Hui-Ying Luk

Introduction: Glycemic dysregulation is a hallmark of type 2 diabetes (T2D) and contributes to skeletal muscle (SKM) loss and frailty risk, especially in older adults. Glycemic control and physical function are supported by SKM capillarization and mitochondrial function, and their impairment contributes to T2D development. While high-intensity interval training (HIIT) is a promising intervention, adherence and effectiveness remain concerns for prescribing HIIT among older adults at risk for T2D. Local heat therapy (LHT) may be a more practical initial strategy to improve SKM architectural factors and precondition SKM, enhancing physiological adaptations to exercise in this population. Methods and analysis: Heat and Exercise in Aging as Therapy (HEAT) is a two-phase, randomized, sham-controlled clinical trial investigating the efficacy of LHT to improve glycemic control and decrease frailty risk via improved SKM architecture among older adults with prediabetes. LHT is tested as a standalone intervention and as a means to precondition SKM for subsequent HIIT, improving exercise adaptations. In Phase 1, LHT and sham (CON) groups apply heat pads for 90 minutes/day, 6 days/week, for 12 weeks. A separate HIIT group completes 4x4-minute cycling intervals at 90–95% VO₂peak, 3 days/week. In Phase 2, LHT and CON groups begin HIIT. Participants (≥50 years) have impaired fasting glucose (100–125 mg/dL) and/or HbA1c (5.7–6.4%). Biospecimen collection and clinical assessments occur at baseline (T1), after Phase 1 (T2), and Phase 2 (T3). To our knowledge, this is the first study to determine the use of local heat pad on pre-diabetic older population. If successful, LHT may be a practical, scalable, non-invasive intervention to improve glycemic control and reduce frailty risk in older adults with prediabetes, preventing progression to T2D.

This study examined how 24-h fluid restriction affects mitochondrial stress and inflammatory responses in peripheral blood mononuclear cells (PBMCs) after high-volume resistance exercise in young men. Dehydration increased markers of mitophagy (PINK1 and p-DRP1 ^S616 ), autophagosome formation (LC3-II and p62), and oxidative stress (H ₂ O ₂ ), while systemic IL-6 and CRP were elevated compared with euhydration. Findings suggest dehydration amplifies mitochondrial stress and proinflammatory signaling, highlighting the importance of proper hydration during exercise and its implications on cellular homeostasis.

Dehydration is prevalent and adversely affects exercise performance; however, its influence on cellular responses to exercise remains unclear. Thus, this study examined the intramuscular responses to resistance‐exercise (RE) in RE‐trained men under dehydrated and euhydrated states. Eleven men (21 ± 1 years, 175.9 ± 6.2 cm, 79.2 ± 12.3 kg, 18.4% ± 6.7% fat) completed two identical lower‐body RE sessions, either with (DEHY) or without (EUHY) fluid‐restriction from 24 h before to 3 h after RE. At pre‐RE (PRE), 1 h, and 3 h post‐RE, muscle samples were collected and analysed for protein content of AKT/mTOR/p70S6K/rpS6 and their corresponding phosphorylation sites, REDD1 and selected autophagy markers, cathepsin L, H ₂ O ₂ concentration, fibre cross‐sectional area (CSA), and muscle water content. Significant time × condition interaction effects revealed that p‐rpS6 ^S240/244 was greater in DEHY than EUHY at PRE and increased from PRE to 1 h and 3 h in both conditions. In DEHY, REDD1 increased from PRE to 1 h and 3 h, active‐cathepsin L decreased from 1 h to 3 h and was greater than EUHY at 1 h, and muscle water content increased from 1 h to 3 h. Significant condition main effects revealed that p‐S6K ^T389 and H ₂ O ₂ were greater, and CSA was smaller, in DEHY versus EUHY. Significant time main effects revealed that p‐AKT ^S473 and p‐mTOR ^S2448 increased from PRE to 1 h and 3 h, LC3‐I decreased from PRE and 1 h to 3 h, LC3‐II decreased from PRE to 1 h and 3 h, and LC3‐II/LC3‐I decreased from PRE to 1 h and increased from 1 h to 3 h. These results suggest that performing RE in a dehydrated state imposes additional stress on the muscle, leading to greater cellular stress and growth signalling. image Key points: Dehydration can negatively impact exercise performance, overall health, and cognitive function in humans. Water makes up about 70% of muscle mass, and dehydration has been shown to decrease muscle size in humans. However, the mechanisms by which dehydration affects muscle response on anabolic and catabolic signalling have only been observed in in vitro studies, leaving the processes in humans still not fully understood. Following 24 h of dehydration, there was an increase in the activation of rpS6 at rest. Additionally, young men exhibited greater activation of S6K during resistance exercise (RE) while dehydrated compared to when they were adequately hydrated. Concurrently, stress (H ₂ O ₂ and REDD1) and proteolytic (active‐cathepsin L) responses were elevated after RE in a dehydrated state compared to an adequately hydrated state. Our research offers new insights into the importance of hydration in muscle responses to exercise, particularly for individuals who are frequently dehydrated.

