Intermuscular and intramuscular adipose tissues: Bad vs. good adipose tissues

Abstract

Human studies of the influence of aging and other factors on intermuscular fat (INTMF) were reviewed. Intermuscular fat increased with weight loss, weight gain, or with no weight change with age in humans. An increase in INTMF represents a similar threat to type 2 diabetes and insulin resistance as does visceral adipose tissue (VAT). Studies of INTMF in animals covered topics such as quantitative deposition and genetic relationships with other fat depots. The relationship between leanness and higher proportions of INTMF fat in pigs was not observed in human studies and was not corroborated by other pig studies. In humans, changes in muscle mass, strength and quality are associated with INTMF accretion with aging. Gene expression profiling and intrinsic methylation differences in pigs demonstrated that INTMF and VAT are primarily associated with inflammatory and immune processes. It seems that in the pig and humans, INTMF and VAT share a similar pattern of distribution and a similar association of components dictating insulin sensitivity. Studies on intramuscular (IM) adipocyte development in meat animals were reviewed. Gene expression analysis and genetic analysis have identified candidate genes involved in IM adipocyte development. Intramuscular (IM) adipocyte development in human muscle is only seen during aging and some pathological circumstance. Several genetic links between human and meat animal adipogenesis have been identified. In pigs, the Lipin1 and Lipin 2 gene have strong genetic effects on IM accumulation. Lipin1 deficiency results in immature adipocyte development in human lipodystrophy. In humans, overexpression of Perilipin 2 (PLIN2) facilitates intramyocellular lipid accretion whereas in pigs PLIN2 gene expression is associated with IM deposition. Lipins and perilipins may influence intramuscular lipid regardless of species.

Keywords:

• adipocytes
• adipose depot physiology
• intramuscular adipose tissue
• intermuscular adipose tissue
• genetic markers
• regulation
• development
• metabolism
• growth

Introduction

By their very nature, problems associated with obesity, diabetes, and metabolic syndrome are being itemized, categorized, and legitimized by the amount of research efforts directed in elucidating resolutions to adverse symptoms associated with these and other metabolic dysfunctions.^1-5 Type I diabetes is known, in part, to be caused by a lack of insulin due to faulty production by β cells of the pancreas, autoantibody removal of insulin in circulation causing sequestration/inactivation, and inhibition or failure of insulin to bind to somatic cells such as skeletal muscle due to a decrease in insulin receptor number or an increase in anti-receptor antibodies binding to the insulin receptor.^6,7 Type II diabetes, metabolic syndrome, and obesity, however, are highly researched but not thoroughly resolved, and result in health issues that are becoming elevated to world-wide crises.^1-5 Resident in these issues are research efforts to determine cellular and molecular control of depot-specific adipocytes, interaction, and association of adipocytes to other somatic cells, disproportionate propensities of depot-specific adipocytes to produce adipokines that function to control numerous types of body cells, and adipocyte propensity to (seemingly) disassociate themselves from normal regulatory function/control during metabolic dysfunctions such as obesity—likely as a consequence of hypoxia in the adipose depot.^2-4,8-12

Adipocytes found in different anatomical adipose depots express divergent physiologies throughout one's lifespan.^9,10,13,14 The five traditional adipose depots in animals and humans are subcutaneous (SQF), visceral (VAT), intermuscular (INTMF), intramuscular (IM), and bone adipose depots. The SQF depot develops and fills with lipid first, followed by the VAT, INTMF, and IM adipose depots, and considerable research efforts are focused on depot differences in the structure, function and/or regulation of all cells contained within the depots.^4,9,10,14,15 In terms of anatomical location, considerable interest has been shown in the abdominal adipocytes of humans due to the possible production of adipokines that appear to function in physiologies such as control of satiety.^8-10 Issues associated with adipose depot include disruption of normal function of organs due to infiltration with lipid-filled adipocytes—for example, non-alcoholic fatty livers are not capable of functioning as properly as normal livers, and is likely a precursor of fatty infiltration into other organs/tissues.^9,16 An interest of this paper is on the specific differences in INTMF vs. IM adipose tissues.

Adipocytes associated with skeletal muscle include the INTMF adipose depot, which associates itself with available spaces in between skeletal muscles, and the IM adipose depot which includes all adipocytes interjecting themselves between and among viable skeletal muscle fibers in the skeletal muscle bed.^9,10,17,18 In meat animals, the IM adipose depot is termed marbling fat.^9,10,17-19 Early development of these two adipose depots occurs during embryogenesis, becomes distinct and committed in the early part of the third trimester in utero, becomes evident during rapid periods of postnatal growth, and slows in the INTMF adipose depot while increasing in the IM adipose depot during late adolescence.^17,19-21 Considerable knowledge exists on stem cell determination and commitment during in utero development, whereas relatively less is known about cell regulation in these two adipose depots during postnatal in vivo growth.^9,10,18-21

A considerable number of reviews are available directing attention to preadipocyte proliferation, conversion to adipocytes and adipocyte ability to conduct lipid metabolism, produce adipokines, and become refractory to systemic signals during periods of abnormal metabolism.^{4,6,8-11,14,15,17-21} Much of this knowledge was generated from studies utilizing cell lines, adipose tissue stromal vascular (SV) cell cultures, or adipose tissue slices–essentially piecing together purported mechanisms of regulation and/or marker expression during cellular conversion processes.^10,13,15,22

