Table 2.

Summary data table of glucagon and TNF alpha effects.

(i) ORO staining density in 6 d insulin treated cell subsequently exposed to glucagon or TNF alpha average percentage ORO+ cell area, normalized to starting percentage, triplicate z discs from n=50 cells
Time Course				Dose Response
Time (d)	Control	Glucagon	TNF alpha	9 d Glucagon	ORO	9 d TNF alpha	ORO
0	100	100	100	0	100	0	100
1	96	96	89	100 ng/mL	92***	0.1 nM	101
2	98	92**	66	1 µg/mL	81***	1 nM	96
4	101	84*	42	10 µg/mL	74***	10 nM	77**
8	96	80**	38			100 nM	52**
12	100	79***	N.D.			1 µM	41***
p value relative to day 0, * p<0.05; **, p<0.01; ***, p<0.001; no symbol, >0.05.				p value relative to zero dose 0, * p<0.05; **, p< 0.01; ***, p<0.001; no symbol, p>0.05.
(ii) Reversal of steatosis average percentage ORO+ cell area, triplicate z discs from n=50 cells ± SD, cells exposed to 6 d insulin then 9 d glucagon or TNF alpha)
6 d insulin+9 day vehicle			+9 d glucagon		+9 d TNF alpha
ORO%	% reversal		ORO%	% reversal	ORO%	% reversal
100	−3		100	−11**	100	−31***
(iii) LTC4 secretion pg/1million cells, in response to IgE (l µg/ml 16h then 250 ng/ml KLH-DNP ± SD
6 d insulin+9 day vehicle			+9 d glucagon		+9 d TNF alpha
220 ± 11.2			198 ± 13.6*		106 ± 21.4***
(iv) LTC4 secretion pg/1 million cells, in response to IgE (1µg/ml 16 h) then KLH-DNP ± SD				(v) LB and SG relative abundance in response to TNF alpha treatment ORO+ % cell area and anti-tryptase+ structures/cell, mean o n=50 cells ± SD
KLH-DNP (ng/nl)	vehicle	6 d TNF alpha		control		6 d TNF alpha
0	13.3	12.4		ORO+% cell area
20	15.6	19.3		15.6		7.7**
200	204.4	164.7**		anti-tryptase+ structures/cell
2000	309.6	212.5**		16 ± 4		13 ± 9
(vi) Degranulation responses Betahexoseaminidase release as % of PMA-Ionomyin stimulated release (% max)
6 d insulin+9 day vehicle			+9 d glucagon		+9 d TNF alpha
66.8%			64.9%		63.7%

(i) ORO staining density in 6 d insulin treated cells subsequently exposed to glucagon or TNF alpha. LB content was assessed using quantification of ORO-positive area in triplicate z discs from 50 cells per point. Stimuli comprised 6 day exposure to insulin (10 µg/ml) for 6 days followed by 9 day exposure (with matched passaging) to glucagon (10 µg/ml) or TNF alpha (250 nM) (unless concentrations otherwise specified). ORO-positive area at initial timepoint or zero dose was treated as 100%, then effects of stimuli were expressed as % normalized to the starting value. (ii) Comparison of the percentage reversal of 6 d insulin-induced steatosis at 9 day exposure to vehicle, versus glucagon (10 µg/ml) or TNF alpha (250 nM). (iii) Leukotriene C₄ (LTC₄) release in response to antigenic stimulation of the RBL2H3 via FcεRI (1 µg/ml IgE anti-DNP for 16 h followed by 250 ng/ml KLH-DNP) in cells exposed to 6 d insulin then 9 d vehicle, glucagon or TNF alpha. (iv) Leukotriene C₄ (LTC₄) release in response to a dose response of antigenic stimulation of the RBL2H3 via FcεRI in cells treated with no insulin, but 6 d vehicle or TNF alpha as indicated. (v) LB and SG abundance measured in cells treated with 6 d insulin or 6 d TNF alpha by ORO-positive z disc area (LB) and major anti-tryptase positive structures of >180 nM diameter and not co-localized either ER-or Golgi-specific stains (SG). (vi) Betahexoseaminidase release in response to PMA/Ionomycin expressed as percentage of maximal release in cells exposed to 6 d insulin followed by 9 d vehicle, 9 d glucagon or 9 d TNF alpha.