Live cell assays and image-based cell analyses require data normalization for accurate interpretation. A commonly used method is to stain and quantify nuclei, followed by data normalization to nuclei count. This nuclei count is often expressed as cell count for uninucleate cells. While manual quantification can be laborious and time-consuming, available automated methods may not be preferred by all users, may lack validation for this specific application, or may be cost-prohibitive. Here, we provide step-by-step instructions for capturing quantifiable images of nuclei stained with fluorescent DNA stains and subsequently quantifying the nuclei using an automated object counting software program developed using Python computer vision libraries. We also validate this program across a range of cell densities. Although the exact time for program execution varies based on the number of images and computer hardware, this program consolidates hours of work counting nuclei into seconds for the program to run. While this protocol was developed using images of fixed, stained cells, images of stained nuclei in live cells and immunofluorescence applications can also be quantified using this program. Ultimately, this program provides an option that does not require a high degree of technological skill and is a validated, open-source alternative to aid cell and molecular biologists in streamlining their workflows, automating the tedious and time-consuming task of nuclei quantification.

Live cell assays and image-based cell analyses require data normalization for accurate interpretation. A commonly used method is to stain and quantify nuclei, followed by data normalization to nuclei count. This nuclei count is often expressed as cell count for uninucleate cells. While manual quantification can be laborious and time-consuming, available automated methods may not be preferred by all users, may lack validation for this specific application, or may be cost-prohibitive. Here, we provide step-by-step instructions for capturing quantifiable images of nuclei stained with fluorescent DNA stains and subsequently quantifying the nuclei using an automated object counting software program developed using Python computer vision libraries. We also validate this program across a range of cell densities. Although the exact time for program execution varies based on the number of images and computer hardware, this program consolidates hours of work counting nuclei into seconds for the program to run. While this protocol was developed using images of fixed, stained cells, images of stained nuclei in live cells and immunofluorescence applications can also be quantified using this program. Ultimately, this program provides an option that does not require a high degree of technological skill and is a validated, open-source alternative to aid cell and molecular biologists in streamlining their workflows, automating the tedious and time-consuming task of nuclei quantification.

Muscle recovery after damage is mediated by circulating factors and intracellular signaling pathways. Our previous studies have demonstrated that resistance exercise (RE)‐induced circulating factors elicited sex‐differential responses in damaged muscle. However, the global effects of these circulating factors on damaged muscle are largely understudied. We examined the differential effects of RE‐induced circulating factors and sex on the damaged muscle proteome. Damaged vastus lateralis muscle from 3 men and 3 women from a parent study were analyzed. Participants completed 2 identical bouts of unilateral eccentric knee extensions immediately followed by either upper body RE to induce circulating factors (EXE) or 20‐min seated rest (CON). Muscle biopsies collected from the damaged leg at 24 h were used. 900 proteins were identified by LC–MS/MS analysis. Ingenuity Pathway Analysis was used to detect activation prediction using z‐scores for functional and pathway analyses. In men, 79 proteins were downregulated and 15 were upregulated in EXE versus CON. These differentially expressed proteins were associated with immunological and inflammatory signaling pathways. Biological functions of the differentially expressed proteins in EXE vs. CON in men include inactivating acute inflammatory signaling, neutrophil extracellular trap signaling, ROS production, and activating IL‐12 signaling. These results underline that RE‐induced circulating factors have a sex‐specific effect on the damaged muscle proteome, where immune signaling is altered in men but not women. Given that the immune response is critical for recovery from muscle damage, these results highlight the potential role of RE‐induced circulating factors that could be essential in mediating muscle recovery.

