Recent findings have shown that muscle contraction evokes an exaggerated pressor response in type 1 diabetes mellitus (T1DM) rats; however, it is not known whether the mechanoreflex, which is commonly stimulated by stretching the Achilles tendon, contributes to this abnormal response. Furthermore, the role of mechano-gated Piezo channels, found on thin-fiber afferent endings, in evoking the mechanoreflex in T1DM is also unknown. Therefore, in male and female streptozotocin (STZ, 50 mg/kg)-induced T1DM and healthy control (CTL) rats, we examined the pressor and cardioaccelerator responses to tendon stretch during the early stage of the disease. To determine the role of Piezo channels, GsMTx-4, a selective Piezo channel inhibitor, was injected into the arterial supply of the hindlimb. At 1 wk after STZ injection in unanesthetized, decerebrate rats, we stretched the Achilles tendon for 30 s and measured pressor and cardioaccelerator responses. We then compared pressor and cardioaccelerator responses to tendon stretch before and after GsMTx-4 injection (10 µg/100 ml). We found that the pressor (change in mean arterial pressure) response [41 ± 5 mmHg ( n = 15) for STZ and 18 ± 3 mmHg ( n = 11) for CTL ( P < 0.01)] and cardioaccelerator (change in heart rate) response [18 ± 4 beats/min for STZ ( n = 15) and 8 ± 2 beats/min ( n = 11) for CTL ( P < 0.05)] to tendon stretch were exaggerated in STZ rats. Local injection of GsMTx-4 attenuated the pressor [55 ± 7 mmHg ( n = 6) before and 27 ± 9 mmHg ( n = 6) after GsMTx-4 ( P < 0.01)], but not the cardioaccelerator, response to tendon stretch in STZ rats and had no effect on either response in CTL rats. These data suggest that T1DM exaggerates the mechanoreflex response to tendon stretch and that Piezo channels play a role in this exaggeration.

Patients with type-2 diabetes mellitus (T2DM) have exaggerated sympathetic activity and blood pressure responses to exercise. However, the underlying mechanisms for these responses, as well as how these responses change throughout disease progression, are not completely understood. For this study, we examined the effect of the progression of T2DM on the exercise pressor reflex, a critical neurocardiovascular mechanism that functions to increase sympathetic activity and blood pressure during exercise. We also aimed to examine the effect of T2DM on reflexive cardiovascular responses to static contraction, as well as those responses to tendon stretch when an exaggerated exercise pressor reflex was present. We evoked the exercise pressor reflex and mechanoreflex by statically contracting the hindlimb muscles and stretching the Achilles tendon, respectively, for 30 s. We then compared pressor and cardioaccelerator responses in unanesthetized, decerebrated University of California Davis (UCD)-T2DM rats at 21 and 31 wk following the onset of T2DM to responses in healthy nondiabetic rats. We found that the pressor response to static contraction was greater in the 31-wk T2DM [change in mean arterial pressure (∆MAP) = 39 ± 5 mmHg] but not in the 21-wk T2DM (∆MAP = 24 ± 5 mmHg) rats compared with nondiabetic rats (∆MAP = 18 ± 2 mmHg; P < 0.05). Similarly, the pressor and the cardioaccelerator responses to tendon stretch were significantly greater in the 31-wk T2DM rats [∆MAP = 69 ± 6 mmHg; change in heart rate (∆HR) = 28 ± 4 beats/min] compared with nondiabetic rats (∆MAP = 14 ± 2 mmHg; ∆HR = 5 ± 3 beats/min; P < 0.05). These findings suggest that the exercise pressor reflex changes as T2DM progresses and that a sensitized mechanoreflex may play a role in exaggerating these cardiovascular responses. NEW & NOTEWORTHY This is the first study to provide evidence that as type-2 diabetes mellitus (T2DM) progresses, the exercise pressor reflex becomes exaggerated, an effect that may be due to a sensitized mechanoreflex. Moreover, these findings provide compelling evidence suggesting that impairments in the reflexive control of circulation contribute to exaggerated blood pressure responses to exercise in T2DM.

Emerging evidence suggests the exercise pressor reflex is exaggerated in early-stage type 1 diabetes mellitus (T1DM). Piezo channels may play a role in this exaggeration since blocking these channels attenuates the exaggerated pressor response to tendon stretch in T1DM rats. However, tendon stretch constitutes a different mechanical and physiological stimuli than that occurring during muscle contraction. Therefore, the purpose of this study was to determine the contribution of Piezo channels in evoking the pressor reflex during an intermittent muscle contraction in T1DM. In unanaesthetized, decerebrate rats we compared the pressor and cardioaccelerator responses to intermittent muscle contraction before and after locally injecting GsMTx-4 (0.25 µM) into the hindlimb vasculature. Although GsMTx-4 has a high potency for Piezo channels, it has also been suggested to block TRPC channels. We, therefore, performed additional experiments to control for this possibility by also injecting SKF 96365 (10 µM), a TRPC channel blocker. We found that local injection of GsMTx-4, but not SKF 96365, attenuated the exaggerated peak pressor (ΔMAP before: 33±3 mmHg, after: 22±3 mmHg, p=0.007) and pressor index (ΔBPi before: 668±91 mmHg·s, after: 418±81 mmHg·s, p=0.021) response in STZ rats (n=8). GsMTx-4 attenuated the exaggerated early-onset pressor as well as the pressor response over time, which eliminated peak differences as well as those over time between T1DM and healthy controls. These data suggest that Piezo channels are an effective target to normalize the exercise pressor reflex in T1DM.

