Adult sheep characteristics
As previously reported (10), LOW sheep were born with lower birth weights compared to HIGH and NORM. Compared to NORM, both LOW and HIGH sheep deposited a greater amount of MES and PER than SUB mass, when they became obese as adolescents upon exposure to the HCHF diet. At the age of 2½ years, all sheep had been exposed to the same low-fat hay-based diet for 2 years; LOW-HCHF sheep (99.0 kg) became heavier as adults compared to LOW-CONV and NORM-CONV sheep (91.1-91.2 kg) with others groups in between. Males were heavier (99.4 kg) than females (92.9 kg). The adult LOW-HCHF sheep had markedly increased plasma levels of cholesterol, urea, creatinine, and lactate compared to other groups [16].
Overall tissue and sex-specific differences in adipose cell size distribution
Distribution of adipocytes on size classes followed a unimodal pattern in SUB and EPI (Fig. 1A and 1B), whereas a clear bimodal pattern was observed in PER with the second peak falling in in different cell-size classes depending on treatment group (Fig. 1C). In MES, there was a plateau across the lower (<800 µm2) cell-size classes, which continued in NORM♀ (unimodal pattern) or was followed by a more or less clear second peak in the other groups (bimodal pattern) (Fig. 1D) .
As shown in Table 1-2 and Fig. 1, females had the highest fat mass (not determined in EPI), adipocyte CSA (except for HIGH♀ in PER), CNI (PER only), and proportions of very small (<40 µm2; PER and MES only) and large adipocytes (SUB: >3200 µm2; MES and PER: >6400 µm2), but lowest proportions of smaller-medium sized adipocytes (SUB: 40-1600 µm2, PER: 800-6400 µm2; MES: 800-3200 µm2). However, in EPI, only few sex effects were seen, and only as interactions with the postnatal nutrition (see below).
In all tissues, positive correlations existed between adipocytes numbers within the smaller cell-size classes (SUB and MES: 200-1600 µm2, P<0.001 and P<0.001-0.05; PER: 200-3200, P<0.01-0.001 µm2; EPI: 40-1600 µm2, P<0.001-0.05) and within the large cell-size classes (SUB: 3200-36000 µm2, P<0.001-0.01; PER and MES: >12800 µm2, P<0.001-0.01; EPI: >6400 µm2, P<0.001-0.05). Correlations between the small and large cell size classes were generally negative (Additional file 2: Fig. S2A-2E), except for the very smallest (<40 µm2) adipocytes in PER and MES, which (unlike other small cells) were positively correlated with numbers of the largest adipocytes (PER: 12800-36000 µm2, P<0.001; MES: >36000 µm2, P<0.05) (Additional file 2: Fig. S2B and S2C).
Long-term impacts of early nutrition history on adipose tissue histology
Implications of malnutrition in early life on adipose morphology and gene expression patterns were, in general, more predominant in males than females, but in a tissue dependent way.
Subcutaneous adipose tissue
There were generally no prolonged effects of early life nutrition history on SUB mass, CNI, average adipocyte CSA (Table 1) or adipocyte size distribution (Fig. 1A). The only exceptions were for very small adipocytes (<40 µm2), where NORM♀ and HIGH♂ had a higher percentage compared to NORM♂ and with other groups in between (P=0.01). The proportion of 40-200 µm2 adipocytes was increased by HCHF compared to CONV in HIGH, but decreased by HCHF in NORM and LOW sheep (Additional file 1: Table S4; P=0.02).
Perirenal adipose tissue
This was the adipose tissue most affected by the prenatal nutrition history, but in a sex-specific way. LOW♀ had the highest and LOW♂ (followed by NORM♂) had the lowest PER mass, average CSA of adipocytes and adipose cell coverage (P=0.01, 0.002, and 0.01 respectively) with other groups in between.
In the first cell size peak (40-400 µm2), NORM♀ had the highest and NORM♂ the lowest ratio of adipocytes, (P<0.01). In the second cell size peak, the NORM♂ and LOW♂ peaked earlier and had a higher ratio of cells in the 1600-6400 µm2 classes and lowest proportions of cells in the largest adipocyte classes, whereas HIGH♂ together with all females peaked in the 6400-12800 µm2 cell size classes (P<0.01). LOW♀ had a markedly higher proportion of large adipocytes (>12800 µm2) (P<0.001) compared to other groups. Interestingly, HIGH♂, unlike NORM♂ and LOW♂, had a phenotype similar to female sheep for most of the studied parameters (adipocyte CSA, tissue composition, and cell size distribution).