Objectives: The WUT (weight, urine color, thirst) Venn diagram has been demonstrated as a practical tool for hydration assessment. However, no investigations have examined if there are sex differences in the accuracy of this method. Therefore, the purpose of this study was to explore potential sex differences in the accuracy of the WUT Venn diagram for hydration status determination. Methods: Twelve men (21 ± 2 years; 81.0 ± 15.9 kg) and twelve women (22 ± 3 years; 68.8 ± 15.2 kg) completed the study. Body mass, urine color, urine specific gravity (USG), urine osmolality (UOSM), thirst level, and plasma osmolality (POSM) were collected at first-morning and afternoon visits for three consecutive days in free-living and euhydrated states. The number of markers indicating dehydration levels were categorized into either 3, 2, 1, or 0 WUT markers. Receiver-operating characteristics analysis calculated the sensitivity and specificity of 1, 2, or 3 hydration markers in detecting dehydration or euhydration. Results: WUT2 and WUT3 resulted in high specificity values in comparison to USG (WUT2—male: 0.912, female: 0.949; WUT3—male: 0.991, female: 1.000), UOSM (WUT2—male: 0.901, female: 0.946; WUT3—male: 0.991, female: 1.000), and POSM (WUT2—male: 0.859, female: 0.896 WUT3; male: 0.977, female: 0.991). WUT1 resulted in high sensitivity values compared to USG (male: 0.931, female: 0.857), but varied between sexes for UOSM (male: 0.875, female: 0.794) and POSM (male: 0.469, female:0.536). Conclusions: The WUT Venn diagram accurately detects dehydration when two or three WUT markers are met in both males and females. The WUT Venn diagram accurately assesses hydration status in males and females, thus providing medical personnel and athlete support teams a cost-effective and time-efficient hydration testing tool.

Denervation-induced calcium/calmodulin-dependent protein kinase II (CaMKII) activation and inflammation can result in muscle atrophy. Curcumin and bisdemethoxycurcumin are well known to exhibit an anti-inflammatory effect. In addition, curcumin has been shown to attenuate CaMKII activation in neuronal cells. This study aimed to examine the effect of curcumin or bisdemethoxycurcumin on CaMKII activation, inflammation, and muscle cross-sectional area (CSA) in spinal nerve ligated rats. Sixteen female rats were assigned to sham (CON), spinal nerve ligation (SNL), SNL+ curcumin 100 mg/kg BW (100CUR), and SNL+ bisdemethoxycurcumin 50 mg/kg BW (50CMO) for 4 weeks. Ipsilateral (surgical) soleus and tibialis anterior (TA) muscles was stained for dystrophin to measure CSA. Ipsilateral and contralateral (non-surgical) plantaris muscles were analyzed for protein content for acetylcholine receptor (AChR), CaMKII, CaMKII^Thr286, nuclear factor-κB (NF-κB), NF-κB^Ser536, and interleukin-1β (IL-1β) and normalized to α-tubulin and then CON. A significant (p < 0.050) group effect was observed for TA CSA where CON (11,082.25 ± 1617.68 μm²; p < 0.001) and 100CUR (9931.04 ± 2060.87 μm²; p = 0.018) were larger than SNL (4062.25 ± 151.86 μm²). In the ipsilateral plantaris, the SNL (4.49 ± 0.69) group had greater CaMKII activation compared to CON (1.00 ± 0.25; p = 0.010), 100CUR (1.12 ± 0.45; p = 0.017), and 50CMO (0.78 ± 0.19; p = 0.009). The ipsilateral plantaris (2.11 ± 0.66) had greater IL-1β protein content than the contralateral leg (0.65 ± 0.14; p = 0.041) in the SNL group. In plantaris, the SNL (1.65 ± 0.51) group had greater NF-κB activation compared to CON (1.00 ± 0.29; p = 0.021), 100CUR (0.61 ± 0.10; p = 0.003), 50CMO (0.77 ± 0.25; p = 0.009) groups. The observed reduction in Ca²⁺ signaling and inflammation in type II plantaris muscle fibers might reflect the changes within the type II TA muscle fibers which may contribute to the mitigation of TA mass loss with curcumin supplementation.

