Obesity has emerged as a major public health issue and a global epidemic. This problem is particularly severe in the middle and older age groups, with data showing that a majority of middle and older age people in Thailand and other Asian countries are overweight and obese (Sakboonyarat et al., 2020). Obesity primarily regulates bone metabolism and protein synthesis (primarily in myocytes), resulting in osteoporosis and increased bone fractures due to diminished muscular strength (Lampropoulos et al., 2012). Obesity has been linked to lower bone mineral density (BMD) and bone mineral content (BMC) (Kim et al., 2010), as well as rapid muscle loss and weakness (Roy et al., 2016). Moreover, obese people are more likely than normal-weight people to suffer from osteoporosis and fractures (Halade et al., 2010; Cao, 2011).

Chronic inflammation (Cao, 2011; Kim et al., 2015), along with skeletal muscle loss (Roy et al., 2016), has been regarded as a major risk factor for osteoporosis. Proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and C-reactive protein (CRP), play crucial roles in bone resorption and osteoclast differentiation (Cao, 2011; Kim et al., 2015), as well as muscle atrophy (Roy et al., 2016). Additionally, the monocyte-to-lymphocyte ratio (MLR), neutrophil-to-lymphocyte ratio (NLR), and platelet-to-lymphocyte ratio (PLR) have become more noticeable because they are simple, inexpensive, and useful inflammatory markers that have been linked to various inflammatory conditions, particularly osteoporosis and bone-derived diseases (Öztürk et al., 2013; Koseoglu, 2017; Gao et al., 2019). Obesity-related increases in proinflammatory cytokines may stimulate osteoclast activity, bone resorption, and catabolic pathways, leading to muscle atrophy (Roy et al., 2016).

Furthermore, in obesity, increased leptin secretion and/ or decreased adiponectin production by adipocytes may directly or indirectly affect bone formation via upregulated proinflammatory cytokine production (Cornish et al., 2002). Obesity has also been associated with rapid muscle loss and decreased muscle strength by modulating the activities of proinflammatory cytokines like TNF-α and leptin, as well as other factors that inhibit protein synthesis and induce proteolysis (Roy et al., 2016). This causes rapid muscle mass loss, followed by muscle weakness and, eventually, bone fractures (Roy et al., 2016).

Regular exercise is a highly effective non-pharmacological approach for improving BMD, muscle strength, and lean mass, as well as preventing future fractures and falls in obese people (Campanha-Versiani et al., 2017; Gandham et al., 2021; Mesinovic et al., 2021; Yarizadeh et al., 2021). Accordingly, aerobic training (AT) has been widely recommended for obese individuals due to its positive effects on body composition, blood pressure, lipids, glycemic control, and aerobic fitness (Vasconcellos et al., 2014; Sultana et al., 2019). In contrast, resistance training (RT) improves muscle mass, strength, and BMD (Mesinovic et al., 2021; Yarizadeh et al., 2021). In obese adults, AT decreases the circulating concentration of proinflammatory cytokines (TNF-α and CRP), whereas RT increases the concentration of anti-inflammatory cytokines (Gonzalo-Encabo et al., 2021). The recommendations of the World Health Organization emphasize the significance of engaging in both AT and RT on a regular basis (Bull et al., 2020), whereas the American College of Sports Medicine recommends moderate- to high-intensity aerobic exercises (AEs) or weight-bearing activities to preserve or increase BMD (Kohrt et al., 2004). As previously stated, combining AT, and RT appears to be one of the most efficient treatment strategies for the obese population.

Various nutritional supplementation strategies have also been proposed given the anti-inflammatory properties of certain supplements, potentially benefiting overweight, and obese adults by reducing the amount of inflammation that manifests as obesity. Numerous studies have previously demonstrated the advantages of polyunsaturated fatty acid (PUFA) consumption (Kruger et al., 2010; Kelly et al., 2013; Bird et al., 2021). Accordingly, one study showed that the consumption of PUFAs has direct anti-inflammatory effects and may have indirect effects on the differentiation of adipocytes and osteoblasts, which may be crucial in obesity (Kelly et al., 2013). Moreover, recent evidence suggests that PUFA, namely α-linolenic acid, supplementation may directly activate anabolic processes in the skeletal muscle (Bird et al., 2021). Furthermore, PUFAs derived from the Eri silkworm have been shown to reduce abdominal obesity and improve resting energy expenditure and fat oxidation in middle-aged adults suffering from obesity (Klomklorm et al., 2020).