Surprisingly, sheep fed the HCHF diet in early postnatal life had a lower adult PER mass than CONV sheep (P=0.02) (Table 2). This was associated with a slightly smaller adipocyte average CSA and a lower proportion of the largest (25600-36000 µm2), but increased proportion of medium sized adipocytes (3200-6400 µm2) in HCHF compared to CONV (P=0.03 and 0.04, respectively; Supplementary Table 3). For medium-large adipocytes (6400-25600 µm2), the proportion was increased by HCHF in NORM and LOW sheep, but decreased by HCHF in HIGH sheep (P=0.01 and 0.005; Additional file 1: Table S4). The CNI was not affected by early life nutrition history (Table 1).
Mesenteric adipose tissue
There were no systematic long-term impacts of the early postnatal nutrition exposure in this tissue. Impacts of the prenatal nutrition history were sex-specific. Hence, LOW♂ and NORM♂ had clear peaks in the lower cell size classes (1600-3200 and 3200-6400 µm2) and lowest proportion of the largest adipocytes compared to other animals; the adipocyte distribution pattern for HCHF♂ was very similar to that of females rather than other males, and in the largest cell size classes (>12800 µm2), LOW♀ had by far the highest proportions of adipocytes and with LOW♂ at the other extreme (lowest proportion) (Fig. 1D; P=0.00001-0.008).
Epicardial adipose tissue
The PER was, in contrast to the other tissues, strongly affected by the early postnatal nutrition history with hardly any effects of prenatal nutrition (Table 1). HCHF sheep had larger average adipocyte CSA (Additional file 1: Table S5) and a shift in cell size distribution (Fig. 1B) towards higher proportions of both small (40-200 µm2; P=0.01) and large (>6400 µm2; P=0.003 to <0.0011) at the expense of medium sized adipocytes (1600-3200 µm2; P<0.0001). The HCHF diet increased and decreased proportions of medium-large (3200-6400 µm2) and medium (800-1600 µm2) sized adipocytes, respectively, in females, whereas males had the opposite responses to HCHF (P=0.02 and 0.01, respectively).
Correlations between cell-number-index and numbers of very small (<40 µm2) and very large adipocytes (25000-36000 µm2) across tissues
Within SUB and MES, CNI was negatively correlated to adipocytes numbers in the largest cell size class (25600-36000 µm2) (r=-0.39 and -0.31, respectively; P<0.05) (Additional file 2: Fig. S2E). Across tissues, CNI in PER was positively correlated to numbers of very small adipocytes in SUB (r=0.37, P<0.05) and to numbers of very large adipocytes in SUB (r=0.42, P<0.05) and MES (r=0.48, P<0.01). Numbers of very large adipocytes in MES were positively correlated to numbers of very small adipocytes in PER (r=0.28, P<0.05) and to very large adipocytes in SUB (r=0.33, P<0.05) and PER (r=0.50, P<0.01).
Systematic sex differences in mRNA expression levels
Across the four tissues studied, males had consistently and markedly higher expression levels than females for almost all genes in all adipose tissues (Additional file 2: Fig. S3), although males only had around half or less fat mass in these tissues (not determined in EPI) compared to females (Table 2). The only genes, where females had the highest expression level, were LPL in SUB and EPI, LEPTIN and CD68 in PER and CGI58 in EPI.
Long-term impacts of early nutrition history on mRNA expression levels
The ADRB1, FTO, and LEPTIN were the only genes, for which expression levels were unaffected by the early life nutrition history.
Subcutaneous adipose tissue
LPL was the only gene affected independently of other factors by the prenatal nutrition, and LOW had the lowest expression levels followed by NORM and HIGH (Fig. 2A; P=0.031). The mRNA expression of 17 genes involved in adipose development and metabolism were affected by the prenatal nutrition in a sex-specific way (Fig. 2B). For all except one gene (ATGL), NORM♂ had higher mRNA expression levels (Fig. 2B; P<0.0001-0.05), whereas for ATGL, the expression level was higher in HIGH♂ and LOW♂ than other groups. In females, if anything, expression levels for several genes were consistently different (higher or lower) in LOW♀ compared to NORM♀ and HIGH♀.
