ABSTRACT OBJECTIVE Through dynamic means, etiological factors, including chronic inflammation and insulin resistance have the potential to perpetuate metabolic incidences such as type 2

diabetes and obesity. Abatement of such syndromes can be achieved by complex mechanisms initiated through bioactive compounds such as polyphenols derived from fruits. Using a whole-fruit

approach, the effects of dietary red raspberry, which is rich in polyphenols, on inflammatory responses and insulin resistance in the skeletal muscles of _Mus musculus_ were studied along

with the potential role of AMP-activated protein kinase (AMPK) to act as a key mediator. SUBJECTS Wild-type (WT) mice and mice deficient in the catalytic subunit (α1) of AMPK (AMPKα1−/−)

mitochondrial biogenesis in the skeletal muscle of WT mice, but not AMPKα1−/− mice. CONCLUSIONS AMPKα1 is an important mediator for the beneficial effects of raspberry through alleviating

inflammatory responses and sensitizing insulin signaling in skeletal muscle of HFD-fed mice. SIMILAR CONTENT BEING VIEWED BY OTHERS ABSCISIC ACID ENRICHED FIG EXTRACT PROMOTES INSULIN

SENSITIVITY BY DECREASING SYSTEMIC INFLAMMATION AND ACTIVATING LANCL2 IN SKELETAL MUSCLE Article Open access 26 June 2020 SHORT-TERM _CUDRANIA TRICUSPIDATA_ FRUIT VINEGAR ADMINISTRATION

ATTENUATES OBESITY IN HIGH-FAT DIET-FED MICE BY IMPROVING FAT ACCUMULATION AND METABOLIC PARAMETERS Article Open access 03 December 2020 BERBERINE ATTENUATES OBESITY-INDUCED SKELETAL MUSCLE

ATROPHY VIA REGULATION OF FUNDC1 IN SKELETAL MUSCLE OF MICE Article Open access 10 February 2025 INTRODUCTION Red raspberry is widely recognized for its high levels of vitamin C and

bioactive polyphenols, including ellagitannins and anthocyanins, which have strong antioxidant capacities1. Several animal studies have shown that supplementation of raspberry extracts

exhibited beneficial effects for the prevention of obesity, inflammation and other metabolic diseases2, 3. However, the impacts of dietary raspberry fruit on skeletal muscle insulin

resistance and the underlying mechanisms remain largely unexplored. Obesity induces ectopic lipid accumulation and desensitizes insulin signaling in skeletal muscle, thus resulting in

systematic insulin resistance and type 2 diabetes4. AMP-activated protein kinase (AMPK) is a key sensor of energy status in skeletal muscle through the control of glucose and fatty acid

metabolism5. The structure of AMPK has been described as a heterotrimeric complex comprised of the catalytic α-subunit and the regulatory β- and γ- subunits6. Activation of AMPK prevents

obesity and associated metabolic diseases through the promotion of glucose utilization, fatty acid oxidation, and mitochondrial biogenesis in skeletal muscle6. Dietary polyphenols, such as

resveratrol, are strong activators of AMPK, which can then promote the browning of white adipose and subsequently alleviate obesity7. Due to the high levels of polyphenols found in the red

raspberry, it is postulated that AMPK plays an essential role in mediating the beneficial effects of red raspberry on metabolic health. The catalytic subunit of AMPK has 2 isoforms (α1 and

α2). Although there is a compensatory mechanism between these two isoforms, their expression shows tissue-specific patterns8, 9 with differential metabolic functions10, 11. The isoform α2 of

AMPK is indispensable for increased glucose uptake by skeletal muscle induced by 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and hypoxia5, 12, 13. Meanwhile, the AMPKα1 isoform

can be activated during skeletal muscle contraction14 and at low caffeine concentrations10. Indeed, AMPKα1 also plays an essential role in myogenin expression and myogenesis15. Previous

studies in our lab have shown the dominant expression of AMPKα1 in satellite cells, which when deleted, impeded muscle regeneration after injury15. Deletion of AMPKα1 in macrophages during

the transition from a proinflammatory (M1) to an anti-inflammatory (M2) phenotype impairs the resolution of inflammation and muscle regeneration after injury16. Altogether, these studies

suggested that AMPKα1 could mediate the alleviation of insulin resistance and metabolic syndromes in skeletal muscle of obese mice consuming raspberry. Thus, we explored the influence of red

raspberry on insulin sensitivity and inflammatory responses in skeletal muscles, along with the potential role of AMPKα1 to act as a key mediator. MATERIALS AND METHODS ANIMAL AND