Knowledge pertaining to normal regulation, dysfunctional regulation, endocrine/adipokine production, and resistance of specific adipose depots to respond to metabolic signals is growing.^2,5-9 Integration of these concept areas to provide a mechanism with which to target for clinical remediation of the adverse symptoms of adipose tissue manifestations is lacking, however, possibly due to conflicting messages about adipose origins, the obese phenotype, its causes, and repercussions.^10-14 Indeed, it appears that the preponderance of evidence states that SQF adiposity supplies a measure of protection against metabolic dysregulation, whereas VAT is considered a source of dysregulation (especially insulin resistance).^2,15-23 However, this message is further muddied by new reports that demonstrate that truncal SQF adipose tissue may actually aid in the metabolic changes that occur with obesity as this depot supplies a greater percentage of adipose tissue compared with the VAT and that the protective effects of SQ fat is limited more to the gluteal femoral SQ depot (or the so-called “pear-shape”).^11,15,24-28 The protective action of gluteal femoral SQF seems to be further limited to the femoral subcutaneous region and not necessarily with the gluteal region in black South African women further confounding the issue with ethnicity differences.^{11,16,25,27,29-32} Indeed, even gender appears to have a profound influence on the responses by different adipose depots.^16,32-34 One mechanism that seems universal in human adipose depot regulation of adipose tissue is insulin, although, with as much variation within one species (human) one can only imagine the differences which exist between species. Knowledge of insulin receptivity, regulation—or lack thereof—and resilience in retaining normal adipose depot physiology in the face of metabolic disturbance is vitally needed.

For hundreds of years, domestic (meat) animals have been intensely selected for a variety of carcass and quality variables. One end point of this selection pressure is IM.³⁵ Meat animals such as beef cattle are fed a grain diet in a feedlot setting prior to slaughter in order to allow the IM to form.³⁶ This feeding regimen is costly, but provides consumers a meat product with adipose cells interspersed among skeletal muscle fibers in the final meat product resulting in a meat product that is flavorful and juicy. Alternate feeding regimes have been studied with little effect on IM.^37-40 Such interspersed groupings of adipocytes in the musculature bed of humans are rare, and only blatantly seen during some pathological circumstance, such as lipotrophic muscular atrophy. The focus of this review is to provide detailed information about INTMF vs IM adipose tissues in humans and animals. We are particularly interested in (1) positive and negative physiological effects of these adipose depots, (2) the association of these depots with abnormal physiologies such as diabetes and metabolic syndrome, (3) gene regulation/expression and marker production by adipocytes within the two adipose depots, and (4) whether some commonalities might exist among animal and human depot-specific gene orthologs.

Molecular Regulation of Adipogenesis

Up to now, most studies on INTMF/IM adipogenesis have been conducted in mice. Due to the small size of mice, it is almost impossible to distinguish intermuscular and intramuscular fat thus all available studies of IM adipogenesis in mice refer to both INTMF and IM fat. In mice, intramuscular adipogenesis initiates later at mid to late gestation, slightly later than fetal muscle development which initiates in the mesoderm at the early fetal stage.^41,42 Wnt signaling plays a crucial role in activating myogenic differentiation, while inhibiting adipogenic differentiation.^43,44 Recently, it has been shown that brown adipocytes through involvement of PRDM16 (PR-domain-containing 16) transcription factor are derived from Myf5 positive cells which are also precursors for myogenic cells.^45,46 Bone morphogenetic protein 7 (BMP7) promotes brown adipogenesis through promoting PRDM16 expression in mice.⁴⁷ In bovine fetuses, these INTMF brown adipocytes appear to convert into white INTMF adipocytes later in life.^47-49 In these two studies, INTMF brown adipogenesis was not examined due to the absence of distinguishable IM fat in bovine fetal muscle. There is no evidence of INTMF/IM brown adipogenesis in pigs.

Compared with brown adipogenesis, white adipogenesis appears to have a dominant role in the formation of inter/intramuscular adipocytes.^48,49 Adipogenesis is briefly separated into two stages: determination and differentiation.⁵⁰ Recently, zinc-finger protein Zfp423 was identified as a transcriptional factor responsible for the adipogenic commitment of progenitor cells, or adipocyte determination.⁵¹ The expression of Zfp423 commits progenitor cells to pre-adipocytes, which further induces peroxisome proliferator-activated receptor γ (PPARγ) expression and terminal adipogenic differentiation of cultured NIH 3T3 cells and in SQ fat.^51,52 It was recently observed that Xfp423 also regulates adipogenic commitment in beef stromal vascular cells.⁵³ Compared with adipogenic commitment, the differentiation of preadipocytes into mature adipocytes is much better studied. Most studies conducted in mice and 3T3-L1 cell line clearly showed that adipogenic differentiation is mediated by PPARγ and CCAAT/enhancer-binding proteins (C/EBPs).⁵⁴ C/EBPβ/δ, which is expressed at a very early stage of adipogenesis, triggers the expression of PPARγ, an essential and indispensable transcription factor for the late stage of adipogenesis; the co-expression of PPARγ and C/EBPα induce genes specific to adipocytes during adipogenesis.^55-57 As a result, cells accumulate lipid droplets and become mature adipocytes.⁵⁸

Animal Studies of Intermuscular Fat

In contrast to human studies, studies of INTMF fat in pigs and cattle are generally more invasive and either multiple sites or the entire INTMF fat depot is examined. Early work on the cellularity, development, and metabolism of INTMF and other adipose tissue depots in cattle, pigs, and sheep showed that INTMF cells in growing pigs, cattle, and sheep hypertrophied much less than SQF and perirenal fat cells but increased in number more than perirenal fat cells in sheep.⁵⁹ Furthermore, cattle and sheep INTMF fat cell cellularity were affected least by a restricted or maintenance diet than SQF and perirenal fat.^60,61 In a recent comparison, the porcine INTMF depot lipid content was lower and adipocytes smaller than in the SQF and perirenal depots.⁶² Growth rates of INTMF and SQF adipose tissue are similar (relative to carcass weight) but in some locations like the belly the INTMF fat grows more rapidly.⁶² The INTMF depot develops before 20 kg of body weight especially in lean pig breeds such as the Pietrain.⁶² In cattle, the INTMF depot is the largest fat depot beginning at 11 mo and remains the largest until slaughter (19 mo) with the amount of SQF representing 60% of the INTMF fat mass throughout growth.⁶³ In the pig INTMF fat represents 18–13% of total fat throughout growth compared with SQF representing 78–84% of total fat.⁶² Classical selection for increased carcass leanness, essentially based on backfat depth, appears to have been less successful for reducing INTMF accretion than for reducing subcutaneous fat in pigs, especially in lean animals. In a study of 94 pigs, seven combinations of pig genotypes and sex were serially slaughtered throughout growth between 12 and 110 kg BW and it was found that the genetically leaner animals, had a higher proportion of INTMF fat as a percent of total fat.⁶⁴ Among common pig breeds, the relative levels of INTMF were dependent on carcass cuts or anatomical location which were highest for the fattest pigs, i.e., Meishans and relative levels of INTMF were similar for Landrace, Large White, Pietrain, and Duroc pigs.⁶⁵ This discrepancy may reflect that many more pigs were used by Gispert et al.⁶⁵ than were examined by Kouba et al.⁶⁴ In a study of INTMF of four primal cuts from high and low growth rate pigs no effect of genetic selection was detected on the INTMF to SQF ratio for the primal cuts.⁶⁶ Feeding 5% beef tallow to pigs up to 135 kg had no influence on INTMF to SQF ratios for individual primal cuts.⁶⁶ The effect of a low protein diet on INTMF content and percent muscle was found to be breed specific and only significant for the Berkshire and Large White breeds.⁶⁷

