Bull Vet Inst Pulawy 51, 655-659, 2007
SPONTANEOUS AMYLOIDOSIS AND DIABETES MELLITUS
IN A RHESUS MACAQUE (MACACA MULATTA)
HANDAN HILAL ARSLAN, CEVAT NISBET1, AND TOLGA GUVENC2
Department of Internal Medicine, 1Department of Biochemistry,
2
Department of Pathology, Faculty of Veterinary Medicine,
University of Ondokuz Mayis, 55139, Samsun, Turkey
hharslan@omu.edu.tr
Received for publication May 30, 2007
Abstract
Clinical signs and necropsy findings were described
in a four-year-old female rhesus macaque with diabetes
mellitus, resulting from spontaneous amyloidosis. Weakness,
polyphagia, polydipsia, and polyuria, which lasted for a few
weeks, were determined from anamnesis and clinical
investigations. Highly increased serum BUN, AST, ALP
levels, glycosuria, and proteinuria was detected in the
biochemical analysis. According to these clinical,
haematological, and biochemical data, it was thought that the
monkey was affected with diabetes mellitus. Despite all efforts
to cure the animal, the patient died. Diffuse amyloidosis was
detected in the kidney and pancreas at the necropsy and
histopathological examinations.
Key
words:
monkey,
spontaneous
amyloidosis, diabetes.
Diabetes mellitus has been described in
numerous non-human primates. Most of these
descriptions derived from experimental data, in which
diabetes was induced by β-cell destruction either
chemically with streptozotocin or surgically (15, 20).
However, spontaneous diabetes has been reported in
cynomolgus and rhesus monkeys but availability of
these reports is limited (16).
Amyloidosis is a term used for the description
of a group of diseases, which are characterised by the
excessive deposition of ultrastructurally identical but
biochemically different protein fibrils either focal or
multifocal in various tissues (12). Amyloid may be
deposited in many organs and tissues but it is most
frequently found in the spleen, liver, kidneys, and
adrenals. Generally, it is a chronic, progressive,
insidious disease of unknown origin. The disease may
not become clinically apparent until major organ
dysfunction develops due to displacement, atrophy, or
necrosis (1, 12). Thus, generally, the disease is usually
diagnosed histopathologically (7). If critical organs such
as the kidneys, liver, or heart are involved, the disease
may be fatal (1). Spontaneously occurred deposition of
amyloid and gradual degeneration of all cells have been
detected in pancreatic islets of Langerhans in some
macaque species and the lesions have been evaluated as
a reason of diabetes mellitus (9). Similar spontaneous
amyloidosis occurs in many non-human primate species,
including rhesus monkeys, but the clinical diagnosis and
treatment is difficult. The main clinical signs of
amyloidosis are hyperglycaemia and cachexia, but these
signs cannot be detected in all cases (10, 20).
In this case, diabetes mellitus was diagnosed
clinically and spontaneous pancreatic amyloidosis was
demonstrated histopathologically in a four-year-old
rhesus macaque. It was a very unusual case for Turkey,
because Turkey is a subtropical country in the northern
hemisphere of the world, and there are no normal living
examples of this species. Therefore, it is known that this
is the first report of spontaneous pancreatic amyloidosis
and diabetes mellitus in a rhesus macaque in Turkey.
Material and Methods
A four-year-old female rhesus macaque who
was brought with complaints of polyphagia, polyuria,
and polydipsia that lasted for a couple of weeks was the
subject of the investigations. The blood samples were
collected from the jugular vein into EDTA-containing
and empty tubes for haematological and biochemical
evaluations. The serum was separated by centrifugation
of the tubes at 1 550 x g for 10 min. The blood cells
were counted by a cell-counter (Ermax-18, Erma Inc.,
Japan). Serum concentrations of glucose and aspartate
aminotransferase (AST), alanine aminotransferase
(ALT), and blood urea nitrogen (BUN) levels were
analysed with commercially available kits according to
the manufacturer’s instructions by autoanalyser
(Autolab, AMS Srl, Selective Access, Netherlands).
The early diagnosis was diabetes mellitus and
the treatment was immediately started. Firstly, 0.5 IU/kg
of insulin (Humulin M 70/30, Eli Lilly and Company)
was administered subcutaneously (the total dose was
divided into two equal parts and was given at 12 h
656
intervals). Additionally, intravenous 0.9 % NaCl and
lactated Ringer’s solutions were administered.
Macroscopic,
histopathological,
and
immunohistochemical investigations were performed
after the patient died. After the necropsy, tissue samples
were fixed in 10% neutral buffered formalin, cut into 4
to 5 µm thick sections that were then stained with
haematoxylin-eosin (HE) and Congo-red technique.