EXPERIMENTAL DESIGN R26Cre/AMPKα1fl/fl mice were generated through the cross-breeding of AMPKα1fl/fl mice (Stock No: 014141, Jackson Lab, Bar Harbor, Maine) with tamoxifen-inducible R26-Cre

mice (Stock No: 004847, Jackson Lab, Bar Harbor, Maine) at Washington State University. To induce the AMPKα1 knockout (AMPKα1−/−), 2-month-old male R26Cre/AMPKα1fl/fl mice were

intraperitoneally injected with tamoxifen (75 mg/kg body weight) for 4 continuous days17. AMPKα1fl/fl mice treated with tamoxifen were used as controls (Wild-type, WT). To minimize possible

confounding changes, dietary treatments started 3 days after the last tamoxifen injection15. All experimental procedures of animal use were performed according to the guidelines of National

Institutes of Health and approved by the Animal Use and Care Committee of Washington State University (Permit No. 04719). Twelve wild-type and AMPKα1−/− mice, respectively, were randomly

separated into two sub-groups and fed either a high-fat diet (HFD; 60% energy from fat, D12492; Research Diets, New Brunswick, NJ, USA) or a HFD diet supplemented with freeze-dried raspberry

(5% of dry feed weight, red raspberry powder). The concentration of the raspberry supplementation was determined by preliminary studies in our lab18. Raspberry powder was prepared as

previously described, which contains polyphenols at ~11 g gallic acid equivalent (GAE)/kg of dry weight, 4.24 ± 0.12% protein, 1.91 ± 0.03% fat, 0.81 ± 0.02% ash, 16.14 ± 0.45% moisture, and

the remaining to be mainly carbohydrates19. Mice were housed in a temperature-controlled environment (23 ± 2 °C, alternating 12-h light/dark cycle) with _ad libitum_ access to food and

water. Feed intake and body weights were monitored weekly until the mice were killed 10 weeks later. Samples of blood, the _Gastrocnemius_ muscle (GA), and the _Tibialis anterior_ muscle

(TA) were rapidly isolated. TA were fixed in 4% paraformaldehyde for sectioning and staining, and GA were rapidly frozen in liquid nitrogen and stored at −80 °C until further analyses.

HISTOCHEMICAL ANALYSES Paraffin-embedded TA muscle sections (5-μm thick) were rehydrated through a series of incubations in xylene and ethanol solutions, and then used for Masson trichrome

staining20. At least four fields per section and four sections per sample were randomly selected for quantification of fat area and collagen area using the Image J 1.46r software (National

Institutes of Health). The average data per biological sample were used for calculations. TOTAL TRIACYLGLYCEROL ANALYSES As previously described, total triacylglycerol determination was

performed using the Folch method20, 21. The frozen GA muscle was powdered under liquid nitrogen and a 30 mg sample was weighed. After adding 0.75 ml of chloroform-methanol 2:1 (v/v), the

samples were left at 4 °C for 48 h. Then, 187.5 µl 0.9% NaCl was added and the mixture was kept at room temperature overnight and then centrifuged at 10,000 × _g_ for 5 min at 4 °C. The

lower phase (20 µl) was transferred into a fresh tube and evaporated until dry for 1 h under the hood. Total triacylglycerols were measured using a kit from Sigma following the

manufacturer’s instructions (cat. no. TR0100). The results were displayed by dividing the total triacylglycerol content by the initial muscle powder weight. QUANTITATIVE REAL-TIME PCR

(QRT-PCR) ANALYSES Total RNA was isolated using TRIzol reagent (Sigma, Saint Louis, MO, USA), followed by reverse-transcription to cDNA using the iScriptTM cDNA Synthesis Kit (Bio-Rad,

Hercules, CA, USA). The mRNA levels were measured by qRT-PCR carried out by the CFX RT-PCR detection system (Bio-Rad). After normalization to _18s_ rRNA content, relative mRNA expression was

determined using the method of 2-ΔΔCt22. Table 1 shows the primer sequences. IMMUNOBLOTTING ANALYSES Immunoblotting analyses were performed as previously described using the Odyssey

Infrared Image System (LI-COR Biosciences, Lincoln, NE, USA)15. Band densities of target proteins were normalized to β-tubulin content. The following antibodies were purchased from Cell