Regarding early postnatal nutrition, HCHF sheep had decreased expression levels for GLUT1, PGC1A, and TNFA, and increased PPARG expression (Additional file 2: Fig. S4A; P=0.004-0.03). For 5 other genes (CD44, CEBPB, FBPASE, IL6, and MCP1), higher expression levels were observed in CONV♂ compared to other groups. For 15 genes (ADIPOQ, CD44, CEBPB, CGI58, FBPASE, GADPH, HSL, IGF1R, IL6, IRS1, MCP1, PLIN1, PGC1A, VEGF, and VEGFA) expression levels depended on the pre- and postnatal interaction, and highest expression levels were observed in NORM-CONV compared to all other groups (P=0.003-0.05). The only deviation from this pattern was that HIGH-HCHF had the highest LPL expression (P=0.02).
Perirenal adipose tissue
In PER, 17 genes were affected by the prenatal nutrition and 6 of them independently of other factors: LOW had the highest expression levels compared to other groups for CGI58, FABP4, GLUT1, IRS1, and VEGFA, or compared to HIGH for IGF1R (Fig. 3A; P=0.0.03-0.03). For 6 genes, the prenatal impacts were sex-specific (Fig. 3B), where the general pattern was that LOW♂ attained the highest expression levels compared to other groups (CD44, GcR, HSL, and TGFB1) except that NORM♂ achieved the highest levels for CEBPB and GLUT4 (Fig. 3B; P=0.0001-0.02). For 5 genes (HSL, IL6, MCP1, UCP2, and VEGF), significant pre- and early postnatal nutrition interactions were found, but no consistent patterns of changes could be deciphered across all groups (Fig. 3C).
Only 3 genes were affected by the early postnatal nutrition independently of other factors, and HCHF sheep had the highest expression levels for CEBPB and PLIN1, but lowest for CD68 (Additional file 2: Fig. S4B; P=0.01-0.02). Similar to SUB, expression levels were highest in CONV♂ compared to other groups for three genes, FABP4, GLUT4, and VEGF (Additional file 2: Fig. S4B; P=0.0007-0.3). Females, if anything, tended to have the opposite response to the postnatal diet compared to males.
Mesenteric adipose tissue
The MES was the depot least affected by early life nutrition history in terms of gene expression patterns. Expression levels of only 4 genes were affected by the prenatal nutrition in either a sex-specific way (CD34, and CEBPB; P=0.01) or depending on the subsequent early postnatal nutrition exposure (CD34, FABP4, and PPARG; P=0.02-0.03), but it was difficult to discern any systematic patterns (results not shown). Furthermore, postnatal nutrition had an impact on expression levels of only one gene, namely ADRA1, where expression levels were highest in HCHF sheep (Additional file 2: Fig. S4C; P=0.03).
Epicardial adipose tissue
The prenatal nutrition history affected the expression of 14 genes in EPI, but only independently of other factors for two genes. Thus, ADRA1 expression levels were reduced in HIGH compared to NORM with LOW in between, and GLUT4 expression levels were reduced in LOW compared to HIGH and NORM (Fig. 4A; P=0.04 and 0.02, respectively). For another 7 genes (ATGL, IGF1R, GcR, LEPR, TGFB1, UCP2, and VEGF), the prenatal impact on expression levels was sex-dependent due to opposite responses in LOW♂ compared to LOW♀ to the prenatal nutrition (Fig. 4B).
Of all the adipose tissues, EPI was affected most by the early postnatal nutrition history. HCHF compared to CONV sheep had increased expression levels for 6 genes (ADIPOQ, FABP4, FAS, HSL, PLIN1, and PPARG) and reduced for 2 (CD68, and PPARA) (Additional file 2: Fig. S4D). For 5 genes (ATGL, CD68, CGI58, GLUT1, and TLR4) postnatal nutrition impacts depended on the prenatal nutrition history, and the general pattern was that LOW-CONV had the highest expression levels for these, and the changes in expression levels induced by HCHF were opposite in LOW compared to CONV and HIGH sheep (Fig. 4C; P=0.05-0.05). CONV♂ had higher expression levels compared to other groups for two genes, namely CD34, and GLUT1 (Additional file 2: Fig. S4D; P=0.04 and 0.01, respectively).