Type 2 diabetes (T2D) is associated with increased inflammation and reactive oxygen species (ROS) in muscles, leading to basal satellite cell (SC) myogenic impairment (i.e., reduction in SC pool), which is critical for maintaining skeletal muscle mass. T2D may contribute to muscle atrophy, possibly due to reductions in the SC pool. Geranylgeraniol (GGOH) and ginger can reduce inflammation and enhance SC myogenesis in damaged muscles, thereby alleviating muscle atrophy; however, their effect on basal SC myogenic state and muscle mass in T2D rats is limited. Rats consumed a control diet (CON), high-fat diet with 35 mg/kg of streptozotocin (HFD), a HFD with 800 mg/kg body weight of GGOH (GG), or a HFD with 0.75% ginger root extract (GRE). In the eighth week, their soleus muscles were analyzed for Pax7, MyoD, and MSTN gene and protein expression, SC myogenic state, and muscle cross-sectional area (CSA). The HFD group had a significantly lower number of Pax7⁺/MyoD⁻ and Pax7⁺/MSTN⁺ cells, less Pax7 and MyoD gene expression, and less MyoD and MSTN protein expression, with a smaller CSA than the CON group. Compared to the GG and GRE groups, the HFD group had a significantly lower number of Pax7⁺/MSTN⁺ cells, less MyoD protein expression, and smaller CSA. The GRE group also had a significantly lower number of Pax7⁻/MyoD⁺ and greater MSTN protein expression than the HFD group. Nevertheless, the CON group had a significantly greater number of Pax7⁺/MyoD⁻ than the GG and GRE groups, and a greater number of Pax7⁻/MyoD⁺ cells than the GRE group with a larger CSA than the GG group. GGOH and ginger persevered muscle CSA, possibly through increased MyoD and the ability to maintain the SC pool in T2D rats.

Purpose: To investigate the effects of thirst on later hydration status, total water intake (TWI-MA), and its potential sex differences. Methods: Twelve men (mean ± standard deviation; age: 21 ± 2 years; mass: 81.0 ± 15.9 kg) and twelve women (age: 22 ± 3 years; mass: 68.8 ± 15.2 kg) visited the laboratory in the morning (first thing in the morning) and afternoon (2:00–4:00 p.m.) for three consecutive days under a free-living condition. At each visit, urine osmolality (U_OSM), urine specific gravity (USG), urine color (U_COL), body mass loss (BML), thirst, and plasma osmolality (P_OSM) were collected and analyzed. The participants recorded their food and fluid intake between the visits to determine TWI-MA. Linear regression was used to predict the effect of morning thirst on the afternoon hydration indices for all the participants, as well as for males and females separately. Results: Higher morning thirst predicted lower U_OSM (r² = 0.056, p = 0.045), USG (r² = 0.096, p = 0.008), U_COL (r² = 0.074, p = 0.021), and higher thirst (r² = 0.074, p = 0.021) in the afternoon. However, morning thirst did not predict afternoon BML, P_OSM, or TWI-MA (p > 0.05). In males, higher morning thirst predicted lower afternoon U_OSM (r² = 0.130, p = 0.031) and USG (r² = 0.153, p = 0.018). Additionally, higher morning thirst predicted higher TWI-MA (r² = 0.154, p = 0.018) in females. Conclusions: Morning thirst had a negligible impact on later hydration status, specifically with afternoon urine indices. Furthermore, higher thirst sensation did not impact BML, P_OSM, or TWI-MA. However, thirst sensation minimally contributed to drinking behavior in females. Overall, individuals may not rely solely on thirst sensation to manipulate their drinking behavior to optimize their fluid balance during their daily lives due to the complexity of thirst mechanisms.