Signaling (Danver, MA, USA): AMPKα (no.2532), phospho-AMPKα at Thr172 (no. 2535), protein kinase B (AKT, no.9272), phospho-AKT at Ser473 (no. 9271), protein kinase C (PKCθ, no.13643),

phospho-PKCθ at Thr538 (no.9377), nuclear factor κB (NFκB) subunit p65 (no.8242), phospho-p65 at Ser536 (no.3033), c-Jun N-terminal kinases (JNK, no. 9252), phospho-JNK at Thr183/Tyr185

(no.9251) and cytochrome C (cyt C, no. 4280). IRDye 800CW goat anti-rabbit (no. 926-32211) and IRDye 680 goat anti-mouse (no. 926-68070) secondary antibodies were purchased from LI-COR

Biosciences (Lincoln, NE, USA). For use, primary antibodies were diluted 1: 1000 using 1× TBST buffer (137 mM Sodium Chloride, 20 mM Tris, 0.1% Tween-20, pH 7.6) with 5% BSA (Bovine Serum

Albumin) and secondary antibodies were diluted 1: 10,000 using TBST buffer. STATISTICAL ANALYSES Within each genotype, the data were analyzed using unpaired two-tailed Student’s _t_ test

using SAS 9.0 (SAS Institute Inc., Cary, NC, USA). All the data were found normally distributed. Results are expressed as mean ± s.d. A significant difference was considered as _P_ < 

0.05. RESULTS RASPBERRY SUPPLEMENTATION ACTIVATED AMPKΑ1 The content of total AMPKα in skeletal muscles was lower in AMPKα1−/− mice (Fig. 1a), which is consistent with successful AMPKα1

knockout induced by tamoxifen. Raspberry supplementation increased the level of p-AMPKα and the ratio of p-/t-AMPK in WT mice, while no difference was found in AMPKα1−/− mice with/without

raspberry (Fig. 1b). The lack of difference in AMPK phosphorylation and ratio of p-/t-AMPK in the absence of AMPKα1 suggests that raspberry supplementation did not activate AMPKα2. RASPBERRY

SUPPLEMENTATION REDUCED LIPID ACCUMULATION IN SKELETAL MUSCLES IN AN AMPKΑ1-DEPENDENT MANNER As described previously in our lab, there was no significant difference of average weekly food

intake between groups (_p_ > 0.05) and dietary raspberry reduced the body weight of wide type mice but not that of the AMPKα1−/− mice (_p_ < 0.01)23. The TA and GA muscle weights were

not altered through raspberry supplementation, nor by AMPK α1 deficiency (Fig. 2a, b). Intramuscular lipid accumulation contributes to obesity-induced insulin resistance by activating

stress-responsive serine kinases and then impeding the activity of downstream insulin signaling molecules such as AKT24, 25. The triacylglycerol content in the GA muscle was elevated due to

the HFD, but partially prevented by dietary raspberry in WT mice. For the AMPKα1 KO mice, no difference was found between HFD and HFD + RAS groups (Fig. 2c), supporting the mediatory role of

AMPK α1. Masson trichrome staining shows the areas of muscle cells in red, collagen in blue, and adipocytes as colorless. More intramuscular adipocytes in TA muscle were observed in the HFD

group compared to the HFD + RAS group of WT mice as shown in Fig. 2d. The areas of fat (Fig. 2e) and collagen (Fig. 2f) in muscle sections were quantified. Fat area was much smaller (_P_ 

< 0.01) in the HFD + RAS group compared to the HFD group of WT mice, consistent with the lower levels of triacylglycerols in the HFD + RAS group of WT mice as shown in Fig. 2c. Raspberry

supplementation also decreased the presence of connective tissues in WT mice. A tendency for a decrease in collagen area was seen in the HFD + RAS group of WT mice (_P_ < 0.10). For

AMPKα1−/− mice, no significant difference was exhibited for either fat and collagen areas. These data suggest that raspberry supplementation reduced lipid accumulation in skeletal muscle of

mice challenged with a HFD diet, a process mediated by AMPK α1. RASPBERRY SUPPLEMENTATION DECREASED THE INFLAMMATORY RESPONSE IN AN AMPKΑ1-DEPENDENT MANNER Ectopic lipid accumulation in

peripheral tissues frequently leads to chronic inflammation. Raspberry intake attenuated HFD-stimulated expression of _Tnfα_, _Il1β_, _Il6_, and _Il18_ in WT mice (Fig. 3a). However, this