The public and private sectors of Thailand are currently promoting Eri silkworm farming. The pupae of the Eri silkworm that are discarded as waste account for approximately 60% of the cocoon’s weight and are rich in PUFAs such as palmitic, oleic, and linoleic acids (Klomklorm et al., 2020), particularly α-linolenic acid (Shanker et al., 2006). Due to its high α-linolenic acid content, Eri silkworm pupae oil has additional health benefits while being safe and nutritionally comparable to common sunflower oils (Longvah et al., 2012). Consequently, promoting silkworm pupae as an alternative food source enhances their value (Gao et al., 2018).

To the best of our knowledge, no study has been conducted to assess the effects of combining concurrent training (CCT) and PUFA supplementation on BMD and BMC, inflammatory biomarkers, and muscle mass, and strength in overweight and obese adults. Therefore, the current study sought to determine the effects of combined CCT and Eri-PUFA consumption on BMD and BMC, inflammatory biomarkers, and muscle mass, and strength in overweight and obese middle-aged adults. We hypothesized that individuals receiving CCT and an Eri-PUFA supplement would have lower inflammatory biomarker levels and greater BMD, BMC, muscle mass, and strength than would those receiving a placebo supplement.

Subjects and study design

From October 2019 to March 2020, overweight, or obese adults from Nakhon Pathom, Thailand were recruited through an advertisement. Written informed consents were obtained from all participants. The Kasetsart University Research Ethics Committee approved the study protocol (no. COA61/084), which was carried out in accordance with the Helsinki Declaration. A total of 33 females aged 41.5±8.7 years who suffered from overweight or obesity [body mass index (BMI): 27.8±2.9 kg/m², body fat percentage (%BF): 41.4±4.3%] were eligible. Among these participants, 57.6% had hypercholesterolemia and 27.3% had hypertriglyceridemia. The participants were inactive and had maintained their weight within a range of 3 kg for at least 6 months before the study. Participants who had a history of type 2 diabetes, peripheral and coronary artery diseases, and uncontrolled hypertension, or were taking chronic-use medications known to affect energy homeostasis were excluded from the study. Participants with diagnostic hypothyroidism or high liver enzymes were also excluded.

To reduce experimental bias, a randomized, placebo-controlled study was conducted with participants blinded to the test results. The sample size was determined by the ability to detect a large effect (1.3), according to a previous report by Solomon et al. (2008). We determined that 80% power was required at a significance level of 0.05. Hence, each group required at least 11 participants to complete the study, as shown in the study flowchart presented in Fig. 1. After initial screening, the participants were matched according to age, BMI, and %BF and then assigned randomly to one of three groups: (1) a placebo-controlled group (CON, n=11); (2) an Eri-PUFA ingestion group (ERI, n=11); or (3) a CCT+ERI group (n=11).

Figure 1. CONSORT flow diagram. CON, control group; ERI, Eri-polyunsaturated fatty acid (PUFA) ingestion; CCT+ERI, combined concurrent training and Eri-PUFA ingestion.

To ensure that the participants in the CON group were unaware of their group assignment, they followed the same study procedures as those in the intervention groups. Each group was instructed to consume a snack bar twice daily. The snack bars were identical in appearance, coloration, and odor. The snack bars contained 5 g of Eri silkworm pupae (approximately 2.5 g of linolenic acid) (European Food Safety Authority, 2009), whereas the placebo bars contained no Eri silkworm pupae. Only the CCT+ERI group engaged in concurrent training three times per week for 60 min under supervision. Participants were instructed to maintain their routine physical activity levels throughout the study. They were also instructed to maintain the same level of food consumption throughout the duration of the study. The dietary intake of the subjects was recorded by monitoring their nutrition logs. Before beginning an exercise program at Sports Science Fitness, participants completed baseline measurements over a 1-week period. At baseline and after the 8-week intervention, blood samples were collected, and anthropometric parameters, body composition, BMD and BMC, and muscle strength were measured.

Preparation of the Eri snack bar

Kasetsart Agricultural and Agro-Industrial Product Improvement Institute of Kasetsart University (Nakhon Pathom, Thailand) provided the Eri snack bars. The snack bars were formulated to contain 19 g of carbohydrates, 3 g of protein, and 5 g of fat with identical macronutrient profiles. Each bar contained linolenic acid (4.80 g/100 g), leucine (3.77 mg/100 g), isoleucine (2.22 mg/100 g), and valine (2.90 mg/100 g), as well as a total of 26.67 g/ 100 g of sugar. Table 1 shows the nutritional information and percentage of total fatty acids for the Eri snack bars. The placebo contained the same number of calories as the Eri snack bar but no silkworm pupae.

Table 1 . Nutritional information and total fatty acid percentage of the Eri snack bars.

