Palmitoleic acid reduces intramuscular lipid and restores insulin sensitivity in obese sheep

Figures & data

Table 1 Primer sequences (5′–3′) for quantitative real-time PCR

Gene abbreviations	Forward	Reverse	Efficiency
ACC	GCTGCTGGTGTTCCTAAAGC	GTCCTCAAATTGTGCGTGAA	1.01
AKT1	AAGAGGCAGGAGGAGGAGAC	CCCAGCAGCTTCAGGTACTC	1.01
AMPK	GTCAAAGTCGGCCAAATGAT	CCTCCGAACACGCAAATAAT	1.03
CPT1a	CCACCTCTTCTGCCTCTACG	TTGAACAGTTCCACCTGCTG	1.04
CPT1b	GGTCGACTTCCAGCTCAGTC	CAGGAGGAACCCACTGTTGT	1.05
ELOVL5	CGCCACACTTAACAGCTTCA	AGCTGGTCTGGATGATGGTC	1.10
ELOVL6	AGTGGATGCAGGAAAACTGG	AAGGGTCAGAGACCAGAGCA	1.05
FABP4	AATTGGGCCAGGAATTTGAT	GCACCAGCTTATCATCCACA	1.00
FASN	TCATCCCCCTGATGAAGAAG	GCAGTGGTCCACCAGGTAGT	1.09
GAPDH	GGGTCATCATCTCTGCACCT	GGTCATAAGTCCCTCCACGA	1.01
GLUT4	CTCTCCACTTGCCCAGAGAC	CTGCCTAGCCACAACACAAA	1.04
HSL	GGCACTCCTGGATGCTGATT	CACTGTCCGCGGAGATACTC	0.98
IR	CTGACTTCCGAGACCTCCTG	GTTCTGTGACTTGGGGTCGT	0.95
IRS	TCCGCCTTTCCTCAAGTTCC	AGCTGTGTCCACCTTTCGAG	0.99
PI3KR1	AAGTCATGGCCATTGTAGGC	ACCTCTTGCTCTTGCGGTAA	0.96
SCD1	CGACGTGGCTTTTTCTTCTC	GATGAAGCACAACAGCAGGA	0.96

Abbreviations: PCR, polymerase chain reaction; ACC, acetyl-CoA carboxylase; AKT1, v-akt murine thymoma viral oncogene homolog 1; AMPK, protein kinase, AMP-activated, alpha 1 catalytic subunit, transcript variant 1; CPT1a, carnitine palmitoyltransferase 1a; CPT1b, carnitine palmitoyltransferase 1b; ELOVL5, fatty acid elongase-5; ELOVL6, fatty acid elongase-6; FABP4, fatty acid binding protein 4; FASN, fatty acid synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT4, glucose transporter type 4; HSL, hormone-sensitive lipase; IR, insulin receptor; IRS, insulin receptor substrate; PI3KR1, phosphoinositide-3-kinase, regulatory subunit 1 (alpha); SCD1, stearoyl-CoA desaturase-1.

Table 2 Gain, carcass traits, and body composition of lambs infused with varying levels of palmitoleic (C16:1) acid

	mg C16:1/kg LW/d			SEM	Contrasts
	0	5	10		Linear	Quadratic
Daily weight gain, kg/d	0.30	0.29	0.07	0.051	0.01	0.18
Carcass weight, kg	58.82	56.68	56.18	2.02	0.38	0.75
Ribeye area, cm^2	22.3	22.4	20.3	1.29	0.31	0.48
Fat thickness, cm	1.98	1.93	1.93	0.25	0.88	0.94
Body wall thickness, cm	4.72	4.98	4.93	0.37	0.70	0.74
Visceral fat depots, kg
Perirenal fat	2.30	2.15	2.41	0.29	0.78	0.58
Omental fat	2.57	3.16	2.68	0.37	0.83	0.26
Mesenteric fat	0.99	0.87	0.83	0.073	0.15	0.67
Body composition, % of carcass weight
Moisture	31.49	–	30.54	0.90	0.48	–
Lipid	39.18	–	40.44	2.25	0.70	–
Protein	14.94	–	14.58	0.78	0.75	–
Bone	14.39	–	14.43	1.09	0.98	–

Note: –, not measured.

Abbreviations: LW, lipid weight; d, days; SEM, standard error of the mean.