Additional
sections,
placed
on
3aminopropyltriethoxysilane (Sigma, USA) coated slides,
were stained by the streptavidin/biotin/peroxidase
complex technique using monoclonal antibody (Clone
Bam-10, Sigma, USA) for β-amyloid. The slides were
then dipped in freshly prepared absolute methanol
containing 3% hydrogen peroxide (v/v) for 5 min to
block endogenous peroxidase activity. All the sections
were then pre-incubated in a 10% normal goat serum for
30 min at room temperature, to block non-specific
binding of the second-step antibody. The sections
reacted with a primary antibody for 60 min at room
temperature, and then they were rinsed with PBS. Next,
the sections reacted with biotin-conjugated second-step
antibody for 10 min at room temperature, and then they
were rinsed in PBS. Afterwards, the sections were
treated with streptavidin/biotin/peroxidase complex for
10 min at room temperature. After another washing with
PBS, the sections were incubated with diaminobenzidine
for 10 min and then counterstained with Mayer’s
haematoxylin. Negative control tissue sections were
incubated with normal rabbit serum.
Results
All findings were summarised in Table 1. At
the clinical examination, the monkey was found to be
lethargic, severely dehydrated, hypothermic, and
cachectic. The mucous membranes were pale and the
capillary refill time was prolonged.
In comparison with reference values (17, 18)
complete blood cell count showed a marked leukopenia
(5.4x103/mm3) and monocytosis (14.8%), and serum
biochemical results showed significant increase in serum
glucose (352 mg/dL), AST (235 U/L), ALT (158 U/L),
and BUN (52 mg/dL) levels. Urine analysis revealed
significant glycosuria and proteinuria. On the basis of
the findings, diabetes mellitus was suspected and the
treatment was immediately started. Despite the all
therapy efforts, the patient died within 24 h after the
treatment started.
The liver and pancreas were found to be
yellowish and crusty at necropsy. The kidneys were
swollen and their capsules were easily peeled.
Homogenic pink coloured amyloid accumulations in
wider areas of the pancreas were observed
microscopically after HE and Congo-red staining (Figs 1
and 2). Immunohistochemically, ß-amyloid deposits
were seen in the pancreas and renal glomeruli (Fig. 3).
Degeneration of hepatocytes and disruption of Remark
cords were found in the liver.
Discussion
Diabetes mellitus has been reported in some
non-human primates. Although most of these
descriptions derived from experimental data, it has been
also noted that diabetes mellitus may occur in nonhuman primates spontaneously (20).
Hubbard et al. (12) observed hyperglycaemia
and cachexia in 12 of the 30 monkeys with pancreatic
islet amyloidosis. Furthermore, the researchers reported
that it was difficult to determine spontaneous pancreatic
islet amyloidosis clinically, but they could do it
histopathologically.
Table 1
Clinical and pathological findings
Anamnesis
Polyphagia, polydipsia and polyuria.
Clinical signs
Lethargy, severe dehydration,
prolonged capillary refill time
Haematological findings
Leukopoenia, monocytosis
Biochemical findings
High glucose, AST, ALT and BUN levels
Urine analysis
Glycosuria, proteinuria
Necropsy
Yellowish and crusty liver and pancreas, swollen kidneys, and
easily peeled kidney capsule
Histopathology and
Immunohistochemistry
Amyloid accumulations in pancreas and glomeruli of kidneys,
ß-amyloid deposits in pancreas and renal glomeruli,
degeneration of hepatocytes and disruption of Remark cords
hypothermia,
cachexia,
657
Fig. 1. Amyloid accumulation in Langerhans islets in the pancreas (HE, 90x).
Fig. 2. Amyloid accumulation in Langerhans islet in the pancreas (Congo-red staining, 180x).
Fig. 3. Amyloid accumulation in renal glomeruli (Streptavidin/biotin/peroxidase complex technique, 180x).
658
Bohm et al. (6) detected significantly decreased
leukocyte number in an immune-suppressed Macaca
mulatta. In addition, pathologic changes, such as tissue
destruction, and acute and chronic inflammations, and
immune disturbances, can be counted among the reasons
of monocytosis. Thus, monocytosis is considered to be a
possible alteration in the blood profile of a patient with
amyloidosis, which is defined as a chronic and immunemediated disorder (4). Leukopoenia and monocytosis
were detected in this case.
The most important clinical and biochemical
findings of diabetes mellitus in monkeys are
hyperglycaemia, glycosuria, hyperlipidaemia, polyuria,
polydipsia, polyphagia, weight loss, anorexia, and
weariness (20). In this case, polyphagia, polydipsia, and
polyuria already existed but anorexia, weariness, and
severe weight loss followed these findings. A severe
hyperglycaemia was found at biochemical analysis.
