It has already been demonstrated that the pancreatic B cells of hypothyroid rats have a reduced capacity to release insulin in response to glucose (l). This impaired B cell function may be partly due to a diminished rate of glucose oxidation and net calcium uptake associated with ultrastructural alteration of the pancreatic islets (2). To identify further other factors responsible for this diminished B cell secretory function, we studied the composition and the turnover rate of phospholipids in islets obtained from hypothyroid rats.Facultad de Ciencias Médica

Cortizo, Ana María

Gagliardino, Juan José

García, María Elisa

Pasquini, Juana M.

SEDICI - Repositorio de la UNLP

IRCS Medical Science: Alimentary System;
Pathology; Physiology. URCS Med. Sci.,12,
Biochemistry; Cell and Membrane Biology;
n l7-1 1 18 (1984)l
Endocrine System; Metabolism and Nutrition;
Effect of hypothyroidism on the composition and turnover rate of islet phospholipids
Ana M. Cortizo, Maria E. Garcia, Juana M. Pasquini and Juan J. Gagliardino
CENEXA - Cento de Endocrinologia Experimentaly Aplicada (UNLP-CONICET), Facultad de Ciencias
Médicas, calles 60 y 120, 1900-La Plata, Argentina
Paper received: 25th September, 1984; amended 20th November, 1984
It has already been demonstrated that the pancreatic B cells of hypothyroid rats have a reduced capacity to release insulin in
response to glucose (l). This impaired B cell function may be partly due to a diminished rate of glucose oxidation and net
calcium uptake associated with ultrastructural alteration of the pancreatic islets (2). To identify further other factors
responsible for this diminished B cell secretory function, we studied the composition and the turnover rate of
phospholipids in islets obtained from hypothyroid rats.
Materials and methods: Wistar male rats about 100 g b.wt were used, rendered hypothyroid by administration of a
single intraperitoneal injection of 700 ¡rCi 131I in saline solution, whilst the remaining animals were used as controls. The
experiments were performed at least 5 weeks after the iodine injection. Thyroid function was assessed by the control of
individual body weight and the circulating levels of T, and T, (Rtl kit, Diagnostic Prod. Corp., Los Angeles). Serum
immunoreactive insulin (IRI) (3) and glucose levels (4) were also measured. Pancreata from both groups of rats were used
to obtain isolated islets by collagenase digestion (5).
Insulin secretion by isolated islets was studied by incubating groups of five islets for 60 min at 37"C in 0.6 ml Krebs
Ringer bicarbonate (KRB) buffer, pH 7.4 with l0lo bovine serum albumin (Sigma Chem. Co., St. Louis, MO), aprotinin
400 IU/ml (Trasylol@kindly provided by Bayer Argentina); and glucose 3.3 or 16.6 mmol/I. The preparation was gassed
with a mixture of 50/o COr:950/o Or. Aliquots from the media were separated for insulin determination by
radioimmunoassay (3).
sH-glycerol incorporation into islet phospholipids was studied using groups of fifty islets incubated for 60 min in I ml
KRB in the presence of 3.3 or 16.6 mM glucose plus the addition of 5 or 25 p.Ci of sH-glycerol (2-sH-glycerol,
l0 mCi/mmol, New England Nuclear, Boston, Ma), respectively. The tubes were gassed with a mixture of 50/o COr:950/o
Or. At the end of the incubation period, the islets were sonicated and the lipids extracted with 2:l chloroform:methanol
and the washed lower phase was used for the analysis of different lipids. Individual phospholipids were separated by thin