	Amount per serving
Calories from fat (kcal)	40
Total fat (g)	5
Saturated fat (g)	2
Saturated fatty acid
Steric acid (%)	4.7
Arachidic acid (%)	0.7
Polyunsaturated fat (g)	3
Polyunsaturated fatty acid
Palmitoleic acid (%)	0.8
Oleic acid (%)	11.3
Linoleic acid (%)	5.8
Linolenic acid (%)	48.0
Cholesterol (mg)	<5
Protein (g)	3
Carbohydrate (g)	19
Dietary fiber (g)	1
Sugars (g)	8
Sodium (mg)	60

Serving size=1 bar (30 g, 130 kcal)..

Concurrent training program

The training regimen included three 60-min sessions per week. After a 10-min warm-up with stretching, participants performed 40 min of aerobic and resistance exercises (REs), followed by a 10-min cooldown with stretching. The training program was developed based on reviewed studies (Bartlett et al., 2011; Gibala et al., 2012; Kang and Ratamess, 2014). The content validity of the training program was good, with an index of item-objective congruence of 0.80. Each training session was supervised by experienced trainers. A HR monitor (H10, Polar Electro Inc.) was used to monitor the intensity of the exercise to ensure consistency and safety. During the 10-min warm-up and cooldown phases, participants stretched the major muscle groups. Each stretch was held for 15 to 30 s to the point of discomfort and then repeated three times.

During each concurrent exercise session, AE was performed first, followed by a 30-s rest period before RE was performed. The AE consisted of low-impact movement patterns performed for 4 min. The prescribed movement pattern in this study was based on our previous research (Klomklorm et al., 2020). AE was performed at an intensity between 70% and 80% of the participants’ heart rate reserve.

REs were performed using an elastic band and body weight. For each exercise, participants were instructed to utilize their full range of motion. During each RE session, participants completed two REs for two sets of 8∼12 repetitions. Each set and exercise were separated by a 30-s active recovery period. The participants were instructed to exhale during the concentric phase and inhale during the eccentric phase of the exercises, which they performed at their own pace. Prior to the exercise sessions, detailed instructions were provided despite all participants having prior RT experience.

Measurements

Anthropometric, body composition, and bone densitometry measurements: Body weight was measured using an electronic scale (Filizzola PL 150, Filizzola^Ⓡ Ltda). BMI was calculated by dividing body weight by height squared. Body composition and bone densitometry were measured using dual-energy X-ray absorptiometry (Lunar iDXA V17 software, GE Healthcare). The outcome variables included fat-free mass, fat mass, %BF, and bone mass for the entire body. In addition, the lumbar spine (L1∼L4 vertebrae) and hip (femoral neck and trochanter) were evaluated for BMD and BMC. Measurements were performed in a temperature-controlled room according to the manufacturer’s recommendations. All analyses were conducted by the same investigator to minimize interobserver variation. The coefficients of variation for repeated measurements at our laboratory were 0.5% for fat-free mass, 3% for fat mass, 0.5% for whole-body BMD, and 0.7% for BMD of the lumbar spine and hip.

Muscle strength measurements: The one repetition maximum (1RM) was determined using an indirect method involving the leg press followed by bench presses. The warm-up consisted of one set of 10 repetitions with a load light enough that the participants could perform 12 to 15 repetitions. If the participant completed more than 10 repetitions, the load was increased by 30 lb for the leg press and 10 lb for the bench press. Each attempt was separated by 3 min. The 1RM workload of each participant was determined based on the loads and repetitions they could perform using the 1RM table. Participants were instructed to control the eccentric and concentric movement speeds but not the cadence. All measurements were supervised by two qualified strength and conditioning professionals.

Blood samples and analysis: To minimize any acute effects of exercise, blood samples were collected under standardized fasting conditions (12 h) in the morning between 7:00 and 9:00 am at baseline and 7 days after the last training session. Blood was collected in a vacuum-sealed test tube and centrifuged for 15 min at 1,500 g and 4°C. Plasma aliquots and serum samples were subsequently frozen at −80°C prior to analysis. Plasma TNF-α concentrations were determined using an enzyme-linked immunosorbent assay (ELISA, Cayman Chemical Co.) according to the manufacturer’s instructions. The concentration of hs-CRP was determined using an immunophelometric assay (BioTécnica Indústria e Comércio Ltda.). Leptin concentrations were determined using an ELISA (DuoSetÒ, R&D Systems). Complete blood count analysis was performed using a Beckman Coulter Gen-S automated analyzer (Beckman Coulter UK Ltd.) within 2 h. Monocyte/lymphocyte, neutrophil/lymphocyte, and platelet/ lymphocyte counts were used to calculate the MLR, NLR, and PLR, respectively.