Limited studies have examined the genetic control of INTMF fat development and composition. Genetic and phenotypic correlations between SQF and INTMF percentages were approximately 0.50 in a comparison of a lean and a fat breed of pigs.⁶² In contrast, genetic and phenotypic correlations between INTMF cross sectional areas and IM content were smaller and ranged from 0.2 to 0.4.⁶² Residual feed intake is strongly genetically correlated with SQF in pigs but is weakly correlated with IM.⁶⁸ In a later study, strong genetic correlations of residual feed intake with INTMF fat accumulations were identified at several sites in pigs indicating that reducing residual feed intake is linked to INTMF accumulations at these sites.⁶⁹ The genetic correlations between daily feed intake and INTMF fat accumulations at several of these sites were strong (0.60, 0.76, and 0.56 for INTMF fat at fifth to sixth thoracic vertebra, one-half body length, and last thoracic vertebra, respectively). In another study, image analysis of abdominal fat, INTMF and SQF from adipose tissue samples from Duroc pigs slaughtered at 105 kg indicated that within anatomical sites the genetic correlations between SQ and INTMF were high whereas, correlations between IM and INTMF fat were much lower.⁷⁰ Heritability estimates for INTMF varied between locations and were lower than SQF area heritabilities at all three anatomical sites.⁷⁰ In finished feed lot steers, heritability estimates for INTMF and SQF were similar (0.40, 0.42).⁷¹ The absence of higher genetic correlations indicated that INTMF, SQF, and IM are regulated by different genetic means.⁷¹

Molecular studies have revealed that porcine perilipin 2 (PLIN2) gene expression analyses showed a positive correlation with higher INTMF.⁷² However, a 3′-UTR mutation PLIN2 genotype was not significantly associated with INTMF in association analysis studies.⁷² Gene expression profiling of SQF, VAT, and INTMF adipose tissues in male and female pigs of three pig breeds with different degrees of adiposity was coupled with Gene Ontology-Biological Processes and KEGG pathway analysis.⁷³ Differentially expressed genes were identified and clustered which revealed that VAT as well as the INTMF were mainly associated with impaired inflammatory and immune response whereas SQ fat was mainly associated with metabolism modulators.⁷³ Gene modules of co-expressed genes substantiated the distinction between depots.⁷³ Furthermore, recent studies demonstrated depot dependent intrinsic DNA methylation differences in the pig and therefore demonstrated epigenetic evidence that both VAT and INTMF are associated with an impaired immune response in the pig.⁷⁴ In a study of miRNA transcriptomes of porcine INTMF and SQF miRNAs, INTMF transcriptomes were enriched in inflammation- and diabetes-related miRNAs compared with SQ fat transcriptomes⁷⁵. Moreover, functional enrichment analysis of the genes predicted to be targeted by the enriched miRNAs indicated that INTMF adipose tissue was associated with immune and inflammatory responses.⁷⁵ Collectively, these results indicated that SQF mainly modulates metabolism, whereas INTMF and VAT are associated with inflammatory and immune responses.^73-75

Human Studies of Intermuscular Fat

Recently, attention has focused on the content, localization, and composition of fat within human skeletal muscle as determinants of insulin resistance and involvement in the metabolic syndrome. Metabolic syndrome is a cluster of conditions, increased blood pressure, elevated insulin levels, excess body fat around the waist, or abnormal cholesterol levels that occur together, increasing the risk of heart disease, stroke and diabetes.⁷⁶ Up until now it has been thought that visceral fat or visceral obesity was responsible for the metabolic syndrome.⁷⁶ In humans INTMF tissue is located between muscle groups and clearly separated from SQF by a well-defined fascia. IM is located within individual muscles as observed on MRI images.⁷⁷ Recently, attention has focused on characteristics of fat within skeletal muscle including the intramyocellular triglycerides (IMCL), INTMF, and a small pool of adipocytes present between muscle fascicles which may be involved in insulin resistance.⁷⁷ Triglyceride accumulation within the muscle cell primarily accounts for IMCL.⁷⁸ Perivascular adipose cells and adipocytes in the intermuscular space and between muscle fascicles account for the perivascular or intermyofibrillar lipids.⁷⁷ With the development and adaptation of MRI and CT technology to evaluate human skeletal muscle lipid deposition many studies have examined factors that influence INTMF in humans.⁷⁷ With MRI or CT, INTMF is localized and studied whereas EMCL and IMCL content can be evaluated by proton magnetic resonance spectroscopy (1H MRS) or MRSI.⁷⁷ Additionally, IMCL can be quantified with lipid histochemistry of muscle biopsies with or without 1H MRS.⁷⁸