layer chromatography (6). Phospholipid areas were visualized with iodine vapour, scraped off from the plates and
transferred to counting vials to measure the corresponding radioactivity in a liquid scintillation counter.
Islets phospholipid composition was studied in groups of 600-700 isolated islets disrupted by sonication in l ml of
0.05 mM phosphate buffer, pH 7 .4. The islet homogenates were extracted and the individual phospholipid fraction isolated
as described above. The spots corresponding to each phospholipid fraction were scraped off and eluted with
1.0 M methanol: HCI at 60'C. The eluates were evaporated and the organic phosphorus was mineralized (7) and later
measured by a colorimetric reaction (8).
Table l: Body weights, serum glucose and thyroid hormone levels in control and hypothyroid rats (means -+ SEM, n in parentheses)
Body weight (g) Serum hormone levels
T' (ngldl) T. (¡¿gldl) IRI (¡¿Ulml)
Serum glucose
(me/dl)Animals Initial Final
Controls
Hypothyroid
125 +- 0.9 (l l) 245 + 4.3
127 + 5.0 (10) 170 i 5.4 (l l) 60.3 + 4.30 (28) 2.5 + 0.13 (28) 35.5 ¿(10)** 27.5 + 2.90 (37)** 0.7 + 0.09 (37)** 24.7 + 4.40 (19) 126.2 + 6.90 (24)2.60 (19)* 119.0 + 6.00 Q4)
In comparison with controlsp <: *0.05: **0.001.
Results and discussion: The final body weight values as well as the serum T, and T. levels demonstrate the deficient
thyroid function in the rats treated with 13rI (Table l). These animals also showed significantly lower serum IRI levels than
their corresponding controls. However, there were no signihcant differences in serum glucose levels between the two
groups. This could be due to an enhanced effect of insulin in these animals in the peripheral tissues (9) .
Table 2: Insulin secretion by islets, isolatedfrom contol and hypothyroid ruts, incubated with
two di.fferent glucose concentrations (means ! SEM, n in parentheses)
Insulin secreted (¡rUlislet/h)
Glucose
concentration Control Hypothyroid
3.3 mM
16.6 mM
10.8 + l.l0 (18)
317 .8 -r 32.5 (20) * *
16.8 r- 2.86 0)
153.9 +- t7 .2 (g).
*ln comparison with the valuefor the 3.3 mM glucosep <: *0.05: **0.00L
The insulin release by isolated islets was not significantly different in the hypothyroid rats as compared to the controls in
the presence of 3.3 mM glucose. However, the amount of insulin released by islets from hypothyroid rats in the presence
of 16.6 mM glucose was signiftcantly lower than that secreted by control islets (Table 2). These results confirm the
diminished B cell capacity to release insulin observed in hypothyroid animals (1).
The phosphorus content of islet phospholipids was significantly lower in hypothyroid rats. This difference was mainly
due to changes in the phosphatidylinositol (PI) and phosphatidylcholine (PC) fractions (Table 3).
1117
Composition and turnover rate of islet phospholipids Ana M. Cortizo et al.
The [3Hl-glycerol incorporation into islet phospholipids was similar in both groups when the islets were incubated in the
presence of 3.3 mM glucose (267.4 -r 13.9 for controls, vs 254.5 -f 18.6 for hypothyroid rats (dpm/islet/h)). Conversely,
in the presence of 16.6 mM glucose, control islets incorporated significantly larger amounts of SH-glycerol than islets from
hypothyroid rats (486.2 -f 30.3 vs 340.9 ! 12.4 dpm/islet/h respectively; p < 0.001).
Table 3: Phosphorus content of phospholipids from islets isolated from contro! and hypothyroid
rats (means ¡ SEM,n: 7I)