Dietary assessment: Before and after the 8-week intervention, participants were required to complete a 3-day food diary (Yang et al., 2010) to determine whether there were differences in dietary intake throughout the study period. Participants were instructed to record their diet on two weekdays and one weekend day. Energy and macronutrient intake analysis was performed using INMUCAL V.3 software (Institute of Nutrition, Mahidol University, Nakhon Pathom, Thailand).

Statistical analyses

Data were presented as means and standard deviations. The normality of the data was determined using the Shapiro-Wilk test. A one-way analysis of variance (ANOVA) was used to compare baseline measurements between each group, whereas a two-way ANOVA with repeated measures [group (CON, ERI, and CCT+ERI)×time (baseline and after 8 weeks of intervention)] was used to determine the combined effects of concurrent training and Eri-PUFA supplementation and time on the dependent variables. Post-hoc comparisons were conducted using univariate analysis if a significant interaction or main effect was observed. For the two-way ANOVA, an effect size (ES) analysis was conducted using eta-squared (η²), with values 0.01, 0.06, and 0.14 indicating small, moderate, and large ES, respectively. Statistical significance was set at P<0.05. Statistical analyses were conducted using SPSS software 26.0 for Windows (IBM Corp.).

Daily energy intake and physical activity levels

The average amount of energy consumed by each group during the experiment did not differ significantly. The CON, ERI, and CCT+ERI groups had mean energy intakes of 1,797±279, 1,868±377, and 1,859±223 kcal/d at baseline and 1,858±285, 1,891±431, and 1,785±508 kcal/d at the end of the interventions, respectively. In all groups, carbohydrates, fat, and protein accounted for approximately 51%, 30%, and 19% of the daily energy intake, respectively (Table 2). These percentages remained constant both before and after the intervention. No significant changes in routine physical activity levels were observed over time, and no differences were noted between the groups. The exercise attendance rate in the CCT+ERI group was 85.6±4.8%. During the training period, participants completed 99.8±2.6% of the prescribed exercise duration and exercised at 96.7±7.2% of the prescribed exercise intensity. Throughout the study, Eri silkworm pupae did not cause any obvious adverse effects.

Table 2 . Dietary intake of participants at baseline and after 8 weeks of experimental study, excluding the supplement ingested.

	CON			ERI			CCT+ERI		Time effect η2 (P-value)	Group×time interaction η2 (P-value)
	Baseline	Post-test		Baseline	Post-test		Baseline	Post-test
Energy intake (kcal/d)	1,797±279	1,858±285		1,868±377	1,891±431		1,859±223	1,785±508	0.009 (0.614)	0.093 (0.242)
Energy intake (kcal/d/kg BW)	26.1±5.5	26.7±5.1		29.1±4.3	29.4±4.9		25.1±2.7	23.7±5.0	0.018 (0.470)	0.102 (0.210)
Protein (g)	86.7±18.7	94.9±21.9		82.0±18.5	81.3±24.4		92.4±17.5	84.0±23.0	0.000 (0.911)	0.117 (0.163)
Carbohydrate (g)	235.0±38.6	247.6±34.7		236.2±66.7	243.9±73.4		222.3±46.9	216.8±93.3	0.008 (0.642)	0.017 (0.252)
Fat (g)	56.8±13.6	53.6±13.1		66.1±13.3	64.6±21.7		66.6±10.6	62.6±11.7	0.075 (0.136)	0.010 (0.868)
Saturated fat (g)	29.5±7.7	31.1±7.2		30.4±4.3	33.8±10.8		34.9±3.6	30.4±6.0	0.000 (0.916)	0.170 (0.068)
Unsaturated fat (g)	30.0±9.0	29.4±7.0		32.1±8.1	30.8±11.8		31.7±9.2	29.8±7.9	0.030 (0.351)	0.005 (0.928)

Data are expressed as mean±SD (n=11)..

CON, control group; ERI, Eri-polyunsaturated fatty acid (PUFA) ingestion; CCT+ERI, concurrent training and Eri-PUFA ingestion; BW, body weight..

Body composition and bone densitometry

Table 3 shows the body composition and bone densitometry at baseline and after the intervention. The characteristics of the participants did not differ significantly between the groups. Following the intervention, no significant interaction between group and time was observed for body weight, BMI, fat-free mass, or fat mass.

Table 3 . Body composition and bone densitometry at baseline and after the intervention.