Studies have shown that early-stage type 1 diabetes mellitus (T1DM) leads to an exaggerated reflex pressor response to both static muscle contraction and tendon stretch. However, whether similar responses are present during dynamic exercise (i.e., intermittent contraction) is not known. Therefore, the purpose of this study was to determine whether T1DM leads to an exaggerated reflex pressor response to intermittent muscle contraction. We measured the exercise pressor reflex in unanesthetized, decerebrated T1DM (Streptozotocin 50 mg/kg; STZ) and healthy control (CTL) Sprague Dawley rats by intermittently contracting the hindlimb muscles for 30 s while measuring mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA), and heart rate (HR). Intermittently contracting the hindlimb muscles evoked exaggerated mean RSNA (STZ: Δ109 ± 21%, n=4; CTL: Δ61 ± 8%, n=5, p<0.05), peak MAP (STZ: Δ32 ± 2 mmHg, n=9; CTL: Δ12 ± 2 mmHg, n=6, p<0.05), blood pressure index (STZ: Δ625 ± 60 mmHg·s, n=9; CTL: Δ241 ± 46 mmHg·s, n=6, p<0.05), and HR (STZ: Δ24 ± 3 bpm, n=9; CTL: Δ9 ± 3 bpm, n=6, p<0.05) responses to similar developed tensions (p>0.05) in T1DM compared to CTL rats. T1DM rats also exhibited exaggerated early-onset sympathetic (onset: 1 s) and pressor (onset: 5 s) responses. These data show that early-stage T1DM leads to an exaggerated pressor reflex evoked by intermittent muscle contraction. The early onset and greater blood pressure index suggest that cardiovascular strain during dynamic exercise may be significantly higher in individuals with T1DM.

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Type 2 diabetes is associated with neuropathic symptoms, including development of peripheral neuropathy and an exaggerated exercise pressor reflex, both of which have been demonstrated in UC Davis Type 2 diabetes mellitus (UCD‐T2DM) rats. This novel animal model more closely mimics the human disease state than traditional T2DM rodent models. Several studies have demonstrated changes in nerve growth factor (NGF) in diabetic patients, with some showing increases and others decreases, and this has been proposed as a potential driver of the development of peripheral neuropathy. Additionally, increased expression of purinergic 2X3 (P2X3) receptors has been suggested as a potential mechanism underlying peripheral nerve sensitization. However, interactions between these factors during diabetes progression are not fully understood. Therefore, the objective of this study was to investigate the relations between NGF, P2X3, and peripheral neuropathy during diabetes progression in UCD‐T2DM rats. We hypothesized that NGF levels would be correlated with traditional indexes of diabetes (glucose and HbA1c) as well as P2X3 expression and mechanical allodynia as measured by paw withdrawal threshold. Methods Blood was collected monthly from the tail vein of male UCD‐T2DM rats from 4 to 8 months of age. Serum levels of NGF, leptin, and insulin were measured by ELISA. Development of mechanical allodynia was tracked monthly using Von Frey filaments to measure paw withdrawal threshold. At 8 months of age, L4‐5 dorsal root ganglia were harvested and P2X3 receptor protein levels were quantified using the Jess automated western blotting platform. Pearson correlations were calculated using Graphpad Prism 9. Results Consistent with wasting and loss of pancreatic beta cell function during diabetes progression, glucose and HbA1c increased significantly over time whereas body weight, insulin, and leptin levels decreased. Circulating NGF concentration was negatively correlated with glucose (r=‐0.61, P<0.01) and HbA1c (r=‐0.79, p<0.01) demonstrating that NGF decreased with the progression of diabetes. Circulating NGF concentration was positively correlated with body weight (r=0.61, p<0.01), insulin (r=0.77, p<0.01), and leptin (r=0.82, p<0.01) thereby showing the same decreasing trend with diabetes progression. Interestingly, NGF was negatively correlated with P2X3 receptor expression (r=‐0.81, p=0.03) and neither NGF nor P2X3 were significantly associated with mechanical allodynia (NGF: r=0.14, p=0.45; P2X3: r= 0.15, p=0.75). Conclusion Circulating NGF is significantly associated with several indexes of diabetes progression in UCD‐T2DM rats, including glucose, HbA1c, leptin, and insulin levels. Though decreasing concentrations of NGF are associated with increases in P2X3 receptor protein expression, neither NGF nor P2X3 appear to be associated with the development of mechanical allodynia.