Phosphorus (nglislet)
Phospholipid Control Hypothyroid
PS
PI
PC
PE
TPL
0.8 + 0.16
0.7 + 0.02
2.0 + 0.04
1.4 + 0.06
4.9 ¡ 0.14c
0.5 + 0.02
0.5 r- 0.02**
1.4 + 0.09*
0.9 + 0.12
3.3 + 0.03r
PE, phosphatidylethanolamine; TPL, total phospholipids. fn represents the number of
acperiments performed in tilplicate with islets obtainedfiom at least six animals. In comparison
with valuefor controlsp 1: *0.05; *"0.0L
The percentages of leH]-glycerol incorporation into the different islet phospholipid fractions also showed a similar
pattern (Table 4). No differences were seen in the presence of low glucose, but with 16.6 mM glucose, control islets
showed a significant increase in the PI fraction, a modest one in the phosphatidylserine (PS), and a marked decrease at the
PC level. High glucose levels did not induce changes in islets from hypothyroid rats.
Table 4: Percentage disÜibution of PIII-glycercl incorporation into phosphotipids in islets isolatedfrom control and hypothyroid rats incubated with
two dffirent concentrations of glucose (means -+- SEM, n : 3l )
o/o [3H] -glycerol incorporated
Group PS PI PC PE
3.3 mM glucose
Control
Hypothyroid
16.6 mM glucose
Control
Hypothyroid
2.5 + 0.33
2.1 + 0.22
3.7 + 0.59
1.7 + 0.04a
5.9 + 0.51**
6.1 + 0.85
13.3 + 0.21
6.4 + 0.43c
66.2 + 2.34*
66.3 + 3.1 I
58.8 + 2.04
65.8 + 0.56b
25.4 + 2.65
25.5 r- 0.82
24.3 + 2.81
26.0 + 0.42
ln represents number of experiments performed by triplicate with islets obta¡ned from at least six animals. In comparison with contrcl group with
16.6 mM glucose p 1 : '0.05; *'0.001. In comparison with corresponding control group p < : a0.05: t0.02; ,0.00 1.
The importance of the islet phospholipid metabolism, particularly the PI fraction, as a regulator of the glucose-induced
insulin secretion has been described in detail in a recent review (10). Moreoever, we have demonstrated that PS turnover
rate is also involved in this mechanism (l l). According to our results, the reduced effect of glucose on insulin secretion in
the hypothyroid state may be due partly to the altered phospholipid composition and the impaired phospholipid turnover
rate observed in the pancreatic islets. Otherwise, these results are the first evidence of a hormonal control of the islet
phospholipid composition and metabolism. Further studies are needed in order to explain the underlying mechanism of
such effects.
l. Ahren, B. and Lundquist, I. (1981) Acta Endocrinol., 97, 508-5132. Gagliardino,l.J. et al. (1982) Proceedings of the First Latin American Thyroid Congress, Mar del Plata, Oct. 9-12, pp. 139-1523. Herbert, Y . et al. (196il J. Clin. Endocrinol. Metab.,25, I 375- I 3844. Dubowsky, K.M. (1962) Clin. Chem.,8,215-2355. Lacy, P. and Kostianovskv, M. (1967) Diobetes,16,35-396. Neskovic, N.M. and Kostic, D.M. (1968) 
"/. Chromatogr.,35, 297-3007. Seminario, L., Soto, E.F. and Cohan, T. (1965) J. Chromatogr., f 7, 513-5198. Chen, P.S. Jr, Toribara, T.Y.and Warner, H. (1956) Analyt. Chem.,28, 175ó-17589. Shah, I.H. et al. (79'15) D¡abetes,24,922-925
10. Best, L. and Malaisse, W.J. (1983) Diabetologia,25,299-305
I l. Gagliardino, J.J. (1982) Proceedings of the I lth Congress of the International Diabetes Federation, Nairobi, Nov. 10-17, pp.19-27
The authors are indebted to Dr. R.E. Hernández for the insulin assays. We also thank Mrs. Silvia Lunati for secretarial support. This
work was supported by funds from CONICET, CIC, Pcia. de Bs.As. and Fundación Lucio Cherny, Argentina.
1118