	CON			ERI			CCT+ERI		Time effect η2 (P-value)	Group×time interaction η2 (P-value)
	Baseline	Post-test		Baseline	Post-test		Baseline	Post-test
Body weight (kg)	70.2±11.1	70.7±11.6		64.0±7.6	64.1±7.8		73.7±6.9	72.2±6.4	0.005 (0.701)	0.031 (0.628)
Body mass index (kg/m2)	28.3±3.2	28.4±3.0		26.5±2.9	26.4±2.9		29.1±3.0	28.4±2.9	0.040 (0.270)	0.051 (0.457)
Fat-free mass (kg)	41.1±3.6	41.6±4.3		38.7±5.3	38.6±5.5		44.7±6.8	44.4±6.9	0.000 (0.959)	0.046 (0.497)
Arm mass (kg)	4.4±0.4	4.4±0.5		4.0±0.8	4.1±0.8		4.6±1.1	4.6±1.2	0.009 (0.601)	0.008 (0.888)
Leg mass (kg)	14.0±1.6	14.0±1.9		13.0±1.6	13.1±1.7		15.4±2.4	15.7±2.4	0.022 (0.415)	0.147 (0.093)
Trunk mass (kg)	17.4±1.9	17.8±2.1		16.7±2.7	16.7±2.8		19.0±3.3	18.7±3.1	0.001 (0.874)	0.099 (0.211)
Fat mass (kg)	29.0±7.4	29.4±7.9		27.1±5.0	25.8±3.5		29.6±4.0	28.3±4.7	0.024 (0.400)	0.031 (0.623)
Total BMC (g)	2,231.5±196.9	2,244.3±186.8		2,127.1±299.6	2,138.5±300.5		2,288.2±348.0	2,312.9±341.5**	0.372 (0.000)1)	0.074 (0.313)
Total BMD (g/cm2)	1.2±0.1	1.2±0.1		1.2±0.1	1.2±0.1		1.2±0.1	1.2±0.1	0.005 (0.694)	0.055 (0.430)
Lumbar spine BMC (g)	59.0±8.3	58.4±7.6		60.2±9.3	60.5±9.4		56.6±15.5	60.0±11.5	0.059 (0.180)	0.157 (0.078)
Lumbar spine BMD (g/cm2)	1.2±0.1	1.2±0.1		1.3±0.1	1.3±0.1		1.2±0.2	1.2±0.1**†‡	0.157 (0.025)1)	0.286 (0.006)1)
Lumbar spine T-score	0.9±1.2	0.9±1.1		1.2±0.1	1.2±0.1		0.7±1.3	0.7±1.3	0.036 (0.300)	0.037 (0.567)
Hip BMC (g)	29.7±4.6	29.3±3.5		28.2±3.4	28.1±3.5		29.3±4.1	31.0±3.7**	0.213 (0.027)1)	0.038 (0.282)
Hip BMD (g/cm2)	1.0±0.1	1.1±0.1		1.0±0.1	1.0±0.1		1.0±0.1	1.1±0.1**	0.248 (0.004)1)	0.144 (0.097)
Hip T-score	0.7±1.1	0.7±1.1		0.5±0.8	0.6±0.8		0.5±0.9	0.6±0.9**	0.331 (0.001)1)	0.048 (0.478)

Data are expressed as mean±SD (n=11)..

¹⁾η²≥0.14..

**P<0.01 for within-group comparison (baseline vs. after the intervention)..

P<0.05 for ^†between-group comparison and ^‡a comparison to the CON..

CON, control group; ERI, Eri-polyunsaturated fatty acid (PUFA) ingestion; CCT+ERI, combined concurrent training and Eri-PUFA ingestion; BMC, bone mineral content; BMD, bone mineral density..

For bone densitometry, no significant differences in BMD or BMC were noted in the CON or ERI groups following the intervention. For lumbar spine BMD, a two-way ANOVA with a large η² ES revealed interactions between group and time [F_(2,30)=6.022, P=0.006, η²=0.286]. Pairwise post-hoc tests revealed that only the CCT+ERI group experienced a significant increase in lumbar spine BMD (5.1%, P<0.01) after the intervention. Moreover, the CCT+ERI group had a greater lumbar spine BMD than the CON group (P<0.05). Furthermore, the main effects analysis revealed that time had a significant effect on total BMC [F_(1,30)=17.738, P=0.000, η²=0.372], hip BMC [F_(1,30)=4.064, P=0.027, η²=0.213], hip BMC [F_(1,30)= 9.914, P=0.040, η²=0.248], and hip T-score [F_(1,30)= 14.830, P=0.001, η²=0.331]. Pairwise post-hoc tests revealed that only the CCT+ERI group had a significant increase in total BMC (1.2%, P<0.01), hip BMC (6.2%, P<0.01), hip BMC (5%, P<0.01), and hip T-score (8.5%, P<0.01) after the intervention. However, no significant differences in total BMC, hip BMC, hip BMC, or hip T-score were observed between the groups (Table 3).