The influence of race, aging, physical activity and obesity on INTMF and muscle changes are summarized in Table 1. In a study of a cohort of a large number of older subjects, INTMF increased in those who either maintained, lost or gained weight indicating that INTMF is regulated independent of SQ fat and other fat depots (Table 1).⁷⁹ Another study demonstrated that physical activity can prevent losses in muscle strength and prevent increases in INTMF in the absence of changes in SQF (Table 1).⁸⁴ In a study of older individuals with metabolic and mobility impairments, 12 wks of resistance training decreased INTMF and increased lean tissue (Table 1).⁸¹ Subjecting younger subjects to the influence of 4 wk of unilateral lower limb suspension significantly increased INTMF accretion and decreased muscle volumes with no change in SQF (Table 1).⁸⁰ This is a clear demonstration of how physical activity controls muscle mass and fat deposition in muscle. Healthy obese subjects (56–64 y old), diabetic subjects, and diabetic subjects with peripheral neuropathy when subjected to a 6 min walk test and a physical performance test showed muscle specific INTMF accretion (Table 1)⁸³ Evaluation of race and obesity (i.e., 1105 Caucasian and 518 Afro-Caribbean men aged 65) demonstrated that INTMF accretion was greater in African men and showed association with type 2 diabetes in both ethnic groups (Table 1).⁸⁵

Table 1. Influence of race, aging, reduced physical activity, and physical activity on INTMF in humans

Topic, subjects	Approach	Results	Conclusions	Reference
Aging, 73 y old men and women, over 5 y in the Health, Aging, and Body Composition study cohort (n = 1678).	A single CT 10-mm axial image of the right thigh, CSA muscle and fat, leg muscle strength, and muscle quality	Weight gain did not prevent loss of muscle strength. Age increased INTMF in men and women and, remarkably, INTMF increased with weight loss, weight gain, or with no weight change.	Aging is associated with decreases in muscle strength and quality regardless of body weight changes. The persistence of increases in INTMF indicates a distinct regulation of INTMF.	^79
Physical activity, 6 men and 12 women aged 19–28 y.	A 4-wk control period followed by 4 wk of unilateral lower limb suspension. Multiple axial images with MRI of muscle, SQF, and INTMF, in the thigh and calf.	Reduced physical activity decreased thigh and calf muscle volumes by 7.4–7.9%, associated with increases in INTMF but no change in SQF.	Reduced physical activity in healthy young adults increases INTMF which may contribute to losses in muscle strength.	^80
Aging and physical training, participants represented wide range of ages, mobility levels and co-morbid diseases.	INTMF fat and lean tissue CSA's obtained from bilateral MRI scans of the thigh. 55 and older individuals subjected to a 12 wk resistance training program.	Significant and positive relationship between age and percent thigh INTMF in individuals with a variety of co-morbid conditions. Resistance training decreased INTMF and increased lean tissue.	Resistance training decreases INTMF accretion in older individuals with metabolic and mobility impairments.	^81
Race and obesity, 1105 Caucasian and 518 Afro-Caribbean men aged 65.	Body mass index, total body fat by DEXA, and calf skeletal muscle composition by pQCT	Greater INTMF and lower SQF in Afro-Caribbean men at all levels of total adiposity. Differences in INTMF and SQF fat were independent of age, height, calf skeletal muscle, and total adipose tissue.	INTMF is greater among African than among Caucasian ancestry men despite lower total adiposity. INTMF associated with T2D in both ethnic groups.	^82
Exercise and muscle location, 45 subjects, 56–64 y old, composed of a healthy obese group, a group with diabetes and a group with diabetes and peripheral neuropathy	INTMF fat over the right calf muscle quantified using MRI. Calf muscle divided into two muscles and three compartments. Six minute walk test and physical performance test (PPT) employed.	The gastrocnemius had highest ratio of INTMF /muscle volume. Groups had similar calf muscle or INTMF volumes. Calf INTMF was inversely related to performance on the 6 min walk test and PPT.	Calf INTMF accretion is muscle specific and associated with poor physical performance.	^83
Physical activity in older adults, 70 -89 y old. Eleven men and 31women in a randomized trial, either physical activity (PA; n = 22) or aging health educational control (SA; n = 20) group.	Physical activity intervention included primarily walking and three phases, adoption, transition, and maintenance (week 25 to end). Skeletal muscle and INTMF CSA's calculated from axial CT images of the midthigh level.	Decreased strength adjusted for muscle mass decreased in SA prevented in PA. A significant increase in INTMF was nearly prevented in PA. No change in SQF in either group.	Physical activity can prevent losses of muscle strength and increases in INTMF in older age.	^84

SQF, subcutaneous adipose tissue; INTMF, intermuscular adipose tissue; CT, computed tomography; CSA, cross-sectional area; MRI, magnetic resonance imaging; pQCT, quantitative CT; DEXA, dual-energy X-ray absorptiometry; T2D, type 2 diabetes; PPT, physical performance test; PA, physical activity group; SA, control group.

Several of these studies indicated that changes in muscle mass, strength and quality with aging may dictate INTMF accretion in humans which was independent of total body weight or total adiposity (Table 1). In the animal studies, INTMF is regulated by genetic means distinct from the other fat depots. The relationship between leanness in pigs and higher proportions of INTMF fat was not observed in human studies and was not corroborated by several pig studies.⁶⁴ Gene expression profiling and intrinsic methylation differences in pigs demonstrated that INTMF and primarily VAT are primarily associated with inflammatory and immune processes and insulin resistance.^73,86 In humans INTMF is closely linked to VAT and is a good predictor for insulin sensitivity nearly equal to that of VAT.⁸⁷ Therefore, it seems that in the pig and humans, INTMF and VAT share a similar pattern in distribution and association of components dictating insulin sensitivity.