Table 3 Serum fatty acid content from lambs infused with varying levels of palmitoleic (C16:1) acid

Fatty acids, μg/mL	mg C16:1/kg LW/d			SEM	Contrasts
	0	5	10		Linear	Quadratic
C14:0	3.47	3.28	3.35	0.17	0.62	0.55
C14:1	3.93	3.68	3.30	0.26	0.09	0.84
C16:0	107.94	102.68	110.53	5.72	0.75	0.35
C16:1 cis-9	8.93	11.69	14.28	1.16	0.001	0.95
C18:0	150.92	124.35	154.18	9.50	0.81	0.02
C18:1 trans	13.00	16.21	15.66	1.30	0.15	0.24
C18:1 cis-9	147.07	132.18	151.00	9.04	0.76	0.13
C18:1 cis-11	5.05	5.89	7.66	0.39	0.001	0.32
C18:2 cis-9,12	201.21	198.33	236.55	13.71	0.07	0.22
C18:3 cis-9,12,15	7.15	7.29	9.69	1.12	0.11	0.41
C20:4 cis-5,8,11,14	28.73	28.15	38.58	2.00	0.001	0.02
C20:5 cis-5,8,11,14,17	3.78	4.16	4.94	0.27	0.001	0.56
C22:5 cis-7,10,13,16,19	8.76	8.12	9.22	0.49	0.51	0.15
C22:6 cis-4,7,10,13,16,19	8.69	9.17	9.02	0.64	0.71	0.38
Total, μg/mL	757.60	716.29	837.11	43.45	0.22	0.10
Saturated	262.33	230.31	268.06	15.13	0.79	0.06
Monounsaturated	12.86	15.37	17.58	1.26	0.01	0.92
Polyunsaturated n-6	229.94	226.48	275.13	14.95	0.04	0.16
Polyunsaturated n-3	28.38	27.74	32.87	2.09	0.13	0.26
Ratio n-6:n-3	8.43	8.77	8.98	0.41	0.34	0.90
C16:1–C16:0	0.083	0.12	0.13	0.01	0.001	0.48
C18:1–C18:0	0.99	1.12	0.99	0.04	0.86	0.01
C16:1–C18:1c11	0.83	1.06	0.98	0.11	0.35	0.28

Abbreviations: LW, lipid weight; d, days; SEM, standard error of the mean.

Figure 1 Effects of palmitoleic acid infusion on serum cis-11 vaccenic acid levels.

Notes: Changes in serum cis-11 vaccenic (C18:1 cis-11) acid concentration (μg/mL) by palmitoleic acid infusion level (0 mg/kg LW/d, 5 mg/kg LW/d, or 10 mg/kg LW/d) at 5 minutes post-dosing by time of the experiment.
Abbreviations: LW, lipid weight; d, days.

Figure 2 Changes in plasma insulin concentration and HOMA-IR values with palmitoleic acid infusion at three levels.

Notes: Plasma insulin concentrations (μIU/mL) (A) and HOMA-IR (B) in lambs infused with varying levels of palmitoleic (C16:1) acid levels (0 mg/kg LW/d, 5 mg/kg LW/d, or 10 mg/kg LW/d) by day of experiment.
Abbreviations: LW, lipid weight; d, days; HOMA-IR, Homeostatis Model Assessment-Insulin Resistance.

Table 4 Fatty acid composition of tissues (subcutaneous adipose tissue, mesenteric adipose tissue, liver, longissimus muscle, ST muscle) from lambs infused with varying levels of palmitoleic (C16:1) acid

Fatty acid, % of total	mg C16:1/kg LW/d			SEM	Contrasts
	0	5	10		Linear	Quadratic
C14:0	1.50	1.68	1.45	0.04	0.35	0.001
C15:0	0.31	0.36	0.35	0.03	0.32	0.29
C16:0	20.74	21.35	20.08	0.28	0.11	0.01
C16:1 cis-9	0.89	1.13	1.02	0.04	0.04	0.01
C17:0	1.31	1.43	1.31	0.04	0.96	0.01
C18:0	26.01	23.15	25.09	0.45	0.16	0.001
C18:1 trans	2.69	3.87	3.24	0.26	0.15	0.01
C18:1 cis-9	33.71	33.68	33.24	0.39	0.41	0.67
C18:2 cis-9,12	4.22	4.40	4.63	0.15	0.06	0.91
C18:3 cis-9,12,15	0.45	0.48	0.54	0.02	0.02	0.57
C20:4 cis-5,8,11,14	2.76	2.68	2.88	0.12	0.52	0.40
C20:5 cis-5,8,11,14,17	0.14	0.22	0.31	0.041	0.001	0.90
C22:5 cis-7,10,13,16,19	0.90	0.92	1.09	0.061	0.03	0.35
C22:6 cis-4,7,10,13,16,19	1.05	1.11	1.24	0.11	0.25	0.81
Saturated fatty acids, %	48.25	46.18	46.62	0.52	0.03	0.05
Odd chain fatty acids, %	1.62	1.79	1.66	0.05	0.60	0.01
Monounsaturated fatty acids, %	34.60	34.81	34.27	0.40	0.57	0.45
Polyunsaturated fatty acids, n-6, %	6.42	6.55	6.93	0.16	0.04	0.55
Polyunsaturated fatty acids, n-3, %	2.12	2.26	2.65	0.10	0.001	0.38
Ratio, n-6:n-3	4.06	4.53	3.97	0.25	0.79	0.09
C16:1–C16:0 ratio	0.046	0.057	0.057	0.003	0.01	0.11
C18:1–C18:0 ratio	1.53	1.71	1.55	0.047	0.69	0.01
C16:1–C18:1c11 ratio	1.42	1.84	1.73	0.072	0.001	0.001