Upper- and lower-body muscle strengths

No significant differences in upper- or lower-body muscle strengths were found following the interventions in the CON and ERI groups. The two-way ANOVA with a large η² ES revealed interactions between group and time for relative bench press strength [F_(2,30)=3.807, P=0.034, η²=0.202]. Pairwise post-hoc tests revealed that only the CCT+ERI group experienced a significant increase in relative bench press strength (16.9%, P<0.01) after the intervention. Moreover, the CCT+ERI group had a greater relative bench press strength than the ERI group (P< 0.05). Furthermore, the main effects analysis revealed that time had a significant effect on the 1RM bench press [F_(1,30)=8.816, P=0.006, η²=0.227]. Pairwise post-hoc tests revealed that after the 8-week intervention, only the CCT+ERI group showed a significantly increase in 1RM bench press (14.6%, P<0.01; Table 4).

Table 4 . Upper- and lower-body muscle strength at baseline and after the intervention.

	CON			ERI			CCT+ERI		Time effect η2 (P-value)	Group×time interaction η2 (P-value)
	Baseline	Post-test		Baseline	Post-test		Baseline	Post-test
Bench press 1RM (kg)	24.2±2.1	26.5±4.5		23.9±4.9	24.0±4.3		25.1±8.3	28.4±8.1**	0.227 (0.006)1)	0.109 (0.178)
Bench press 1RM (kg/BW)	0.39±0.06	0.41±0.08		0.36±0.07	0.36±0.07		0.35±0.09	0.40±0.09**	0.216 (0.017)1)†‡	0.202 (0.034)1)
Leg press 1RM (kg)	233.5±38.9	233.9±58		193.7±35.6	183.5±44.5		227.5±53.5	237.6±39.8	0.000 (0.989)	0.023 (0.702)
Leg press 1RM (kg/BW)	3.4±0.5	3.4±0.9		3.0±0.5	2.8±0.6		3.1±0.7	3.3±0.6	0.001 (0.861)	0.053 (0.445)

Data are expressed as mean±SD (n=11)..

¹⁾η²≥0.14..

**P<0.01 for within-group comparison (baseline vs. after the intervention)..

P<0.05 to ^†between-group comparison and ^‡a comparison to the CON..

CON, control group; ERI, Eri-polyunsaturated fatty acid (PUFA) ingestion; CCT+ERI, combined concurrent training and Eri-PUFA ingestion; 1RM, one repetition maximum; BW, body weight..

Inflammatory markers and leptin concentration

Fig. 2 shows the results for inflammatory markers and leptin concentrations at baseline and after the intervention. The CON group showed no significant differences in MLR, NLR, PLR, TNF-α, hs-CRP, or leptin concentrations following the intervention. The two-way ANOVA with a large η² ES revealed interactions between group and time for MLR [F_(2,30)=6.875, P=0.003, η²=0.314] and TNF-α concentrations [F_(2,30)=4.568, P=0.019, η²= 0.233]. Pairwise post-hoc tests revealed that the ERI and CCT+ERI groups showed a significant decrease in the MLR (−25%, P<0.01 and −21.4%, P<0.05, respectively) and TNF-α concentrations (−21.6%, P<0.05 and −19.4%, P<0.05, respectively) after the intervention. In addition, the ERI group had lower MLR (P<0.05) and TNF-α (P<0.05) concentrations than the CON group, whereas the CCT+ERI group had lower TNF-α (P<0.05) concentrations than the CON group. Furthermore, the main effects analysis revealed that time had a significant effect on NLR [F_(1,30)=4.881, P=0.015, η²=0.246] and hs-CRP concentrations [F_(1,30)=6.325, P=0.017, η²= 0.174]. Pairwise post-hoc tests revealed that after the 8-week intervention, only the CCT+ERI group showed a significant decrease in NLR (−16.4%, P<0.05), whereas only the ERI group showed a significant decrease in hs-CRP concentrations (−32.2%, P<0.01). However, no significant differences were observed between the groups (Fig. 2).

Figure 2. Comparison of the monocyte/lymphocyte ratio (A), neutrophil/lymphocyte ratio (B), platelet/lymphocyte ratio (C), tumor necrosis factor-alpha (TNF-α) levels (D), hs-C-reactive protein (CRP) levels (E), and leptin levels (F) at baseline and after the intervention. Data are expressed as mean±SD (n=11). *P<0.05 and **P<0.01 for within-group comparison (baseline vs. after the intervention). ^†P<0.05 and ^††P<0.01 for between-group comparison. CON, control group; ERI, Eri-polyunsaturated fatty acid (PUFA) ingestion; CCT+ERI, combined concurrent training and Eri-PUFA ingestion.