Intramuscular Adipogenesis

Overview of adipose tissue prenatal development in skeletal muscle

During the prenatal stage, skeletal muscle development mainly involves the formation of muscle fibers (i.e., myogenesis), but also the formation of IM adipocytes (adipogenesis) and fibroblasts (i.e., fibrogenesis). These cells are derived from a common pool of multipotent cells, mesenchymal progenitor cells. Based on recent discoveries, it appears that during fetal skeletal muscle development, mesenchymal multipotent cells first diverge to either myogenic or fibro/adipogenic lineages. Myogenic lineage cells further develop into muscle fibers, INTMF/IM brown adipocytes, and satellite cells, while fibro/adipogenic lineage cells develop into the stromal vascular fraction of muscle where white adipocytes, fibroblasts and resident fibro/adipogenic progenitors reside (FAPs, the counterpart of satellite cells).^48,53,88 These resident FAPs become largely quiescent and form the stem cell pool for later adipogenesis and fibrogenesis in mature muscle.^89-91 Because both myogenic and fibro/adipogenic cells are from the same pool of progenitor cells, the initial myogenic or fibro/adipogenic commitment can be considered as a competitive process, with enhancing myogenesis reducing fibro/adipogenesis, and vice versa. Indeed, both IM fat and collagen accumulation is lower while muscle mass is higher in the largest compared with the smallest littermates of pigs, indicating the shifting of myogenesis to fibro/adipogenesis in the smallest piglets.^92,93 Consistently, previous studies demonstrated both IM accumulation and fibrosis in fetal and offspring sheep due to maternal over-nutrition, which correlates with downregulated myogenesis^94-96 and suggests that IM adipogenesis and fibrogenesis are correlated due to a common cell lineage. Predictably, in genetically high marbling Wagyu cattle, both IM adipogenesis and collagen accumulation are higher than in relatively low marbling Angus cattle.⁹⁷

Intramuscular Adipogenesis in Livestock Species

Existing evidence points to the similarities in adipogenesis between INTMF/IM and SQF adipogenesis. The overall expression pattern of PPARγ and C/EBPα appears similar between IM and SQF SV cells, despite a temporal differences.⁹⁸ The expression of these markers in preadipocytes of IM is similar with those in SQF despite at  a lower level, indicating that the SV cells of IM are at a relatively earlier differentiation stage compared with that of SQF.⁹⁹ In addition, IM preadipocytes respond to dexamethasone treatments, similar to SQ preadipocytes but the response is greater for SQF preadipocytes in line with a recent report that the expression profiles of microRNAs between intramuscular and SQ SV cells are similar.^100,101 Similarly, in bovine SV cell cultures, both IM and SQF cells are responsive to dexamethasone, but the responsiveness is greater in subcutaneous cells, echoing the more advanced differentiation of SQF compared with IM SV cells.¹⁰² In genetically obese pig breeds, higher expression of PPARγ in the IM preadipocytes was observed than in genetically lean breeds, indicating enhanced adipogenic differentiation.⁸⁶ Compared with the difference between breeds, there is more obvious difference among species. For example, the expression of both PPARγ and C/EBPα were undetectable in bovine IM preadipocytes after 4 d of adipogenic differentiation while they were expressed in swine IM SV cell cultures early on and are highly induced in mouse 3T3-L1 cell preadipocyte cultures after 4 d.^99,103,104 It appears that the adipogenic differentiation of SV cells is the fastest in mice, followed by swine and the slowest in cattle.

Adipocyte Development within IM

The rate of accretion and mode of cellular development of meat animal adipose tissue is depot and species dependent.¹⁰⁵ In contrast to other depots, IM adipocyte hyperplasia in meat animals apparently continues on with either a delayed plateau or no plateau in hyperplasia.¹⁰⁶ Intramuscular adipocyte size and number dictate skeletal muscle lipid content in several meat animals, including cattle, rabbits, and pigs.¹⁰⁷ Long-term selection for decreased backfat in pigs was highly associated with decreased IM as well.¹⁰⁸ Selection for lean growth in sheep also significantly reduced IM.¹⁰⁹ A study of cattle selected for high growth indicated no change in IM lipid despite a significant increase in backfat accretion.¹¹⁰ Therefore, changes in growth with increased leanness may not be involved in the link between leanness and IM lipid. Intramuscular fat can be influenced by many factors (Table 2) including age, diet, gender, fasted state, genetics, and physical activity.^113,128 These factors influence IM lipid accretion in a muscle- and species-dependent manner.¹¹¹ For example, somatotropin treatment in pigs produced depot dependent changes in fatty acid composition in a comparison of SQF and IM adipose tissue.¹²⁹ Dietary induced obesity increases IM lipid content in several species.¹³⁰ There is considerable evidence that diet/dietary components can influence IM lipid in production animals (Table 2). Reduced protein diets may increase IM lipid by preferentially increasing steroyl-CoA desaturase enzyme activity.¹³¹ Muscle lipid content is increased by 40% when pigs are fed low lysine diets (Table 2). Changes in muscle lipid content could be due to age or altered muscle metabolism.

Table 2. Intramuscular lipid (IML) accretion that occurs naturally is species and muscle dependent

Factor	Species/muscle	Summary
Age	Cattle and pigs, several muscles	Lipid accretion dependent on muscle and species^59,111
	Cattle, LD	Lipid increase between 11 and 15 mo—no change after 15 mo^63
	Cattle	Linear increase in marbling to 200–400 kg—no further increase after 420 kg^112
Gender	Cattle, LD	Marbling lipid highest in heifers, intermediate in steers, and lowest in bulls^113
Diet	Japanese black cattle, LD	Reduced vitamin A serum levels increases marbling^113,114
	Holstein steers, LD	Vitamin A restriction does not influence marbling FCS and FCN^115
	Pigs, LD and BF	Lysine-deficient diet increases lipid^116
	Holstein steers, LD	Marbling FCS influenced by energy level and FCN influenced by energy source^117
	Pigs, BF and LD	Lipid decreased with feed restriction (30–70 kg), not influenced by compensatory growth^118
	Pigs, LD, RF, deep and superficial ST.	Feed restriction reduced lipid in muscle-dependent manner (ST > RF,LD) on a constant age basis^119
	Pigs, LD	Reduced protein and energy diet reduced lipid and FCS, long-term feed restriction reduced lipid and FCN^120
	Yucatan mini-pigs, BF	Overfeeding (4–16 mo) increased FCS and lipid^121
	All meat animals, overall muscularity	Variability of IML may be more efficiently linked when evaluating multiple markers (nutrition, diet frequency, specific metabolites, genetics)^111,122,123
Endocrine	Pigs	Porcine GH decreases overall lipid load^124
	Pigs, trapezius	No influence of IGF-II genotype on FCS and lipid^107
Birth weight	Pigs, LD and ST	Low birth wt increases ST lipid and FCS, but not LD^125
Muscle fiber types	Chickens and glycolytic fibers	Lower lipid content^111
Obesity	Pigs, LD	Increased lipid^126,127

Abbreviations: BF, biceps femoris; LD, longissimus dorsi; ST, semitendinosus; RF, rectus femoris; FCN, fat cell number; FCS, fat cell size.