Abbreviations: ST, semitendinosus; LW, lipid weight; d, days; SEM, standard error of the mean.

Figure 3 Effects of palmitoleic acid infusion on intramuscular lipid content.

Notes: Total lipid content (g/100 g of tissue) of liver, LM, and ST muscle (A), and SQ and MS fat (B) in lambs infused with varying levels of palmitoleic (C16:1) acid levels (0 mg/kg LW/d, 5 mg/kg LW/d, or 10 mg/kg LW/d) for 28 d.
Abbreviations: LM, longissimus muscle; ST, semitendinosus; SQ, subcutaneous; MS, mesenteric; LW, lipid weight; d, days.

Figure 4 Effects of palmitoleic acid infusion on mean adipocyte size and distribution.

Notes: Mean adipocyte size (μm) (A) for both IM and SQ adipose tissues, and adipocyte size distribution (μm) (B) for IM adipose tissues by palmitoleic acid infusion level. IM adipocyte size was smaller (P<0.05) in palmitoleic acid infusion for dose level 10 mg/kg BW/d.
Abbreviations: LW, lipid weight; d, days; IM, intramuscular; SQ, subcutaneous; BW, body weight.

Figure 5 Fold-change in lipogenic gene expression for 10 mg C16:1/kg LW/d versus control (0 mg/kg BW/d) averaged over the three tissues (subcutaneous adipose tissue, ST muscle, and liver).

Notes: *P<0.05. **P<0.01.
Abbreviations: BW, body weight; LW, lipid weight; d, days; ST, semitendinosus; SCD, stearoyl-CoA desaturase; FASN, fatty acid synthase; ACC, acetyl-CoA carboxylase; ELOVL5, fatty acid elongase-5; ELOVL6, fatty acid elongase-6; CPT1a, carnitine palmitoyltransferase 1a; HSL, hormone-sensitive lipase; FABP4, fatty acid binding protein 4.

Figure 6 Effects of palmitoleic acid infusion on gene expression and protein content of tissues.

Notes: Fold-change in glucose/insulin-related gene expression (A) for 10 mg C16:1/kg LW/d versus control (0 mg/kg BW/d) averaged over the three tissues (subcutaneous adipose tissues, ST muscle, and liver). Density of AMPKα1 protein density (B), pAMPKα1 protein density (C), and ratio of pAMPKα1 to AMPKα1 for 10 mg C16:1/kg LW/d versus control (0 mg/kg BW/d) averaged over the three tissues (subcutaneous adipose tissues, ST muscle, and liver). *P<0.05.
Abbreviations: LW, lipid weight; d, days; BW, body weight; ST, semitendinosus; AMPK, protein kinase, AMP-activated, alpha 1 catalytic subunit, transcript variant 1; P13KR1, phosphoinositide-3-kinase, regulatory subunit 1 (alpha); IR, insulin receptor; AKT1,v-akt murine thymoma viral oncogene homolog 1; IRS1, insulin receptor substrate 1.

Figure 7 Palmitoleic acid infusion alters gene expression differentially by tissue.

Notes: Fold-change in gene expression for 10 mg C16:1/kg LW/d versus control (mg/kg LW/d) by tissue (subcutaneous adipose, liver, and ST muscle). The interaction between palmitoleic acid infusion and tissue (ST, SQ, liver) was significant (P<0.05) for GLUT4 and CPT1b. *P<0.05. **P<0.01.
Abbreviations: LW, lipid weight; d, days; ST, semitendinosus; SQ, subcutaneous; GLUT4, glucose transporter type 4; CPT1b, carnitine palmitoyltransferase 1b.