We earlier hypothesized that combining concurrent training with Eri-PUFA supplementation would significantly increase BMD and BMC, muscle mass, and strength, as well as significantly decrease inflammatory biomarkers in overweight and obese adults. The current study demonstrated that combining concurrent training with the Eri-PUFA supplement promoted greater lumbar spine BMD and lesser inflammation than did the placebo control condition. Although Eri-PUFA supplementation had no effect on BMC or BMD, it did reduce inflammation, with significant differences observed when compared to the placebo control condition. In addition, our findings revealed that concurrent training and Eri-PUFA consumption had a positive effect on upper-body but not lower-body muscular strength or muscle mass. For overweight and obese individuals, increased BMD and muscular strength and decreased inflammatory markers may be beneficial in preventing the development of osteoporosis and sarcopenic obesity.

The effects of concurrent training and Eri-PUFA supplementation on BMD and BMC remain unclear. A majority of the studies conducted to date have focused on either concurrent training (Svendsen et al., 1993; Lester et al., 2009; Marques et al., 2011) or PUFA supplementation (Mangano et al., 2013). A high ratio of PUFA to saturated fatty acid consumption has been reported to improve BMD. PUFA is abundant in Eri silkworm pupae, representing approximately 70% of total fatty acids (Longvah et al., 2012). Our findings indicate that the combined intervention is more effective than either Eri-PUFA supplementation alone or the placebo condition. Following the 8-week intervention, the combined intervention promoted a significant increase in lumbar spine BMD (5.1%). However, Eri-PUFA supplementation had no effect on BMC or BMD when administered alone. Our findings contradict those presented in previous reports that PUFA consumption increases BMD (Martin-Bautista et al., 2010; Salari Sharif et al., 2010). A previous study suggested that total PUFA intake was modestly associated with greater BMD via an increase in bone turnover, particularly decreased bone resorption. Before changes in BMD become noticeable, the optimal duration of total PUFA intake should be at least 6 months (Mangano et al., 2013). Because our intervention lasted only 8 weeks, it is possible that their sensitivity to Eri-PUFA supplementation was insufficient in the short term.

Physical activity appears to directly and indirectly affect all bone cells, as well as many aspects of bone remodeling. Weight-bearing and lower-limb exercises in the concurrent exercise program, such as side-taps, leg curls, knee-ups, and side-to-side rocking in the AE session and squats and lunges in the RE session, may induce changes in peak strain energy (Martelli et al., 2014) that are likely to stimulate bone formation at these skeletal sites. Previous studies on weight-bearing and lower-limb exercise interventions in older adults (Daly et al., 2020) and postmenopausal women (Kelley et al., 2002) have also reported increases in femoral neck and lumbar spine BMD, which is consistent with our findings. Furthermore, although our intervention was only 8 weeks long, the optimal duration of a single exercise session should be longer than 6 months before changes in BMD become noticeable (Ma et al., 2013). It is possible that our participants were sedentary, which causes bones to become more sensitive to mechanical loading. Individuals who change their lifestyle show a significantly higher BMD during exercise compared to those who make no changes.

The immunosuppressive effects of concurrent exercise or PUFA consumption have been documented previously. The current study found that MLR and TNF-α concentrations were decreased in the ERI (−25% and −21.6%, respectively) and CCT+ERI groups (−21.4% and −19.4%, respectively) without a change in body weight or BMI. Although notable changes in hs-CRP levels were observed after 8 weeks of Eri-PUFA consumption, no differences were found between the groups. It is possible that their sensitivity to short-term Eri-PUFA supplementation was insufficient. Our findings indicated that both PUFA consumption alone and concurrent training and Eri-PUFA ingestion reduced inflammatory markers such as MLR and TNF-α in obese and overweight adults. Similarly, previous studies have demonstrated that postmenopausal women (Tartibian et al., 2011) and patients with metabolic syndrome (Ortega et al., 2016) had reduced levels of proinflammatory cytokines (TNF-α and CRP) after a combination of exercise training and PUFA supplementation. Furthermore, consuming PUFA on its own has direct anti-inflammatory effects (Kelly et al., 2013). PUFA supplementation had potentially beneficial effects on TNF-α (Guebre-Egziabher et al., 2008) and was linked to lower adipose tissue inflammation (Kalupahana et al., 2010).