Type 2 diabetes (T2DM) is a metabolic disorder that can lead to many neuropathic complications such as mechanical allodynia, which is perceived pain caused by a normally non‐painful stimulus. T2DM is also associated with exaggerated pressor and cardioaccelerator responses to skeletal muscle contraction, namely the exercise pressor reflex. The mechanisms by which diabetes elicits these effects are not fully understood; however, both mechanical allodynia and the exercise pressor reflex are evoked by thin‐fiber afferent activity. The purpose of this study was to utilize noninvasive techniques to monitor the development and progression of diabetes‐induced neuropathy in thin‐fiber afferents. We used male UC Davis T2DM (n=9) rats from the age of 4 to 7 months to model the progression of T2DM. We took monthly measurements of blood glucose, HbA1c, and body weight. We also measured changes in mechanical sensitivity using von Frey filaments. Filaments were applied to the L5 dermatome on the plantar side of the left and right hind paws, between the footpads; the lowest force eliciting an abrupt paw withdrawal was measured and averaged between paws. We found that blood glucose (4mo: 255±28 mg/dl, 5mo: 366±50 mg/dl, 6mo: 417±58 mg/dl, 7mo: 453±49 mg/dl) and HbA1c (4mo: 5.7±0.2%, 5mo: 6.6±0.4%, 6mo: 9.3±1.2%, 7mo: 10.3±1.2%) were above the diabetic threshold (300 mg/dl, 6.5%) at 5, 6, and 7 months of age; they were significantly higher at 6 and 7 months, compared to 4 months, p<0.05. Body weight was significantly higher at 5, 6, and 7 months compared to 4 months (4mo: 525±4g, 5mo: 609±3g, 6mo: 620±21g, 7mo: 610±31g; p<0.05). Sensitivity to von Frey filaments was significantly greater, indicating mechanical allodynia, at 6 and 7 months compared to 4 months (4mo: 29±3.2 g, 5mo: 26.3±4.5 g, 6mo: 14.1±3.3 g, 7mo: 9.7±1.7 g; p<0.05). We conclude that mechanical allodynia progresses in severity as the rats develop and progress with T2DM. Moreover, these observations may provide insight to diabetes‐induced changes in thin‐fiber dependent reflexes, such as the exercise pressor reflex.Support or Funding InformationN/AThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Previous work has shown that the exercise pressor reflex is exaggerated in type 2 diabetic rats that present with hyperglycemia. Although it is likely that chronic hyperglycemia plays a role, no studies have isolated the acute effects of high blood glucose levels on this reflex. Therefore, the purpose of the current study was to determine the acute effects of hyperglycemia on the exercise pressor reflex. We first determined the glucose concentration needed to increase local blood glucose level in the hindlimb vasculature to a similar level seen in our type 2 diabetic rats that had an exaggerated exercise pressor reflex. To do this, healthy Sprague‐Dawley rats were infused over 20 min with one of two different concentrations of glucose (125 mg/ml, male n=3 or 250 mg/ml, male n=3) into the arterial supply of the left hindlimb with blood flow to and from the hindlimb restricted. At the same time somatostatin (3.9 ug/100 ul) was infused systemically in order to prevent an endogenous insulin response. Blood glucose was measured every two minutes from both hindlimbs to determine how long it would take to bring blood glucose concentration up to the desired level. We then determined the acute effect of glucose on the pressor and cardioaccelerator responses during static contraction of the hindlimb. The exercise pressor reflex was evoked in healthy, unanesthetized, decerebrated Sprague‐Dawley rats by statically contracting the hindlimb muscle for 30 seconds before and after 15 minutes of either glucose (250 mg/ml) or saline infusion, while mean arterial pressure and heart rate were recorded. We found that infusing 250 mg/ml of glucose significantly raised blood glucose levels to 486 ± 84 mg/dl by the 3rd minute compare to the control leg. However, infusing 125 mg/ml of glucose did not raise blood glucose concentration to 546 ± 33 mg/dl until the 11th minute compare to the control leg (p<0.05). Therefore, we decided to use 250 mg/ml glucose for the exercise pressor experiment. We then found that acutely infusing 250 mg/ml of glucose into the arterial supply of the hindlimb did not significantly affect the pressor (ΔMAP before glucose: 13 ± 0.4 mmHg; after glucose: 12 ± 0.8 mmHg, n=4; before saline: 11 ± 2 mmHg; after saline: 10 ± 2 mmHg, n=3; p>0.05) or cardioaccelerator (ΔHR before glucose: 14 ± 1 bpm; after glucose: 12 ± 2 bpm, n=4; before saline: 14 ± 3 bpm; after saline: 12 ± 4 bpm, n=3; p>0.05) responses to static contraction. We conclude that the acute presence of glucose in the circulation of the hindlimb muscles plays no role in exaggerating the exercise pressor reflex.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.