beneficial role of raspberry supplementation was not present in AMPKα1−/− mice. Inflammatory responses are mediated by the activation of NF-κB (nuclear factor kappa B) and JNK/MAPK

pathways26,27,28. Protein p65 is a key component of the NF-κB pathway with obesity up-regulating its phosphorylation20. Although the total contents of p65 did not change, a much lower

phosphorylation level of p65 and a low phospho to total ratio of p65 (p-p65/t-p65) were detected in the HFD + RAS group of WT mice (Fig. 3b). In AMPKα1−/− mice, raspberry supplementation did

not reduce the phosphorylation level of p65. In addition, raspberry supplementation also decreased the phosphorylation level of JNK in WT mice (Fig. 3c). Although the total level of JNK and

the ratio of p-/t-JNK showed a decreasing tendency, changes were not significant. However, these benefits disappeared in AMPKα1−/− mice, showing the mediatory role of AMPKα1. RASPBERRY

IMPROVED INSULIN SENSITIVITY IN AN AMPKΑ1-DEPENDENT MANNER Previous studies in our laboratory have reported that raspberry supplementation increased glucose tolerance, and decreased lipids

and insulin levels in the serum of WT mice but not in AMPKα1−/− mice, which reflected improved insulin sensitivity by raspberry supplementation through regulating AMPKα123. Glucose

transporter 4 (GLUT4) is indispensable for whole-body glucose homeostasis and its deficiency leads to insulin resistance and ectopic lipid accumulation29, 30. Consistently raspberry

supplementations increased _Glut4_ mRNA and protein contents in WT mice but not in AMPKα1−/− mice (Fig. 4a, b). Because increased lipid accumulation and inflammation are correlated with

insulin resistance, insulin signaling pathways were further analyzed. In WT mice, the contents of PKCθ and its phosphorylation were down-regulated by 19.5% (_p_ < 0.1) and 27.5% (_p_ <

 0.01) in raspberry supplemented group, respectively. In the absence of AMPKα1, however, these differences disappeared (Fig. 4c). Although the total level of AKT was not different, its

phosphorylation was higher (_P_ < 0.01) in RAS supplemented WT mice when compared to those fed only HFD (Fig. 4c). Consequently, the HFD + RAS group of WT mice had a significantly higher

p-/t-AKT ratio (_P_ < 0.05). Ablation of AMPKα1 abolished these changes induced by raspberry supplementation. Therefore, AMPKα1 is required for the beneficial effects of raspberry on

insulin signaling in skeletal muscle of mice under the challenge of HFD. RASPBERRY PROMOTED MITOCHONDRIAL BIOGENESIS IN AN AMPKΑ1-DEPENDENT MANNER The mitochondria play an indispensable role

in cellular energy metabolism while its dysfunction in skeletal muscle is associated with decreased insulin sensitivity and the development of type 2 diabetes31. Raspberry supplementation

increased the protein level of cytochrome C (Cyt C) in skeletal muscle (_p_ < 0.01), suggesting increased contents of mitochondria (Fig. 5a). Meanwhile, the mRNA expression levels for

_Pgc1α_, _Nrf1_, and _Cpt1_ were up-regulated in the HFD + RAS group of WT mice (Fig. 5b). However, in AMPK α1−/− mice, no such differences were observed. The mRNA expression of _Cycs_ and

_Tfam_ did not differ between WT and AMPK α1−/− groups. In summary, increased mitochondrial biogenesis could be responsible for the reduced lipid accumulation elicited by raspberry

supplementation in WT mice challenged with HFD in an AMPKα1-dependent manner. DISCUSSION Obesity and associated chronic inflammations induce a state of insulin resistance in adipose tissue,

skeletal muscles, and the liver, which is indispensable for the development of type 2 diabetes32. Numerous pharmaceutical approaches aimed at preventing obesity and inflammation have shown

positive results, but with various side effects and risks33. Nutritional interventions have the advantage of being natural and safe, providing a more suitable alternative for long-term

therapy. Raspberries contain high amounts of polyphenols and other bioactive compounds and have been shown to have beneficial effects in treating obesity and metabolic diseases3, 34.