Epidemiological studies have indicated that the prevalence of fractures increases as BMI progresses from overweight to obese (Gonnelli et al., 2014). Poor bone quality caused by obesity-related systemic inflammation contributes to the development of osteoporosis and increased fracture risk (Das, 2001). The reduction in inflammatory markers caused by the combined intervention may thus be attributed to improvements in BMD, especially in the lumbar spine of obese adults. Reduced MLR and TNF-α concentrations may account for the increased lumbar BMD in the CCT+ERI group.

The immune system and immune factors play vital roles in osteoporosis progression. In fact, evidence suggested that MLR could be a reliable, inexpensive, and novel potential osteoporosis predictor (Gao et al., 2019). Although the exact mechanism by which osteoporosis increases MLR is unknown, patients with osteoporosis had a higher MLR than healthy subjects (Gao et al., 2019). Furthermore, TNF-α is linked to increased bone resorption and decreased bone formation (Osta et al., 2014; Kim et al., 2015). However, the current study showed that the relationship between increased BMD and decreased MLR and TNF-α concentrations was not statistically significant. The anti-osteoporosis mechanism of the combined intervention should be different. These findings require additional research with a larger sample size. Another possible mechanism may involve elevated levels of estrogen, osteocalcin, and calcitonin, along with decreased levels of parathyroid hormone and prostaglandin E2 (Tartibian et al., 2011).

Although a notable increase in upper-body muscle strength (16.9% increase) was observed after the combined intervention, no changes in lower-body muscle strength or muscle mass were observed. However, consuming Eri-PUFA alone did not improve these variables. These findings imply that the combined intervention may be effective in preserving muscle strength, particularly upper-body muscle strength. Overweight and obese adults may be at increased risk for sarcopenia due to the hyperinflammation state associated with excess BF. Increased levels of TNF‐a have been shown to inhibit the expression of anabolic hormones and activate catabolic signaling for death cell receptors on myocytes, promoting an increase in apoptosis (Li et al., 2022).

The current study discovered that lowering TNF-α levels can contribute to muscle strength gain during combined intervention, which is consistent with previous findings (Stewart et al., 2007; Libardi et al., 2012; Ruangthai and Phoemsapthawee, 2019). Although neural adaptations were not assessed, evidence has suggested that RT in concurrent training programs promotes neural adaptations (Behm, 1995), which may have contributed to the improvements in upper-body muscular strength observed in our study. Our findings, however, revealed that CCT+ERI did not improve lower-body muscle strength. Several factors, including session volume, intensity, and frequency, participant training status, and intervention duration, can explain this disparity. Furthermore, Eri-PUFA alone had no effect on muscular strength. Hence, the synergistic effects of Eri-PUFA supplementation and concurrent training on strength and muscle mass might have gone unnoticed.

The strengths of our study include the randomized controlled trial design, the high adherence to exercise regimens, and the comprehensive evaluation of BMD, inflammatory markers, and leptin. However, the current study is limited by the relatively small sample size. Consequently, it might be difficult to generalize our findings to other populations. The next critical step appears to be isolating the effects of each PUFA. Finally, our study design lacked a concurrent training group, indicating the absence of trained control groups.

The present study concludes that 8 weeks of combining concurrent training with Eri-PUFA intake increases BMD in the lumbar spine and decreases inflammatory markers. Although Eri-PUFA consumption has no direct effect on BMD, reducing inflammatory markers may enhance the effects of concurrent training on BMD. These findings suggest that concurrent training and Eri-PUFA consumption may improve bone quality and prevent the development of osteoporosis in obese or overweight middle-aged adults.

The authors would like to thank Dr. Waraporn Apiwatanapiwat, Miss Prapassorn Rukthaworn, and Miss Phornpimol Janchai of the Kasetsart Agricultural and Agro-Industrial Product Improvement Institute for the product preparation and analysis. We would also like to thank Dr. Supannika Kawvised of the Radiological Technology School, Faculty of Health Science Technology, HRH Princess Chulabhorn College of Medical Science, Chulabhorn Royal Academy for the BMD analysis. We would also like to thank the participants for their enthusiastic participation in this study. Finally, we would like to express our gratitude to the Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, for determining blood chemistry measurements.

This research was funded by the Thailand Research Fund, Thailand Science Research and Innovation (Grant no. RDG62T0041/2019).

The authors declare no conflict of interests.

Concept and design: JP, PV. Analysis and interpretation: JP, AK, RR. Data collection: AK, US, PT, PP. Writing the article: JP. Critical revision of the article: JP. Final approval of the article: all authors. Statistical analysis: JP, AK. Obtained funding: PV. Overall responsibility: JP.