English

Effect of hypothyroidism on the composition and turnover rate of islet phospholipids

Servicio de Difusión de la Creación Intelectual

IRCS Medical Science: Alimentary System;Pathology; Physiology. URCS Med. Sci.,12,Biochemistry; Cell and Membrane Biology;n l7-1 1 18 (1984)lEndocrine System; Metabolism and Nutrition;Effect of hypothyroidism on the composition and turnover rate of islet phospholipidsAna M. Cortizo, Maria E. Garcia, Juana M. Pasquini and Juan J. GagliardinoCENEXA - Cento de Endocrinologia Experimentaly Aplicada (UNLP-CONICET), Facultad de CienciasMédicas, calles 60 y 120, 1900-La Plata, ArgentinaPaper received: 25th September, 1984; amended 20th November, 1984It has already been demonstrated that the pancreatic B cells of hypothyroid rats have a reduced capacity to release insulin inresponse to glucose (l). This impaired B cell function may be partly due to a diminished rate of glucose oxidation and netcalcium uptake associated with ultrastructural alteration of the pancreatic islets (2). To identify further other factorsresponsible for this diminished B cell secretory function, we studied the composition and the turnover rate ofphospholipids in islets obtained from hypothyroid rats.Materials and methods: Wistar male rats about 100 g b.wt were used, rendered hypothyroid by administration of asingle intraperitoneal injection of 700 ¡rCi 131I in saline solution, whilst the remaining animals were used as controls. Theexperiments were performed at least 5 weeks after the iodine injection. Thyroid function was assessed by the control ofindividual body weight and the circulating levels of T, and T, (Rtl kit, Diagnostic Prod. Corp., Los Angeles). Serumimmunoreactive insulin (IRI) (3) and glucose levels (4) were also measured. Pancreata from both groups of rats were usedto obtain isolated islets by collagenase digestion (5).Insulin secretion by isolated islets was studied by incubating groups of five islets for 60 min at 37"C in 0.6 ml KrebsRinger bicarbonate (KRB) buffer, pH 7.4 with l0lo bovine serum albumin (Sigma Chem. Co., St. Louis, MO), aprotinin400 IU/ml (Trasylol@kindly provided by Bayer Argentina); and glucose 3.3 or 16.6 mmol/I. The preparation was gassedwith a mixture of 50/o COr:950/o Or. Aliquots from the media were separated for insulin determination byradioimmunoassay (3).sH-glycerol incorporation into islet phospholipids was studied using groups of fifty islets incubated for 60 min in I mlKRB in the presence of 3.3 or 16.6 mM glucose plus the addition of 5 or 25 p.Ci of sH-glycerol (2-sH-glycerol,l0 mCi/mmol, New England Nuclear, Boston, Ma), respectively. The tubes were gassed with a mixture of 50/o COr:950/oOr. At the end of the incubation period, the islets were sonicated and the lipids extracted with 2:l chloroform:methanoland the washed lower phase was used for the analysis of different lipids. Individual phospholipids were separated by thinlayer chromatography (6). Phospholipid areas were visualized with iodine vapour, scraped off from the plates andtransferred to counting vials to measure the corresponding radioactivity in a liquid scintillation counter.Islets phospholipid composition was studied in groups of 600-700 isolated islets disrupted by sonication in l ml of0.05 mM phosphate buffer, pH 7 .4. The islet homogenates were extracted and the individual phospholipid fraction isolatedas described above. The spots corresponding to each phospholipid fraction were scraped off and eluted with1.0 M methanol: HCI at 60'C. The eluates were evaporated and the organic phosphorus was mineralized (7) and latermeasured by a colorimetric reaction (8).Table l: Body weights, serum glucose and thyroid hormone levels in control and hypothyroid rats (means -+ SEM, n in parentheses)Body weight (g) Serum hormone levelsT' (ngldl) T. (¡¿gldl) IRI (¡¿Ulml)Serum glucose(me/dl)Animals Initial FinalControlsHypothyroid125 +- 0.9 (l l) 245 + 4.3127 + 5.0 (10) 170 i 5.4 (l l) 60.3 + 4.30 (28) 2.5 + 0.13 (28) 35.5 ¿(10)** 27.5 + 2.90 (37)** 0.7 + 0.09 (37)** 24.7 + 4.40 (19) 126.2 + 6.90 (24)2.60 (19)* 119.0 + 6.00 Q4)In comparison with controlsp <: *0.05: **0.001.Results and discussion: The final body weight values as well as the serum T, and T. levels demonstrate the deficientthyroid function in the rats treated with 13rI (Table l). These animals also showed significantly lower serum IRI levels thantheir corresponding controls. However, there were no signihcant differences in serum glucose levels between the twogroups. This could be due to an enhanced effect of insulin in these animals in the peripheral tissues (9) .Table 2: Insulin secretion by islets, isolatedfrom contol and hypothyroid ruts, incubated withtwo di.fferent glucose concentrations (means ! SEM, n in parentheses)Insulin secreted (¡rUlislet/h)Glucoseconcentration Control Hypothyroid3.3 mM16.6 mM10.8 + l.l0 (18)317 .8 -r 32.5 (20) * *16.8 r- 2.86 0)153.9 +- t7 .2 (g).