarinic antagonists, AF-DX 116 and gallamine, on membrane potential (Vm), input resistance (Ri), responses induced by methacholine, muscarinic slow postsynaptic potentials and action potentials in the superior cervical ganglion of the rabbit. Gallamine or AF-DX 116 antagonized methacholine-induced or synaptically-evoked muscarinic hyperpolarization, without having significant effect on depolarization induced by methacholine or synaptically. The drug AF-DX 116 reduced evoked muscarinic hyperpolarizing potentials, without significant change in Vm or Ri, recorded in the absence of muscarinic stimulation. In contrast to AF-DX 116, gallamine elicited a concentration-dependent depolarization of the membrane, with a corresponding increase in Ri, when tested in the absence of muscarinic stimulation. These effects of gallamine were accompanied by an increase in duration and decrease in the slope of the descending phase of the action potential. Blockade by gallamine of evoked hyperpolarization was independent of membrane depolarization and readily occurred when gallamine-induced depolarization was prevented by clamping Vm at its pre-gallamine level. The effects of gallamine were maintained during its presence and reversed upon washing with gallamine-free physiological solution. These results indicate that AF-DX 116 and gallamine have a specificity for antagonism of muscarinic responses, mediated by receptors of the M2 type in the superior cervical ganglion. However, gallamine, while an effective antagonist of M2 responses, also has the ability to modify the electrical characteristics of ganglion cells and thus may modify ganglionic transmission by mechanisms other than antagonism of receptors.

Choice and stimulus-response compatibility affect duration of response selection.

In general, for movements to visual targets, response times increase with the number of possible response choices. However, this rule only seems to hold when an incompatibility exists between the stimulus and response, and is absent when stimulus and response are highly compatible (e.g., when reaching toward the location of the stimulus). Stimulus-response (S-R) compatibility can be manipulated either at the level of stimulus and response characteristics, or at the level of the mapping between elements of the stimulus and response sets. The current study was undertaken to determine the extent of the interaction between choice and each of these two levels of S-R compatibility. Subjects used a joystick to move a cursor in response to two, four or eight possible cues, with S-R compatibility manipulated along two dimensions (type of stimulus, and mapping between stimulus and response sets) in separate blocks of trials. Choice effects were absent when S-R relationships were highly compatible, moderate when incompatible in either of the two dimensions, and greatest when incompatible in both dimensions. These results indicate that choice affects response selection at each stage in the decoding of S-R relationships. Similar but smaller effects were seen for trials in which the stimulus was the same as that presented in the immediately preceding trial, suggesting that repeated stimulus-response transformations are faster and more efficient due to the priming effects of previous trials.

Neuropeptide Y receptor agonists: multiple effects on spontaneous activity in the paraventricular hypothalamus.

In vitro rat hypothalamic slices were used to examine the ability of neuropeptide Y (NPY), and the putative Y1 and Y2 receptor agonists [Pro34]NPY and [C2]NPY, to modify spontaneous single-neuron discharge in the paraventricular nucleus (PVN). NPY and [Pro34]NPY, at high concentrations (1500 nM), decreased discharge rates. At intermediate concentrations (150 nM) these peptides produced multiple effects, including increases, decreases, and biphasic changes. At lower concentrations (0.15-15 nM), they typically increased discharge rates. In contrast, [C2]NPY, at all concentrations (1.5-1500 nM), predominantly increased discharge rates. Thus, these NPY subtype agonists have multiple effects on discharge rate, which may be due to actions on multiple NPY receptor subtypes.

Effects of movement predictability on cortical motor activation.

Humans have the ability to make motor responses to unpredictable visual stimuli, and do so as a matter of course on a daily basis. We used functional magnetic resonance imaging (fMRI) to examine the neural substrate of this behavior in six cortical motor areas. We found that five of these areas (premotor, cingulate, supplementary motor area, pre-supplementary motor area, and superior parietal lobule) showed increased activation in association with an unpredictable behavior compared to a predictable one; only the motor cortex remained unchanged. There was also a quantitative relation between the response time and functional activation in the premotor and cingulate cortex. There was less activation across all the motor areas with repetition of the motor tasks. With the exception of the pre-supplementary motor area, all areas were significantly lateralized, with a greater volume of activation in the hemisphere contralateral to the performing hand. In addition, a left hemisphere dominance was found in the activation of motor cortex and supplementary motor areas. Our results suggest that activation in motor areas is differentially and quantitatively related to higher order aspects of motor behavior such as movement predictability.

Force and the motor cortex.

The relation between the activity of cells in the motor cortex and static force has been studied extensively. Most studies have concentrated on the relation to the magnitude of force; this relation is more or less monotonic. The slope of the relation, however, shows considerable variation among different studies and seems to be inversely associated with the range of forces over which the cell activity has been studied. Cells in the motor cortex also show variation in activity with the direction of static force. When both the direction and the magnitude of static force are allowed to vary, a majority of cells show significant changes in activity with direction of force alone, an intermediate number relate to both direction and magnitude, while a small number relate purely to the magnitude. This suggests that the direction of static force can be controlled independently of its magnitude and that this directional signal is especially prominent in the motor cortex. In general, it has been more difficult to study the relations to dynamic force. There is a correlation between motor cortex cell activity and the rate of change of force. The direction of dynamic force is also an important determinant of cell activity. When both static and dynamic force output are required (for example, with arm movement in the presence of gravity) it is the dynamic signal that is most clearly reflected in motor cortex activity. The relations between motor cortex activity and static or dynamic force are not invariant, but may be modified by the behavioral context of the motor output.

Force and the motor cortex.

The relation between the activity of cells in the motor cortex and static force has been studied extensively. Most studies have concentrated on the relation to the magnitude of force; this relation is more or less monotonic. The slope of the relation, however, shows considerable variation among different studies and seems to be inversely associated with the range of forces over which the cell activity has been studied. Cells in the motor cortex also show variation in activity with the direction of static force. When both the direction and the magnitude of static force are allowed to vary, a majority of cells show significant changes in activity with direction of force alone, an intermediate number relate to both direction and magnitude, while a small number relate purely to the magnitude. This suggests that the direction of static force can be controlled independently of its magnitude and that this directional signal is especially prominent in the motor cortex. In general, it has been more difficult to study the relations to dynamic force. There is a correlation between motor cortex cell activity and the rate of change of force. The direction of dynamic force is also an important determinant of cell activity. When both static and dynamic force output are required (for example, with arm movement in the presence of gravity) it is the dynamic signal that is most clearly reflected in motor cortex activity. The relations between motor cortex activity and static or dynamic force are not invariant, but may be modified by the behavioral context of the motor output.