Generally, leptin gene expression marks SQF and distinguishes adipose depots from one another in several species.¹⁰⁵ Throughout growth, IM adipocytes are less metabolically activity when compared with SQF and perirenal adipocytes.¹⁰⁵ Lipogenesis distinguished SQF from IM adipose tissue explants and SV cell cultures from several breeds of cattle.¹³² Gene expression for enzymes follows this pattern suggesting marked metabolic differences in adipocytes from different depots may exist at the gene level.¹⁰⁷ There may be a developmental delay in metabolic efficiency, hyperplasic or hypertrophic growth which contributes to these metabolic differences between IM and SQF.¹⁰⁵

Intramyocellular Lipid Dynamics

The idea that skeletal muscle, itself, may adsorb lipid and form intramyocellular lipid droplets is not new, but rarely constitutes a major source of lipid storage/available energy. Moreover, slow-oxidative skeletal muscle is the myofiber type that possesses sufficient metabolic and biochemical machinery (mitochondria) to process internal lipid droplets. As such, dramatic rises in intramyocellular lipid is commonly associated with pathological conditions such as insulin resistance.⁷⁸ Due to its presence in obesity, metabolic syndrome and other metabolic pathologies, intramyocellular lipid accumulation is beginning to be looked at with some interest. Few reports exist whereby this phenomenon even is evident in domesticated (meat) animals, but three studies are available evaluating this event in pigs.^133-135 In a study utilizing pigs of different body composition, Reiter et al. demonstrated that genetic preference for leanness resulted in lower genetic markers for variables of adiposity and lipid metabolism within adipose tissue over pigs of more conventional body composition.¹³⁴ However, select skeletal muscle markers for oxidative metabolism of lipids were higher in the leaner pig group; suggesting that intramyocellular lipid is processed more rapidly in lean-type pigs over conventional pigs of similar age and such metabolism is heightened by application of commercially available β-adrenergic agents.¹³⁴ Utilizing genetically small pigs, which are isolated from other breeds, it was shown that diets to mimic dyslipidemic metabolic syndrome resulted in greater accumulation of intramyocellular lipid, but did not result in greater overall numbers of lipid droplets in pigs.¹³³ Moreover, skeletal muscle contractile proteins appear altered when excess lipid was incorporated within afflicted skeletal muscle fibers.¹³³ At a more molecular level, Gandolfi et al. demonstrated that PLIN protein 2, was associated with lipid droplets in skeletal muscles of pigs displaying high IM lipid content, and that the expression of PLIN was associated with extra-cellular lipid availability to skeletal muscle cells.¹³⁵

Gene Expression Associations/Interactions in the Deposition Process of IM in Animals

Elucidation of common adipogenic mechanisms regulating depot-specific partitioning of lipids in animals and humans is important. In domestic animals, deposition of IM is regulated by complex interactions involving muscle, fat, and connective tissue and is associated with the genetic background, nutrition and development of an animal, as described in previous excellent reviews.^{41,111,136-140} Comprehensive high throughput genomic technologies have revealed large numbers of differentially expressed (DE) genes, and signaling pathways including adipogenic and lipogenic related genes, metabolic enzymes, and cholesterol and bile acid homeostasis (Table 3) related to IM in livestock species.^111,158-161 The transcriptome profiling of marbling LM tissue of heifers from Wagyu × Hereford indicated a strong positive correlation between expression of several adipogenic genes, adiponectin, C1Q, and collagen domain containing (ADIPOQ), SCD, thyroid hormone responsive (THRSP), and Fatty acid synthase (FAS), with IM.¹⁶¹

Table 3. Common genes/genetic markers associated with INTMF and IM fat in humans and animals

Genes/genetic markers	Humans	Animals	References
Adipogenic and lipogenic related	Increased Stearoyl-CoA desaturase SCD1 - SNPs in the 3¢UTR (G > A; C > T)	Increased SCD1 - SNPs in the 3¢UTR (G > A; C > T)—in cattle and pig	^141
		Increased SCD1- LM tissues of Wagyu × Hereford cattle	^142
	PPARγ and coactivator 1α (PGC1α) regulation of pyruvate dehydrogenase (PDK4)	PPARγ, C/EBPα, FABP4, Zfp423—Angus cattle	^97,143
		PPARγ and MAPK signaling—higher expression with IM deposition in Beijing-you chicken	^144
	FLJ20920 through its interaction with PPARγ	Novel porcine FLJ36031 (pFLJ)	^145,146
	Perilipin 2 (PLIN2) facilitates intramyocellular lipid (IMCL) storage	Lipin 1 and Lipin-β - IM deposition in obese pigs	^147,148
Metabolic enzymes	Increased expression of Insulin growth factor-1 (IGF-1) and IGFBP3 with higher body fat	Increased expression of IGF-1 and IGFBP3 with increased muscle growth of cattle	^149,150
Cholesterol and bile acid homeostasis	Lipopolysaccharide (LPS) induces MAPK-dependent proinflammatory cytokine/expression® suppresses PPARγ and insulin responsiveness.	Liver transcriptomics in porcine using RNA seq - differential expression of LPS endotoxin/pro-inflammatory cytokines (LPS/IL-1)	^151,152
Fat mass and obesity-associated (FTO) gene	Homozygous for the risk alleles of SNP rs993939—linked with increased risk for obesity	Mapped between SW1302 and SWR1130 on SSC6 in the porcine adjacent to the region with QTLs for IM percentage	^153,154
Agouti signaling protein (ASIP)	ASIP through interaction with STAT 1/3 (signal transducer and activator of transcription) and PPARγ	ASIP/melanocortin signaling system in chickens	^155,156
		ASIP mRNA >9-fold increase in IM of Japanese Black cattle vs. Holstein	^157