*ln comparison with the valuefor the 3.3 mM glucosep <: *0.05: **0.00LThe insulin release by isolated islets was not significantly different in the hypothyroid rats as compared to the controls inthe presence of 3.3 mM glucose. However, the amount of insulin released by islets from hypothyroid rats in the presenceof 16.6 mM glucose was signiftcantly lower than that secreted by control islets (Table 2). These results confirm thediminished B cell capacity to release insulin observed in hypothyroid animals (1).The phosphorus content of islet phospholipids was significantly lower in hypothyroid rats. This difference was mainlydue to changes in the phosphatidylinositol (PI) and phosphatidylcholine (PC) fractions (Table 3).1117Composition and turnover rate of islet phospholipids Ana M. Cortizo et al.The [3Hl-glycerol incorporation into islet phospholipids was similar in both groups when the islets were incubated in thepresence of 3.3 mM glucose (267.4 -r 13.9 for controls, vs 254.5 -f 18.6 for hypothyroid rats (dpm/islet/h)). Conversely,in the presence of 16.6 mM glucose, control islets incorporated significantly larger amounts of SH-glycerol than islets fromhypothyroid rats (486.2 -f 30.3 vs 340.9 ! 12.4 dpm/islet/h respectively; p < 0.001).Table 3: Phosphorus content of phospholipids from islets isolated from contro! and hypothyroidrats (means ¡ SEM,n: 7I)Phosphorus (nglislet)Phospholipid Control HypothyroidPSPIPCPETPL0.8 + 0.160.7 + 0.022.0 + 0.041.4 + 0.064.9 ¡ 0.14c0.5 + 0.020.5 r- 0.02**1.4 + 0.09*0.9 + 0.123.3 + 0.03rPE, phosphatidylethanolamine; TPL, total phospholipids. fn represents the number ofacperiments performed in tilplicate with islets obtainedfiom at least six animals. In comparisonwith valuefor controlsp 1: *0.05; *"0.0LThe percentages of leH]-glycerol incorporation into the different islet phospholipid fractions also showed a similarpattern (Table 4). No differences were seen in the presence of low glucose, but with 16.6 mM glucose, control isletsshowed a significant increase in the PI fraction, a modest one in the phosphatidylserine (PS), and a marked decrease at thePC level. High glucose levels did not induce changes in islets from hypothyroid rats.Table 4: Percentage disÜibution of PIII-glycercl incorporation into phosphotipids in islets isolatedfrom control and hypothyroid rats incubated withtwo dffirent concentrations of glucose (means -+- SEM, n : 3l )o/o [3H] -glycerol incorporatedGroup PS PI PC PE3.3 mM glucoseControlHypothyroid16.6 mM glucoseControlHypothyroid2.5 + 0.332.1 + 0.223.7 + 0.591.7 + 0.04a5.9 + 0.51**6.1 + 0.8513.3 + 0.216.4 + 0.43c66.2 + 2.34*66.3 + 3.1 I58.8 + 2.0465.8 + 0.56b25.4 + 2.6525.5 r- 0.8224.3 + 2.8126.0 + 0.42ln represents number of experiments performed by triplicate with islets obta¡ned from at least six animals. In comparison with contrcl group with16.6 mM glucose p 1 : '0.05; *'0.001. In comparison with corresponding control group p < : a0.05: t0.02; ,0.00 1.The importance of the islet phospholipid metabolism, particularly the PI fraction, as a regulator of the glucose-inducedinsulin secretion has been described in detail in a recent review (10). Moreoever, we have demonstrated that PS turnoverrate is also involved in this mechanism (l l). According to our results, the reduced effect of glucose on insulin secretion inthe hypothyroid state may be due partly to the altered phospholipid composition and the impaired phospholipid turnoverrate observed in the pancreatic islets. Otherwise, these results are the first evidence of a hormonal control of the isletphospholipid composition and metabolism. Further studies are needed in order to explain the underlying mechanism ofsuch effects.l. Ahren, B. and Lundquist, I. (1981) Acta Endocrinol., 97, 508-5132. Gagliardino,l.J. et al. (1982) Proceedings of the First Latin American Thyroid Congress, Mar del Plata, Oct. 9-12, pp. 139-1523. Herbert, Y . et al. (196il J. Clin. Endocrinol. Metab.,25, I 375- I 3844. Dubowsky, K.M. (1962) Clin. Chem.,8,215-2355. Lacy, P. and Kostianovskv, M. (1967) Diobetes,16,35-396. Neskovic, N.M. and Kostic, D.M. (1968) "/. Chromatogr.,35, 297-3007. Seminario, L., Soto, E.F. and Cohan, T. (1965) J. Chromatogr., f 7, 513-5198. Chen, P.S. Jr, Toribara, T.Y.and Warner, H. (1956) Analyt. Chem.,28, 175ó-17589. Shah, I.H. et al. (79'15) D¡abetes,24,922-92510. Best, L. and Malaisse, W.J. (1983) Diabetologia,25,299-305I l. Gagliardino, J.J. (1982) Proceedings of the I lth Congress of the International Diabetes Federation, Nairobi, Nov. 10-17, pp.19-27The authors are indebted to Dr. R.E. Hernández for the insulin assays. We also thank Mrs. Silvia Lunati for secretarial support. Thiswork was supported by funds from CONICET, CIC, Pcia. de Bs.As. and Fundación Lucio Cherny, Argentina.1118

http://sedici.unlp.edu.ar/bitstream/handle/10915/67396/Documento_completo.pdf?sequence=1

Effect of hypothyroidism on the composition and turnover rate of islet phospholipids

Abstract

Similar works

Full text

Available Versions

SEDICI - Repositorio de la UNLP

Servicio de Difusión de la Creación Intelectual