Walzer (20) reported that serum glucose values were
persistently higher than 150 mg/dL in diabetic primates
and higher than 300 mg/dL in severely diabetic
primates. In this case, serum glucose value was 352
mg/dL. Glycosuria is one of the very important classical
clinical symptoms of diabetes mellitus (16). Severe
glycosuria was found in the urine analysis. Blood and
Studdert (5) noted that the kidneys are the organ most
often affected by amyloidosis and the amyloid is most
often deposited in glomeruli. Glomerular amyloidosis
usually leads to marked proteinuria (8). In this case,
proteinuria was detected and this was connected with
amyloid accumulation in the glomeruli. All these
clinical and laboratory data, which supported the
diagnosis of diabetes mellitus were compatible with the
previously reported findings.
BUN increases in the kidneys with chronic
amyloidosis (4). The BUN level was determined high in
this case, and amyloid accumulations were seen in the
periphery of capillary balls of the glomerulus in the
histopathological examinations. The cause of a high
BUN level resulted from chronic amyloidosis of the
kidneys.
Intensive amyloid accumulation was observed
in the pancreas after necropsy (Fig. 1). Similarly, with
our findings, Palotay and Howard (14) found amyloid
accumulation in Langerhans islets of the pancreas of 21
monkeys with diabetes mellitus. Howard and Palotay
(11) also noted β-cell loss in Langerhans islets, due to
massive amyloid infiltration in the pancreas in two
monkeys with hyperglycaemia. Wagner et al. (19)
reported that due to pancreatic amyloidosis, type II
diabetes mellitus occurred in monkeys.
There is a connection between diabetes and
liver destruction. It has also been reported that
hepatomegaly and abnormalities of liver enzymes could
occur as a result of hepatocellular glycogen
accumulation (2). AST is an enzyme, localised in
cytosol and mitochondria, and it is not organ specific. Its
highest concentrations are in hepatocytes, myocardium,
skeletal muscles, kidney tissue, and placenta cells.
Serum AST concentrations increase due to necrosis or
destruction of these tissues (2, 3). ALT is a cytoplasmic
enzyme and its serum level increases in hepatic diseases
with hepatocyte destructions (16).
Serum ALT and AST levels were found to be
remarkably higher in this rhesus macaque than the
reported reference levels (17, 18). In addition,
degeneration in hepatocytes and disruption of Remark
cords were seen in the histopathological examinations of
the liver. These findings were considered compatible
with the hepatic degeneration, due to the disorders in
carbohydrate metabolism in diabetes mellitus related to
pancreatic amyloidosis.
Wagner et al. (19) examined diabetes mellitus
and islet amyloidosis in 6 cynomolgus monkeys aged
between 15 and 20 years. They concluded that
amyloidosis was the common symptom in the old age.
Similarly, to the case described, pancreatic islet
amyloidosis was determined as the cause of diabetes
mellitus in those cases. However, in contradiction with
that report, the monkey in our study was 4-year-old.
Thus, this is a valuable finding; because it is proof that
spontaneous severe islet amyloidosis in the pancreas is
not only seen in old monkeys.
Naumenko and Krylova (13) detected
amyloidosis in 88 monkeys of 2 588 autopsied
macaques. In their study, amyloidois was more often
seen in the medullary stroma and vascular walls and
more rarely under tubular basal membranes and in
glomeruli of the kidneys. Amyloidosis was detected in
glomeruli of the kidneys in this case. The prevalence of
amyloidosis was detected only in 3.2% in 1-4-year-old
Macaca mulatta and this ratio increased to 23.2% in 1720-year-old ones and to 44.1% in those older than 20
years. (13). Therefore, this data also showed that
spontaneous glomerular and pancreatic amyloidosis is
unusual in a 4-year-old Macaca mulatta.
In summary, diabetes mellitus was observed in
a 4-year-old rhesus macaque with typical history and
clinical and laboratory findings. Similarly, to the
previous data, increased blood glucose levels,
glycosuria,
polyuria,
polydipsia,
polyphagia,
dehydration, weight loss, anorexia, and weariness were
detected. Unfortunately, the patient was lost despite all
therapy efforts. Evaluation of this case can be valuable
due to spontaneous occurrence of diabetes mellitus
following severe pancreatic islet amyloidosis.
Additionally, the case showed that although it
contradicted with many reports, the disease could take
place in early courses of life in monkeys. Furthermore, it
is known that this is the first reported case of
spontaneous pancreatic amyloidosis and diabetes
mellitus of a monkey in Turkey.
Acknowledgments: We would like to thank
Dr Yucel Meral for their clinical support.
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