Global analysis with GeneChip Bovine Genome array in Japanese Black and Holstein steers revealed that vast majority of DE genes were downregulated in IM and the mRNA abundance of adipogenic key regulators peroxisome proliferator-activated receptor gamma (PPARγ) and fatty acid binding protein (FABP)4, was neither directly associated with the size of adipocytes in a tissue nor with the amount of IM content.¹⁶² On the contrary, higher expression of Zfp423, PPARγ, C/EBPα, and FABP4 in Wagyu with high marbling was observed as compared with the Angus cattle, indicating enhanced proliferation and/or adipogenic differentiation. Recently, Zfp423 was demonstrated as a very early marker for adipogenesis, which induces adipogenic commitment through upregulation of PPARγ expression.^51,52,97 The PPAR and mitogen-activated protein kinase (MAPK) signaling genes have also been positively correlated with the IM deposition in Beijing-you, a Chinese chicken breed possessing high IM as compared with the commercial broiler chicken Arbor Acres.^144,163

In pigs, positive association between the IM content and the expressions of myosin (MYL1), adipose-specific phospholipase A2 (AdPLA), melanocortin 4 receptor (MC4R), phosphoenolpyruvated carboxykinase (PEPCK), and SCD, and novel porcine FLJ36031 (pFLJ) genes are suggestive of their role in fast glycolysis and lipid deposition.^142,145 Genome-wide-association studies (GWAS) and the liver RNA-Seq on female pigs with extreme phenotypes for IM showed differential expression of transposable elements, long non-coding RNAs.^151,164,165 Furthermore, these studies established the role of putative protein-coding genes including Endotoxin lipopolysaccharide/pro-inflammatory cytokines (LPS/IL-1) in mediating inhibition of retinoid X receptors (RXR) function to regulate cholesterol homeostasis and bile acid homeostasis (Table 3). Besides changes in abundance of markers for lipid synthesis and transport, alterations have also been observed in protein and amino acid metabolism with differences in IM content.^111,166,167 Insulin growth factor-1 (IGF-1) and IGFBP3 have been associated with increased muscle growth of cattle resulting in elevation in metabolic enzymes with greater IM, suggesting an increased nutrient utilization through amino acid metabolism.¹⁴⁹

Genetic Variations/QTL and Epigenetic Changes Associated with IM Deposition in Animals

Several quantitative trait loci (QTL) regions and genetic markers for IM have been identified in pigs, chickens and cattle.^111,136 Some of excellent indicators for predicting IM content in livestock species include the SNPs in various genes e.g., DGAT1, DGAT2, serine-arginine-rich protein (SFRS18), adipogenic genes (ADRP, PPARγ), lipogenic genes (FASN, SREBP1, and SCD1), FABP4, PNAS-4, and calpain-system.^111,168-170 QTL analysis has identified the region between S0228 and SW1881on pig chromosome 6 (SSC6) that influences IM.^171,172

Recent studies have suggested that the effect of polymorphisms influencing IM, cholesterol, and fatty acid contents are modulated by several factors related to muscle location, metabolism and function.¹⁷³ Stearoyl-CoA desaturase (SCD1) activity has been shown to increase with higher fat accumulation in skeletal muscle in humans and animals possibly related to the SNPs in 3¢-UTR that contains G > A and C > T in the bovine gene (which are also highly conserved in humans and pigs).¹⁴¹ Furthermore, the polymorphism in the PPARγ (TA haplotype favorable over CG haplotype) upstream transcriptional regulatory region has been associated with higher IM.¹⁷⁴ On the contrary, a few other GWA have had limited success in the identification of the genetic drivers of IM percentage which could be due to variations including treatments, genetics etc., resulting in the similar phenotype.^175,176 High throughput genomic technologies using powerful SNP panels will have to be combined with phenomics approach to further understand the complexities of IM.

During the past decade, various miRNAs, endogenous small non-coding RNAs, have been shown to impact on epigenetic regulatory mechanisms with implications to the global gene expression in adipogenesis in cattle, pigs, and lipid metabolism disorders.^101,177-183 In pigs, eight inflammation-related and nine diabetes-related miRNAs were present in higher abundance in the INTMF transcriptome compared with the SQF indicating the metabolic role of IM in obesity-related metabolic dysfunction.⁷⁵ The expansion of INTMF could induce some changes in muscle metabolism resulting in insulin sensitivity because of the release of adipokines and metabolites from fat cells surrounding muscle fibers.⁷⁷ Future studies focusing on the identification of the factors that modulate miRNAs expression could further expand our current understanding of the environment and genetic factors that influence gene expression during adipogenesis.