However, the effects of raspberry in insulin resistance of skeletal muscle and the mediatory role of AMPK have not been examined. Obesity induces ectopic lipid storage and inflammatory

response, accompanied by the secretion of proinflammatory cytokines such as TNFα, IL1β and IL631. The bioactive polyphenols in red raspberry occur primarily as ellagitannins and

anthocyanins, which have anti-inflammatory effects1, 35. In the current study, raspberry supplementation promoted insulin signaling, reduced lipid accumulation, and alleviated the

inflammatory response in skeletal muscle. These benefits disappeared in AMPKα1 knockout mice, which showed the indispensable role of AMPKα1 in mediating the beneficial effects of dietary

raspberry. Increased mitochondrial biogenesis in WT mice due to raspberry consumption could be a causative reason for these beneficial effects. Following AMPKα1 knockout,

raspberry-stimulated mitochondrial biogenesis disappeared, supporting the mediatory role of AMPKα1. AMPK is a promising drug target for preventing and treating obesity and associated

metabolic disease36. Increasing the activity of AMPK in skeletal muscles is associated with enhanced mitochondrial biogenesis and lipid oxidation7. The two catalytic α isoforms (α1 and α2)

of AMPK have different tissue expression patterns. AMPKα1 is widely expressed in all tissues while predominately in brain and adipose tissues, whereas both α1 and α2 isoforms are expressed

in skeletal muscles and the heart37. Their difference in subcellular localization and substrate specificity also suggest their differential roles in the regulation of metabolic processes8,

9. AMPK is normally activated in response to an energy-depleting state17. Due to allosteric activation by AMP and covalent activation by upstream kinases, AMPKα2 activation is more dependent

on AMP and energy depletion than the α1 isoform8, 38. Isoforms of AMPK are activated according to the intensity of exercise: low-intensity exercise preferentially activates the α1 isoform

while moderate intensity exercise preferentially activates the α2 isoform11. In obesity and insulin resistance models, endurance training (treadmill running) increased the activity of AMPKα1

but not the α2 isoform39, 40. The mediating role of caffeine (1,3,7-trimethylxanthine) on skeletal muscle metabolism is also achieved through AMPK; low concentrations (1 mM) of caffeine

predominantly activate AMPKα1 via an energy-independent manner while AMPKα2 was activated at high concentrations (3 mM) of caffeine, depending on energy depletion10. The polyphenols in red

raspberries, such as anthocyanins, activate AMPKα141, consistent with our observation in this study that dietary raspberry did not significantly activate AMPKα2 in the skeletal muscle of

obese mice. In conclusion, we found that raspberry supplementation reduced lipid accumulation, alleviated the inflammatory response, improved insulin sensitivity, and promoted mitochondrial

biogenesis in the skeletal muscle of HFD-fed mice. These beneficial effects depended on the indispensable mediator: AMPKα1. Further studies should focus on the signaling mechanisms of

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Google Scholar  Download references ACKNOWLEDGEMENTS This study was supported by grants from the National Institutes of Health (R01-HD067449 and R21-AG049976) and a grant from the National

Processed Raspberry Council to M.D. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Animal Sciences, Nutrigenomics and Growth Biology laboratory, Washington State University,

Pullman, WA, 99164, USA Liang Zhao, Tiande Zou, Noe Alberto Gomez, Bo Wang & Min Du * Jiangxi Province Key Laboratory of Animal Nutrition, College of Animal Science and Technology,

Jiangxi Agricultural University, Nanchang, Jiangxi, 330045, China Tiande Zou * School of Food Sciences, Washington State University, Pullman, WA, 99164, USA Mei-Jun Zhu * Beijing Advanced

Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing, 100194, China Min Du Authors * Liang

Zhao View author publications You can also search for this author inPubMed Google Scholar * Tiande Zou View author publications You can also search for this author inPubMed Google Scholar *

Noe Alberto Gomez View author publications You can also search for this author inPubMed Google Scholar * Bo Wang View author publications You can also search for this author inPubMed Google

Scholar * Mei-Jun Zhu View author publications You can also search for this author inPubMed Google Scholar * Min Du View author publications You can also search for this author inPubMed 

Google Scholar CONTRIBUTIONS L.Z. and M.D. designed the study and wrote the manuscript. L.Z. and T.Z. performed the experiments. L.Z., B.W., M.D. analyzed and interpreted the data. N.A.G,

M-J.Z. and M.D. revised the manuscript. CORRESPONDING AUTHOR Correspondence to Min Du. ETHICS DECLARATIONS CONFLICT OF INTEREST The authors declare that they have no conflict of interest.

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ARTICLE Zhao, L., Zou, T., Gomez, N.A. _et al._ Raspberry alleviates obesity-induced inflammation and insulin resistance in skeletal muscle through activation of AMP-activated protein kinase

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