Common Genes/Genetic Markers Associated with IM and IM Fat in Humans and Animals

Emerging evidence suggests that INTMF accumulation surrounding skeletal muscle in humans has significant negative impacts on health, causing obesity/type 2 diabetes and their associated conditions.^138,184 To understand the genetic complexity of these conditions, many studies have focused on human subjects or on the mouse as a model organism.⁷⁷ Accumulation of INTMF fatty acid metabolites in human skeletal muscle plays an important role in the regulation of pyruvate dehydrogenase kinase (PDK4) gene expression possibly through interaction with PPARγ and PPARγ coactivator 1α (PGC1α).^143,185 Association of human minor alleles for nonsynonymous coding variants (G531L, I66V) in the carnitine palmitoyltransferase-1B gene (CPT1B, the rate limiting enzyme in mitochondrial β-oxidation of long-chain fatty acids), has been associated with lower IM and higher SQ, independent of overall adiposity.⁸²

Several studies have established the role of human agouti signaling protein (ASIP) expressed in adipose tissue, in the development of metabolic disorders like obesity and insulin resistance through influencing the expression of STAT 1/3 (signal transducer and activator of transcription) and PPARγ transcription factors.¹⁵⁵ ASIP/melanocortin signaling system has also been found to regulate lipid metabolism in chickens and ASIP mRNA abundance showed more than 9-fold increase in IM of Japanese Black cattle compared with Holstein (Table 3).^156,157 Three independent whole genome association studies in humans provided evidence that three different SNPs in the FTO (fat mass and obesity-associated) gene intron 1 are significantly associated with both childhood and adult obesity.^153,186,187 FTO gene participates in fat tissue and energy homeostasis and adults homozygous for the risk alleles of SNP rs993939 are linked with increased risk for obesity.¹⁵³ Interestingly, the FTO gene was assigned between SW1302 and SWR1130 on SSC6 in the porcine (Table 3), adjacent to the region with a number of QTLs for average daily gain, IM percentage and body lipid content.¹⁵⁴ Comprehensive database search revealed that FLJ, which are involved in fat deposition and adipogenesis are highly conserved among different species and pig FLJ (pFLJ) shares 93%, 83%, 92%, and 92% homology with human, mouse, chimpanzee and rhesus monkey FLJ, respectively.¹⁴⁵ FLJ20920 is an important gene in human adipogenesis through its interaction with PPARγ.¹⁴⁶ In pigs, the Lipin1 gene may have a crucial effect on body lipid accumulation, whereas the Lipin-β isoform may play an important role in IM deposition in obese pigs.¹⁴⁷ Overexpression of PLIN2 has been shown to result in decreased gene expression of several PPARα target genes and reduced transcriptional activity of mitochondrial genes facilitating intramyocellular lipid (IMCL) storage.¹⁴⁸ A better understanding of the molecular pathways or genetic markers involved in INTMF and IM deposition could potentially result in novel therapies to treat obesity.

Conclusions

As summarized in Figure 1, review of human INTMF studies indicated that INTMF is influenced by aging, physical activity, race and obesity and induced limb inactivity, and is increased with weight loss, weight gain, or with no weight change. As such, INTMF is clearly a threat to type 2 diabetes and projects many physiologies similar to VAT. Studies of INTMF in animals are not numerous and covered different topics such as quantitative deposition and genetic relationships with other fat depots. Therefore, in growing animals, INTMF is regulated by genetic means distinct from the other fat depots. The relationship between leanness in pigs and higher proportions of INTMF fat was not similarly observed in humans. More specifically, in humans, changes in muscle mass, strength, and quality with aging are associated with INTMF accretion whereas muscle characteristics do not change with age pigs. It seems as though pigs are not “old” relative to humans. Even so, gene expression profiling and intrinsic methylation differences in pigs demonstrated that INTMF and VAT are primarily associated with inflammatory and immune processes. In humans INTMF is closely linked to VAT and INTMF was a good predictor for insulin sensitivity nearly equal to that of VAT. Therefore, it seems that in the pig and humans, INTMF and VAT share a similar pattern in distribution and association of components dictating insulin sensitivity.

Figure 1. Major metabolic, physiological, and genetic changes associated with different Intermuscular and Intramuscular fat in animals and humans.

Intramuscular adipocyte development affects the quality of meat, and studies in meat animals have included the influence of aging, gender, diet, obesity, and birth weights. Gene expression analysis and genetic analysis have identified candidate genes involved in IM adipocyte development in these animals. Alternatively, IM adipocyte development in muscle of humans is rare, and rarely seen except during some pathological circumstance, such as lipotrophic muscular atrophy. Therefore the cellularity and regulation of human IM are rarely studied.

Several genetic links between human and meat animal adipogenesis have been identified. The human agouti signaling protein (ASIP) is expressed in adipose tissue (Fig. 1) and may be involved in the development of obesity and insulin resistance by influencing the expression of STAT 1/3 and PPARγ transcription factors.¹⁵⁵ ASIP mRNA was increased in IM more than 9-fold in Japanese Black cattle compared with Holstein.¹⁵⁷ Whole genome association studies in humans provided evidence that three different SNPs in the FTO gene are associated with both childhood and adult obesity.^153,186,187 The FTO gene is located on porcine SSC6 (Table 3), adjacent to a number of QTLs for IM percentage and body lipid content.¹⁵⁴

Comprehensive database search revealed that FLJ, which are involved in fat deposition and adipogenesis are highly conserved among different species including the pig FLJ (pFL).¹⁴⁵ FLJ20920 is involved in human adipogenesis through its interaction PPARγ.¹⁴⁶ In pigs, the PLIN1 and PLIN2 genes have strong genetic effects on fat depot lipid accretion including IM accumulation, and the PLIN-β isoform may influence IM deposition in obese pigs (Table 3).¹⁴⁷ PLIN1 deficiency results in immature adipocyte development human lipodystrophy.¹⁸⁸ In humans, overexpression of PLIN 2 decreases gene expression of several PPARα target genes and facilitates intramyocellular lipid.¹⁴⁸ In pigs, PLIN2 gene expression was associated with IM deposition.^72,135 It is clear that the PLIN family may influence intramuscular lipid regardless of species.

The literature clearly indicated that there is very little IMCL in meat animal muscle and very little evidence of intramuscular adipocytes in human muscle. In meat animals IMCL is present in newborn and young animals but dissipates as intramuscular adipocytes develop and increase in number.¹⁰⁶ Possibly, intramuscular adipocytes prevent or antagonize IMCL in animals to prevent lipotoxicity in muscle.¹³⁰ The virtual absence of intramuscular adipocytes in human muscle may augment IMCL and associated dire consequences.¹³⁰ Future studies are dictated to validate these possibilities.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.